Dynamics of Phosphates and Normal Biological Function

**23**

**Chapter 2**

**Abstract**

the Environment?

aquatic plants such as *Pistia stratiotes.*

**1. Phosphorus: an introduction**

volume of 17 cm3

*D. Sayantan and Sumona Sanyal Das*

Phosphorus: A Boon or Curse for

Phosphorus, a limiting nutrient of biosphere, exists as dissolved inorganic phos-

phorus (DIP), dissolved organic phosphorus (DOP), particulate inorganic phosphorus (PIP) and particulate organic phosphorus (POP) in water of soil as well as ponds, lakes, etc. The only available phosphorus for plants are DIP, while the other forms need to be converted to DIP by the decomposing microorganisms of the soil. The heavy metals (such as arsenic and chromium), which are the menace of both terrestrial and aquatic environment, are taken up by the plants and animals causing toxicity at physiological level. However, the metal (Cr and As) toxicity can be mitigated competitively by phosphorus, since the latter is a structural analogue. Since, phosphorus is an essential nutrient, plants prefer it over Cr or As. At the same time, if excess of phosphorus is applied in the soil in the form of fertilisers, it gets discharged into the water bodies (ponds, lakes, etc.) through agricultural runoff, causing eutrophication followed by harming the health of the water bodies. This can be further mitigated by employing the phenomenon of luxury uptake by the

**Keywords:** arsenic, chromium, eutrophication, luxury uptake, phosphorus

number of phosphorus is 15, and it has a density of 1.823 g/cm3

and black phosphorus, which commonly exists [5].

Phosphorus (P) (*fos'furus* = light-bearing) is a reactive, non-metallic, multivalent chemical element of the nitrogen family group 15 [Va] of the periodic table, along with arsenic, antimony and bismuth and moscovium [1]. The atomic

(−3, +3 and +5) and one naturally occurring isotope (31P) (http://epathshala.nic. in/QR/books/12Chemistry/7.1.pdf). Phosphorus has 15 protons and there are 17 known isotopes. It is the 11th most abundant element in the earth's crust, consisting approximately of 0.1% by weight [2]. It was discovered and named by Hennig Brand who was a Hamburg merchant and alchemist by profession in 1669 by boiling and evaporating about 1100 l of urine and heating the residue with sand (which yield hot gases and vapour) and condensing it with cold water. The final substance to condense was soft, waxy white solid material which glowed in the dark. This element was named phosphorus [3, 4]. Phosphorus exists in several allotropic forms showing different properties, three of which are white phosphorus, red phosphorus

/mol, an atomic mass of 30.97 g/mol, three main oxidation states

(298 K), an atomic

#### **Chapter 2**

## Phosphorus: A Boon or Curse for the Environment?

*D. Sayantan and Sumona Sanyal Das*

#### **Abstract**

Phosphorus, a limiting nutrient of biosphere, exists as dissolved inorganic phosphorus (DIP), dissolved organic phosphorus (DOP), particulate inorganic phosphorus (PIP) and particulate organic phosphorus (POP) in water of soil as well as ponds, lakes, etc. The only available phosphorus for plants are DIP, while the other forms need to be converted to DIP by the decomposing microorganisms of the soil. The heavy metals (such as arsenic and chromium), which are the menace of both terrestrial and aquatic environment, are taken up by the plants and animals causing toxicity at physiological level. However, the metal (Cr and As) toxicity can be mitigated competitively by phosphorus, since the latter is a structural analogue. Since, phosphorus is an essential nutrient, plants prefer it over Cr or As. At the same time, if excess of phosphorus is applied in the soil in the form of fertilisers, it gets discharged into the water bodies (ponds, lakes, etc.) through agricultural runoff, causing eutrophication followed by harming the health of the water bodies. This can be further mitigated by employing the phenomenon of luxury uptake by the aquatic plants such as *Pistia stratiotes.*

**Keywords:** arsenic, chromium, eutrophication, luxury uptake, phosphorus

#### **1. Phosphorus: an introduction**

Phosphorus (P) (*fos'furus* = light-bearing) is a reactive, non-metallic, multivalent chemical element of the nitrogen family group 15 [Va] of the periodic table, along with arsenic, antimony and bismuth and moscovium [1]. The atomic number of phosphorus is 15, and it has a density of 1.823 g/cm3 (298 K), an atomic volume of 17 cm3 /mol, an atomic mass of 30.97 g/mol, three main oxidation states (−3, +3 and +5) and one naturally occurring isotope (31P) (http://epathshala.nic. in/QR/books/12Chemistry/7.1.pdf). Phosphorus has 15 protons and there are 17 known isotopes. It is the 11th most abundant element in the earth's crust, consisting approximately of 0.1% by weight [2]. It was discovered and named by Hennig Brand who was a Hamburg merchant and alchemist by profession in 1669 by boiling and evaporating about 1100 l of urine and heating the residue with sand (which yield hot gases and vapour) and condensing it with cold water. The final substance to condense was soft, waxy white solid material which glowed in the dark. This element was named phosphorus [3, 4]. Phosphorus exists in several allotropic forms showing different properties, three of which are white phosphorus, red phosphorus and black phosphorus, which commonly exists [5].

White phosphorus consists of tetrahedral P4 molecules, in which each atom is bound to the other three atoms by a single bond. This form generally exists (in liquid and gaseous) as tetrahedral P4 molecules at temperature up to 800°C that change to P2 molecules at temperature higher than 800°C [6]. White phosphorus is the most reactive and least stable; for this reason it should be stored under water, as it is dangerously reactive in air, and it must be handled with forceps, as contact with the skin may result in severe burns. Due to its high reactivity, white phosphorus is never found as a free element on earth. It is also very poisonous, 50 mg of which constitutes an approximate fatal dose. Exposure to white phosphorus should not exceed 0.1 mg/m3 (8-h time weighted average—40-h work week) [7]. It is the most volatile and least dense of all allotropes [8]. Because of its dehydrating nature, it has a corrosive property [9].

Red phosphorus is polymeric in structure. It is non-poisonous, odourless and insoluble in water. It can be viewed as a derivative of P4. It can be produced by heating white phosphorus to around 250°C within the sight of daylight. Freshly prepared, bright red phosphorus is highly reactive and ignites at about 300°C, though it is still more stable than white phosphorus, which ignites at about 30°C.

Black phosphorus is a layered semiconducting material similar in appearance to graphite with numerous uses in optoelectronic, semiconductor and photovoltaic applications. In a two-dimensional form, black phosphorus is known as phosphorene and has similar properties to other 2D semiconductor materials such as graphene. Among other allotropes, this form is the most thermodynamically stable and densest allotropes under ambient temperature and pressure.

#### **2. Sources of phosphorus**

Phosphorus is a naturally occurring element that exists in minerals, soil, living organisms and water. Phosphorus occurs in nature as orthophosphate (PO4 <sup>3</sup><sup>−</sup>), principally in the mineral apatite Ca5(PO4)3(F, Cl, OH) but also in monazite (Ce, La, Nd, Th) (PO4, SiO4) and xenotime YPO4. It is widely dispersed at trace levels in minerals such as olivine, pyroxene, amphibole and mica and is also present in biological materials such as the bone. Phosphorus is a major element in all plants. In the natural world, phosphorus is never encountered in its elemental form but only as phosphates, which consists of a phosphorus atom bonded to four oxygen atoms. This can exist as the negatively charged phosphate ion (PO4 <sup>3</sup><sup>−</sup>), which is how it occurs in minerals, or as organophosphates in which there are organic molecules attached to one, two or three of the oxygen atoms. Phosphorus exists in many different forms in soil. In general, these sources can be grouped into three general forms: (i) organic phosphorus, (ii) soluble phosphorus and (iii) adsorbed phosphorus. Phosphorus (in the forms hydrogen phosphate [HPO4 <sup>2</sup><sup>−</sup>] and dihydrogen phosphate [H2PO4 <sup>−</sup>]) is least mobile in soil. The comprehension of forms of phosphorus will enable to understand mobility of phosphorus in soil and the extent to which phosphorus can move within the environment.

Organic phosphorus account for 15–80% of the phosphorus in soils, the exact amount being dependent upon the nature of the soil and its composition. It is the principal form of phosphorus in the manure of most animals. About two-thirds of the phosphorus in fresh manure is in the organic form. Nucleic acid constitutes 1–5% of total organic phosphorus in soil.

Soluble phosphorus or available inorganic phosphorus include small amounts of organic phosphorus, as well as the orthophosphates, H2PO4 <sup>−</sup> and HPO4 <sup>2</sup><sup>−</sup> (the primary forms of phosphorus), taken up by algae and plants (aquatic and terrestrial). The soluble form accounts for the smallest proportion of the total phosphorus

**25**

**Figure 1.**

*Phosphorus cycle (image source: Ref. [11]).*

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

is adsorbed by plants in the ionic forms H2PO4

surface runoff concentrations [10].

cally available phosphorus enters the ecosystem.

**3. Presence of phosphorus in environment**

and "fixed," or tightly bound, phosphorus compounds.

in most of the soils. When fertiliser or manure (both containing mostly soluble phosphorus) is added to soil, the soil's pool of soluble phosphorus increases. With time, soluble phosphorus is transformed slowly to less-soluble forms. The effluents of sewage treatment plants contain mostly soluble phosphorus. Phosphorus

phosphorus does not leach. In fact, it moves very little, even with large amounts of precipitation or irrigation. Attached phosphorus includes labile or loosely bound,

Agricultural fields lose adsorbed phosphorus in a mechanism to transport phosphorus to water bodies. Phosphorus travel to surface water attached to particles of soil or manure. Phosphorus can also dissolve into runoff water as it passes over the surface of the field. Soil particles strip soluble phosphorus from the water as it moves through the soil profile. Leaching of phosphorus usually is not a significant concern. The concentration of phosphorus in soil leachate is significantly less than

However, special situations can produce higher concentrations of phosphorus in groundwater. The capacity of soil to absorb phosphorus can be overwhelmed on sandy soils or when the water table is close to the soil surface. Also, cracking in soils

Increased use of phosphorus in fertiliser had unsustainable consequences. Since phosphorus remains a finite, diminishing and irreplaceable resource, this is affecting global phosphorus cycles. Phosphorus in rocks is unavailable to organisms, but now increased used of phosphorus in fertilisers has tripled the rate at which biologi-

The phosphorus cycle (**Figure 1**) is the process by which phosphorus moves through the lithosphere, hydrosphere and biosphere. Phosphorus being a constituent of nucleic acids as well as cell membranes and phosphate/phosphorylated compounds is a nutrient of major importance for the biosphere. Calcium phosphate is also the primary component of mammalian bones and teeth and is used in a variety of other biological functions. The phosphorus cycle is an extremely slow process, as various weather conditions (e.g. rain and erosion) help to wash the phosphorus

creates channels allowing surface water to travel directly to groundwater.

<sup>−</sup> and HPO4

<sup>−</sup> [10]. The adsorbed

#### *Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

*Contemporary Topics about Phosphorus in Biology and Materials*

exceed 0.1 mg/m3

a corrosive property [9].

**2. Sources of phosphorus**

phosphate [H2PO4

White phosphorus consists of tetrahedral P4 molecules, in which each atom is bound to the other three atoms by a single bond. This form generally exists (in liquid and gaseous) as tetrahedral P4 molecules at temperature up to 800°C that change to P2 molecules at temperature higher than 800°C [6]. White phosphorus is the most reactive and least stable; for this reason it should be stored under water, as it is dangerously reactive in air, and it must be handled with forceps, as contact with the skin may result in severe burns. Due to its high reactivity, white phosphorus is never found as a free element on earth. It is also very poisonous, 50 mg of which constitutes an approximate fatal dose. Exposure to white phosphorus should not

volatile and least dense of all allotropes [8]. Because of its dehydrating nature, it has

Red phosphorus is polymeric in structure. It is non-poisonous, odourless and insoluble in water. It can be viewed as a derivative of P4. It can be produced by heating white phosphorus to around 250°C within the sight of daylight. Freshly prepared, bright red phosphorus is highly reactive and ignites at about 300°C, though

Black phosphorus is a layered semiconducting material similar in appearance to graphite with numerous uses in optoelectronic, semiconductor and photovoltaic applications. In a two-dimensional form, black phosphorus is known as phosphorene and has similar properties to other 2D semiconductor materials such as graphene. Among other allotropes, this form is the most thermodynamically stable and

Phosphorus is a naturally occurring element that exists in minerals, soil, living

principally in the mineral apatite Ca5(PO4)3(F, Cl, OH) but also in monazite (Ce, La, Nd, Th) (PO4, SiO4) and xenotime YPO4. It is widely dispersed at trace levels in minerals such as olivine, pyroxene, amphibole and mica and is also present in biological materials such as the bone. Phosphorus is a major element in all plants. In the natural world, phosphorus is never encountered in its elemental form but only as phosphates, which consists of a phosphorus atom bonded to four oxygen

it occurs in minerals, or as organophosphates in which there are organic molecules attached to one, two or three of the oxygen atoms. Phosphorus exists in many different forms in soil. In general, these sources can be grouped into three general forms: (i) organic phosphorus, (ii) soluble phosphorus and (iii) adsorbed phos-

phorus will enable to understand mobility of phosphorus in soil and the extent to

Organic phosphorus account for 15–80% of the phosphorus in soils, the exact amount being dependent upon the nature of the soil and its composition. It is the principal form of phosphorus in the manure of most animals. About two-thirds of the phosphorus in fresh manure is in the organic form. Nucleic acid constitutes

Soluble phosphorus or available inorganic phosphorus include small amounts

primary forms of phosphorus), taken up by algae and plants (aquatic and terrestrial). The soluble form accounts for the smallest proportion of the total phosphorus

<sup>−</sup>]) is least mobile in soil. The comprehension of forms of phos-

<sup>3</sup><sup>−</sup>),

<sup>3</sup><sup>−</sup>), which is how

<sup>2</sup><sup>−</sup>] and dihydrogen

<sup>−</sup> and HPO4

<sup>2</sup><sup>−</sup> (the

organisms and water. Phosphorus occurs in nature as orthophosphate (PO4

atoms. This can exist as the negatively charged phosphate ion (PO4

phorus. Phosphorus (in the forms hydrogen phosphate [HPO4

of organic phosphorus, as well as the orthophosphates, H2PO4

which phosphorus can move within the environment.

1–5% of total organic phosphorus in soil.

it is still more stable than white phosphorus, which ignites at about 30°C.

densest allotropes under ambient temperature and pressure.

(8-h time weighted average—40-h work week) [7]. It is the most

**24**

in most of the soils. When fertiliser or manure (both containing mostly soluble phosphorus) is added to soil, the soil's pool of soluble phosphorus increases. With time, soluble phosphorus is transformed slowly to less-soluble forms. The effluents of sewage treatment plants contain mostly soluble phosphorus. Phosphorus is adsorbed by plants in the ionic forms H2PO4 <sup>−</sup> and HPO4 <sup>−</sup> [10]. The adsorbed phosphorus does not leach. In fact, it moves very little, even with large amounts of precipitation or irrigation. Attached phosphorus includes labile or loosely bound, and "fixed," or tightly bound, phosphorus compounds.

Agricultural fields lose adsorbed phosphorus in a mechanism to transport phosphorus to water bodies. Phosphorus travel to surface water attached to particles of soil or manure. Phosphorus can also dissolve into runoff water as it passes over the surface of the field. Soil particles strip soluble phosphorus from the water as it moves through the soil profile. Leaching of phosphorus usually is not a significant concern. The concentration of phosphorus in soil leachate is significantly less than surface runoff concentrations [10].

However, special situations can produce higher concentrations of phosphorus in groundwater. The capacity of soil to absorb phosphorus can be overwhelmed on sandy soils or when the water table is close to the soil surface. Also, cracking in soils creates channels allowing surface water to travel directly to groundwater.

Increased use of phosphorus in fertiliser had unsustainable consequences. Since phosphorus remains a finite, diminishing and irreplaceable resource, this is affecting global phosphorus cycles. Phosphorus in rocks is unavailable to organisms, but now increased used of phosphorus in fertilisers has tripled the rate at which biologically available phosphorus enters the ecosystem.

#### **3. Presence of phosphorus in environment**

The phosphorus cycle (**Figure 1**) is the process by which phosphorus moves through the lithosphere, hydrosphere and biosphere. Phosphorus being a constituent of nucleic acids as well as cell membranes and phosphate/phosphorylated compounds is a nutrient of major importance for the biosphere. Calcium phosphate is also the primary component of mammalian bones and teeth and is used in a variety of other biological functions. The phosphorus cycle is an extremely slow process, as various weather conditions (e.g. rain and erosion) help to wash the phosphorus

**Figure 1.** *Phosphorus cycle (image source: Ref. [11]).*

#### *Contemporary Topics about Phosphorus in Biology and Materials*

found in rocks into the soil. In the soil, the organic matter (e.g. plants and fungi) absorbs the phosphorus to be used for various biological processes [11].

Phosphorus is an important element for all forms of life. As phosphate (PO4 <sup>3</sup><sup>−</sup>), it makes up an important part of the structural framework that holds DNA and RNA together. Phosphates are also a critical component of ATP, the cellular energy carrier, as they serve as an energy release for organisms to use in building proteins or contacting muscles. Like calcium, phosphorus is important to vertebrates; in the human body, 80% of phosphorus is found in teeth and bones [11].

The phosphorus cycle differs from the other major biogeochemical cycles in that it does not include a gas phase, although small amounts of phosphoric acid (H3PO4) may make their way into the atmosphere, contributing, in some cases, to acid rain. The water, carbon, nitrogen and sulphur cycles all include at least one phase in which the element is in its gaseous state. Very less amount of phosphorus circulates in the atmosphere because under normal temperatures and pressures condition of the earth, phosphorus and its various compounds are not gases. The largest reservoir of phosphorus is found in the lithosphere [11].

Weathering: since the main source of phosphorus is found in rocks, the first step of the phosphorus cycle involves the extraction of phosphorus from the rocks by weathering. From weathering the released phosphorus is transported to the soil by wind or water as inorganic phosphate. Absorption by plant and animal: inorganic phosphate is absorbed and assimilated by plants. In most soil, the amount of available phosphorus is about 0.01% of the total phosphorus in soil. From the plant, phosphorus moves through the food chain in the organic form. Once the living organisms die, the organically bound P is returned to the environment by the process of degradation by the decomposition. Organically the dead organic matter is acted upon by the phosphatising bacteria to release inorganic phosphorus from bound organic form. Phosphorus is also returned from shallow marine deposits in fish harvest and guano deposits in fish-eating birds and geological uplift. Turnover of organic phosphorus occurs due to phosphatase activity associated with root activity and microbial populations. The precipitation of phosphorus in marine habitats limit primary productivity [1, 11].

#### **4. Role of phosphorus in mitigating arsenic and chromium toxicity from plants**

Since phosphorus is a limiting nutrient for terrestrial biological productivity, it commonly plays a key role in net carbon uptake in terrestrial ecosystems. Although the inorganic phosphorus of soil causes harm to the environment in the form of eutrophication, it has some beneficial effects too. The phosphorus present in soils helps in mitigation of heavy metal toxicity in plants. Increasing population; rapid urbanisation; rapidly expanding industrial areas; use of fertilisers, pesticides and manures; and atmospheric decomposition have added lots of pollutants in the soil [12, 13]. Heavy metals like arsenic (As) and chromium (Cr) are highly toxic and cause ill effects at very low concentrations [14]. They are posing a major environmental challenge; since they do not undergo microbial and chemical degradation, they become persistent and bioaccumulative in nature [15, 16]. They affect soil chemistry by altering pH and conductivity of soil and also cause oxidative and physiological toxicity in the plants [17]. Many technologies like electrokinetic (EK) technique [18], electrokinetic-geosynthetic approach [19] and excavation and physical removal of the soil [20] are used to clean heavy metal pollutants. Each remediation technology has its specific benefits and limitations, but in general none of them is cost-effective [21]. Phytoremediation has the benefit of being a relatively

**27**

**Figure 2.**

*Ref. [23]).*

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

was amended along with the AsO4

total P stored in root and shoot tissues.

and 64%, respectively, with respect to control (0 μM PO4

low-cost, natural solution to an environmental problem [22]. Some plants are hyperaccumulators of metal. If the hyperaccumulator plants are edible, the roots will take up the heavy metals from the soil, leading to bioaccumulation of heavy metals in the entire plant body, which will ultimately enter into the food chain and pose health hazards to higher organisms. So, in order to stop the entry of heavy metals into the food chain, it is excellent to apply phosphorus in the soil that will alleviate the uptake and accumulation of heavy metals in food crops growing in the soil contaminated with heavy metals like arsenic and chromium (**Figure 2**) [23–25]. Literatures suggest that arsenate and phosphate share a common transport pathway via roots of the higher plants [23, 26, 27]. Since phosphate is a growthsupporting nutrient, plants prefer its uptake over arsenate. With the understanding of the above molecular analogy between arsenate and phosphate, the most cost-effective method for amelioration of arsenic toxicities, at the oxidative and physiological levels, in the agriculture would be the prevention of its entry in the food chain using phosphate amendments. Thus, a study by Sayantan and Shardendu [23] has been designed to determine the effect of arsenate-phosphate amendments on the variation in the physiological and oxidative toxicities in the root and shoot tissues of *Amaranthus viridis L*. By differentiating the total As accumulation at the tissue level, it was found in the present study that the roots of *A. viridis* accumulated up to twofold higher amount of total As than that in shoot. When the PO4

and shoot tissues was significantly (p < 0.001) reduced by the maximum of 68.18

observations were further supported statistically by obtaining significant (p < 0.05) negative correlation coefficient values between the total As accumulation and the

Sayantan and Shardendu [24] showed the role of different levels of phosphorus amendments on chromium toxicity in *Raphanus sativus L.* The experiments were done with the design in which five different concentrations of Cr have been taken and at each Cr level; there were five amendments of P concentration. After completion of experiment, results showed chromium accumulation in plant roots

*Relationship between total As and P accumulations in root and shoot tissues of* A. viridis*, each with representations of Pearson's correlation coefficient (r-value) and level of significance (p value) (image source:* 

3−

<sup>3</sup><sup>−</sup> supplies, the total As accumulated in the root

<sup>3</sup><sup>−</sup> amendment). These

#### *Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

*Contemporary Topics about Phosphorus in Biology and Materials*

found in rocks into the soil. In the soil, the organic matter (e.g. plants and fungi)

Phosphorus is an important element for all forms of life. As phosphate (PO4

The phosphorus cycle differs from the other major biogeochemical cycles in that it does not include a gas phase, although small amounts of phosphoric acid (H3PO4) may make their way into the atmosphere, contributing, in some cases, to acid rain. The water, carbon, nitrogen and sulphur cycles all include at least one phase in which the element is in its gaseous state. Very less amount of phosphorus circulates in the atmosphere because under normal temperatures and pressures condition of the earth, phosphorus and its various compounds are not gases. The largest reser-

Weathering: since the main source of phosphorus is found in rocks, the first step of the phosphorus cycle involves the extraction of phosphorus from the rocks by weathering. From weathering the released phosphorus is transported to the soil by wind or water as inorganic phosphate. Absorption by plant and animal: inorganic phosphate is absorbed and assimilated by plants. In most soil, the amount of available phosphorus is about 0.01% of the total phosphorus in soil. From the plant, phosphorus moves through the food chain in the organic form. Once the living organisms die, the organically bound P is returned to the environment by the process of degradation by the decomposition. Organically the dead organic matter is acted upon by the phosphatising bacteria to release inorganic phosphorus from bound organic form. Phosphorus is also returned from shallow marine deposits in fish harvest and guano deposits in fish-eating birds and geological uplift. Turnover of organic phosphorus occurs due to phosphatase activity associated with root activity and microbial populations. The precipitation of phosphorus in marine

**4. Role of phosphorus in mitigating arsenic and chromium toxicity** 

Since phosphorus is a limiting nutrient for terrestrial biological productivity, it commonly plays a key role in net carbon uptake in terrestrial ecosystems. Although the inorganic phosphorus of soil causes harm to the environment in the form of eutrophication, it has some beneficial effects too. The phosphorus present in soils helps in mitigation of heavy metal toxicity in plants. Increasing population; rapid urbanisation; rapidly expanding industrial areas; use of fertilisers, pesticides and manures; and atmospheric decomposition have added lots of pollutants in the soil [12, 13]. Heavy metals like arsenic (As) and chromium (Cr) are highly toxic and cause ill effects at very low concentrations [14]. They are posing a major environmental challenge; since they do not undergo microbial and chemical degradation, they become persistent and bioaccumulative in nature [15, 16]. They affect soil chemistry by altering pH and conductivity of soil and also cause oxidative and physiological toxicity in the plants [17]. Many technologies like electrokinetic (EK) technique [18], electrokinetic-geosynthetic approach [19] and excavation and physical removal of the soil [20] are used to clean heavy metal pollutants. Each remediation technology has its specific benefits and limitations, but in general none of them is cost-effective [21]. Phytoremediation has the benefit of being a relatively

it makes up an important part of the structural framework that holds DNA and RNA together. Phosphates are also a critical component of ATP, the cellular energy carrier, as they serve as an energy release for organisms to use in building proteins or contacting muscles. Like calcium, phosphorus is important to vertebrates; in the

<sup>3</sup><sup>−</sup>),

absorbs the phosphorus to be used for various biological processes [11].

human body, 80% of phosphorus is found in teeth and bones [11].

voir of phosphorus is found in the lithosphere [11].

habitats limit primary productivity [1, 11].

**from plants**

**26**

low-cost, natural solution to an environmental problem [22]. Some plants are hyperaccumulators of metal. If the hyperaccumulator plants are edible, the roots will take up the heavy metals from the soil, leading to bioaccumulation of heavy metals in the entire plant body, which will ultimately enter into the food chain and pose health hazards to higher organisms. So, in order to stop the entry of heavy metals into the food chain, it is excellent to apply phosphorus in the soil that will alleviate the uptake and accumulation of heavy metals in food crops growing in the soil contaminated with heavy metals like arsenic and chromium (**Figure 2**) [23–25].

Literatures suggest that arsenate and phosphate share a common transport pathway via roots of the higher plants [23, 26, 27]. Since phosphate is a growthsupporting nutrient, plants prefer its uptake over arsenate. With the understanding of the above molecular analogy between arsenate and phosphate, the most cost-effective method for amelioration of arsenic toxicities, at the oxidative and physiological levels, in the agriculture would be the prevention of its entry in the food chain using phosphate amendments. Thus, a study by Sayantan and Shardendu [23] has been designed to determine the effect of arsenate-phosphate amendments on the variation in the physiological and oxidative toxicities in the root and shoot tissues of *Amaranthus viridis L*. By differentiating the total As accumulation at the tissue level, it was found in the present study that the roots of *A. viridis* accumulated up to twofold higher amount of total As than that in shoot. When the PO4 3− was amended along with the AsO4 <sup>3</sup><sup>−</sup> supplies, the total As accumulated in the root and shoot tissues was significantly (p < 0.001) reduced by the maximum of 68.18 and 64%, respectively, with respect to control (0 μM PO4 <sup>3</sup><sup>−</sup> amendment). These observations were further supported statistically by obtaining significant (p < 0.05) negative correlation coefficient values between the total As accumulation and the total P stored in root and shoot tissues.

Sayantan and Shardendu [24] showed the role of different levels of phosphorus amendments on chromium toxicity in *Raphanus sativus L.* The experiments were done with the design in which five different concentrations of Cr have been taken and at each Cr level; there were five amendments of P concentration. After completion of experiment, results showed chromium accumulation in plant roots

#### **Figure 2.**

*Relationship between total As and P accumulations in root and shoot tissues of* A. viridis*, each with representations of Pearson's correlation coefficient (r-value) and level of significance (p value) (image source: Ref. [23]).*

was dose-dependent and increased as the concentration of Cr supply is enhanced. However, the impact of amendment of phosphorus concentration on Cr accumulation was notable. At each Cr supply, accumulation of Cr decreased as the levels of P increased, showing a reciprocal correlation (−0.960 ≤ r ≤ −0.762) among these two parameters.

Qian et al. [28] showed that chromium could damage the thylakoid structure and impinge on the chloroplast, therefore affecting algal growth. P availability can alleviate Cr toxicity in *C. vulgaris* by decreasing the absorption of Cr and changing the absorption of other metal ions. It is, therefore, necessary to consider the phosphorus availability when the toxicity of metal compounds is evaluated.

It is known that the nutrients present in the soil influence the Cr uptake by *Raphanus sativus* [24]. Sayantan and Shardendu [25] examine the role of Cr toxicity on root and shoot tissues of *Spinacia oleracea* L. (spinach) and its variation when different levels of P were amended with the Cr supply. The path of movement of Cr and P is similar in plants; however, P is preferred over Cr. Thus, P competitively inhibits the Cr uptake. With the phosphorus amendment in the growth medium, accumulation of chromium decreased up to 55% in root and 50% in shoot tissues. These results showed two general observations, i.e. increasing Cr concentrations induced an enhancement in toxicity at both physiological and oxidative levels; and the amendment of P at each Cr supply ameliorated the toxic effects in both kinds of tissues of *S. oleracea.*

Phosphorus is essential to all living beings also; it is one of the main nutrients for animal and plant growth. Phosphorus is used by plants in photosynthesis. When there is less phosphorus during photosynthesis, the production of sugars is restricted. This directly affects the colour of the plant. Phosphorus-deficient plants will not be green but have a purple hue to them. A lack of sugar in the plants will restrict the growth of the plant since sugar is the main source of energy in a plant.

Phosphorus acts as a catalyst. When fertiliser is added to plants, it is often thought of to assist in allowing them to grow quicker and stronger. This is due to the phosphorus in the fertiliser. Phosphorus speeds up plant growth as well as the quality of plant growth.

Phosphorus is also a key component in the structures of life. It is found linking DNA and RNA together and being found in bones of animals. Phosphorus is also involved in ATP that forms during photosynthesis.

Phosphorus is vital to the environment because it allows plant growth that is necessary to keep the ecosystem balanced and flourishing. It allows plants to grow robust to feed the animals that eat them. Healthier plants also allow more oxygen to be released in the air. In manageable amounts, phosphorus can help any ecosystem thrive by providing more food and oxygen. The only measure to check the use of phosphorus for the environment is to reduce the usage of chemical fertiliser and shift towards organic farming. This will reduce eutrophication and, in other words, improve the health of lakes.

#### **5. Eutrophication**

When sewage and agricultural runoff containing phosphates or other nutrients enter water bodies, they cause overnutrition, leading to eutrophication. "Eutrophication is an enrichment of water by nutrient salts that causes structural changes to the ecosystem such as: increased production of algae and aquatic plants, depletion of fish species, general deterioration of water quality and other effects that reduce and preclude use" (Organisation for Economic Cooperation and Development). Eutrophication is a major environmental issue

**29**

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

medium within 60 days at 50mg/L supply.

as it causes degradation in the quality of water and is one of the major hindrances for achieving the quality objectives established by Designated Best Use Water Quality Criteria by Central Pollution Control Board, India, 2019 [29]. As per the International Lake Environment Committee Foundation, Japan, about 54% of lakes are affected by the phenomenon of eutrophication in Asia ("http://www. eniscuola.net/en/2016/11/03/what-is-eutrophication-causes-effects-andcontrol/"; "https://www.ilec.or.jp/en/") [30]. Eutrophication occurs naturally over centuries as lakes age and is filled in with sediments [31]. However, anthropogenic activities like discharge of growth-limiting nutrients like phosphorus have accelerated the rate and extent of eutrophication. Any aquatic body starts its life cycle as oligotrophic, i.e. clear body of water. Eutrophication is distinguished by a remarkable increase of algae (very simple, non-flowering aquatic plant) due to the considerable availability of one or more growth factors obligatory for photosynthesis, such as sunlight, carbon dioxide and nutrients (primarily phosphorus) [32]. With the introduction of nutrients through runoff, algae start to grow in an uncontrolled manner. With this growth, increasingly large biomass is formed which is destined to decay. In deep water, pond collects a good amount of organic substance, represented by the algae having reached the end of their life cycle. Eventually, there is algal bloom when the lake becomes marsh or debris. An excessive consumption of oxygen is required by microorganisms to destroy all the dead algae. This created an oxygen-free environment in the lake bottom, anaerobic organisms being responsible for the degradation of the biomass [33]. The microorganisms, decomposing the organic substance in the absence of oxygen-free compounds that are toxic, such as ammonia and hydrogen sulphide (H2S), were formed. The absence of oxygen reduces biodiversity causing, in certain cases, even the death of animal and plant species. All this happens when the rate of degradation of the algae by microorganisms is greater than that of oxygen regeneration, which in summer is already present in low concentrations. The stage is eutrophic, when the lake is filled with sediment, while aquatic animal life will perish. It will then turn into dry land. The rate of eutrophication strikes a balance between the production of aquatic life and its destruction by bacterial decomposition. With large input of nutrients from human sources, bacterial decomposition cannot keep pace with productivity and sedimentation is accelerated whereby eutrophication is favoured. Lakes can be protected from eutrophication by providing measures for sewage treatment and preventing the sewage and agricultural runoff from entering the water bodies. Another method is to use aquatic plants as their high relative growth rates efficiently absorb nutrients from their surrounding media, thereby providing a simple and inexpensive solution for phosphorus-polluted water bodies. Experiments done by Shardendu et al. [34] proved that *Pistia stratiotes* L. accumulated the highest amount of tissue P (1 .06 ± 0 .22mg/g dw) than other common wetland species like *Eichhornia*, *Phragmites* and *Typha*. They further found out that up to 91% phosphate was removed from the surrounding

#### *Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

*Contemporary Topics about Phosphorus in Biology and Materials*

parameters.

tissues of *S. oleracea.*

quality of plant growth.

improve the health of lakes.

**5. Eutrophication**

involved in ATP that forms during photosynthesis.

was dose-dependent and increased as the concentration of Cr supply is enhanced. However, the impact of amendment of phosphorus concentration on Cr accumulation was notable. At each Cr supply, accumulation of Cr decreased as the levels of P increased, showing a reciprocal correlation (−0.960 ≤ r ≤ −0.762) among these two

Qian et al. [28] showed that chromium could damage the thylakoid structure and impinge on the chloroplast, therefore affecting algal growth. P availability can alleviate Cr toxicity in *C. vulgaris* by decreasing the absorption of Cr and changing the absorption of other metal ions. It is, therefore, necessary to consider the phos-

It is known that the nutrients present in the soil influence the Cr uptake by *Raphanus sativus* [24]. Sayantan and Shardendu [25] examine the role of Cr toxicity on root and shoot tissues of *Spinacia oleracea* L. (spinach) and its variation when different levels of P were amended with the Cr supply. The path of movement of Cr and P is similar in plants; however, P is preferred over Cr. Thus, P competitively inhibits the Cr uptake. With the phosphorus amendment in the growth medium, accumulation of chromium decreased up to 55% in root and 50% in shoot tissues. These results showed two general observations, i.e. increasing Cr concentrations induced an enhancement in toxicity at both physiological and oxidative levels; and the amendment of P at each Cr supply ameliorated the toxic effects in both kinds of

Phosphorus is essential to all living beings also; it is one of the main nutrients for animal and plant growth. Phosphorus is used by plants in photosynthesis. When there is less phosphorus during photosynthesis, the production of sugars is restricted. This directly affects the colour of the plant. Phosphorus-deficient plants will not be green but have a purple hue to them. A lack of sugar in the plants will restrict the growth of the plant since sugar is the main source of energy in a plant. Phosphorus acts as a catalyst. When fertiliser is added to plants, it is often thought of to assist in allowing them to grow quicker and stronger. This is due to the phosphorus in the fertiliser. Phosphorus speeds up plant growth as well as the

Phosphorus is also a key component in the structures of life. It is found linking DNA and RNA together and being found in bones of animals. Phosphorus is also

Phosphorus is vital to the environment because it allows plant growth that is necessary to keep the ecosystem balanced and flourishing. It allows plants to grow robust to feed the animals that eat them. Healthier plants also allow more oxygen to be released in the air. In manageable amounts, phosphorus can help any ecosystem thrive by providing more food and oxygen. The only measure to check the use of phosphorus for the environment is to reduce the usage of chemical fertiliser and shift towards organic farming. This will reduce eutrophication and, in other words,

When sewage and agricultural runoff containing phosphates or other nutrients enter water bodies, they cause overnutrition, leading to eutrophication. "Eutrophication is an enrichment of water by nutrient salts that causes structural changes to the ecosystem such as: increased production of algae and aquatic plants, depletion of fish species, general deterioration of water quality and other effects that reduce and preclude use" (Organisation for Economic Cooperation and Development). Eutrophication is a major environmental issue

phorus availability when the toxicity of metal compounds is evaluated.

**28**

as it causes degradation in the quality of water and is one of the major hindrances for achieving the quality objectives established by Designated Best Use Water Quality Criteria by Central Pollution Control Board, India, 2019 [29]. As per the International Lake Environment Committee Foundation, Japan, about 54% of lakes are affected by the phenomenon of eutrophication in Asia ("http://www. eniscuola.net/en/2016/11/03/what-is-eutrophication-causes-effects-andcontrol/"; "https://www.ilec.or.jp/en/") [30]. Eutrophication occurs naturally over centuries as lakes age and is filled in with sediments [31]. However, anthropogenic activities like discharge of growth-limiting nutrients like phosphorus have accelerated the rate and extent of eutrophication. Any aquatic body starts its life cycle as oligotrophic, i.e. clear body of water. Eutrophication is distinguished by a remarkable increase of algae (very simple, non-flowering aquatic plant) due to the considerable availability of one or more growth factors obligatory for photosynthesis, such as sunlight, carbon dioxide and nutrients (primarily phosphorus) [32]. With the introduction of nutrients through runoff, algae start to grow in an uncontrolled manner. With this growth, increasingly large biomass is formed which is destined to decay. In deep water, pond collects a good amount of organic substance, represented by the algae having reached the end of their life cycle. Eventually, there is algal bloom when the lake becomes marsh or debris. An excessive consumption of oxygen is required by microorganisms to destroy all the dead algae. This created an oxygen-free environment in the lake bottom, anaerobic organisms being responsible for the degradation of the biomass [33]. The microorganisms, decomposing the organic substance in the absence of oxygen-free compounds that are toxic, such as ammonia and hydrogen sulphide (H2S), were formed. The absence of oxygen reduces biodiversity causing, in certain cases, even the death of animal and plant species. All this happens when the rate of degradation of the algae by microorganisms is greater than that of oxygen regeneration, which in summer is already present in low concentrations. The stage is eutrophic, when the lake is filled with sediment, while aquatic animal life will perish. It will then turn into dry land. The rate of eutrophication strikes a balance between the production of aquatic life and its destruction by bacterial decomposition. With large input of nutrients from human sources, bacterial decomposition cannot keep pace with productivity and sedimentation is accelerated whereby eutrophication is favoured. Lakes can be protected from eutrophication by providing measures for sewage treatment and preventing the sewage and agricultural runoff from entering the water bodies. Another method is to use aquatic plants as their high relative growth rates efficiently absorb nutrients from their surrounding media, thereby providing a simple and inexpensive solution for phosphorus-polluted water bodies. Experiments done by Shardendu et al. [34] proved that *Pistia stratiotes* L. accumulated the highest amount of tissue P (1 .06 ± 0 .22mg/g dw) than other common wetland species like *Eichhornia*, *Phragmites* and *Typha*. They further found out that up to 91% phosphate was removed from the surrounding medium within 60 days at 50mg/L supply.

*Contemporary Topics about Phosphorus in Biology and Materials*

#### **Author details**

D. Sayantan1 \* and Sumona Sanyal Das2

1 Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru, Karnataka, India

2 Department of Zoology, Patna University, Patna, Bihar, India

\*Address all correspondence to: sayantan.d@christuniversity.in

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**31**

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

> [9] Aviv U, Kornhaber R, Harats M, Haik J. The burning issue of white phosphorus: A case report and review of the literature. Disaster and Military Medicine. 2017;**3**:6. DOI: 10.1186/

[10] Phosphorus in Soil Crop System. The National Academics of Sciences Engineering Medicine. The National Academics Press. Washington DC, USA: Soil and Water Quality. An Agenda for Agriculture; 1993. p. 284. Available from: https://nap.edu/read/2132/

[11] Lappalainen HK et al. Pan-

a holistic understanding of the

2016;**16**:14421-14461

2010;**41**(7):820-831

Eurasian Experiment (PEEX): Towards

feedbacks and interactions in the landatmosphere-ocean-society continuum in the northern Eurasian region. Atmospheric Chemistry and Physics.

[12] Zhang MK, Liu ZY, Wang H. Use of single extraction methods to predict bioavailability of heavy metals in polluted soils to rice. Communications in Soil Science and Plant Analysis.

[13] Khan S, Cao Q, Zheng YM, Huang YZ, Zhu YG. Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environmental Pollution. 2008;**152**(3):686-692

[14] Sood A, Uniyal PL, Prasanna R, Ahluwalia AS. Phytoremediation potential of aquatic macrophytes, Azolla. Ambio. 2012;**41**(2):122-137

[15] Ali H, Khan E, Ilahi I. Environmental

hazardous heavy metals: Environmental persistence, toxicity, and bioaccumulation. Journal of Chemistry. 2019; Article ID 6730305. DOI: 10.1155/2019/

chemistry and ecotoxicology of

6730305

s40696-017-0034-y

chapter11#284

[1] Ahemad M, Zaidi A, Khan MS, Oves M. Biological importance of phosphorus and phosphate solubilizing microbes. In: Khan MS, Zaidi A, editors. Phosphate Solubilizing Microbes for Crop Improvement. NY, USA: Nova

Science Publishers; 2011

**References**

December 2019]

1404219609

jennystanford.com

December 2019]

pediapress.com

[8] Oliveira P. The Elements:

[2] Helmenstine AM. Chemical Composition of the Earth's

Crust—Elements. ThoughtCo. 2019. Available from: thoughtco.com/ chemical-composition-of-earthscrust-elements-607576 [Accessed: 06

[3] Parkes GD, Mellor JW. Mellor's Modern Inorganic Chemistry. London, UK: Longman's Green and Co.; 1939

[4] Sommers AM. Phosphorus (Understanding the Elements of the Periodic Table). NY, USA: The Rosen Publishing Group; 2007. p. 25. ISBN:

[5] Cai Y, Zhang G, Zhang Y-W. Phosphorene: Physical Properties, Synthesis and Fabrication. NY, USA: Jenny Stanford Publishing Pte. Ltd.; 2019. ISBN 978-981-4774-64-2 (Hardcover), 978-0-203-71061-6 (e Book). Available from: www.

[6] Lumen. Introduction to Chemistry. Properties of Phosphorus. 2019. [Accessed: 06 December 2019]

[7] Los Alamos National Laboratory. Periodic Table of the Elements. 2019. Available from: https://depts. washington.edu/eooptic/linkfiles/ The%20Elements.pdf [Accessed: 06

Periodic Table Reference. Boppstrasse 64, Mainia, Germany: Pedia Press GmbH; 2011. Available from: http://

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

#### **References**

*Contemporary Topics about Phosphorus in Biology and Materials*

**30**

**Author details**

Karnataka, India

\* and Sumona Sanyal Das2

provided the original work is properly cited.

2 Department of Zoology, Patna University, Patna, Bihar, India

\*Address all correspondence to: sayantan.d@christuniversity.in

1 Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

D. Sayantan1

[1] Ahemad M, Zaidi A, Khan MS, Oves M. Biological importance of phosphorus and phosphate solubilizing microbes. In: Khan MS, Zaidi A, editors. Phosphate Solubilizing Microbes for Crop Improvement. NY, USA: Nova Science Publishers; 2011

[2] Helmenstine AM. Chemical Composition of the Earth's Crust—Elements. ThoughtCo. 2019. Available from: thoughtco.com/ chemical-composition-of-earthscrust-elements-607576 [Accessed: 06 December 2019]

[3] Parkes GD, Mellor JW. Mellor's Modern Inorganic Chemistry. London, UK: Longman's Green and Co.; 1939

[4] Sommers AM. Phosphorus (Understanding the Elements of the Periodic Table). NY, USA: The Rosen Publishing Group; 2007. p. 25. ISBN: 1404219609

[5] Cai Y, Zhang G, Zhang Y-W. Phosphorene: Physical Properties, Synthesis and Fabrication. NY, USA: Jenny Stanford Publishing Pte. Ltd.; 2019. ISBN 978-981-4774-64-2 (Hardcover), 978-0-203-71061-6 (e Book). Available from: www. jennystanford.com

[6] Lumen. Introduction to Chemistry. Properties of Phosphorus. 2019. [Accessed: 06 December 2019]

[7] Los Alamos National Laboratory. Periodic Table of the Elements. 2019. Available from: https://depts. washington.edu/eooptic/linkfiles/ The%20Elements.pdf [Accessed: 06 December 2019]

[8] Oliveira P. The Elements: Periodic Table Reference. Boppstrasse 64, Mainia, Germany: Pedia Press GmbH; 2011. Available from: http:// pediapress.com

[9] Aviv U, Kornhaber R, Harats M, Haik J. The burning issue of white phosphorus: A case report and review of the literature. Disaster and Military Medicine. 2017;**3**:6. DOI: 10.1186/ s40696-017-0034-y

[10] Phosphorus in Soil Crop System. The National Academics of Sciences Engineering Medicine. The National Academics Press. Washington DC, USA: Soil and Water Quality. An Agenda for Agriculture; 1993. p. 284. Available from: https://nap.edu/read/2132/ chapter11#284

[11] Lappalainen HK et al. Pan-Eurasian Experiment (PEEX): Towards a holistic understanding of the feedbacks and interactions in the landatmosphere-ocean-society continuum in the northern Eurasian region. Atmospheric Chemistry and Physics. 2016;**16**:14421-14461

[12] Zhang MK, Liu ZY, Wang H. Use of single extraction methods to predict bioavailability of heavy metals in polluted soils to rice. Communications in Soil Science and Plant Analysis. 2010;**41**(7):820-831

[13] Khan S, Cao Q, Zheng YM, Huang YZ, Zhu YG. Health risks of heavy metals in contaminated soils and food crops irrigated with wastewater in Beijing, China. Environmental Pollution. 2008;**152**(3):686-692

[14] Sood A, Uniyal PL, Prasanna R, Ahluwalia AS. Phytoremediation potential of aquatic macrophytes, Azolla. Ambio. 2012;**41**(2):122-137

[15] Ali H, Khan E, Ilahi I. Environmental chemistry and ecotoxicology of hazardous heavy metals: Environmental persistence, toxicity, and bioaccumulation. Journal of Chemistry. 2019; Article ID 6730305. DOI: 10.1155/2019/ 6730305

[16] Masindi V, Muedi KL. In: Saleh HE-DM, Aglan RF, editors. Environmental Contamination by Heavy Metals, Heavy Metals. IntechOpen; 2018. DOI: 10.5772/ intechopen.76082. Available from: https://www.intechopen.com/ books/heavy-metals/environmentalcontamination-by-heavy-metals

[17] Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry. 2010;**48**:909-930

[18] Maini G, Sharman AK, Knowles CJ, Sunderland G, Jackman SA. Electrokinetic remediation of metals and organics from historically contaminated soil. Journal of Chemical Technology and Biotechnology. 2000;**75**:657-664. DOI: 10.1002/1097-4660(200008)75:8<657:: AID-JCTB263>3.0.CO;2-5

[19] Jones CJEP. Earth Reinforcement and Soil Structures. London: Thomas Telford Publishing; 1996. p. 379

[20] Lambert M, Leven BA, Green RM. New Methods of Cleaning Up Heavy Metal in Soils and Water. Available from: https://cfpub.epa.gov/ ncer\_abstracts/index.cfm/fuseaction/ display.files/fileID/14295

[21] Comparing Costs of Remediation Technologies. National Research Council. Innovation in Groundwater and Soil Cleanup: From Concept to Commercialization. Washington DC: The National Academies Press; 1997. p. 10.17226/5781

[22] Peuke AD, Rennenberg H. Phytoremediation. EMBO Reports. 2005;**6**(6):497-501. DOI: 10.1038/ sj.embor.7400445

[23] Sayantan D, Shardendu. Phosphate amendments moderate the arsenate accumulation and its subsequent oxidative and physiological toxicities

in *Amaranthus viridis* L. Proceedings of the National Academy of Sciences, India, Section B: Biological Sciences. 2017;**87**:1343-1353. DOI: 10.1007/ s40011-016-0711-5

[24] Sayantan D, Shardendu. Amendment in phosphorus levels moderate the chromium toxicity in *Raphanus sativus* L. as assayed by antioxidant enzymes activities. Ecotoxicology and Environmental Safety. 2013;**95**:161-170

[25] Sayantan D, Shardendu. Phosphorus amendment competitively prevents chromium uptake and mitigates its toxicity in *Spinacea oleracea* L. Indian Journal of Experimental Biology. 2015;**53**:395-405

[26] Elias M, Wellner A, Goldin-Azulay K, Chabrier E, et al. The molecular basis of phosphate discrimination in arsenate-rich environments. Nature. 2012;**491**:134-137. DOI: 10.1038/ nature11517

[27] Zhao FJ, Ma JF, Meharg AA, McGrath SP. Arsenic uptake and metabolism in plants. The New Phytologist. 2009;**181**:777-794

[28] Qian H, Sun Z, Sun L, Jiang Y, Wei Y, Xie J, et al. Phosphorus availability changes chromium toxicity in the freshwater alga *Chlorella vulgaris*. Chemosphere. 2013;**93**(6):885-891. DOI: 10.1016/j.chemosphere.2013.05.035

[29] Central Pollution Control Board. Designated Best Use Water Quality Criteria. Forest and Climate Change, Government of India: Ministry of Environment; 2019. Available from: https://cpcb.nic.in/wqstandards/

[30] Available from: http://www. eniscuola.net/wp-content/uploads/ 2016/11/Pdf\_eutrophication.pdf

[31] Carpenter SR. Submersed vegetation: An internal factor in lake

**33**

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

ecosystem succession. The American

[32] De AK. Environmental Chemistry. 7th ed. Delhi: New Age International

[33] Penelope RV, Charles RV. Water Resources and the Quality of Natural Waters. London: Jones and Bartbett

Sharma D, Irfan S. Luxury uptake and removal of phosphorus from water column by representative aquatic plants and its implication for wetland management. ISRN Soil Science. 2012;**2012**:Article ID 516947, 9 pages.

[34] Shardendu S, Sayantan D,

DOI: 10.5402/2012/516947

Naturalist. 1981;**118**:372-383

Publishers; 2010. p. 214

publishers; 1992

*Phosphorus: A Boon or Curse for the Environment? DOI: http://dx.doi.org/10.5772/intechopen.91250*

ecosystem succession. The American Naturalist. 1981;**118**:372-383

*Contemporary Topics about Phosphorus in Biology and Materials*

in *Amaranthus viridis* L. Proceedings of the National Academy of Sciences, India, Section B: Biological Sciences. 2017;**87**:1343-1353. DOI: 10.1007/

[25] Sayantan D, Shardendu. Phosphorus amendment competitively prevents chromium uptake and mitigates its toxicity in *Spinacea oleracea* L. Indian Journal of Experimental Biology.

[26] Elias M, Wellner A, Goldin-Azulay K,

Chabrier E, et al. The molecular basis of phosphate discrimination in arsenate-rich environments. Nature. 2012;**491**:134-137. DOI: 10.1038/

[27] Zhao FJ, Ma JF, Meharg AA, McGrath SP. Arsenic uptake and metabolism in plants. The New Phytologist. 2009;**181**:777-794

[28] Qian H, Sun Z, Sun L, Jiang Y, Wei Y, Xie J, et al. Phosphorus

availability changes chromium toxicity in the freshwater alga *Chlorella vulgaris*. Chemosphere. 2013;**93**(6):885-891. DOI: 10.1016/j.chemosphere.2013.05.035

[29] Central Pollution Control Board. Designated Best Use Water Quality Criteria. Forest and Climate Change, Government of India: Ministry of Environment; 2019. Available from: https://cpcb.nic.in/wqstandards/

[30] Available from: http://www. eniscuola.net/wp-content/uploads/ 2016/11/Pdf\_eutrophication.pdf

[31] Carpenter SR. Submersed vegetation: An internal factor in lake

s40011-016-0711-5

[24] Sayantan D, Shardendu. Amendment in phosphorus levels moderate the chromium toxicity in *Raphanus sativus* L. as assayed by antioxidant enzymes activities. Ecotoxicology and Environmental

Safety. 2013;**95**:161-170

2015;**53**:395-405

nature11517

[16] Masindi V, Muedi KL. In: Saleh HE-DM, Aglan RF, editors. Environmental Contamination by Heavy Metals, Heavy Metals. IntechOpen; 2018. DOI: 10.5772/ intechopen.76082. Available from: https://www.intechopen.com/ books/heavy-metals/environmentalcontamination-by-heavy-metals

[17] Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry.

[18] Maini G, Sharman AK, Knowles CJ, Sunderland G, Jackman SA. Electrokinetic remediation of metals and organics from historically contaminated soil. Journal of Chemical Technology and Biotechnology. 2000;**75**:657-664. DOI: 10.1002/1097-4660(200008)75:8<657::

[19] Jones CJEP. Earth Reinforcement and Soil Structures. London: Thomas Telford Publishing; 1996. p. 379

Green RM. New Methods of Cleaning Up Heavy Metal in Soils and Water. Available from: https://cfpub.epa.gov/ ncer\_abstracts/index.cfm/fuseaction/

[21] Comparing Costs of Remediation Technologies. National Research Council. Innovation in Groundwater and Soil Cleanup: From Concept to Commercialization. Washington DC: The National Academies Press; 1997.

[22] Peuke AD, Rennenberg H. Phytoremediation. EMBO Reports. 2005;**6**(6):497-501. DOI: 10.1038/

[23] Sayantan D, Shardendu. Phosphate amendments moderate the arsenate accumulation and its subsequent oxidative and physiological toxicities

2010;**48**:909-930

AID-JCTB263>3.0.CO;2-5

[20] Lambert M, Leven BA,

display.files/fileID/14295

p. 10.17226/5781

sj.embor.7400445

**32**

[32] De AK. Environmental Chemistry. 7th ed. Delhi: New Age International Publishers; 2010. p. 214

[33] Penelope RV, Charles RV. Water Resources and the Quality of Natural Waters. London: Jones and Bartbett publishers; 1992

[34] Shardendu S, Sayantan D, Sharma D, Irfan S. Luxury uptake and removal of phosphorus from water column by representative aquatic plants and its implication for wetland management. ISRN Soil Science. 2012;**2012**:Article ID 516947, 9 pages. DOI: 10.5402/2012/516947

**35**

(porous/spongy bone namely trabecular) [1].

**Chapter 3**

*Pinki Dey*

**Abstract**

**1. Introduction**

Bone Mineralisation

The mineralisation term mentions the development of inorganic precipitation over an organic background. This process occurs in a life span of biological organism for the formation of bone, teeth, exoskeletons, egg shells, etc. So, basically bone mineralisation is defined as the process of deposition of minerals on the bone matrix for the development of bone. The human bone is made up of 60–70% minerals which include calcium phosphate in the form of hydroxyapatite followed by 20–40% organic matrix containing type I collagen fibres and less than 5% of water and lipids. During bone mineralisation process osteoblasts which are also known as bone forming cells, aids to the production of calcium phosphate crystals which are then aligned in the collagen based fibrous matrix. The bone mineralisation proce-

Bone is a multifaceted system which behaves as mechanical shield for providing support and security. Bone also plays an important role in haemostasis. Recently, it came to observation that bone also aids in functioning of endocrine glands. To fulfil all these purposes, bone is a well architecture behaving as a functionally graded structure from millimetre to nanometre range. As a result of this gradation bone shows an unusual amalgamation of high stiffness and toughness which are frequently inversely associated. Broadly, bone is made up of organic and mineral part where the organic part is comprised of type I collagen whereas the mineral part contains the calcium deficient hydroxyapatite. The mineral part of the bone is interlinked with collagen in such an organised manner that it not only provides flexibility and ductility to the structure but also shows stiffness. This organisation of bone when observed at nanoscale range it behaves as a composite which acts as a shield to the brittle hydroxyapatite from damaging and also helps in carrying load by transferring forces around the bond hence thereby reducing the stress formation in the composite matrix. The fibres of the collagen intermingled with mineral part are arranged in very different manner when observed on microscopic range. This arrangement at micrometre level completely depends on the rate of bone formation or bone location on the substrate. This is due to the various functions played by the bone tissue, i.e., during fracture rapid bone formation development of bone during growth, unhurried bone formation in order to adjust in accordance to mechanical requirements so as to avoid any damage to its structure as well as maintaining its mechanical assets. Bone can be divided into two major categories depending on its mechanical and biological aspects i.e. cortical bone (compact/dense) and cancellous

dure also known as calcification is a lifelong activity of a human being.

**Keywords:** bone, mineralisation, calcium phosphates and collagen fibres

## **Chapter 3** Bone Mineralisation

*Pinki Dey*

### **Abstract**

The mineralisation term mentions the development of inorganic precipitation over an organic background. This process occurs in a life span of biological organism for the formation of bone, teeth, exoskeletons, egg shells, etc. So, basically bone mineralisation is defined as the process of deposition of minerals on the bone matrix for the development of bone. The human bone is made up of 60–70% minerals which include calcium phosphate in the form of hydroxyapatite followed by 20–40% organic matrix containing type I collagen fibres and less than 5% of water and lipids. During bone mineralisation process osteoblasts which are also known as bone forming cells, aids to the production of calcium phosphate crystals which are then aligned in the collagen based fibrous matrix. The bone mineralisation procedure also known as calcification is a lifelong activity of a human being.

**Keywords:** bone, mineralisation, calcium phosphates and collagen fibres

### **1. Introduction**

Bone is a multifaceted system which behaves as mechanical shield for providing support and security. Bone also plays an important role in haemostasis. Recently, it came to observation that bone also aids in functioning of endocrine glands. To fulfil all these purposes, bone is a well architecture behaving as a functionally graded structure from millimetre to nanometre range. As a result of this gradation bone shows an unusual amalgamation of high stiffness and toughness which are frequently inversely associated. Broadly, bone is made up of organic and mineral part where the organic part is comprised of type I collagen whereas the mineral part contains the calcium deficient hydroxyapatite. The mineral part of the bone is interlinked with collagen in such an organised manner that it not only provides flexibility and ductility to the structure but also shows stiffness. This organisation of bone when observed at nanoscale range it behaves as a composite which acts as a shield to the brittle hydroxyapatite from damaging and also helps in carrying load by transferring forces around the bond hence thereby reducing the stress formation in the composite matrix. The fibres of the collagen intermingled with mineral part are arranged in very different manner when observed on microscopic range. This arrangement at micrometre level completely depends on the rate of bone formation or bone location on the substrate. This is due to the various functions played by the bone tissue, i.e., during fracture rapid bone formation development of bone during growth, unhurried bone formation in order to adjust in accordance to mechanical requirements so as to avoid any damage to its structure as well as maintaining its mechanical assets. Bone can be divided into two major categories depending on its mechanical and biological aspects i.e. cortical bone (compact/dense) and cancellous (porous/spongy bone namely trabecular) [1].

#### **2. Bone composition**

The mineral portion of the bone which basically comprises of hydroxyapatite contributes to the 65% of weight of bone. The remaining 20–30% of the bone weight composed of collagen of Type I. And, the last 10% of weight includes water molecules which are present collagen-mineral structure. Also, there are some free water molecules in the bone composite which gets redistributed during load bearing phase of bone. These free molecules pass through the vascular channels of bones and hence play a pivotal role in detection of signals by cells thus transferring information regarding load bearing environment. The interaction between bone mineral and unbound water molecules in a ratio of 1:1 helps in the process of bone mineralisation i.e. when the amount of water declines the mineralisation of bone starts to proceed and vice versa. The bone mineralisation is directly related to the stiffness of the bone as bone tends to grow stiffer since it contains higher amounts of mineral and lesser quantity of water. And as a result of this stiffened bone becomes more prone to brittleness and hence fracture easily. Approximately, 90% of organic part of bone is made up of collagen of type I category also collagen of type III and V are present in very minor quantities. And, the last 10% belong to non-collagenous proteins which help in the regulation of collagen development and management of fibre size, resistance to micro-crack, cellular adhesion and mineralisation. Around 85% of non-collagenous proteins are present in extracellular matrix and rest resides with the bone cells.

#### **2.1 Collagen**

The fibres of collagen are intertwined with the plate like structure of bone. Each and every collagen molecule exists as a triple helical structure formed from two chains of α 1 and one chain of α 2. Individual chain has around 1000 amino acids lengthwise and the centre part of the helix contains triplets of gly-X-Y in repeating sequence. The glycine molecules help in the formation of helical structure of collagen. Basically, all the amino acids are incorporated in collagen but the X and Y units are often composed of residue of hydroproline and proline. The maintenance of helical rigidity of the chain is due to the proline and hydroproline residues. The hydroxyl part of the hydroproline amino acid is important for interaction with water molecules via hydrogen bonding. The triple helix structure of collagen retained by the water molecules is affined toward hydroxyproline. At intercellular stage, peptides of non-helical region, i.e., N-propeptide as well as C-propeptides hold the chains at one place by cross- linking with sulphur. The propeptides present at the terminal point of triple helix are called procollagen molecule. During the exocytosis of molecules, the propeptide parts are broken down enzymatically resulting in non-helical parts at the molecular end N or C-terminal respectively. The enzymatically cleaved peptides result in the development of mature collagen molecule which has a pattern of non-helical N- and C-terminal peptides and helical nature of triple helix region. The microfibrils of collagen are semi-hexagonal system of five collagen molecules. The lateral and longitudinal combination of microfibrils leads to the formation of fibres approximately 10 μm in length and around 150 nm in diameter. The collagen fibres when observed under electron microscope showed a band pattern of around 67 nm. This band pattern is known as D-banding and it demonstrates the area between the neighbouring ends of collagen molecules as well as the overlapping between the lateral neighbouring molecules present at the end sections. In an osteoporotic bone the average diameter between the collagen fibrils and their spacing is very less as compared to a normal bone. The collagen fibrils are joined with the help of various types of cross-links which may have influential

**37**

*Bone Mineralisation*

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

**2.2 Non collagenous proteins**

turn controls mineralisation.

effects on the materialistic properties of the surrounding tissues and thereby affecting the mechanical traits of the whole bone system. The crosslinks bonds which connects the collagen fibrils are broadly categorised into enzymatic crosslinks and non-enzymatic crosslinks which forms a AGEs (advanced glycation end products).

There are various proteins of non-collagenous nature that helps in the regulation of formation and preservation of the extracellular matrix. Even though they comprise only of 2% of bone in weight but NCPs do a very important job during embryogenesis and formation as well as establishment of fibrils of collagen. It also regulates mineral of formation of bone and deliver channels for all signalling

1.*Proteoglycans*: They are basically consisting of various heparin sulphate, hyaluronan, small leucine rich proteoglycans called SLRPs. These are wide range of molecules where the main proteins are covalently bonded to the lateral chains of sulphated glycosaminoglycan. The proteoglycans which are present in the bone are smaller in size as compared to the other non-bone region proteoglycans. It manages the nucleation of apatite and the growth of apatite which in

2.*Glycoproteins*: It consists of fibronectin, thrombospondin (TSP1 and TSP2), vitronectin and lastly, alkaline phosphatase (ALP). In bones there are huge numbers of glycoproteins, out of which functions of few glycoproteins are not known. The activity of ALP helps in determining mineralisation factors because it undergoes hydrolysis with pyrophosphates which, as a result restricts the deposition of minerals by tagging itself with mineral crystals. If the pyrophosphates are neutralised, then this leads to normal growth of mineral crystals and as a result regulates the bone mineralisation. Since ALP is solely not produced by bone but also by kidney and liver any changes in the levels of ALP will not give any precise results regarding the mineralisation activity. Nonetheless, if the ALP is taken from the bone specific region, then the levels of ALP may determine the activity of bone mineralisation. In fact, lower levels of ALP or dysfunctionality of ALP leads to disorder recognised as hypophosphatemia which results in hypercalcemia causing death in children. TSP1 (Thrombospondin1) and TSP2 (Thrombospondin2) are found in mesenchymal stem cells and chondrocytes in the process of development of cartilage in the primary steps of bone development. TSP2 enhances the process of mineralisation as well as it escalates the osteoid process during mineralisation. Both vitronectin and fibronectin attaches to the cells where the vitronectin helps in attachment of cells which are found in plasma membrane of osteoclast and works with the osteopontin for binding osteoclasts to the matrix of mineral. The fibronectin plays an important role in cell proliferation during formation of bone.

3.*SIBLING* also known as small integrin binding ligand N linked glycoprotein which includes dentin matrix acidic phosphoprotein 1 (DMP1), osteopontin, sialoproteins and MEPE (matrix extracellular phosphoglycoprotein). In the preliminary stages of osteogenesis osteopontin is secreted. The ostoponin exists near the periodontal region of teeth as well as the cement line of bone. It restricts the crystal growth during mineralisation, also it attaches itself to the osteoclast in order to enhance the binding of osteoclast to the mineral surface of bone in the course of bone resorption. DEMP1 has an immense inclination

regarding the attachment of cells. The NCPs are divided into

effects on the materialistic properties of the surrounding tissues and thereby affecting the mechanical traits of the whole bone system. The crosslinks bonds which connects the collagen fibrils are broadly categorised into enzymatic crosslinks and non-enzymatic crosslinks which forms a AGEs (advanced glycation end products).

#### **2.2 Non collagenous proteins**

*Contemporary Topics about Phosphorus in Biology and Materials*

The mineral portion of the bone which basically comprises of hydroxyapatite contributes to the 65% of weight of bone. The remaining 20–30% of the bone weight composed of collagen of Type I. And, the last 10% of weight includes water molecules which are present collagen-mineral structure. Also, there are some free water molecules in the bone composite which gets redistributed during load bearing phase of bone. These free molecules pass through the vascular channels of bones and hence play a pivotal role in detection of signals by cells thus transferring information regarding load bearing environment. The interaction between bone mineral and unbound water molecules in a ratio of 1:1 helps in the process of bone mineralisation i.e. when the amount of water declines the mineralisation of bone starts to proceed and vice versa. The bone mineralisation is directly related to the stiffness of the bone as bone tends to grow stiffer since it contains higher amounts of mineral and lesser quantity of water. And as a result of this stiffened bone becomes more prone to brittleness and hence fracture easily. Approximately, 90% of organic part of bone is made up of collagen of type I category also collagen of type III and V are present in very minor quantities. And, the last 10% belong to non-collagenous proteins which help in the regulation of collagen development and management of fibre size, resistance to micro-crack, cellular adhesion and mineralisation. Around 85% of non-collagenous proteins are present in extracellular matrix and rest resides

The fibres of collagen are intertwined with the plate like structure of bone. Each

and every collagen molecule exists as a triple helical structure formed from two chains of α 1 and one chain of α 2. Individual chain has around 1000 amino acids lengthwise and the centre part of the helix contains triplets of gly-X-Y in repeating sequence. The glycine molecules help in the formation of helical structure of collagen. Basically, all the amino acids are incorporated in collagen but the X and Y units are often composed of residue of hydroproline and proline. The maintenance of helical rigidity of the chain is due to the proline and hydroproline residues. The hydroxyl part of the hydroproline amino acid is important for interaction with water molecules via hydrogen bonding. The triple helix structure of collagen retained by the water molecules is affined toward hydroxyproline. At intercellular stage, peptides of non-helical region, i.e., N-propeptide as well as C-propeptides hold the chains at one place by cross- linking with sulphur. The propeptides present at the terminal point of triple helix are called procollagen molecule. During the exocytosis of molecules, the propeptide parts are broken down enzymatically resulting in non-helical parts at the molecular end N or C-terminal respectively. The enzymatically cleaved peptides result in the development of mature collagen molecule which has a pattern of non-helical N- and C-terminal peptides and helical nature of triple helix region. The microfibrils of collagen are semi-hexagonal system of five collagen molecules. The lateral and longitudinal combination of microfibrils leads to the formation of fibres approximately 10 μm in length and around 150 nm in diameter. The collagen fibres when observed under electron microscope showed a band pattern of around 67 nm. This band pattern is known as D-banding and it demonstrates the area between the neighbouring ends of collagen molecules as well as the overlapping between the lateral neighbouring molecules present at the end sections. In an osteoporotic bone the average diameter between the collagen fibrils and their spacing is very less as compared to a normal bone. The collagen fibrils are joined with the help of various types of cross-links which may have influential

**2. Bone composition**

with the bone cells.

**2.1 Collagen**

**36**

There are various proteins of non-collagenous nature that helps in the regulation of formation and preservation of the extracellular matrix. Even though they comprise only of 2% of bone in weight but NCPs do a very important job during embryogenesis and formation as well as establishment of fibrils of collagen. It also regulates mineral of formation of bone and deliver channels for all signalling regarding the attachment of cells. The NCPs are divided into


for hydroxyl apatite and N-telopeptide region of type I collagen, also is indicated by osteocytes and osteoblasts. It helps in local regulation of bone mineralisation. Although, it is quite an unknown fact whether DMP1 is involved in the differentiation process of osteoblasts to osteocytes. But in adequate DMP1 results in hypophosphatemia rickets due to increased level of FGF23. MEPE also belongs to SIBLING genre which functions to locally regulate Mineralisation. It is mainly found in osteocytes and odontoblasts, where it is supremely demonstrated in tissues which are still under Mineralisation such as modification of intramembrane and endochondral plaited bone of your fractured callus. *In vivo* results show that lack of MEPE gives increased bone mass as well as decreased in bone loss.


#### **3. Bone mineral**

Bone mineral is made up of carbonate apatite which has poor crystalline structure. The apatite undergoes nucleation in the space between collagen fibrils end called hole zones and it spreads longitudinal over the collagen fibrils. In the beginning, mineral is settled in the form of amorphous calcium phosphate followed by calcium carbonate in good amount. The carbonate proportion reduces during bone tissue maturation, also mineral crystals form disc like structure growing laterally while aligning themselves parallel to the fibrils of collagen. The l-axis (long axis) of mineral disc oriented with the longitudinal line of bone. The typical size of mineral crystals has less than 10 nm thickness. Gradually, the mineral disc merge with other crystals to form a large polycrystalline structure, which becomes indeed larger than the thickness of fibrils. The mineral crystals grow more in size during bone ageing due to changes in ion contents of mineral composition. The age of the tissue is directly proportional to the size of the crystals. Although, it is tough to differentiate amid small crystals bearing numerous defects and large crystals having less defects, as both shows likewise crystalline behaviour. The soluble carbonate which adheres to surface of crystals can also be filled in by hydroxyl and phosphates groups of carbonate apatite. As a result, it helps in easy resorption of mineral. During the incidents of acid load, bicarbonate (-HCO3) is being absorbed so as to maintain the pH of blood. The deficiency of -HCO3 is balanced by the presence of carbonate and phosphate ions in bone mineral. When there is an abnormal production of acid, the

**39**

*Bone Mineralisation*

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

vary from months to year [1–19].

**4. Bone cells**

bone mineral tank helps in the maintenance of acid–base balance, which also many times leads to loss in bone mass. There are certain cations such as Mg, Na, strontium in place of calcium ions and fluoride ions in place of hydroxyl ions in apatite matrix. Also, these kinds of substitutions can cause changes in mechanical properties of bone as well as in the behaviour of osteoclast and osteoblasts. Previously, for osteoporosis sodium fluoride used as an anabolic remedy. It was observed that sodium fluoride promoted pre-osteoblasts and osteoprogenitor cells hence stimulating uninterrupted formation of bone without the initialization of resorption. However, it has been seemed that carbonate apatite is less unaffected by resorption than fluoro-apatite. Also, replacement of fluoride ions into the mineral leads to escalated brittleness of bone thus causing the bone prone to fracture. The poor mechanical behaviour of bone is only due to the replacement of fluoride ions from bone as compared to the occurrence of substitutions of other ions from the bone mineral. The accumulation of bone mineralisation takes place in two consecutive phases. The first phase is the rapid nucleation of the primary mineral crystals. This phase is also known as primary Mineralisation. And, second phase relates to the slow proliferation and development of the primary crystals up to a size of 40 × 3 × 7.5 nm. In the course of primary mineralisation crystals are very quickly accumulated in the collagen network thus attaining 65–70% of total mineralisation within 3 weeks approximately. During the second step, the mineral is deposited at a steady rate but in a more efficient way, till the mineral attains the required bodily limit, which may

Bone is a structurally and metabolically very complicated organ which is a composite of mineral, collagen material and bone cells [20]. The bone cells basically include osteoblasts, osteoclasts and osteocytes, which are found in mesenchymal stem cells known to accumulate osteiod before the mineralisation process takes off, thereby helping in bone formation [21, 22]. Osteoblasts are known for regulating mineralisation and in the formation of extracellular matrix. They are originated from bone and are in cuboidal form especially found at bone surface and carries out the function of resorption [21, 23]. The quantity and function of osteoclasts are dependent on many factors such as proliferation, differentiation, rate of resorption by already developed osteoclasts and lastly cell lineage allocation [24]. They are the derivative of multinucleated polarised cells which are migratory in nature with good source of lysozyme enzymes [25]. They consist of mitochondria of pleomorphic type, vacuoles and lysosomes [26]. The formation and resorption of bone is the joint activity of osteoclasts and osteoblasts. And the factors which are involved during this process are prostaglandin E2 (PGE2), transforming growth factor beta 1 (TGF-β1), fibroblast growth factor, parathyroid hormone (PTH), osteoprotegerin ligand OPGL also known as RANKL (receptor activator of nuclear factor kappa B [(NF-ĸB) ligand]) and TRANCE i.e. TNF related activation induced cytokine. Resorption can also effect the biomechanical activities of bone for instance, formation of strong bone from a weak one [24]. Majority of bone cells are in the nature of osteocytes, thus comprising of 90–95% in the skeleton of an adult. The mature osteoblasts in the bone matrix are recognised as osteocytes. And they help in responding to the mechanical strain thus generating signals which can further coordinate the bone resorption and its formation [27]. Osteocytes that are present in mature bone are joined together with long extensions of cytoplasm that form small capillary like structure called lacunae or canalcali for the transfer of nutrients and wastes. Osteocytes are spread across the mineral matrix and connect to the

#### *Bone Mineralisation DOI: http://dx.doi.org/10.5772/intechopen.92065*

*Contemporary Topics about Phosphorus in Biology and Materials*

ance between resorption and formation of bone.

as decreased in bone loss.

**3. Bone mineral**

for hydroxyl apatite and N-telopeptide region of type I collagen, also is indicated by osteocytes and osteoblasts. It helps in local regulation of bone mineralisation. Although, it is quite an unknown fact whether DMP1 is involved in the differentiation process of osteoblasts to osteocytes. But in adequate DMP1 results in hypophosphatemia rickets due to increased level of FGF23. MEPE also belongs to SIBLING genre which functions to locally regulate Mineralisation. It is mainly found in osteocytes and odontoblasts, where it is supremely demonstrated in tissues which are still under Mineralisation such as modification of intramembrane and endochondral plaited bone of your fractured callus. *In vivo* results show that lack of MEPE gives increased bone mass as well

4.*Osteocalcin (Gla bone protein)*: The osteocalcin is indicated in osteoblasts as well as osteocytes. It helps in binding o calcium and deposition of mineral. As a result, it is considered as an indicator of bone formation. However sometimes It may also behave as a precursor in regulation of osteoclasts. It has been seen that mice in absentia of osteocalcin suffers from grave osteoporosis. Hence, it can be seen why osteocalcin is considered as one of the important marker for bone remodelling. It has been also observed that post-menopausal osteoporosis that its increased level increases bone remodelling rate causing acute imbal-

5.*Osteonectin* are also known as SPARC, i.e., secreted proteins acidic rich in cysteine. It is present at mineral deposited location where it bonds with collagen, hydroxyapatite and vitronectin. It helps proliferation of freshly nucleated mineral crystals. Since its plays a vital part in osteoblasts growth, its non-existence causes osteopenia i.e. low bone density. It bandages itself to various growth factors such as FEF2, PDGF (platelet derived growth factor), VEGF (vascular

Bone mineral is made up of carbonate apatite which has poor crystalline structure. The apatite undergoes nucleation in the space between collagen fibrils end called hole zones and it spreads longitudinal over the collagen fibrils. In the beginning, mineral is settled in the form of amorphous calcium phosphate followed by calcium carbonate in good amount. The carbonate proportion reduces during bone tissue maturation, also mineral crystals form disc like structure growing laterally while aligning themselves parallel to the fibrils of collagen. The l-axis (long axis) of mineral disc oriented with the longitudinal line of bone. The typical size of mineral crystals has less than 10 nm thickness. Gradually, the mineral disc merge with other crystals to form a large polycrystalline structure, which becomes indeed larger than the thickness of fibrils. The mineral crystals grow more in size during bone ageing due to changes in ion contents of mineral composition. The age of the tissue is directly proportional to the size of the crystals. Although, it is tough to differentiate amid small crystals bearing numerous defects and large crystals having less defects, as both shows likewise crystalline behaviour. The soluble carbonate which adheres to surface of crystals can also be filled in by hydroxyl and phosphates groups of carbonate apatite. As a result, it helps in easy resorption of mineral. During the incidents of acid load, bicarbonate (-HCO3) is being absorbed so as to maintain the pH of blood. The deficiency of -HCO3 is balanced by the presence of carbonate and phosphate ions in bone mineral. When there is an abnormal production of acid, the

endothelial growth factor) for the regulation of mineralisation.

**38**

bone mineral tank helps in the maintenance of acid–base balance, which also many times leads to loss in bone mass. There are certain cations such as Mg, Na, strontium in place of calcium ions and fluoride ions in place of hydroxyl ions in apatite matrix. Also, these kinds of substitutions can cause changes in mechanical properties of bone as well as in the behaviour of osteoclast and osteoblasts. Previously, for osteoporosis sodium fluoride used as an anabolic remedy. It was observed that sodium fluoride promoted pre-osteoblasts and osteoprogenitor cells hence stimulating uninterrupted formation of bone without the initialization of resorption. However, it has been seemed that carbonate apatite is less unaffected by resorption than fluoro-apatite. Also, replacement of fluoride ions into the mineral leads to escalated brittleness of bone thus causing the bone prone to fracture. The poor mechanical behaviour of bone is only due to the replacement of fluoride ions from bone as compared to the occurrence of substitutions of other ions from the bone mineral. The accumulation of bone mineralisation takes place in two consecutive phases. The first phase is the rapid nucleation of the primary mineral crystals. This phase is also known as primary Mineralisation. And, second phase relates to the slow proliferation and development of the primary crystals up to a size of 40 × 3 × 7.5 nm. In the course of primary mineralisation crystals are very quickly accumulated in the collagen network thus attaining 65–70% of total mineralisation within 3 weeks approximately. During the second step, the mineral is deposited at a steady rate but in a more efficient way, till the mineral attains the required bodily limit, which may vary from months to year [1–19].

#### **4. Bone cells**

Bone is a structurally and metabolically very complicated organ which is a composite of mineral, collagen material and bone cells [20]. The bone cells basically include osteoblasts, osteoclasts and osteocytes, which are found in mesenchymal stem cells known to accumulate osteiod before the mineralisation process takes off, thereby helping in bone formation [21, 22]. Osteoblasts are known for regulating mineralisation and in the formation of extracellular matrix. They are originated from bone and are in cuboidal form especially found at bone surface and carries out the function of resorption [21, 23]. The quantity and function of osteoclasts are dependent on many factors such as proliferation, differentiation, rate of resorption by already developed osteoclasts and lastly cell lineage allocation [24]. They are the derivative of multinucleated polarised cells which are migratory in nature with good source of lysozyme enzymes [25]. They consist of mitochondria of pleomorphic type, vacuoles and lysosomes [26]. The formation and resorption of bone is the joint activity of osteoclasts and osteoblasts. And the factors which are involved during this process are prostaglandin E2 (PGE2), transforming growth factor beta 1 (TGF-β1), fibroblast growth factor, parathyroid hormone (PTH), osteoprotegerin ligand OPGL also known as RANKL (receptor activator of nuclear factor kappa B [(NF-ĸB) ligand]) and TRANCE i.e. TNF related activation induced cytokine. Resorption can also effect the biomechanical activities of bone for instance, formation of strong bone from a weak one [24]. Majority of bone cells are in the nature of osteocytes, thus comprising of 90–95% in the skeleton of an adult. The mature osteoblasts in the bone matrix are recognised as osteocytes. And they help in responding to the mechanical strain thus generating signals which can further coordinate the bone resorption and its formation [27]. Osteocytes that are present in mature bone are joined together with long extensions of cytoplasm that form small capillary like structure called lacunae or canalcali for the transfer of nutrients and wastes. Osteocytes are spread across the mineral matrix and connect to the

surface of bone and bone marrow via dendrites which involve osteoclast precursors for the stimulation of bone resorption and regulation of differentiation of mesenchymal stem cells [28–32]. The roles which are played by osteocytes and lacunae/ canalcali involves restriction of fatigue cracks, exchange of mineral, hormonal stimulation for detection of stress or strain, mending of microdamage, modelling or remodelling of bone under mechanical criteria, osteocytic osteolysis, regulation of osteoclastic cutting cone during exchange of mineral and reformed remodelling act after the resorption [27].

During the bone mineralisation, the crystals of mineral are accumulated in a systematic manner over the extracellular matrix, where the cells surrounding the mineral matrix prepares a pattern for mineral accumulation thus commencing the location for mineralisation and fixing the final dimensions of mineral crystals. Although, various studies have been conducted throughout the world for determining the mechanism for formation of mineral crystals in every organism, but the exact explanation related to this mechanism remains unclear [33–35]. In accordance to the conventional theories about biomineralization, NCPs were actively engaged in the process of matrix mineralisation. In 1994, Hunter and Goldberg postulated that the effects of mineralisation for BSPs were completely connected to the glutamate and aspartate-rich sequences of peptides [36]. Later around 1997, Stubbs et al. carried out studies to consider the involvement of other groups such as sulphate, phosphate sialic acidic groups in the process of mineralisation [37]. According to earlier reports, nucleation of mineral takes place in their principal ionic solution which is supersaturated in nature. During nucleation in solid phase, a critical size of crystal is required in order to initiate the nucleation. This mechanism is known as stochastic solute clustering [38, 39].

At present, two important models have been considered for bone mineralisation process. The first model which involves mineralisation with the help of collagen template and the second model include the matrix vesicles for mineralisation purpose. It has been widely accepted that mineral formation is a systematic procedure which can never take place in the absence of matrix. Basically, the matrix gives an ordered pattern of deposition of mineral, thus directly participating in the mineralisation by behaving as a nuclear. It has been also seen that different mineralised tissues have different matrices [40]. Alternatively, matrix vesicles are the particles derived from extracellular matrix having a diameter of 100 nm, precisely positioned inside the bone matrix and the matrix of cartilage and peridentin. They provide the initial location for the calcification of all skeletal tissues. They are generally formed from a polarised bud which gets discharged from surface of the chondrocytes, osteoblasts and odontoblasts [41, 42]. The matrix vesicles are considered for preliminary location for mineral build-ups in bone tissue [43, 44]. In course of mineralisation involving cells, the formation of primary hydroxyapatite crystals takes place inside the vesicle membrane matrix [45].

a.*Collagen-moderated mineralisation*: In this type of mineralisation, the template for accumulation of mineral is provided by the collagen present in the bone tissue. And, these very collagen fibrils decide the sizes of crystals that can attained for the process of mineralisation. On the other hand, mineralisation does not take place in a deficiency of NCPs because they behave as molecules that generate signals all through the course of mineralisation [40, 46]. It was observed that BSPs role as a crystal nucleator, affected the osteocalcin recognition and remodelling of mineralised surfaces [36, 37, 47–51]. However, osteopontin and osteonectin helped in regulation of crystal formation on the basis of size, type and growth [52, 53]. During the growth phase, the crystals nucleates from an amorphous phase were the intervallic pattern of 67 nm cross-striated

**41**

*Bone Mineralisation*

levels of PO4

surface of fibrils is carried out [40].

**5. Pathological mineralisation**

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

collagen fibres [54, 55] carryover the nucleation in the 40 nm long gap sparsely dense zone. The aforementioned process was recommended to be guided by heavily acidic NCPs [56–59]. The basic principal of collagen based template mineralisation focuses mainly on the job of collagen fibrils during bone Mineralisation. When the mineralising fibre were observed under cryo-transmission electron microscope, it was found that polyaspartic acid which has soluble behaviour plays an integral part in collagen mineralisation [56, 60]. The calcium triphosphate ions complex is formed from the prenucleated clusters of acidic polypeptide [38]. These clusters are negatively charged; as a result, they get attracted towards the positive part on collagen [56]. Consequently, these ionic complex gets fused inside the collagen fibrils and then transform into a solid amorphous mass which further grows into an ordered apatite crystal

complex regulated by the arrangement of collagen fibrils [40].

b.*Matrix-vesicle moderated mineralisation*: In this type of mechanism, the preliminary phase starts off at the mineral visceral where the Ca2+ ions and the inorganic phosphate (Pi) are formed *in vivo* [41, 61, 62]. The annexins and phosphatidylserine are the calcium binding molecules which tag them with BSPs so as to invite and regulate the deposition of calcium and phosphate ions previous to the creation of crystals of insoluble hydroxyapatite [62, 63]. During, this phase the pH of the intravesicules rises above due to the activity of carbonic anhydrase found in mineral vesicles [64], causing the stabilisation of primary mineral crystals [42]. In the second phase of matrix moderated mineralisation, the breakdown of mineral vesicles membranes takes place where the already formed hydroxyapatite are exposed to extracellular fluid [61]. The extracellular fluid comprises of matrix vesicles with homeostatically regulated

<sup>3</sup><sup>−</sup> and Ca2+ in order to help in proliferation of new hydroxyapatite

crystals onto the already formed hydroxyapatite crystals. The perforations in the matrix vesicle membrane are carried out by proteases [65] and phospholipases [66]. The metalloproteinases of matrix vesicles which have the capability of degenerating mineral deficient proteoglycans helps in transferring of mineral towards itself [67]. In the recent studies, it has been observed that collagen type II and X bind to the outside surfaces of matrix vesicles thus acting as a channel for the transfer of crystals into the extravesicular matrix [68]. Three promising functions of matrix vesicles have been identified during the course of mineralisation. The first function involves the control of ion concentration by the matrix vesicles inside the matrix so as to start off the mineralisation around the collagen fibrils. Also, it controls the compositions of ions necessary for the formation of intravesicular apatite crystals thus starting the process of mineralisation with the transfer of ions to the collagen. And lastly, when the mineral vesicles interact with the collagen, the deposition of mineral onto the

Mineralisation is categorised into physiological or pathological types depending on the type of bone tissues i.e. hard bone tissue or soft bone tissue. Physiological mineralisation is required for the development of skeletal tissues in order to carry out daily functions of a normal human life. The second category of mineralisation is pathological mineralisation also known as ectopic which involves the mineralisation of soft bone tissues such as cartilages (articular cartilage) and tissues surrounding cardiac vessels that causes diseases and death. Recently, it has been found that the

*Contemporary Topics about Phosphorus in Biology and Materials*

after the resorption [27].

stochastic solute clustering [38, 39].

crystals takes place inside the vesicle membrane matrix [45].

surface of bone and bone marrow via dendrites which involve osteoclast precursors for the stimulation of bone resorption and regulation of differentiation of mesenchymal stem cells [28–32]. The roles which are played by osteocytes and lacunae/ canalcali involves restriction of fatigue cracks, exchange of mineral, hormonal stimulation for detection of stress or strain, mending of microdamage, modelling or remodelling of bone under mechanical criteria, osteocytic osteolysis, regulation of osteoclastic cutting cone during exchange of mineral and reformed remodelling act

During the bone mineralisation, the crystals of mineral are accumulated in a systematic manner over the extracellular matrix, where the cells surrounding the mineral matrix prepares a pattern for mineral accumulation thus commencing the location for mineralisation and fixing the final dimensions of mineral crystals. Although, various studies have been conducted throughout the world for determining the mechanism for formation of mineral crystals in every organism, but the exact explanation related to this mechanism remains unclear [33–35]. In accordance to the conventional theories about biomineralization, NCPs were actively engaged in the process of matrix mineralisation. In 1994, Hunter and Goldberg postulated that the effects of mineralisation for BSPs were completely connected to the glutamate and aspartate-rich sequences of peptides [36]. Later around 1997, Stubbs et al. carried out studies to consider the involvement of other groups such as sulphate, phosphate sialic acidic groups in the process of mineralisation [37]. According to earlier reports, nucleation of mineral takes place in their principal ionic solution which is supersaturated in nature. During nucleation in solid phase, a critical size of crystal is required in order to initiate the nucleation. This mechanism is known as

At present, two important models have been considered for bone mineralisation process. The first model which involves mineralisation with the help of collagen template and the second model include the matrix vesicles for mineralisation purpose. It has been widely accepted that mineral formation is a systematic procedure which can never take place in the absence of matrix. Basically, the matrix gives an ordered pattern of deposition of mineral, thus directly participating in the mineralisation by behaving as a nuclear. It has been also seen that different mineralised tissues have different matrices [40]. Alternatively, matrix vesicles are the particles derived from extracellular matrix having a diameter of 100 nm, precisely positioned inside the bone matrix and the matrix of cartilage and peridentin. They provide the initial location for the calcification of all skeletal tissues. They are generally formed from a polarised bud which gets discharged from surface of the chondrocytes, osteoblasts and odontoblasts [41, 42]. The matrix vesicles are considered for preliminary location for mineral build-ups in bone tissue [43, 44]. In course of mineralisation involving cells, the formation of primary hydroxyapatite

a.*Collagen-moderated mineralisation*: In this type of mineralisation, the template for accumulation of mineral is provided by the collagen present in the bone tissue. And, these very collagen fibrils decide the sizes of crystals that can attained for the process of mineralisation. On the other hand, mineralisation does not take place in a deficiency of NCPs because they behave as molecules that generate signals all through the course of mineralisation [40, 46]. It was observed that BSPs role as a crystal nucleator, affected the osteocalcin recognition and remodelling of mineralised surfaces [36, 37, 47–51]. However, osteopontin and osteonectin helped in regulation of crystal formation on the basis of size, type and growth [52, 53]. During the growth phase, the crystals nucleates from an amorphous phase were the intervallic pattern of 67 nm cross-striated

**40**

collagen fibres [54, 55] carryover the nucleation in the 40 nm long gap sparsely dense zone. The aforementioned process was recommended to be guided by heavily acidic NCPs [56–59]. The basic principal of collagen based template mineralisation focuses mainly on the job of collagen fibrils during bone Mineralisation. When the mineralising fibre were observed under cryo-transmission electron microscope, it was found that polyaspartic acid which has soluble behaviour plays an integral part in collagen mineralisation [56, 60]. The calcium triphosphate ions complex is formed from the prenucleated clusters of acidic polypeptide [38]. These clusters are negatively charged; as a result, they get attracted towards the positive part on collagen [56]. Consequently, these ionic complex gets fused inside the collagen fibrils and then transform into a solid amorphous mass which further grows into an ordered apatite crystal complex regulated by the arrangement of collagen fibrils [40].

b.*Matrix-vesicle moderated mineralisation*: In this type of mechanism, the preliminary phase starts off at the mineral visceral where the Ca2+ ions and the inorganic phosphate (Pi) are formed *in vivo* [41, 61, 62]. The annexins and phosphatidylserine are the calcium binding molecules which tag them with BSPs so as to invite and regulate the deposition of calcium and phosphate ions previous to the creation of crystals of insoluble hydroxyapatite [62, 63]. During, this phase the pH of the intravesicules rises above due to the activity of carbonic anhydrase found in mineral vesicles [64], causing the stabilisation of primary mineral crystals [42]. In the second phase of matrix moderated mineralisation, the breakdown of mineral vesicles membranes takes place where the already formed hydroxyapatite are exposed to extracellular fluid [61]. The extracellular fluid comprises of matrix vesicles with homeostatically regulated levels of PO4 <sup>3</sup><sup>−</sup> and Ca2+ in order to help in proliferation of new hydroxyapatite crystals onto the already formed hydroxyapatite crystals. The perforations in the matrix vesicle membrane are carried out by proteases [65] and phospholipases [66]. The metalloproteinases of matrix vesicles which have the capability of degenerating mineral deficient proteoglycans helps in transferring of mineral towards itself [67]. In the recent studies, it has been observed that collagen type II and X bind to the outside surfaces of matrix vesicles thus acting as a channel for the transfer of crystals into the extravesicular matrix [68]. Three promising functions of matrix vesicles have been identified during the course of mineralisation. The first function involves the control of ion concentration by the matrix vesicles inside the matrix so as to start off the mineralisation around the collagen fibrils. Also, it controls the compositions of ions necessary for the formation of intravesicular apatite crystals thus starting the process of mineralisation with the transfer of ions to the collagen. And lastly, when the mineral vesicles interact with the collagen, the deposition of mineral onto the surface of fibrils is carried out [40].

#### **5. Pathological mineralisation**

Mineralisation is categorised into physiological or pathological types depending on the type of bone tissues i.e. hard bone tissue or soft bone tissue. Physiological mineralisation is required for the development of skeletal tissues in order to carry out daily functions of a normal human life. The second category of mineralisation is pathological mineralisation also known as ectopic which involves the mineralisation of soft bone tissues such as cartilages (articular cartilage) and tissues surrounding cardiac vessels that causes diseases and death. Recently, it has been found that the

reasons and aspects which cause physiological mineralisation can be similar to that of pathological. Lately, it has been reported both the mineralisation are instigated by the matrix vesicles where the particles which resides inside the membrane are released from the plasma membrane of mineralisation cells. The activators and regulators which cause pathological mineralisation are the same activators and regulators which will cause pathological mineralisation. It has been also reported that apoptosis causes physiological mineralisation and it has been seen that if physiological mineralisation happens to happen take place after injury of tissue, can prompt pathological mineralisation around the damaged or the injured tissue [69]. The mineralisation process is important for imparting mechanical properties in the bone [70]. As a result, when not regulated properly can lead to the inadequate or extreme mineralisation. Therefore, bone tissue quality gets jeopardised and becoming the cause for many bone related diseases. Osteomalacia is one such condition where the disease is caused in adults due to deficiency of bone mineral or excessive bone resorption. Rickets is the osteomalacia in children. As already explained, in a fit and mature bone, osteoclasts eliminate bone whereas osteoblasts accumulate osteoid and thus carrying out the mineralisation. During osteomalacia, calcification rate is decreased while the bone surface is being increased due to the building up of non-mineralised osteoid. The common symptoms of osteomalacia involves brittle bone, weakened muscles along with severe body ache [71–73]. Another known disease caused by pathological mineralisation is fibrous osteodystropy. In various findings, it has been seen that the flexibility and deformation of bone depends because of constant and extreme contact with the PTH, thus hampering with the bone load bearing capacity [74–78]. In osteocalcia, bone tissue is destroyed and resorped osteoclasts where the remodelling area, previously taken up by calcified bone is then occupied by fibrous connective tissue. As the disease progresses, non-mineralised formed bone takes up the place of cortical bone. And also, mineralised osteoid in the remodelling space was previously filled up by osseous tissue [79]. Paget's disease is a very commonly known disorder of bone in adults which is chronic in nature. This is also known as osteitis deformans [80, 81]. This is mainly found in middle aged men as compared to women [80, 82, 83]. In this disease, the resorption mechanism of bone gets speed up which results in formation of thick unarranged bone mineral matrix. As a result, producing weakened bone structure, painful fragile bone, joints arthritis inside the targeted bone. Sometimes, Paget's disease transform into a preliminary cancer of bone identified as Paget's sarcoma. The large multinucleated osteoclasts initiate the pathological Mineralisation of bones with high resorption causing Paget's disease [84]. This hastened resorption results in an unorganised deposition of mineral by osteoblasts during remodelling [85]. Thus causing irregularities in cortical thickness, coarsening of trabecular and vascularization of fibrous tissues, which then produces fatigue during high stress condition [81, 86, 87]. Another very well-known bone disease is osteoporosis, where the affected bone has depleted mass with structural degradation as well as amplified porosity of bone tissue [88]. Usually, in osteoporosis all bones are affected as compared to the Paget's disease where only a part of bone is targeted. When the mineral content of the bone goes below the critical value, the bone becomes more brittle in nature thus the load bearing capacity along with other mechanical properties of bone gets deteriorated [89]. The BMD also known as bone mineral density is directly proportional to the mechanical strength of bone. The patients with ongoing history of osteoporosis has reduced BMD, hence are more prone to fracture [90–93]. There are basically three main reasons behind the cause of osteoporosis. The first reason postulates that the commencing of the osteoporosis may be due to the underdevelopment of bone during the growth period of individuals. The second reason focuses on the bone development due to heightened resorption process.

**43**

biomaterial [105].

**6.3 Bioactive glasses**

*Bone Mineralisation*

ment is very crucial [20].

**6.1 Inorganic materials**

**6.2 Calcium phosphates**

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

And lastly, osteoporosis may happen due to the lack of new bone growth during the course of remodelling [94]. Earlier, it was believed that osteoporosis is an outbreak of ageing process but with the recent studies it has been observed that it may be caused because of malnutrition, alterations in biomechanical loading, production

For many years, biomaterials are being experimented in such a way so as to choose them as a replacement for damaged or diseased tissues. As the exclusive qualities of bone tissues are solely connected to the bone mineralisation, thereby interpreting and regulating the mineralisation process of artificial bone replace-

Since the mineral part of the bone is inorganic in nature, many biomaterials such as calcium phosphates, hydroxyapatite and bioglass are being used as bone alternatives [96]. It was in 1969, it was observed that bioglass forms bond with the bone thus restricting the development of fibrous tissues surrounding the bone [97]. There are various other ceramic biomaterials available such as hydroxyapatite [98], β-tricalcium phosphate, glass–ceramic [99–101], which are after sintering have displayed the bone bonding ability, hence paving the way for themselves clinically

The first in the list of very commonly used bioceramics are the calcium phosphate based ceramics, broadly used in the field of dentistry and orthopaedics. They are mainly used for purpose of coatings onto the top surface of metal implants such as titanium, etc. The study of mineralisation can be performed in some fluids which imitates the ionic composition of blood plasma. One such kind of fluid is simulated body fluid suggested by Kokubo et al. in 1991. This fluid mimics the ionic concentrations of human blood plasma almost exactly. This fluid is used for the purpose of studying mineralisation behaviour of biomaterials in vitro. The SBF initiates the formation of apatite (bone-like) over the synthetic biomaterial surfaces, thus giving the idea of mineralisation of the biomaterial in vivo [103]. Kokubo et al. also suggested that in vivo bone reaction of an artificial biomaterial can be anticipated by the formation of apatite over its surface in SBF, but this theory now has been challenged [104]. In 2010, Bertazzo et al. suggested that calcium phosphates exhibited the osteoconductive nature of bone guided by bone tissues beside the biomaterial surface at the site of implantation orthotopically. Also, there are other calcium phosphate ceramics present which shows osteoinductive traits i.e. ability to form bone at implantation site ectopically. Although, the exact mechanism behind the osteoinductive nature of certain calcium phosphates depend on various factors such as composition, structure and conformation of the calcium phosphate

In 1969, Hench observed that certain silicon-based glasses formed bond with the bone [106] and named it as Bioactive® glass also known as 45S5 consisting of

of excessive hormones and prolonged history of acidosis [95].

**6. Mineralisation of synthetic biomaterials**

in reconstructive and regenerative medicine fields [102].

#### *Bone Mineralisation DOI: http://dx.doi.org/10.5772/intechopen.92065*

*Contemporary Topics about Phosphorus in Biology and Materials*

reasons and aspects which cause physiological mineralisation can be similar to that of pathological. Lately, it has been reported both the mineralisation are instigated by the matrix vesicles where the particles which resides inside the membrane are released from the plasma membrane of mineralisation cells. The activators and regulators which cause pathological mineralisation are the same activators and regulators which will cause pathological mineralisation. It has been also reported that apoptosis causes physiological mineralisation and it has been seen that if physiological mineralisation happens to happen take place after injury of tissue, can prompt pathological mineralisation around the damaged or the injured tissue [69]. The mineralisation process is important for imparting mechanical properties in the bone [70]. As a result, when not regulated properly can lead to the inadequate or extreme mineralisation. Therefore, bone tissue quality gets jeopardised and becoming the cause for many bone related diseases. Osteomalacia is one such condition where the disease is caused in adults due to deficiency of bone mineral or excessive bone resorption. Rickets is the osteomalacia in children. As already explained, in a fit and mature bone, osteoclasts eliminate bone whereas osteoblasts accumulate osteoid and thus carrying out the mineralisation. During osteomalacia, calcification rate is decreased while the bone surface is being increased due to the building up of non-mineralised osteoid. The common symptoms of osteomalacia involves brittle bone, weakened muscles along with severe body ache [71–73]. Another known disease caused by pathological mineralisation is fibrous osteodystropy. In various findings, it has been seen that the flexibility and deformation of bone depends because of constant and extreme contact with the PTH, thus hampering with the bone load bearing capacity [74–78]. In osteocalcia, bone tissue is destroyed and resorped osteoclasts where the remodelling area, previously taken up by calcified bone is then occupied by fibrous connective tissue. As the disease progresses, non-mineralised formed bone takes up the place of cortical bone. And also, mineralised osteoid in the remodelling space was previously filled up by osseous tissue [79]. Paget's disease is a very commonly known disorder of bone in adults which is chronic in nature. This is also known as osteitis deformans [80, 81]. This is mainly found in middle aged men as compared to women [80, 82, 83]. In this disease, the resorption mechanism of bone gets speed up which results in formation of thick unarranged bone mineral matrix. As a result, producing weakened bone structure, painful fragile bone, joints arthritis inside the targeted bone. Sometimes, Paget's disease transform into a preliminary cancer of bone identified as Paget's sarcoma. The large multinucleated osteoclasts initiate the pathological Mineralisation of bones with high resorption causing Paget's disease [84]. This hastened resorption results in an unorganised deposition of mineral by osteoblasts during remodelling [85]. Thus causing irregularities in cortical thickness, coarsening of trabecular and vascularization of fibrous tissues, which then produces fatigue during high stress condition [81, 86, 87]. Another very well-known bone disease is osteoporosis, where the affected bone has depleted mass with structural degradation as well as amplified porosity of bone tissue [88]. Usually, in osteoporosis all bones are affected as compared to the Paget's disease where only a part of bone is targeted. When the mineral content of the bone goes below the critical value, the bone becomes more brittle in nature thus the load bearing capacity along with other mechanical properties of bone gets deteriorated [89]. The BMD also known as bone mineral density is directly proportional to the mechanical strength of bone. The patients with ongoing history of osteoporosis has reduced BMD, hence are more prone to fracture [90–93]. There are basically three main reasons behind the cause of osteoporosis. The first reason postulates that the commencing of the osteoporosis may be due to the underdevelopment of bone during the growth period of individuals. The second reason focuses on the bone development due to heightened resorption process.

**42**

And lastly, osteoporosis may happen due to the lack of new bone growth during the course of remodelling [94]. Earlier, it was believed that osteoporosis is an outbreak of ageing process but with the recent studies it has been observed that it may be caused because of malnutrition, alterations in biomechanical loading, production of excessive hormones and prolonged history of acidosis [95].

#### **6. Mineralisation of synthetic biomaterials**

For many years, biomaterials are being experimented in such a way so as to choose them as a replacement for damaged or diseased tissues. As the exclusive qualities of bone tissues are solely connected to the bone mineralisation, thereby interpreting and regulating the mineralisation process of artificial bone replacement is very crucial [20].

#### **6.1 Inorganic materials**

Since the mineral part of the bone is inorganic in nature, many biomaterials such as calcium phosphates, hydroxyapatite and bioglass are being used as bone alternatives [96]. It was in 1969, it was observed that bioglass forms bond with the bone thus restricting the development of fibrous tissues surrounding the bone [97]. There are various other ceramic biomaterials available such as hydroxyapatite [98], β-tricalcium phosphate, glass–ceramic [99–101], which are after sintering have displayed the bone bonding ability, hence paving the way for themselves clinically in reconstructive and regenerative medicine fields [102].

#### **6.2 Calcium phosphates**

The first in the list of very commonly used bioceramics are the calcium phosphate based ceramics, broadly used in the field of dentistry and orthopaedics. They are mainly used for purpose of coatings onto the top surface of metal implants such as titanium, etc. The study of mineralisation can be performed in some fluids which imitates the ionic composition of blood plasma. One such kind of fluid is simulated body fluid suggested by Kokubo et al. in 1991. This fluid mimics the ionic concentrations of human blood plasma almost exactly. This fluid is used for the purpose of studying mineralisation behaviour of biomaterials in vitro. The SBF initiates the formation of apatite (bone-like) over the synthetic biomaterial surfaces, thus giving the idea of mineralisation of the biomaterial in vivo [103]. Kokubo et al. also suggested that in vivo bone reaction of an artificial biomaterial can be anticipated by the formation of apatite over its surface in SBF, but this theory now has been challenged [104]. In 2010, Bertazzo et al. suggested that calcium phosphates exhibited the osteoconductive nature of bone guided by bone tissues beside the biomaterial surface at the site of implantation orthotopically. Also, there are other calcium phosphate ceramics present which shows osteoinductive traits i.e. ability to form bone at implantation site ectopically. Although, the exact mechanism behind the osteoinductive nature of certain calcium phosphates depend on various factors such as composition, structure and conformation of the calcium phosphate biomaterial [105].

#### **6.3 Bioactive glasses**

In 1969, Hench observed that certain silicon-based glasses formed bond with the bone [106] and named it as Bioactive® glass also known as 45S5 consisting of 45 wt% of SiO2, 24.5 wt% of CaO, 24.5 wt% of Na2O and 6 wt% of P2O5.it can be synthesised by two methods which are sol-gel and melt-quench. Because of its awesome response towards the bone formation in vivo, it has been considered for more clinical applications in the arena of orthopaedics. To comprehend the behaviour of bioglasses in vivo, they have been submerged in SBF to analyse the physio-chemical route of Mineralisation onto the material surface. It has been found that a carbonated apatite layer is formed which is almost like bone mineral. The mineralisation behaviour of calcium phosphate ceramics and bioglasses follow the same mechanism for the apatite formation, thus establishing a bond (chemically) between the host bone and biomaterial [107].

#### **6.4 Organic materials**

Moreover, along with inorganic biomaterials there are other organic biomaterial in the form of biopolymers are available which also shows excellent properties to be considered for tissue regeneration. Such kind of polymers is PLA (polylactic acid), PGA (polyglycolic acid), collagen, hyaluronic acid and many more. The biopolymers do not help in the formation of bone as that of bioceramics but they act as a supporting matrix for the treatment of damaged bone. The biopolymers are broadly divided into two categories I.e. Hydrated and non-hydrated polymers depending on the water retention ability [20].

#### **6.5 Hydrated biopolymers**

Hydrogels are considered under this label as the water is taken up by the polymeric network making them swollen in shape. Since they can take up water very quickly and can retain it, they are considered mainly for the application of cell culture, drug delivery and tissue engineering. On the other hand, they cannot be considered for bone regeneration purpose as they cannot provide any mechanical stability to the affected site [108, 109]. Also, the water content of hydrogels gets so high that it becomes almost impossible to sterilise them [110]. Furthermore, they fail to develop any with the surrounding tissues. The researchers around the world are trying to initiate mineralisation in the hydrogels by incorporating bioceramic particles such as calcium phosphates, hydroxyapatite or bioglass. Also, introduction of enzymes which catalyses the mineralisation activity into the hydrogels or injecting certain artificial analogues to the matrix vesicles in order to trigger biominerlisation. And, lastly the polymeric hydrogels are charged with negative ions or groups in order to invite positively charged calcium ions and thus stimulating mineralisation in inert hydrogels [110].

#### **6.6 Non-hydrated biopolymers**

This category includes many biopolymers such as PLA, PGA, collagen, chitosan, etc. to be considered broadly in the field of tissue engineering. The scaffolds which are made up of non-hydrated polymers can be developed by various techniques such as 3D printing [111], porogen leaching [112], fibre meshing, microsphere sintering [111], phase separation gas foaming [113], and supercritical fluid processing [114]. The scaffold so developed by aforementioned processing techniques differ in surface properties and porosities [115]. Mineralisation of these polymers can be attained by various ways such as incubating in SBF, modifying surface with anionic groups so as to attract calcium ions over the surface of biomaterial. In non-hydrated polymers the assessment of mineralisation behaviour can be studied using SBF similar to bioceramic scaffolds. The nature of SBF taken for mineralisation activity

**45**

**Author details**

Department of Ceramic Engineering, National Institute of Technology, Rourkela,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: pinkideyvs@gmail.com

provided the original work is properly cited.

Pinki Dey

Odisha, India

*Bone Mineralisation*

**7. Conclusions**

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

proteins so as to trigger biominerlisation [116–120].

functioning when compared to the natural bone.

impacts the ionic composition and the configuration of mineral phase for example more concentrated form of SBF speeds up the bone mineralisation. The negatively charged proteins play integral role in controlling the deposition of mineral phase onto the natural bone, thus affecting the mineralisation in SBF. Hence, the surface of biopolymer is functionalized with negatively charged proteins or bone related

Bone is a very complex organ which has hierarchical organised tissue that helps in giving protection and support mechanically to all the organs including brain because of its mineralised behaviour. The mineralisation of bone is mediated by either collagen or matrix vesicles. Both the pathways are interconnected; but, their interconnectivity is still not extensively studied. By studying the mineralisation pattern of bone, several bone related diseases caused due to pathological mineralisation or other reasons lacking in the mineralisation can be cured with the help of development of advanced biomaterials which may showcase equivalent levels of biological

#### *Bone Mineralisation DOI: http://dx.doi.org/10.5772/intechopen.92065*

impacts the ionic composition and the configuration of mineral phase for example more concentrated form of SBF speeds up the bone mineralisation. The negatively charged proteins play integral role in controlling the deposition of mineral phase onto the natural bone, thus affecting the mineralisation in SBF. Hence, the surface of biopolymer is functionalized with negatively charged proteins or bone related proteins so as to trigger biominerlisation [116–120].

### **7. Conclusions**

*Contemporary Topics about Phosphorus in Biology and Materials*

host bone and biomaterial [107].

the water retention ability [20].

**6.5 Hydrated biopolymers**

tion in inert hydrogels [110].

**6.6 Non-hydrated biopolymers**

**6.4 Organic materials**

45 wt% of SiO2, 24.5 wt% of CaO, 24.5 wt% of Na2O and 6 wt% of P2O5.it can be synthesised by two methods which are sol-gel and melt-quench. Because of its awesome response towards the bone formation in vivo, it has been considered for more clinical applications in the arena of orthopaedics. To comprehend the behaviour of bioglasses in vivo, they have been submerged in SBF to analyse the physio-chemical route of Mineralisation onto the material surface. It has been found that a carbonated apatite layer is formed which is almost like bone mineral. The mineralisation behaviour of calcium phosphate ceramics and bioglasses follow the same mechanism for the apatite formation, thus establishing a bond (chemically) between the

Moreover, along with inorganic biomaterials there are other organic biomaterial in the form of biopolymers are available which also shows excellent properties to be considered for tissue regeneration. Such kind of polymers is PLA (polylactic acid), PGA (polyglycolic acid), collagen, hyaluronic acid and many more. The biopolymers do not help in the formation of bone as that of bioceramics but they act as a supporting matrix for the treatment of damaged bone. The biopolymers are broadly divided into two categories I.e. Hydrated and non-hydrated polymers depending on

Hydrogels are considered under this label as the water is taken up by the polymeric network making them swollen in shape. Since they can take up water very quickly and can retain it, they are considered mainly for the application of cell culture, drug delivery and tissue engineering. On the other hand, they cannot be considered for bone regeneration purpose as they cannot provide any mechanical stability to the affected site [108, 109]. Also, the water content of hydrogels gets so high that it becomes almost impossible to sterilise them [110]. Furthermore, they fail to develop any with the surrounding tissues. The researchers around the world are trying to initiate mineralisation in the hydrogels by incorporating bioceramic particles such as calcium phosphates, hydroxyapatite or bioglass. Also, introduction of enzymes which catalyses the mineralisation activity into the hydrogels or injecting certain artificial analogues to the matrix vesicles in order to trigger biominerlisation. And, lastly the polymeric hydrogels are charged with negative ions or groups in order to invite positively charged calcium ions and thus stimulating mineralisa-

This category includes many biopolymers such as PLA, PGA, collagen, chitosan, etc. to be considered broadly in the field of tissue engineering. The scaffolds which are made up of non-hydrated polymers can be developed by various techniques such as 3D printing [111], porogen leaching [112], fibre meshing, microsphere sintering [111], phase separation gas foaming [113], and supercritical fluid processing [114]. The scaffold so developed by aforementioned processing techniques differ in surface properties and porosities [115]. Mineralisation of these polymers can be attained by various ways such as incubating in SBF, modifying surface with anionic groups so as to attract calcium ions over the surface of biomaterial. In non-hydrated polymers the assessment of mineralisation behaviour can be studied using SBF similar to bioceramic scaffolds. The nature of SBF taken for mineralisation activity

**44**

Bone is a very complex organ which has hierarchical organised tissue that helps in giving protection and support mechanically to all the organs including brain because of its mineralised behaviour. The mineralisation of bone is mediated by either collagen or matrix vesicles. Both the pathways are interconnected; but, their interconnectivity is still not extensively studied. By studying the mineralisation pattern of bone, several bone related diseases caused due to pathological mineralisation or other reasons lacking in the mineralisation can be cured with the help of development of advanced biomaterials which may showcase equivalent levels of biological functioning when compared to the natural bone.

### **Author details**

Pinki Dey Department of Ceramic Engineering, National Institute of Technology, Rourkela, Odisha, India

\*Address all correspondence to: pinkideyvs@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Burr DB. Bone morphology and organization. In: Basic and Applied Bone Biology. 2nd ed. Academic Press, Elsevier; 2019

[2] Bonnucci E, Motta PM. Ultrastructure of skeletal tissues. In: Bone and Cartilage in Health and Disease. Boston: Kluwer Academic Publishers; 1990

[3] Brookes M, Revell WJ. Blood Supply of Bone: Scientific Aspects. London: Springer-Verlag; 1998

[4] Burr DB, Allen MR. Calcified tissue international, special issue: Bone material properties and and skeletal fragility. Calcified Tissue International. 2015;**97**:199-241

[5] Castañeda-Corral G, Jimenez-Andrade JM, Blook AP, Taylor RN, Mantyh WG, Kaczmarska MJ. The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A. Neuroscience. 2011;**178**:196-207

[6] Dempster D, Felsednberg D, van der Geest S. The Bone Quality Book. Amsterdam: Elsevier; 2006

[7] Enlow DH, Brown SO. A comparative histological study of fossil and recent bone tissues. Part III. Mammalian bone tissues. Texas Journal of Science. 1957;**10**:187-230

[8] Fonseca H, Moreira-Gonçalves D, Appell Coriolano HJ, Duarte JA. Bone quality: The determinants of bone strength and fragility. Sports Medicine. 2014;**44**:37-53

[9] Foote JS. A contribution to the comparative histology of the femur. Smithsonian Contrib. Knowl. 1916;**35**:1-242

[10] Fuchs RK, Allen MR, Ruppel ME, Diab T, Phipps RJ, Miller LM. In situ

examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy. Matrix Biology. 2008;**27**:34-41

[11] Fukumoto TJ. Bone as an endocrine organ. Trends Endocrinol. Metab. 2009;**20**:230-236

[12] Gurkan UA, Akkus O. The mechanical environment of bone marrow: A review. Annals of Biomedical Engineering. 2008;**36**:1978-1991

[13] Jee WSS, Weiss L. The skeletal tissues. In: Weiss L, editor. Histology: Cell and Tissue Biology. New York: Elsevier Biomedical; 1983

[14] Kaplan FS, Hayes WC, Keaveny TM, et al. Form and function of bone. In: Simon SR, editor. Orthopaedic Basic Science. Chicago: American Academy of Orthopaedic Surgeons; 1994

[15] Karsenty G, MacDougald O, Rosen CJ. Interactions between bone, adipose tissue and metabolism. Bone. 2012;**50**(Special Issue):429-579

[16] Martin RB, Burr DB, Sharkey NA, Fyhrie DP. Skeletal Tissue Mechanics. 2nd ed. New York: Springer-Verlag; 2015

[17] Reznikov N, Bilton M, Lari L, Stevens MM, Kröger R. Fractual-like hierarchical organization of bone begins at the nanoscale. Science. 2018;**360**:507-517

[18] Ruppel ME, Miller LM, Burr DB. The effect of the micro¬scopic and nanoscale structure on bone fragility. Osteoporos. Int. 2008;**19**:1251-1265

[19] Sivaraj KK, Adams RH. Blood vessel formation and function in bone. Development. 2016;**143**:2706-2715

**47**

*Bone Mineralisation*

pp. 129-146

2001;**10**(2):S86-S95

2000;**15**(2):198-208

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

and differential interference contrast microscopy. Bone. 2001;**28**(2):145-149

D-enhanced osteocytic and osteoclastic bone resorption. The American Journal of Physiology. 1973;**224**(6):1345-1357

Väänänen HK. Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts. Experimental Cell Research. 2004;**294**(2):458-468

[32] Zhao S et al. MLO-Y4 osteocyte-like cells support osteoclast formation and activation. Journal of Bone and Mineral Research. 2002a;**17**(11):2068-2079

[33] Beniash E. Biominerals— Hierarchical nanocomposites: The example of bone. Wiley Interdisciplinary

Reviews - Nanomedicine and Nanobiotechnology. 2011;**3**(1):47-69

[34] Boskey AL. Matrix proteins and mineralization: An overview. Connective Tissue Research.

[35] Boskey AL. Biomineralization: Conflicts, challenges, and opportunities.

Journal of Cellular Biochemistry.

[36] Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoproteins: Role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein. The Biochemical Journal.

[37] Stubbs JT et al. Characterization of native and recombinant bone

sialoprotein: Delineation of the mineralbinding and cell adhesion domains and structural analysis of the RGD domain. Journal of Bone and Mineral Research.

1996;**35**(1-4):357-363

1998;**72**(S30-31):83-91

1994;**302**:175-179

1997;**12**(8):1210-1222

[30] Baylink D et al. Vitamin

[31] Heino TJ, Hentunen TA,

[20] An J, Leeuwenburgh S, Wolke J, Jansen J. Mineralization processes in hard tissue: Bone. In: Biomineralization and Biomaterials. Elsevier; 2016.

[21] Sommerfeldt D, Rubin C. Biology of bone and how it orchestrates the form and function of the skeleton. European Spine Journal.

[22] Compton JT, Lee FY. A review of osteocyte function and the emerging importance of sclerostin. The Journal of Bone and Joint Surgery. American Volume. 2014;**96**(19):1659-1668

N-cadherin in the development of the differentiated osteoblastic phenotype. Journal of Bone and Mineral Research.

[24] Harada S-I, Rodan GA. Control

resorption by osteoclasts. Science. 2000;**289**(5484):1504-1508

[26] Walker DG. Enzymatic and

[27] Lanyon L. Osteocytes, strain detection, bone modeling and remodeling. Calcified Tissue International. 1993;**53**(1):S102-S107

[28] Bonewald LF. Osteocytes as dynamic multifunctional cells. Annals of the New York Academy of Sciences.

[29] Kamioka H, Honjo T, Takano-Yamamoto T. A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy

2007;**1116**(1):281-290

electron microscopic analysis of isolated osteoclasts. Calcified Tissue Research.

of osteoblast function and regulation of bone mass. Nature.

2003;**423**(6937):349-355

[25] Teitelbaum SL. Bone

1972;**9**(1):296-309

[23] Ferrari SL et al. A role for

#### *Bone Mineralisation DOI: http://dx.doi.org/10.5772/intechopen.92065*

[20] An J, Leeuwenburgh S, Wolke J, Jansen J. Mineralization processes in hard tissue: Bone. In: Biomineralization and Biomaterials. Elsevier; 2016. pp. 129-146

[21] Sommerfeldt D, Rubin C. Biology of bone and how it orchestrates the form and function of the skeleton. European Spine Journal. 2001;**10**(2):S86-S95

[22] Compton JT, Lee FY. A review of osteocyte function and the emerging importance of sclerostin. The Journal of Bone and Joint Surgery. American Volume. 2014;**96**(19):1659-1668

[23] Ferrari SL et al. A role for N-cadherin in the development of the differentiated osteoblastic phenotype. Journal of Bone and Mineral Research. 2000;**15**(2):198-208

[24] Harada S-I, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature. 2003;**423**(6937):349-355

[25] Teitelbaum SL. Bone resorption by osteoclasts. Science. 2000;**289**(5484):1504-1508

[26] Walker DG. Enzymatic and electron microscopic analysis of isolated osteoclasts. Calcified Tissue Research. 1972;**9**(1):296-309

[27] Lanyon L. Osteocytes, strain detection, bone modeling and remodeling. Calcified Tissue International. 1993;**53**(1):S102-S107

[28] Bonewald LF. Osteocytes as dynamic multifunctional cells. Annals of the New York Academy of Sciences. 2007;**1116**(1):281-290

[29] Kamioka H, Honjo T, Takano-Yamamoto T. A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone. 2001;**28**(2):145-149

[30] Baylink D et al. Vitamin D-enhanced osteocytic and osteoclastic bone resorption. The American Journal of Physiology. 1973;**224**(6):1345-1357

[31] Heino TJ, Hentunen TA, Väänänen HK. Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts. Experimental Cell Research. 2004;**294**(2):458-468

[32] Zhao S et al. MLO-Y4 osteocyte-like cells support osteoclast formation and activation. Journal of Bone and Mineral Research. 2002a;**17**(11):2068-2079

[33] Beniash E. Biominerals— Hierarchical nanocomposites: The example of bone. Wiley Interdisciplinary Reviews - Nanomedicine and Nanobiotechnology. 2011;**3**(1):47-69

[34] Boskey AL. Matrix proteins and mineralization: An overview. Connective Tissue Research. 1996;**35**(1-4):357-363

[35] Boskey AL. Biomineralization: Conflicts, challenges, and opportunities. Journal of Cellular Biochemistry. 1998;**72**(S30-31):83-91

[36] Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoproteins: Role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein. The Biochemical Journal. 1994;**302**:175-179

[37] Stubbs JT et al. Characterization of native and recombinant bone sialoprotein: Delineation of the mineralbinding and cell adhesion domains and structural analysis of the RGD domain. Journal of Bone and Mineral Research. 1997;**12**(8):1210-1222

**46**

*Contemporary Topics about Phosphorus in Biology and Materials*

examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy.

[11] Fukumoto TJ. Bone as an endocrine organ. Trends Endocrinol. Metab.

Matrix Biology. 2008;**27**:34-41

[12] Gurkan UA, Akkus O. The mechanical environment of bone marrow: A review. Annals of Biomedical Engineering.

[13] Jee WSS, Weiss L. The skeletal tissues. In: Weiss L, editor. Histology: Cell and Tissue Biology. New York:

[14] Kaplan FS, Hayes WC, Keaveny TM, et al. Form and function of bone. In: Simon SR, editor. Orthopaedic Basic Science. Chicago: American Academy of

2009;**20**:230-236

2008;**36**:1978-1991

Elsevier Biomedical; 1983

Orthopaedic Surgeons; 1994

2015

2018;**360**:507-517

[15] Karsenty G, MacDougald O, Rosen CJ. Interactions between bone, adipose tissue and metabolism. Bone. 2012;**50**(Special Issue):429-579

[16] Martin RB, Burr DB, Sharkey NA, Fyhrie DP. Skeletal Tissue Mechanics. 2nd ed. New York: Springer-Verlag;

[17] Reznikov N, Bilton M, Lari L, Stevens MM, Kröger R. Fractual-like hierarchical organization of bone begins at the nanoscale. Science.

[18] Ruppel ME, Miller LM, Burr DB. The effect of the micro¬scopic and nanoscale structure on bone fragility. Osteoporos. Int. 2008;**19**:1251-1265

[19] Sivaraj KK, Adams RH. Blood vessel formation and function in bone. Development. 2016;**143**:2706-2715

[1] Burr DB. Bone morphology and organization. In: Basic and Applied Bone Biology. 2nd ed. Academic Press,

Ultrastructure of skeletal tissues. In: Bone and Cartilage in Health and Disease. Boston: Kluwer Academic

[3] Brookes M, Revell WJ. Blood Supply of Bone: Scientific Aspects. London:

[4] Burr DB, Allen MR. Calcified tissue international, special issue: Bone material properties and and skeletal fragility. Calcified Tissue International.

[5] Castañeda-Corral G, Jimenez-Andrade JM, Blook AP, Taylor RN, Mantyh WG, Kaczmarska MJ. The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A. Neuroscience. 2011;**178**:196-207

[6] Dempster D, Felsednberg D, van der Geest S. The Bone Quality Book.

[7] Enlow DH, Brown SO. A comparative histological study of fossil and recent bone tissues. Part III. Mammalian bone tissues. Texas Journal of Science.

[8] Fonseca H, Moreira-Gonçalves D, Appell Coriolano HJ, Duarte JA. Bone quality: The determinants of bone strength and fragility. Sports Medicine.

[9] Foote JS. A contribution to the comparative histology of the femur. Smithsonian Contrib. Knowl.

[10] Fuchs RK, Allen MR, Ruppel ME, Diab T, Phipps RJ, Miller LM. In situ

Amsterdam: Elsevier; 2006

1957;**10**:187-230

2014;**44**:37-53

1916;**35**:1-242

[2] Bonnucci E, Motta PM.

Elsevier; 2019

**References**

Publishers; 1990

2015;**97**:199-241

Springer-Verlag; 1998

[38] Gebauer D, Völkel A, Cölfen H. Stable prenucleation calcium carbonate clusters. Science. 2008;**322**(5909):1819-1822

[39] Pouget EM et al. The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM. Science. 2009;**323**(5920):1455-1458

[40] Chai YC et al. Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies. Acta Biomaterialia. 2012;**8**(11):3876-3887

[41] Anderson HC. Molecular biology of matrix vesicles. Clinical Orthopaedics and Related Research. 1995;**314**:266-280

[42] Anderson HC. Matrix vesicles and calcification. Current Rheumatology Reports. 2003;**5**(3):222-226

[43] Landis W. Chemistry and Biology of Mineralized Tissues. Toronto: University of Toronto Press; 2005

[44] Aparicio S et al. Optimal methods for processing mineralized tissues for Fourier transform infrared microspectroscopy. Calcified Tissue International. 2002;**70**(5):422-429

[45] Boskey AL. Mineralization of bones and teeth. Elements. 2007;**3**(6):385-391

[46] Palmer LC et al. Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel. Chemical Reviews. 2008;**108**(11):4754-4783

[47] He G et al. Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization. The Journal of Biological Chemistry. 2005;**280**(39):33109-33114

[48] Monfoulet L et al. Bone sialoprotein, but not osteopontin, deficiency impairs the mineralization of regenerating bone during cortical defect healing. Bone. 2010;**46**(2):447-452

[49] Farbod K et al. Interactions between inorganic and organic phases in bone tissue as a source of inspiration for design of novel nanocomposites. Tissue Engineering. Part B, Reviews. 2013;**20**(2):173-188

[50] Meyer U et al. Decreased expression of osteocalcin and osteonectin in relation to high strains and decreased mineralization in mandibular distraction osteogenesis. The Journal of Cranio-Maxillofacial Surgery. 1999;**27**(4):222-227

[51] Hoang QQ et al. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature. 2003;**425**(6961):977-980

[52] Roach H. Why does bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption. Cell Biology International. 1994;**18**:617-628

[53] Rosenthal AK et al. Osteopontin promotes pathologic mineralization in articular cartilage. Matrix Biology. 2007;**26**(2):96-105

[54] Miller BF et al. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise. The Journal of Physiology. 2005;**567**(3):1021-1033

[55] Orgel JP et al. Microfibrillar structure of type I collagen in situ. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(24):9001-9005

[56] Nudelman F et al. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nature Materials. 2010;**9**(12):1004-1009

**49**

*Bone Mineralisation*

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

[57] Glimcher M, Muir H. Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds. Philosophical Transactions of the Royal Society B. 1984;**304**(1121):479-508

from bovine fetal alveolar bone and dog osteosarcoma. Calcified Tissue International. 1983;**35**(1):791-797

phospholipids in biological calcification: Distribution of phospholipase activity in calcifying epiphyseal cartilage. Clinical Orthopaedics and Related Research.

[67] Dean DD et al. Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans. Calcified Tissue International. 1992;**50**(4):342-349

[68] Wu L et al. Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen. The Journal of Biological Chemistry.

1991;**266**(2):1195-1203

2006;**18**(2):174-180

1998;**22**(1):79-84

1990. pp. 329-396

2006;**38**(11):1310-1315

1974;**303**(7851):229-233

[74] Lynch MJ et al. Fibrous

osteodystrophy in dromedary camels

[69] Kirsch T. Determinants of pathological mineralization. Current Opinion in Rheumatology.

[70] Yeni Y, Brown C, Norman T. Influence of bone composition and apparent density on fracture toughness of the human femur and tibia. Bone.

[72] Feng JQ et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nature Genetics.

[73] Aaron J et al. Frequency of osteomalacia and osteoporosis in fractures of the proximal femur. Lancet.

[71] Parfitt A. Osteomalacia and related disorders. In: Metabolic Bone Disease and Clinically Related Disorders. 2nd ed. Philadelphia, PA: WB Saunders;

[66] Wuthier RE. The role of

1973;**90**:191-200

[58] Landis W et al. Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. Journal of Structural Biology. 1993;**110**(1):39-54

[59] Traub W, Arad T, Weiner S. Origin of mineral crystal growth in collagen fibrils. Matrix. 1992;**12**(4):251-255

[60] Cölfen H. Biomineralization: A crystal-clear view. Nature Materials.

[61] Stewart T. The presence of delayed hypersensitivity reactions in patients toward cellular extracts of their malignant tumors. 1. The role of tissue antigen, nonspecific reactions of nuclear material, and bacterial antigen as a cause for this phenomenon. Cancer.

[62] Wu L et al. Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. Evidence of an association between matrix vesicles and collagen. The Journal of Biological Chemistry. 1989;**264**(35):21346-21355

[63] Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences of the United States of America. 1993;**90**(18):8562-8565

[64] Stechschulte Jr, DJ, et al. Presence and specific concentration of carbonic anhydrase II in matrix vesicles. Bone and Mineral. 1992;**17**(2):187-191

[65] Hirschman A et al. Neutral peptidase activities in matrix vesicles

2010;**9**(12):960-961

1969;**23**(6):1368-1379

#### *Bone Mineralisation DOI: http://dx.doi.org/10.5772/intechopen.92065*

*Contemporary Topics about Phosphorus in Biology and Materials*

of regenerating bone during cortical defect healing. Bone.

[49] Farbod K et al. Interactions between inorganic and organic phases in bone tissue as a source of inspiration for design of novel nanocomposites. Tissue Engineering. Part B, Reviews.

[50] Meyer U et al. Decreased expression of osteocalcin and osteonectin in relation to high strains and decreased mineralization in mandibular distraction osteogenesis. The Journal of Cranio-Maxillofacial Surgery. 1999;**27**(4):222-227

[51] Hoang QQ et al. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature. 2003;**425**(6961):977-980

[52] Roach H. Why does bone matrix contain non-collagenous proteins? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone mineralisation and resorption. Cell Biology International.

[53] Rosenthal AK et al. Osteopontin promotes pathologic mineralization in articular cartilage. Matrix Biology.

[54] Miller BF et al. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise. The Journal of Physiology. 2005;**567**(3):1021-1033

[55] Orgel JP et al. Microfibrillar structure of type I collagen in situ. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(24):9001-9005

[56] Nudelman F et al. The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nature Materials.

2010;**9**(12):1004-1009

2010;**46**(2):447-452

2013;**20**(2):173-188

1994;**18**:617-628

2007;**26**(2):96-105

[38] Gebauer D, Völkel A, Cölfen H. Stable prenucleation calcium carbonate clusters. Science. 2008;**322**(5909):1819-1822

[39] Pouget EM et al. The initial stages of template-controlled CaCO3 formation revealed by cryo-TEM. Science. 2009;**323**(5920):1455-1458

[40] Chai YC et al. Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies. Acta Biomaterialia. 2012;**8**(11):3876-3887

[41] Anderson HC. Molecular biology of matrix vesicles. Clinical Orthopaedics and Related Research.

Reports. 2003;**5**(3):222-226

of Toronto Press; 2005

[42] Anderson HC. Matrix vesicles and calcification. Current Rheumatology

[43] Landis W. Chemistry and Biology of Mineralized Tissues. Toronto: University

[44] Aparicio S et al. Optimal methods for processing mineralized tissues for Fourier transform infrared microspectroscopy. Calcified Tissue International. 2002;**70**(5):422-429

[45] Boskey AL. Mineralization of bones and teeth. Elements. 2007;**3**(6):385-391

[46] Palmer LC et al. Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel. Chemical Reviews. 2008;**108**(11):4754-4783

[47] He G et al. Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization. The Journal of Biological Chemistry. 2005;**280**(39):33109-33114

[48] Monfoulet L et al. Bone sialoprotein, but not osteopontin, deficiency impairs the mineralization

1995;**314**:266-280

**48**

[57] Glimcher M, Muir H. Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds. Philosophical Transactions of the Royal Society B. 1984;**304**(1121):479-508

[58] Landis W et al. Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high-voltage electron microscopic tomography and graphic image reconstruction. Journal of Structural Biology. 1993;**110**(1):39-54

[59] Traub W, Arad T, Weiner S. Origin of mineral crystal growth in collagen fibrils. Matrix. 1992;**12**(4):251-255

[60] Cölfen H. Biomineralization: A crystal-clear view. Nature Materials. 2010;**9**(12):960-961

[61] Stewart T. The presence of delayed hypersensitivity reactions in patients toward cellular extracts of their malignant tumors. 1. The role of tissue antigen, nonspecific reactions of nuclear material, and bacterial antigen as a cause for this phenomenon. Cancer. 1969;**23**(6):1368-1379

[62] Wu L et al. Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. Evidence of an association between matrix vesicles and collagen. The Journal of Biological Chemistry. 1989;**264**(35):21346-21355

[63] Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences of the United States of America. 1993;**90**(18):8562-8565

[64] Stechschulte Jr, DJ, et al. Presence and specific concentration of carbonic anhydrase II in matrix vesicles. Bone and Mineral. 1992;**17**(2):187-191

[65] Hirschman A et al. Neutral peptidase activities in matrix vesicles from bovine fetal alveolar bone and dog osteosarcoma. Calcified Tissue International. 1983;**35**(1):791-797

[66] Wuthier RE. The role of phospholipids in biological calcification: Distribution of phospholipase activity in calcifying epiphyseal cartilage. Clinical Orthopaedics and Related Research. 1973;**90**:191-200

[67] Dean DD et al. Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans. Calcified Tissue International. 1992;**50**(4):342-349

[68] Wu L et al. Collagen-binding proteins in collagenase-released matrix vesicles from cartilage. Interaction between matrix vesicle proteins and different types of collagen. The Journal of Biological Chemistry. 1991;**266**(2):1195-1203

[69] Kirsch T. Determinants of pathological mineralization. Current Opinion in Rheumatology. 2006;**18**(2):174-180

[70] Yeni Y, Brown C, Norman T. Influence of bone composition and apparent density on fracture toughness of the human femur and tibia. Bone. 1998;**22**(1):79-84

[71] Parfitt A. Osteomalacia and related disorders. In: Metabolic Bone Disease and Clinically Related Disorders. 2nd ed. Philadelphia, PA: WB Saunders; 1990. pp. 329-396

[72] Feng JQ et al. Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nature Genetics. 2006;**38**(11):1310-1315

[73] Aaron J et al. Frequency of osteomalacia and osteoporosis in fractures of the proximal femur. Lancet. 1974;**303**(7851):229-233

[74] Lynch MJ et al. Fibrous osteodystrophy in dromedary camels (Camelus dromedarius). Journal of Zoo and Wildlife Medicine. 1999;**30**:577-583

[75] Fincham J et al. Mseleni joint disease. Part I. An animal model? South African Medical Journal. 1985;**67**(2):51-57

[76] Long GG et al. Fibrous osteodystrophy in an opossum. Journal of Wildlife Diseases. 1975;**11**(2):221-223

[77] Flom JO, Brown RJ, Jones RE. Fibrous osteodystrophy in a wild dolphin. Journal of the American Veterinary Medical Association. 1978;**173**(9):1124-1126

[78] Jaffe H, Bodansky A, Blair JE. Fibrous osteodystrophy (osteitis fibrosa) in experimental hyperparathyroidism of Guinea-pigs. Archives of Pathology. 1931;**11**:207

[79] Fetter A, Siemering G, Riser W. Osteoporosis and osteopetrosis. In: Newton CD, Nunamaker DM, editors. Textbook of Small Animal Orthopaedics. Philadelphia, PA: Lippincott; 1985. pp. 627-629

[80] Whyte MP. Paget's disease of bone. The New England Journal of Medicine. 2006;**355**(6):593-600

[81] Siris ES. Paget's disease of bone. Journal of Bone and Mineral Research. 1998;**13**(7):1061-1065

[82] Kanis JA. Pathophysiology and Treatment of Paget's Disease of Bone. Oxford: Taylor & Francis; 1998

[83] Altman RD et al. Influence of disodium etidronate on clinical and laboratory manifestations of Paget's disease of bone (osteitis deformans). The New England Journal of Medicine. 1973;**289**(26):1379-1384

[84] Rebel A et al. Osteoclast ultrastructure in Paget's disease. Calcified Tissue Research. 1976;**20**(1):187-199

[85] Meunier PJ et al. Bone histomorphometry in Paget's disease: Quantitative and dynamic analysis of pagetic and nonpagetic bone tissue. Arthritis and Rheumatism. 1980;**23**(10):1095-1103

[86] Merkow R, Lane J. Paget's disease of bone. The Orthopedic Clinics of North America. 1990;**21**(1):171-189

[87] Ooi C, Fraser W. Paget's disease of bone. Postgraduate Medical Journal. 1997;**73**(856):69-74

[88] Kanis J et al. The use of clinical risk factors enhances the performance of BMD in the prediction of hip and osteoporotic fractures in men and women. Osteoporosis International. 2007;**18**(8):1033-1046

[89] Gao H et al. Materials become insensitive to flaws at nanoscale: Lessons from nature. Proceedings of the National Academy of Sciences of the United States of America. 2003;**100**(10):5597-5600

[90] Faibish D, Ott SM, Boskey AL. Mineral changes in osteoporosis: A review. Clinical Orthopaedics and Related Research. 2006;**443**:28

[91] Cefalu CA. Is bone mineral density predictive of fracture risk reduction? Current Medical Research and Opinion. 2004;**20**(3):341-349

[92] McCalden RW, McGeough JA. Age-related changes in the compressive strength of cancellous bone. The relative importance of changes in density and trabecular architecture. The Journal of Bone and Joint Surgery. American Volume. 1997;**79**(3):421-427

[93] Koh L, Ng D. Osteoporosis risk factor assessment and bone densitometry–current status and future trends. Annals of the Academy of Medicine, Singapore. 2002;**31**(1):37-42

**51**

*Bone Mineralisation*

*DOI: http://dx.doi.org/10.5772/intechopen.92065*

pathology: Evidence-Based Perspectives from Molecular Biology to Systems Biology. Dordrecht: Springer; 2011

[104] Bohner M, Lemaitre J. Can bioactivity be tested in vitro with SBF solution? Biomaterials.

[105] Bertazzo S et al. Hydroxyapatite surface solubility and effect on cell adhesion. Colloids surf. B Biointerfaces.

[106] Hench LL, Wilson J. Surfaceactive biomaterials. Science. 1984;**226**(4675):630-636

[107] Gross U, Schmitz HJ, Strunz V. Surface activities of bioactive glass, aluminum oxide, and titanium in a living environment. Annals of the New York Academy of Sciences.

[108] Hoffman AS. Hydrogels for biomedical applications. Advanced Drug Delivery Reviews. 2002;**54**(1):3-12

[109] Driessens F et al. Effective formulations for the preparation of calcium phosphate bone cements. Journal of Materials Science. Materials

in Medicine. 1994;**5**(3):164-170

2010;**16**(6):577-585

[110] Gkioni K et al. Mineralization of hydrogels for bone regeneration. Tissue Engineering. Part B, Reviews.

[111] Sherwood JK et al. A threedimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials. 2002;**23**(24):4739-4751

[112] Mikos AG et al. Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. Journal of Biomedical Materials Research. 1993;**27**(2):183-189

[113] Langer RS, Vacanti JP. Preparation

scaffold for attaching cells to produce vascularized tissue in vivo. Google

of three-dimensional fibrous

Patents; 1998

2009;**30**(12):2175-2179

2010;**78**(2):177-184

1988;**523**(1):211-226

[95] NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy. Osteoporosis prevention,

[94] Chiappelli F. Osteoimmuno-

diagnosis, and therapy. JAMA.

[96] Jarcho M. Calcium phosphate ceramics as hard tissue prosthetics. Clinical Orthopaedics and Related

Research. 1981;**157**:259-278

[97] Hench LL et al. Bonding mechanisms at the interface of ceramic prosthetic materials. Journal of Biomedical Materials Research.

[98] Jarcho M et al. Tissue, cellular and subcellular events at a bone-ceramic hydroxylapatite interface. Journal of Bioengineering. 1977;**1**(2):79-92

[99] Ducheyne P, De Groot K. In vivo surface activity of a hydroxyapatite alveolar bonesubstitute. Journal of Biomedical Materials Research.

[100] LeGeros R et al. Biphasic calcium phosphate bioceramics: Preparation, properties and applications. Journal of Materials Science. Materials in Medicine. 2003;**14**(3):201-209

[101] Kitsugi T et al. Bone bonding behavior of three kinds of apatite containing glass ceramics. Journal of Biomedical Materials Research.

[102] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials.

2001;**285**(6):785-795

1971;**5**(6):117-141

1981;**15**(3):441-445

1986;**20**(9):1295-1307

2006;**27**(15):2907-2915

[103] Kokubo T. Bioactive glass

ceramics: Properties and applications. Biomaterials. 1991;**12**(2):155-163

*Bone Mineralisation DOI: http://dx.doi.org/10.5772/intechopen.92065*

*Contemporary Topics about Phosphorus in Biology and Materials*

[85] Meunier PJ et al. Bone

1980;**23**(10):1095-1103

1997;**73**(856):69-74

2007;**18**(8):1033-1046

2003;**100**(10):5597-5600

2004;**20**(3):341-349

1997;**79**(3):421-427

America. 1990;**21**(1):171-189

histomorphometry in Paget's disease: Quantitative and dynamic analysis of pagetic and nonpagetic bone tissue. Arthritis and Rheumatism.

[86] Merkow R, Lane J. Paget's disease of bone. The Orthopedic Clinics of North

[87] Ooi C, Fraser W. Paget's disease of bone. Postgraduate Medical Journal.

[88] Kanis J et al. The use of clinical risk factors enhances the performance of BMD in the prediction of hip and osteoporotic fractures in men and women. Osteoporosis International.

[89] Gao H et al. Materials become insensitive to flaws at nanoscale: Lessons from nature. Proceedings of the National Academy of Sciences of the United States of America.

[90] Faibish D, Ott SM, Boskey AL. Mineral changes in osteoporosis: A review. Clinical Orthopaedics and Related Research. 2006;**443**:28

[91] Cefalu CA. Is bone mineral density predictive of fracture risk reduction? Current Medical Research and Opinion.

[92] McCalden RW, McGeough JA.

compressive strength of cancellous bone. The relative importance of changes in density and trabecular architecture. The Journal of Bone and Joint Surgery. American Volume.

Age-related changes in the

[93] Koh L, Ng D. Osteoporosis risk factor assessment and bone

densitometry–current status and future trends. Annals of the Academy of Medicine, Singapore. 2002;**31**(1):37-42

(Camelus dromedarius). Journal of Zoo and Wildlife Medicine. 1999;**30**:577-583

osteodystrophy in an opossum. Journal of Wildlife Diseases. 1975;**11**(2):221-223

[77] Flom JO, Brown RJ, Jones RE. Fibrous osteodystrophy in a wild dolphin. Journal of the American Veterinary Medical Association.

[78] Jaffe H, Bodansky A, Blair JE. Fibrous osteodystrophy (osteitis fibrosa) in experimental hyperparathyroidism of Guinea-pigs. Archives of Pathology.

[79] Fetter A, Siemering G, Riser W. Osteoporosis and osteopetrosis. In: Newton CD, Nunamaker DM, editors. Textbook of Small Animal Orthopaedics. Philadelphia, PA: Lippincott; 1985. pp. 627-629

[80] Whyte MP. Paget's disease of bone. The New England Journal of Medicine.

[81] Siris ES. Paget's disease of bone. Journal of Bone and Mineral Research.

[82] Kanis JA. Pathophysiology and Treatment of Paget's Disease of Bone. Oxford: Taylor & Francis; 1998

[83] Altman RD et al. Influence of disodium etidronate on clinical and laboratory manifestations of Paget's disease of bone (osteitis deformans). The New England Journal of Medicine.

[75] Fincham J et al. Mseleni joint disease. Part I. An animal model? South African Medical Journal.

[76] Long GG et al. Fibrous

1978;**173**(9):1124-1126

2006;**355**(6):593-600

1998;**13**(7):1061-1065

1973;**289**(26):1379-1384

[84] Rebel A et al. Osteoclast ultrastructure in Paget's disease. Calcified Tissue Research. 1976;**20**(1):187-199

1931;**11**:207

1985;**67**(2):51-57

**50**

[94] Chiappelli F. Osteoimmunopathology: Evidence-Based Perspectives from Molecular Biology to Systems Biology. Dordrecht: Springer; 2011

[95] NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy. Osteoporosis prevention, diagnosis, and therapy. JAMA. 2001;**285**(6):785-795

[96] Jarcho M. Calcium phosphate ceramics as hard tissue prosthetics. Clinical Orthopaedics and Related Research. 1981;**157**:259-278

[97] Hench LL et al. Bonding mechanisms at the interface of ceramic prosthetic materials. Journal of Biomedical Materials Research. 1971;**5**(6):117-141

[98] Jarcho M et al. Tissue, cellular and subcellular events at a bone-ceramic hydroxylapatite interface. Journal of Bioengineering. 1977;**1**(2):79-92

[99] Ducheyne P, De Groot K. In vivo surface activity of a hydroxyapatite alveolar bonesubstitute. Journal of Biomedical Materials Research. 1981;**15**(3):441-445

[100] LeGeros R et al. Biphasic calcium phosphate bioceramics: Preparation, properties and applications. Journal of Materials Science. Materials in Medicine. 2003;**14**(3):201-209

[101] Kitsugi T et al. Bone bonding behavior of three kinds of apatite containing glass ceramics. Journal of Biomedical Materials Research. 1986;**20**(9):1295-1307

[102] Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials. 2006;**27**(15):2907-2915

[103] Kokubo T. Bioactive glass ceramics: Properties and applications. Biomaterials. 1991;**12**(2):155-163

[104] Bohner M, Lemaitre J. Can bioactivity be tested in vitro with SBF solution? Biomaterials. 2009;**30**(12):2175-2179

[105] Bertazzo S et al. Hydroxyapatite surface solubility and effect on cell adhesion. Colloids surf. B Biointerfaces. 2010;**78**(2):177-184

[106] Hench LL, Wilson J. Surfaceactive biomaterials. Science. 1984;**226**(4675):630-636

[107] Gross U, Schmitz HJ, Strunz V. Surface activities of bioactive glass, aluminum oxide, and titanium in a living environment. Annals of the New York Academy of Sciences. 1988;**523**(1):211-226

[108] Hoffman AS. Hydrogels for biomedical applications. Advanced Drug Delivery Reviews. 2002;**54**(1):3-12

[109] Driessens F et al. Effective formulations for the preparation of calcium phosphate bone cements. Journal of Materials Science. Materials in Medicine. 1994;**5**(3):164-170

[110] Gkioni K et al. Mineralization of hydrogels for bone regeneration. Tissue Engineering. Part B, Reviews. 2010;**16**(6):577-585

[111] Sherwood JK et al. A threedimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials. 2002;**23**(24):4739-4751

[112] Mikos AG et al. Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. Journal of Biomedical Materials Research. 1993;**27**(2):183-189

[113] Langer RS, Vacanti JP. Preparation of three-dimensional fibrous scaffold for attaching cells to produce vascularized tissue in vivo. Google Patents; 1998

[114] Ginty PJ et al. Mammalian cell survival and processing in supercritical CO2. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(19):7426-7431

[115] Stevens MM. Biomaterials for bone tissue engineering. Materials Today. 2008;**11**(5):18-25

[116] Tanahashi M, Matsuda T. Surface functional group dependence on apatite formation on self-assembled monolayers in a simulated body fluid. Journal of Biomedical Materials Research. 1997;**34**(3):305-315

[117] Stephansson SN, Byers BA, García AJ. Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding. Biomaterials. 2002;**23**(12):2527-2534

[118] Thorwarth M et al. Bioactivation of an anorganic bone matrix by P-15 peptide for the promotion of early bone formation. Biomaterials. 2005;**26**(28):5648-5657

[119] Filmon R et al. Effects of negatively charged groups (carboxymethyl) on the calcification of poly(2-hydroxyethyl methacrylate). Biomaterials. 2002;**23**(14):3053-3059

[120] Song J, Malathong V, Bertozzi CR. Mineralization of synthetic polymer scaffolds: A bottom-up approach for the development of artificial bone. Journal of the American Chemical Society. 2005;**127**(10):3366-3372

**53**

**Chapter 4**

**Abstract**

collagen

**1. Introduction**

Proteins in Calcium Phosphates

Biomineralization is a process of creating crystalline structures under biological control. The process takes place in hard tissues, such as bones, cartilages, and teeth. Biominerals are a combination of a crystal phase deposited onto an organic matrix. Inorganic components are mainly responsible for the biomineral hardness, while the organic matrix controls the shape, size, and polymorph of the crystals. Within the organic matrix, proteins exhibit a special biomineralization activity. Among them, one can distinguish insoluble collagen and soluble noncollagenous proteins. It is particularly noteworthy that noncollagenous proteins are intrinsically disordered proteins. High flexibility, acidic nature, and susceptibility to modifications make them especially adapted to the biomineralization control. This review paper is dedicated to the proteins which are involved in biomineralization of bones and teeth.

**Keywords:** biomineralization, bones, teeth, SIBLINGs, intrinsically disordered proteins,

Biomineralization is a process of formation of an inorganic solid within the biological system [1]. Biominerals are organic-inorganic composites, which fulfill various biological functions. In vertebrates, hard tissues provide body support, take part in tearing food, protect organs, and are reservoirs of calcium and phosphate. Understanding of

Human hard tissues are formed of calcium orthophosphates [2]. Among them, most important are amorphous calcium phosphate, octacalcium phosphate, calcium hydrogenphosphate dihydrate, and calcium-deficient apatite and hydroxyapatite (HA) [3]. In organisms, calcium orthophosphates occur mainly in the form of poorly crystallized nonstoichiometric sodium-, magnesium-, and carbonate-

Biomineralization is a multistep process, which requires using the structures of extracellular matrix vesicles, numerous enzymes and glycoproteins. Due to strict control, biominerals differ from pure chemical minerals [6]. In contrast to geological minerals, biominerals are composite materials comprised of both inorganic and bioorganic components. The mineral constituent gives tissues hardness and resistance to mechanical damage. Stiffness of the tissue depends on the amount of inorganic components and organic phase [7]. About 70% of bone tissue is made up of mineral structure, while the rest is water and organic substances [1]. Moreover,

molecular basis of biomineralization is essential to obtain new biomaterials.

containing HA—so-called biological apatite [2–5].

*Marta Kalka, Anna Zoglowek, Andrzej Ożyhar* 

Biomineralization

*and Piotr Dobryczycki*

#### **Chapter 4**

*Contemporary Topics about Phosphorus in Biology and Materials*

[114] Ginty PJ et al. Mammalian cell survival and processing in supercritical CO2. Proceedings of the National Academy of Sciences of the United States of America.

[115] Stevens MM. Biomaterials for bone tissue engineering. Materials Today.

[116] Tanahashi M, Matsuda T. Surface functional group dependence on apatite formation on self-assembled monolayers in a simulated body fluid. Journal of Biomedical Materials Research.

[117] Stephansson SN, Byers BA, García AJ. Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation

and integrin receptor binding. Biomaterials. 2002;**23**(12):2527-2534

2005;**26**(28):5648-5657

methacrylate). Biomaterials. 2002;**23**(14):3053-3059

2005;**127**(10):3366-3372

[118] Thorwarth M et al. Bioactivation of an anorganic bone matrix by P-15 peptide for the promotion of early bone formation. Biomaterials.

[119] Filmon R et al. Effects of negatively charged groups (carboxymethyl) on the calcification of poly(2-hydroxyethyl

[120] Song J, Malathong V, Bertozzi CR. Mineralization of synthetic polymer scaffolds: A bottom-up approach for the development of artificial bone. Journal of the American Chemical Society.

2006;**103**(19):7426-7431

2008;**11**(5):18-25

1997;**34**(3):305-315

**52**

## Proteins in Calcium Phosphates Biomineralization

*Marta Kalka, Anna Zoglowek, Andrzej Ożyhar and Piotr Dobryczycki*

#### **Abstract**

Biomineralization is a process of creating crystalline structures under biological control. The process takes place in hard tissues, such as bones, cartilages, and teeth. Biominerals are a combination of a crystal phase deposited onto an organic matrix. Inorganic components are mainly responsible for the biomineral hardness, while the organic matrix controls the shape, size, and polymorph of the crystals. Within the organic matrix, proteins exhibit a special biomineralization activity. Among them, one can distinguish insoluble collagen and soluble noncollagenous proteins. It is particularly noteworthy that noncollagenous proteins are intrinsically disordered proteins. High flexibility, acidic nature, and susceptibility to modifications make them especially adapted to the biomineralization control. This review paper is dedicated to the proteins which are involved in biomineralization of bones and teeth.

**Keywords:** biomineralization, bones, teeth, SIBLINGs, intrinsically disordered proteins, collagen

#### **1. Introduction**

Biomineralization is a process of formation of an inorganic solid within the biological system [1]. Biominerals are organic-inorganic composites, which fulfill various biological functions. In vertebrates, hard tissues provide body support, take part in tearing food, protect organs, and are reservoirs of calcium and phosphate. Understanding of molecular basis of biomineralization is essential to obtain new biomaterials.

Human hard tissues are formed of calcium orthophosphates [2]. Among them, most important are amorphous calcium phosphate, octacalcium phosphate, calcium hydrogenphosphate dihydrate, and calcium-deficient apatite and hydroxyapatite (HA) [3]. In organisms, calcium orthophosphates occur mainly in the form of poorly crystallized nonstoichiometric sodium-, magnesium-, and carbonatecontaining HA—so-called biological apatite [2–5].

Biomineralization is a multistep process, which requires using the structures of extracellular matrix vesicles, numerous enzymes and glycoproteins. Due to strict control, biominerals differ from pure chemical minerals [6]. In contrast to geological minerals, biominerals are composite materials comprised of both inorganic and bioorganic components. The mineral constituent gives tissues hardness and resistance to mechanical damage. Stiffness of the tissue depends on the amount of inorganic components and organic phase [7]. About 70% of bone tissue is made up of mineral structure, while the rest is water and organic substances [1]. Moreover,

#### *Contemporary Topics about Phosphorus in Biology and Materials*


*Bioinformatic predictions of a disordered structure in proteins were done with PONDR predictor (http://www.pondr.com/) [13].*

#### **Table 1.**

*Proteins involved in bone and teeth biomineralization.*

having formed *in vivo* under well-controlled conditions, biomineral phases often have properties, such as shape, size, crystallinity, isotopic, and trace element compositions, quite unlike their inorganically formed counterparts [2].

The key factors, which determine the size, shape, internal structure and properties of biominerals, are proteins, which control the nucleation and growth of the crystals. Biomineralization involves protein-protein interactions and interactions between proteins and inorganic fraction. Among them, two major groups can be distinguished. Scaffold for a growing mineral phase provides insoluble collagen. In human bones, collagen makes up to 20–30% wt. Nucleation and crystal growth regulation are controlled by soluble, noncollagenous proteins. They are usually highly acidic [8, 9], undergo extensive posttranslational modifications [10], and frequently belong to the group of intrinsically disordered proteins (IDPs) [11]. IDPs are dynamic, flexible, heterogeneous populations of molecules without a well-defined folded structure. The highly charged character, along with the low content of hydrophobic amino acid residues, results in strong electrostatic repulsion and the lack of a well-packed hydrophobic core [12]. High content of IDPs' carboxyl and phosphate acidic groups involved in biomineralization results in high calcium binding capabilities. Examples of proteins engaged in bones and teeth biomineralization are shown in **Table 1**.

#### **2. The basic division of proteins involved in the formation of calcium phosphate biominerals**

Bone and teeth biomineralization takes place within the extracellular matrix (ECM), outside the cells. ECM is a highly organized and complex structure, unique to the specific organ type. It is an organic, noncellular fraction of mineralized tissues, composed of structural and functional proteins [14]. The content of the organic fraction varies depending on the type of tissue and constitutes 30% of bone, 20–25% of dentin, and only 0.5% of mature enamel. The basic division of proteins involved in the formation of calcium phosphate biominerals includes insoluble matrix like collagen and soluble noncollagenous proteins (NCPs).

#### **2.1 Collagenous proteins of ECM**

Among organic phase components, there are insoluble substances, which include structural macromolecules, creating a scaffold for the growing mineral.

**55**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

the mutual arrangement of collagen fibers [20].

disordered proteins [11].

**biomineralization**

proteolytic cleavage sites [24].

**2.2 Noncollagenous proteins (NCPs) of extracellular matrix (ECM)**

**3. Intrinsically disordered proteins in calcium phosphate** 

While structural collagen is the main protein of ECM, NCPs are important for the regulation of biomineralization. NCPs are usually acidic, capable of binding large amounts of calcium ions. Proteins, which directly influence biomineralization, act as nucleators and regulators of crystal growth and orientation [21]. NCPs are frequently posttranslationally modified, e.g., phosphorylated, glycosylated, or proteolytically processed. A large number of noncollagenous proteins have disordered secondary and tertiary structures and belong to a group of intrinsically

IDPs are a group of proteins that have gained a special interest of researchers for over 20 years [12, 22, 23]. Their discovery was especially challenging for traditional protein structure paradigm, stating that protein function depends on a fixed tertiary structure. The IDP group includes proteins with altered secondary and tertiary structures from total random coil structure to some intrinsically disordered regions. They are characterized by considerable plasticity, flexibility, and high conformational dynamics. IDP features are high net charge and low hydrophobicity, which is not conducive to the formation of the hydrophobic core. They often bind low molecular ligands and macromolecules such as ions and proteins. By interacting with ligands, IDPs can undergo local or global structuring. They can also fulfill their functions while remaining completely disordered. Other characteristics of this group are multiple amino acid repeats and susceptibility to posttranslational modifications. An open structure and lack of a packed core increase the availability of potential phosphorylation, glycosylation, and

Extended, flexible structure, highly acidic nature, susceptibility to modifications and especially the ability to interact with many different partners make these proteins particularly adapted to the biomineralization control. Many NCPs belong to IDP group. Family of small integrin-binding ligand N-linked glycoprotein (SIBLING) is the example of IDPs engaged in HA formation and is presented below. In addition to SIBLINGs, enamel matrix proteins such as ameloblastin, amelotin,

They give shape to the resulting crystal and can act as initiators for the creation of crystallization roots. An example of such substance is collagen. Animal tissues primarily contain collagen type I or collagen type II [15]. Collagen is the major ECM molecule that self-assembles into cross-striated fibrils, provides support for cell growth, and is responsible for the mechanical resilience of connective tissues. The term "collagen" defines a whole family of glycoproteins. The most common motifs in the amino acid sequence of collagen are repeating sequence [Gly-X-Y]n, both with and without interruptions. The X and Y positions are occupied by proline and its hydroxylated form, hydroxyproline, respectively. The right-handed triple helix is formed from three left-handed polyproline α-chains of identical length, which gives collagen a unique quaternary structure [16]. Hydroxylation of the rest of the proline in collagen is necessary for the stabilization of the triple helix [17]. Collagen chains create interactions leading to the organization of chains in a four-stage array [18]. Collagen fibers are also cross-linked via lysyl residues [19]. Electron microscopy techniques allow to observe successive regions of overlap and gaps, resulting from

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

*Contemporary Topics about Phosphorus in Biology and Materials*

**Protein name Accession** 

*Proteins involved in bone and teeth biomineralization.*

Matrix extracellular phosphoglycoprotein

(preproprotein)

**Table 1.**

Dentin sialophosphoprotein

having formed *in vivo* under well-controlled conditions, biomineral phases often have properties, such as shape, size, crystallinity, isotopic, and trace element compositions, quite unlike their inorganically formed counterparts [2].

**number**

Osteopontin AAA59974.1 4.4 ~34 70% Bone sialoprotein AAA60549.1 4.1 ~35 59%

Dentin matrix protein-1 AAC51332.1 4.0 ~56 90%

Ameloblastin AAG35772.1 4.9 ~48 58% Amelogenin AAC21581.1 6.5 ~22 65% *Bioinformatic predictions of a disordered structure in proteins were done with PONDR predictor (http://www.pondr.com/) [13].*

**pI Mass (kDa)**

AAK70343.1 8.6 ~58 64%

NP\_055023.2 3.6 ~131 88%

**Overall percent disordered—PONDR**

**2. The basic division of proteins involved in the formation of calcium** 

Bone and teeth biomineralization takes place within the extracellular matrix (ECM), outside the cells. ECM is a highly organized and complex structure, unique to the specific organ type. It is an organic, noncellular fraction of mineralized tissues, composed of structural and functional proteins [14]. The content of the organic fraction varies depending on the type of tissue and constitutes 30% of bone, 20–25% of dentin, and only 0.5% of mature enamel. The basic division of proteins involved in the formation of calcium phosphate biominerals includes insoluble matrix like collagen and soluble noncollagenous proteins (NCPs).

Among organic phase components, there are insoluble substances, which include structural macromolecules, creating a scaffold for the growing mineral.

The key factors, which determine the size, shape, internal structure and properties of biominerals, are proteins, which control the nucleation and growth of the crystals. Biomineralization involves protein-protein interactions and interactions between proteins and inorganic fraction. Among them, two major groups can be distinguished. Scaffold for a growing mineral phase provides insoluble collagen. In human bones, collagen makes up to 20–30% wt. Nucleation and crystal growth regulation are controlled by soluble, noncollagenous proteins. They are usually highly acidic [8, 9], undergo extensive posttranslational modifications [10], and frequently belong to the group of intrinsically disordered proteins (IDPs) [11]. IDPs are dynamic, flexible, heterogeneous populations of molecules without a well-defined folded structure. The highly charged character, along with the low content of hydrophobic amino acid residues, results in strong electrostatic repulsion and the lack of a well-packed hydrophobic core [12]. High content of IDPs' carboxyl and phosphate acidic groups involved in biomineralization results in high calcium binding capabilities. Examples of proteins engaged in bones and teeth biomineralization are shown in **Table 1**.

**54**

**phosphate biominerals**

**2.1 Collagenous proteins of ECM**

They give shape to the resulting crystal and can act as initiators for the creation of crystallization roots. An example of such substance is collagen. Animal tissues primarily contain collagen type I or collagen type II [15]. Collagen is the major ECM molecule that self-assembles into cross-striated fibrils, provides support for cell growth, and is responsible for the mechanical resilience of connective tissues. The term "collagen" defines a whole family of glycoproteins. The most common motifs in the amino acid sequence of collagen are repeating sequence [Gly-X-Y]n, both with and without interruptions. The X and Y positions are occupied by proline and its hydroxylated form, hydroxyproline, respectively. The right-handed triple helix is formed from three left-handed polyproline α-chains of identical length, which gives collagen a unique quaternary structure [16]. Hydroxylation of the rest of the proline in collagen is necessary for the stabilization of the triple helix [17]. Collagen chains create interactions leading to the organization of chains in a four-stage array [18]. Collagen fibers are also cross-linked via lysyl residues [19]. Electron microscopy techniques allow to observe successive regions of overlap and gaps, resulting from the mutual arrangement of collagen fibers [20].

#### **2.2 Noncollagenous proteins (NCPs) of extracellular matrix (ECM)**

While structural collagen is the main protein of ECM, NCPs are important for the regulation of biomineralization. NCPs are usually acidic, capable of binding large amounts of calcium ions. Proteins, which directly influence biomineralization, act as nucleators and regulators of crystal growth and orientation [21]. NCPs are frequently posttranslationally modified, e.g., phosphorylated, glycosylated, or proteolytically processed. A large number of noncollagenous proteins have disordered secondary and tertiary structures and belong to a group of intrinsically disordered proteins [11].

#### **3. Intrinsically disordered proteins in calcium phosphate biomineralization**

IDPs are a group of proteins that have gained a special interest of researchers for over 20 years [12, 22, 23]. Their discovery was especially challenging for traditional protein structure paradigm, stating that protein function depends on a fixed tertiary structure. The IDP group includes proteins with altered secondary and tertiary structures from total random coil structure to some intrinsically disordered regions. They are characterized by considerable plasticity, flexibility, and high conformational dynamics. IDP features are high net charge and low hydrophobicity, which is not conducive to the formation of the hydrophobic core. They often bind low molecular ligands and macromolecules such as ions and proteins. By interacting with ligands, IDPs can undergo local or global structuring. They can also fulfill their functions while remaining completely disordered. Other characteristics of this group are multiple amino acid repeats and susceptibility to posttranslational modifications. An open structure and lack of a packed core increase the availability of potential phosphorylation, glycosylation, and proteolytic cleavage sites [24].

Extended, flexible structure, highly acidic nature, susceptibility to modifications and especially the ability to interact with many different partners make these proteins particularly adapted to the biomineralization control. Many NCPs belong to IDP group. Family of small integrin-binding ligand N-linked glycoprotein (SIBLING) is the example of IDPs engaged in HA formation and is presented below. In addition to SIBLINGs, enamel matrix proteins such as ameloblastin, amelotin,

and enamelin also have disordered structures [25]. More information about the proteins involved in HA biomineralization belonging to IDPs can be found in reviews [11, 25, 26].

#### **3.1 SIBLINGs**

An example of NCPs is the family of small integrin-binding ligand N-linked glycoprotein (SIBLING). It is a group of proteins identified in bone, dentine, and cementum, which includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) [27]. It is believed that they all evolved as a result of gene duplication from extracellular calcium-binding phosphoprotein family. Although proteins differ in composition of amino-acid sequence, SIBLINGs share common features [28]. Their genes are located in a 375 kb region on the 4q21 human and 5q mouse chromosomes. They display a similar exon structure. Their sequence contains an Arg-Gly-Asp (RGD) motif, which mediates cell attachment/signaling by binding to cell-surface integrins [28]. SIBLINGs belong to IDP group and are highly acidic. They share numerous sequence repeats, which are often observed in the case of IDPs [26]. Particularly important for their biomineralization activity may be acidic serine- and aspartate-rich motifs (ASARM), which are involved in the phosphate administration [29, 30]. SILBINGs are frequently posttranslationally medicated. Some of their biological functions depend on phosphorylation, glycosylation, proteolytic processing, sulfonation, or transglutaminase cross linking [31].

#### *3.1.1 Osteopontin*

Osteopontin (OPN), also known as secreted phosphoprotein 1 (SSP1), is a highly phosphorylated and glycosylated sialoprotein. It is a multifunctional protein expressed by several cell types including osteoblasts, osteocytes, as well as osteoclasts or odontoblasts [32]. OPN undergoes many posttranslational modifications such as phosphorylation, glycosylation, sulfonation, or proteolytic processing, and modifications vary depending on a protein role and localization [31]. In bones and dentin, osteopontin is located at the site of biomineral formation. Synthesis of OPN is stimulated by calcitriol, physiologically active form of vitamin D, which is known as a trigger for bone destruction and remodeling [33, 34].

Osteopontin was originally identified as a bridge between the cell and HA in ECM of bone [35]. It is known that OPN increases the adhesion of bone cells by concentrating in mineralized collagen matrix during bone formation and remodeling [36, 37]. The protein is highly produced by developing osteoblasts and osteoclasts, and it has been shown to regulate both cell type adhesions.

Another role of ONP during biomineralization is modulation of osteoclastic function. OPN binding to integrin αvβ3 is essential for regulation of osteoclastic activity and is necessary in the formation of a sealing zone [34]. Through CD44 associated cell signaling, OPN stimulates osteoclast migration [38].

As a highly acidic phosphoprotein, OPN binds to the surface of hydroxyapatite crystals through the electrostatic interactions between crystals and carboxyl and phosphate groups. Furthermore, disordered structure of OPN can promote the binding of calcium ions. The results of *in vitro* and *in vivo* studies suggest that osteopontin has an inhibitory effect on HA formation and growth [39, 40]. *OPN* knockout mice display increased mineral content and size [41]. Therefore, it is suggested that OPN is one of the proteins whose role is to prevent crystal formation in soft tissues. Peptides of OPN obtained after proteolytic processing differ in a biomineralization effect. ASARM peptide of OPN binds to HA crystals and consequently

**57**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

an inhibitory effect [43].

*3.1.2 Bone sialoprotein*

inhibits ECM matrix mineralization [42]. Its inhibition activity is dependent on serine residues' phosphorylation. N- and C-terminal fragments of OPN from milk promote *de novo* HA formation, but at the same time, the central fragment showed

Bone sialoprotein (BSP) is one of the most abundant NCPs of bone. In contrast to OPN, BSP is only localized in tissues that undergo mineralization: bone, dentin and mineralizing cartilage, and cementum [44]. The protein is produced by osteoblasts, osteocytes, osteoclasts, as well as by chondrocytes. Disordered structure of BSP was shown by NMR, CD, and SAXS studies [28, 45–47]. BSP is highly glycosylated especially at C-terminal fragment, and to a lesser extent also phosphorylated and sulfonated [31]. N-terminal region of the protein contains collagen-binding motif. *In vivo* and *in vitro* studies indicated that BSP can be involved in initial stages of hydroxyapatite biomineralization [48]. BSP is intensely expressed by osteoblastic cells in sites of primary bone formation. The protein is present at mineralizing boundaries of bone, dentin, and calcifying cartilage tissues. On the other hand, BSP increases osteoclastogenesis, and in that way, it can initiate bone remodeling. *BSP* knockout mice are characterized by short, hypomineralized bones with high trabecular bone mass [49, 50]. Overexpression of BSP leads to dwarfism, decreased

It was shown that BSP has high affinity to hydroxyapatite and acts as a *de novo* nucleator of HA crystals *in vitro* [52]. The protein also binds to type I collagen, and by interaction with collagen, BSP could regulate HA nucleation [53]. Moreover, control of mineral formation is highly associated with the state of phosphorylation,

When creating skull bones, including the mandible, alveoli, and skull vault, the osteoprogenitor cells secrete a collagen extracellular matrix—osteoid. Skull bones are formed by intracerebral ossification. BSP affects biomineralization of the matrix in the ossification process, hence, the formation, shaping, and growth of hydroxyapatite crystals. In the absence of BSP, bone formation is delayed and osteoblast

Matrix extracellular phosphoglycoprotein (MEPE) is located in mineralized tissues as bone and dentin, but was found also in nonmineralized organs. The protein is primarily expressed by osteoblasts and osteocytes that are embedded within the mineralized matrix in bone and by odontoblasts during odontogenesis [57]. Similar to osteopontin, MEPE seems to be a multifunctional ECM protein. Results of *in vivo* and *in vitro* studies suggest that MEPE is important for bone mineralization, Pi homeostasis and cell attachment [58–60]. The protein was originally identified as interacting with PHEX. PHEX by binding to MEPE protects the protein from proteolytic cleavage by cathepsin B. Cleavage by cathepsin B leads to the release of the ASRAM peptide. The ASRAM motif is a functional domain of MEPE responsible for its inhibitory activity [61]. The peptide may be responsible for phosphate and calcium regulation during the mineralization process. ASARM peptide inhibits hydroxyapatite mineralization by binding free calcium with high avidity, and their inhibitory effect is highly dependent on serine residues phosphorylation. AC100, another MEPE fragment, containing the integrin binding (RGD) and glycosaminoglycan-attachment (SGDG) motifs stimulates new bone

bone mineral density, and decreased trabecular bone volume [51].

sulfonation, and glycosylation of BSP [27, 54, 55].

*3.1.3 Matrix extracellular phosphoglycoprotein*

formation *in vitro* and *in vivo* [62].

activity is impaired [56].

inhibits ECM matrix mineralization [42]. Its inhibition activity is dependent on serine residues' phosphorylation. N- and C-terminal fragments of OPN from milk promote *de novo* HA formation, but at the same time, the central fragment showed an inhibitory effect [43].

#### *3.1.2 Bone sialoprotein*

*Contemporary Topics about Phosphorus in Biology and Materials*

[11, 25, 26].

**3.1 SIBLINGs**

*3.1.1 Osteopontin*

and enamelin also have disordered structures [25]. More information about the proteins involved in HA biomineralization belonging to IDPs can be found in reviews

An example of NCPs is the family of small integrin-binding ligand N-linked glycoprotein (SIBLING). It is a group of proteins identified in bone, dentine, and cementum, which includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) [27]. It is believed that they all evolved as a result of gene duplication from extracellular calcium-binding phosphoprotein family. Although proteins differ in composition of amino-acid sequence, SIBLINGs share common features [28]. Their genes are located in a 375 kb region on the 4q21 human and 5q mouse chromosomes. They display a similar exon structure. Their sequence contains an Arg-Gly-Asp (RGD) motif, which mediates cell attachment/signaling by binding to cell-surface integrins [28]. SIBLINGs belong to IDP group and are highly acidic. They share numerous sequence repeats, which are often observed in the case of IDPs [26]. Particularly important for their biomineralization activity may be acidic serine- and aspartate-rich motifs (ASARM), which are involved in the phosphate administration [29, 30]. SILBINGs are frequently posttranslationally medicated. Some of their biological functions depend on phosphorylation, glycosylation,

proteolytic processing, sulfonation, or transglutaminase cross linking [31].

as a trigger for bone destruction and remodeling [33, 34].

and it has been shown to regulate both cell type adhesions.

associated cell signaling, OPN stimulates osteoclast migration [38].

Osteopontin (OPN), also known as secreted phosphoprotein 1 (SSP1), is a highly phosphorylated and glycosylated sialoprotein. It is a multifunctional protein expressed by several cell types including osteoblasts, osteocytes, as well as osteoclasts or odontoblasts [32]. OPN undergoes many posttranslational modifications such as phosphorylation, glycosylation, sulfonation, or proteolytic processing, and modifications vary depending on a protein role and localization [31]. In bones and dentin, osteopontin is located at the site of biomineral formation. Synthesis of OPN is stimulated by calcitriol, physiologically active form of vitamin D, which is known

Osteopontin was originally identified as a bridge between the cell and HA in ECM of bone [35]. It is known that OPN increases the adhesion of bone cells by concentrating in mineralized collagen matrix during bone formation and remodeling [36, 37]. The protein is highly produced by developing osteoblasts and osteoclasts,

Another role of ONP during biomineralization is modulation of osteoclastic function. OPN binding to integrin αvβ3 is essential for regulation of osteoclastic activity and is necessary in the formation of a sealing zone [34]. Through CD44-

As a highly acidic phosphoprotein, OPN binds to the surface of hydroxyapatite crystals through the electrostatic interactions between crystals and carboxyl and phosphate groups. Furthermore, disordered structure of OPN can promote the binding of calcium ions. The results of *in vitro* and *in vivo* studies suggest that osteopontin has an inhibitory effect on HA formation and growth [39, 40]. *OPN* knockout mice display increased mineral content and size [41]. Therefore, it is suggested that OPN is one of the proteins whose role is to prevent crystal formation in soft tissues. Peptides of OPN obtained after proteolytic processing differ in a biomineralization effect. ASARM peptide of OPN binds to HA crystals and consequently

**56**

Bone sialoprotein (BSP) is one of the most abundant NCPs of bone. In contrast to OPN, BSP is only localized in tissues that undergo mineralization: bone, dentin and mineralizing cartilage, and cementum [44]. The protein is produced by osteoblasts, osteocytes, osteoclasts, as well as by chondrocytes. Disordered structure of BSP was shown by NMR, CD, and SAXS studies [28, 45–47]. BSP is highly glycosylated especially at C-terminal fragment, and to a lesser extent also phosphorylated and sulfonated [31]. N-terminal region of the protein contains collagen-binding motif. *In vivo* and *in vitro* studies indicated that BSP can be involved in initial stages of hydroxyapatite biomineralization [48]. BSP is intensely expressed by osteoblastic cells in sites of primary bone formation. The protein is present at mineralizing boundaries of bone, dentin, and calcifying cartilage tissues. On the other hand, BSP increases osteoclastogenesis, and in that way, it can initiate bone remodeling. *BSP* knockout mice are characterized by short, hypomineralized bones with high trabecular bone mass [49, 50]. Overexpression of BSP leads to dwarfism, decreased bone mineral density, and decreased trabecular bone volume [51].

It was shown that BSP has high affinity to hydroxyapatite and acts as a *de novo* nucleator of HA crystals *in vitro* [52]. The protein also binds to type I collagen, and by interaction with collagen, BSP could regulate HA nucleation [53]. Moreover, control of mineral formation is highly associated with the state of phosphorylation, sulfonation, and glycosylation of BSP [27, 54, 55].

When creating skull bones, including the mandible, alveoli, and skull vault, the osteoprogenitor cells secrete a collagen extracellular matrix—osteoid. Skull bones are formed by intracerebral ossification. BSP affects biomineralization of the matrix in the ossification process, hence, the formation, shaping, and growth of hydroxyapatite crystals. In the absence of BSP, bone formation is delayed and osteoblast activity is impaired [56].

#### *3.1.3 Matrix extracellular phosphoglycoprotein*

Matrix extracellular phosphoglycoprotein (MEPE) is located in mineralized tissues as bone and dentin, but was found also in nonmineralized organs. The protein is primarily expressed by osteoblasts and osteocytes that are embedded within the mineralized matrix in bone and by odontoblasts during odontogenesis [57]. Similar to osteopontin, MEPE seems to be a multifunctional ECM protein. Results of *in vivo* and *in vitro* studies suggest that MEPE is important for bone mineralization, Pi homeostasis and cell attachment [58–60]. The protein was originally identified as interacting with PHEX. PHEX by binding to MEPE protects the protein from proteolytic cleavage by cathepsin B. Cleavage by cathepsin B leads to the release of the ASRAM peptide. The ASRAM motif is a functional domain of MEPE responsible for its inhibitory activity [61]. The peptide may be responsible for phosphate and calcium regulation during the mineralization process. ASARM peptide inhibits hydroxyapatite mineralization by binding free calcium with high avidity, and their inhibitory effect is highly dependent on serine residues phosphorylation. AC100, another MEPE fragment, containing the integrin binding (RGD) and glycosaminoglycan-attachment (SGDG) motifs stimulates new bone formation *in vitro* and *in vivo* [62].

#### *3.1.4 Dentin matrix protein-1*

Dentin matrix protein-1 (DMP1) was the first molecule identified by cloning from the dentin matrix. Besides dentin, the protein is located also in bone and cementum as well as in other nonmineralized tissues [63–65]. It is remarkably acidic and is a hydrophilic protein with many serine, aspartate, and glutamate residues. The protein is characterized by disordered structure that can aggregate in the presence of calcium ions [66].

It was shown that DMP1 is essential for both odontoblasts and osteoblasts maturation. The protein controls odontogenesis, osteogenesis, and Pi homeostasis. The effect of DMP1 on HA biomineralization depends on posttranslational modifications. It was shown that phosphorylated DMP1 inhibits HA formation and growth, while the dephosphorylated form acts as a HA nucleator [67, 68].

It was demonstrated that DMP1 is proteolytically processed into N-terminal 37-K fragment and 57-K fragment from the C-terminal region, and it is likely that full-length DMP1 is a precursor to these functional fragments [69]. It seems that the 57-K fragment plays a key role in the biomineralization process. The 57-K fragment contains 41 phosphate groups, while the 37-K fragment has only 12 phosphate groups. In addition, the 57-K fragment contains many functional sequences and domains such as the RGD motif [70], the ASARM peptide [71], and the peptide functioning as nucleator [72, 73]. In contrast to the full-length DMP1, the 57-K fragment promoted HA nucleation and growth [66]. Furthermore, it was shown that DMP1 N-terminal fragment is able to stabilize the metastable amorphous calcium phosphate. Due to high aspartic acid residual content, the 37-K fragment can bind calcium ions very strongly favoring formation and stabilization of the amorphous nuclei [72]. It was shown that the DMP1 57-K fragment also controls calcium carbonate mineralization *in vitro* [74].

#### *3.1.5 Dentin sialophosphoprotein*

Dentin sialophosphoprotein (DSPP) was discovered by cDNA cloning using a mouse odontoblast cDNA library and was the first believed to be connected only with dentin. Subsequent studies have revealed that the protein is expressed also in bone, cementum, and some nonmineralized tissues [75, 76]. Analysis of *DSPP* knockout mice and mutation in the *DSPP* gene indicated a special role of protein in dentin mineralization. In humans, a mutation in the *DSPP* gene causes dentin hypomineralization and significant tooth decay, named dentinogenesis imperfecta [77]. Additionally, studies of *DSPP*-null mice suggest that DSPP is crucial in the initial mineralization of bone as well as in the remodeling of the skeleton and therefore on bone turnover [78].

It is suggested that DSPP is a precursor protein activated after proteolytic processing. Cleavage of DSPP by zinc metalloproteinase bone morphogenetic protein-1 (BPM1) results in two protein fragments: dentin phosphoprotein (phosphophoryn) (DPP) and dentin sialoprotein (DSP) [79]. Subsequently, cleavage of C-terminal of DSP by matrix metalloproteinase 2 (MMP2) and MMP20 leads to the release of the third fragment called dentin glycoprotein (DGP), which has a strong affinity to hydroxyapatite [80].

Highly acidic DPP is the most abundant NCP of ECM in dentin. Amino acid sequence of DPP is composed mainly of aspartic acid and serine residues, whereas ~80% of serine residues are phosphorylated and phosphorylation is crucial to its function. The protein is also glycosylated [81] and contains ASARM peptide. Due to its amino acid composition and phosphorylation, DPP binds large amounts of calcium ions with high affinity. The protein is involved in nucleation and control of

**59**

receptors [92].

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

**4. Other proteins specific to teeth or bones**

growth [86–88].

dentin organic matrix [89].

**4.1 Tooth**

*4.1.1 Enamel*

the formation and growth of HA crystals during dentin mineralization [82, 83]. DPP binds to collagen fibrils and is present in front of dentin mineralization [84, 85]. There is a hypothesis that in front of mineralization, DPP is promoting the formation of initial apatite crystals [86]. When predentin is converted to dentin, DPP with other proteins binds to the growing HA faces and inhibits or slows down crystal

Dentin, enamel, and cementum are calcified tissues and are major components

The organic part of enamel consists mainly of hydrophobic proteins, known as amelogenins and anionic proteins, which include ameloblastin, enamelin, and tuftelin [90]. Enamel also contains sialophosphoproteins and enamel proteases such as matrix-20 metalloproteinase (MMP-20, matrix metalloproteinase-20, also known as enamelysin) and kallikrein 4 (KLK4). At the end of the crystallization, enamel loses almost entire organic matrix, which is degraded and replaced by hydroxyapatite crystals [90]. Residues, small peptides, and amino acids account for

Amelogenins are a group of heterogeneous proteins present in odontoblasts and identified in enamel of maturing teeth. They are the main proteins of the organic enamel matrix, constituting over 90% of the pool of proteins secreted by ameloblasts into the intercellular matrix during the formation of enamel. These are hydrophobic proteins rich in proline, glutamine, leucine, and histidine. Amelogenins isolated from various organisms are characterized by high homology [91]. Amelogenins belong to IDP groups; conformational analysis by CD and NMR spectroscopy showed that recombinant porcine amelogenin rP172 exists in an extended, unfolded state in the monomeric form [92]. The extended, labile conformation of rP172 amelogenin is compatible with known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell

Amelogenin controls the organization and growth of enamel crystals, and its presence is critical for normal enamel formation. Defects in amelogenin sequence

Ameloblastin, also known as amelin, is a tooth-specific glycoprotein. Unlike amelogenin, ameloblastin is located close to the cell surface [94]. Ameloblastin accounts for about 5–10% of all proteins present in the enamel, and it is the second most abundant protein among the intercellular matrix of the enamel [95]. Ameloblastin is also synthesized in dentin and cement, but its role in these tissues is not determined. Ameloblastin is a molecule of cell adhesion required to maintain a

only 1% of enamel and do not resemble the original matrix [90].

lead to defective enamel crystal formation and organization [93].

of teeth. Dentin is usually covered by enamel on the crown and cementum on the root and surrounds the entire pulp. About 70% (wt) of dentin consists of the mineral phase, 20% (wt) is an organic material, and 10% (wt) is water. Enamel is the hardest substance in the human body and contains 96% (wt) of mineral phase. Enamel and dentin are created by two layers, namely odontoblasts and ameloblasts. Dentin is characterized by a high content of collagen I comprising ~90% of the

the formation and growth of HA crystals during dentin mineralization [82, 83]. DPP binds to collagen fibrils and is present in front of dentin mineralization [84, 85]. There is a hypothesis that in front of mineralization, DPP is promoting the formation of initial apatite crystals [86]. When predentin is converted to dentin, DPP with other proteins binds to the growing HA faces and inhibits or slows down crystal growth [86–88].

#### **4. Other proteins specific to teeth or bones**

#### **4.1 Tooth**

*Contemporary Topics about Phosphorus in Biology and Materials*

Dentin matrix protein-1 (DMP1) was the first molecule identified by cloning from the dentin matrix. Besides dentin, the protein is located also in bone and cementum as well as in other nonmineralized tissues [63–65]. It is remarkably acidic and is a hydrophilic protein with many serine, aspartate, and glutamate residues. The protein is characterized by disordered structure that can aggregate in the pres-

It was shown that DMP1 is essential for both odontoblasts and osteoblasts maturation. The protein controls odontogenesis, osteogenesis, and Pi homeostasis. The effect of DMP1 on HA biomineralization depends on posttranslational modifications. It was shown that phosphorylated DMP1 inhibits HA formation and growth,

It was demonstrated that DMP1 is proteolytically processed into N-terminal 37-K fragment and 57-K fragment from the C-terminal region, and it is likely that full-length DMP1 is a precursor to these functional fragments [69]. It seems that the 57-K fragment plays a key role in the biomineralization process. The 57-K fragment contains 41 phosphate groups, while the 37-K fragment has only 12 phosphate groups. In addition, the 57-K fragment contains many functional sequences and domains such as the RGD motif [70], the ASARM peptide [71], and the peptide functioning as nucleator [72, 73]. In contrast to the full-length DMP1, the 57-K fragment promoted HA nucleation and growth [66]. Furthermore, it was shown that DMP1 N-terminal fragment is able to stabilize the metastable amorphous calcium phosphate. Due to high aspartic acid residual content, the 37-K fragment can bind calcium ions very strongly favoring formation and stabilization of the amorphous nuclei [72]. It was shown that the DMP1 57-K fragment also controls

Dentin sialophosphoprotein (DSPP) was discovered by cDNA cloning using a mouse odontoblast cDNA library and was the first believed to be connected only with dentin. Subsequent studies have revealed that the protein is expressed also in bone, cementum, and some nonmineralized tissues [75, 76]. Analysis of *DSPP* knockout mice and mutation in the *DSPP* gene indicated a special role of protein in dentin mineralization. In humans, a mutation in the *DSPP* gene causes dentin hypomineralization and significant tooth decay, named dentinogenesis imperfecta [77]. Additionally, studies of *DSPP*-null mice suggest that DSPP is crucial in the initial mineralization of bone as well as in the remodeling of the skeleton and therefore on

It is suggested that DSPP is a precursor protein activated after proteolytic processing. Cleavage of DSPP by zinc metalloproteinase bone morphogenetic protein-1 (BPM1) results in two protein fragments: dentin phosphoprotein (phosphophoryn) (DPP) and dentin sialoprotein (DSP) [79]. Subsequently, cleavage of C-terminal of DSP by matrix metalloproteinase 2 (MMP2) and MMP20 leads to the release of the third fragment called dentin glycoprotein (DGP), which has a strong affinity to

Highly acidic DPP is the most abundant NCP of ECM in dentin. Amino acid sequence of DPP is composed mainly of aspartic acid and serine residues, whereas ~80% of serine residues are phosphorylated and phosphorylation is crucial to its function. The protein is also glycosylated [81] and contains ASARM peptide. Due to its amino acid composition and phosphorylation, DPP binds large amounts of calcium ions with high affinity. The protein is involved in nucleation and control of

while the dephosphorylated form acts as a HA nucleator [67, 68].

calcium carbonate mineralization *in vitro* [74].

*3.1.5 Dentin sialophosphoprotein*

bone turnover [78].

hydroxyapatite [80].

*3.1.4 Dentin matrix protein-1*

ence of calcium ions [66].

**58**

Dentin, enamel, and cementum are calcified tissues and are major components of teeth. Dentin is usually covered by enamel on the crown and cementum on the root and surrounds the entire pulp. About 70% (wt) of dentin consists of the mineral phase, 20% (wt) is an organic material, and 10% (wt) is water. Enamel is the hardest substance in the human body and contains 96% (wt) of mineral phase. Enamel and dentin are created by two layers, namely odontoblasts and ameloblasts. Dentin is characterized by a high content of collagen I comprising ~90% of the dentin organic matrix [89].

#### *4.1.1 Enamel*

The organic part of enamel consists mainly of hydrophobic proteins, known as amelogenins and anionic proteins, which include ameloblastin, enamelin, and tuftelin [90]. Enamel also contains sialophosphoproteins and enamel proteases such as matrix-20 metalloproteinase (MMP-20, matrix metalloproteinase-20, also known as enamelysin) and kallikrein 4 (KLK4). At the end of the crystallization, enamel loses almost entire organic matrix, which is degraded and replaced by hydroxyapatite crystals [90]. Residues, small peptides, and amino acids account for only 1% of enamel and do not resemble the original matrix [90].

Amelogenins are a group of heterogeneous proteins present in odontoblasts and identified in enamel of maturing teeth. They are the main proteins of the organic enamel matrix, constituting over 90% of the pool of proteins secreted by ameloblasts into the intercellular matrix during the formation of enamel. These are hydrophobic proteins rich in proline, glutamine, leucine, and histidine. Amelogenins isolated from various organisms are characterized by high homology [91]. Amelogenins belong to IDP groups; conformational analysis by CD and NMR spectroscopy showed that recombinant porcine amelogenin rP172 exists in an extended, unfolded state in the monomeric form [92]. The extended, labile conformation of rP172 amelogenin is compatible with known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell receptors [92].

Amelogenin controls the organization and growth of enamel crystals, and its presence is critical for normal enamel formation. Defects in amelogenin sequence lead to defective enamel crystal formation and organization [93].

Ameloblastin, also known as amelin, is a tooth-specific glycoprotein. Unlike amelogenin, ameloblastin is located close to the cell surface [94]. Ameloblastin accounts for about 5–10% of all proteins present in the enamel, and it is the second most abundant protein among the intercellular matrix of the enamel [95]. Ameloblastin is also synthesized in dentin and cement, but its role in these tissues is not determined. Ameloblastin is a molecule of cell adhesion required to maintain a

diversified state of ameloblasts [96]. Bioinformatic analyzes and molecular modeling of the protein structure suggest that it may belong to IDPs [97], which was confirmed by the CD spectra of recombinant mouse protein [98].

The N-terminal fragment of ameloblastin comprising the self-assembly motif was shown to colocalize with amelogenin across the entire growing enamel, indicating the role of the two proteins in the organization of the linear growth of HA crystallites [99–101]. Both proteins self-assemble into higher order structures from monomeric subunits, similar to type I collagen, the predominant matrix protein of bones, and dentin [102, 103]. Full-length amelogenin undergoes hierarchical stepwise assembly, first forming oligomers, which in turn assemble into higher order structures and stabilize mineral prenucleation clusters, and organize them into parallel arrays of linear chains, yielding the formation of crystallite bundles [102]. There is a hypothesis that the higher order structures of the self-assembled ameloblastin (or most likely its N-terminal moiety) contribute to the oriented growth of the linear chains of amelogenin in the 3D space [104].

#### *4.1.2 Dentin*

Dentin forms a support for hard tissues of a tooth. It covers both crown and root structures and is responsible for its shape. The formation of dentin is closely related to bone. Both bone and dentin are composed of a collagenous matrix and a mineral phase with hydroxyapatite plate-like crystals. Mineralization of dentin and bone extracellular matrix is initiated with the aid of matrix vesicles and later involves secretion of families of a specialized matrix protein [105]. In contrast to bones, dentin is not remodeled and does not participate in the regulation of calcium and phosphate metabolism. The tooth formation takes place throughout the lifetime of a tooth. Unlike enamel, dentin mineral content increases with age due to continuous mineral deposition either as physiological secondary dentin or as tertiary dentin following injury [105].

Dentin is composed of a collagen I, which accounts for about 92% of organic components. In addition to collagen, there is also a group of noncollagen proteins, which include dentin-specific phosphoryls (DPPs) and dentin sialoproteins (DSP), sialic acid, osteopontin, dentin matrix proteins (DMP1, DMP2, DMP3), BSP, acidic bone-75 glycoprotein, osteonectin, and proteins rich in γ-carboxyglutamic acid. Among organic components should also be mentioned proteoglycans, growth factors, phospholipids, and enzymes [106, 107]. Inorganic dentin components make up to about 70% of the tissue mass, making the dentin harder than the bone. The mass of a single hydroxyapatite crystal is about 10 times greater than in bone, but many times less than in enamel, and its size is about 35 × 10 × 100 nm [108].

#### *4.1.3 Cementum*

Cementum protects the dental root dentin with a very thin layer. In many respects, it is very unique: it is not vascularized and is not innervated, it does not undergo constant remodeling like a bone, but it grows all the time. Cellular and acellular cement are distinguished based on the presence of cementocytes in its structure. The structure and composition of cement resembles bone tissue. Inorganic constituents make up to about 65% of tissue mass and consist mainly of hydroxyapatite crystals [109, 110].

Cementum provides contact between roots of the teeth and periodontium ligaments [111]. hrCEMP1 (recombinant human cementum protein 1) is an isolated form of cementum human β-sheet protein. It has been showed that hrCEMP1 forms clusters of 6.5 nm diameter [111]. hrCEMP1 is an inducer of specific

**61**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

**4.2 Bone**

above [27].

hrCEMP1 is an inducer of polymorphic crystals [111].

nucleation and growth of hydroxyapatite crystals [112].

pH in extracellular bone matrix.

ases, which promote bone degradation [114].

University of Science and Technology.

**Acknowledgements**

biomineralization proteins. It stimulates the differentiation of osteoblasts and cementoblasts. It is suggested that hrCEMP1 plays a significant role in octacalcium phosphate biomineralization and helps its binding to hydroxyapatite even without posttranslational modifications. X-ray diffraction measurements showed that

The matrix of bone consists of type I collagen molecule self-assembled into a triple helix consisting of two α1 and one α2 chains (ca. 300 nm in length and 1.5 nm in diameter) and hydroxyapatite (Ca10(PO4)6(OH)2) nanocrystals (plateshaped, 1.5–4 nm in thickness) deposited along collagen fibrils [112, 113]. The collagen fiber does not undergo spontaneous mineralization in the presence of phosphate and calcium ions [20], and the involvement of noncollagen proteins is necessary. The majority of these proteins belong to the SIBLING family described

Bones and dentin are characterized by a similar composition and the mechanism of their formation [112]. Their organic matrix consists of type I collagen and the mineral matrix built of hydroxyapatite. Osteoblasts and odontoblasts, cells involved in osteogenesis and dentinogenesis, first secrete a nonmineralized matrix—a bone osteoid, and in case of dentin—predentin and then with fibers of collagen I form matrix for biomineralization [112]. Calcium and phosphate ions are dislocated from the vascular network into the mineralization matrix. Osteoid and predentin contain numerous noncollagenous proteins called NCPs. On the basis of mutation experiments and suppression of NCP genes, it is suggested that they stimulate the

Bones and dentin differ significantly. Bone is a dynamic tissue because it is constantly remodeled, while dentin is a rather static tissue [112]. Osteoblasts

produce matrix components as a result of controlling growth factors to form a bone. Hydroxyapatite crystals grow above vesicles, thus creating mineralization centers. Bones have a lot of vessels, and they store calcium ions. The reason for bone resorption is the action of lysosomal enzymes produced by osteoclasts and lowering the

Osteoclasts are responsible for the attachment, bone degradation, and subsequent tissue resorption. It was found that this is possible due to the strong polarization of osteoclasts. They have both frontal and posterior abdominal polarization. On the underside of the osteoclasts, there are structures responsible for the degradation of the mineral surface. The adhesion zone is located in the sealing zone [114]—a ring made of actin and podosomes in which extracellular resorption pit is segregated and is used to retain heavy substances (minerals) formed during bone resorption near the bone surface [43, 114]. Podosomes are dynamic structures built of actin. They perform important functions, including adhesion, destruction of the bone and its matrix, and recognition of the appropriate medium [115]. Podosomes contain a large number of V-ATPase molecules that pump protons and secrete prote-

This work was supported by the National Science Center (Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity subsidy from the Polish Ministry of Science and High Education for the Faculty of Chemistry of Wroclaw

biomineralization proteins. It stimulates the differentiation of osteoblasts and cementoblasts. It is suggested that hrCEMP1 plays a significant role in octacalcium phosphate biomineralization and helps its binding to hydroxyapatite even without posttranslational modifications. X-ray diffraction measurements showed that hrCEMP1 is an inducer of polymorphic crystals [111].

#### **4.2 Bone**

*Contemporary Topics about Phosphorus in Biology and Materials*

the linear chains of amelogenin in the 3D space [104].

*4.1.2 Dentin*

following injury [105].

*4.1.3 Cementum*

hydroxyapatite crystals [109, 110].

confirmed by the CD spectra of recombinant mouse protein [98].

diversified state of ameloblasts [96]. Bioinformatic analyzes and molecular modeling of the protein structure suggest that it may belong to IDPs [97], which was

The N-terminal fragment of ameloblastin comprising the self-assembly motif was shown to colocalize with amelogenin across the entire growing enamel, indicating the role of the two proteins in the organization of the linear growth of HA crystallites [99–101]. Both proteins self-assemble into higher order structures from monomeric subunits, similar to type I collagen, the predominant matrix protein of bones, and dentin [102, 103]. Full-length amelogenin undergoes hierarchical stepwise assembly, first forming oligomers, which in turn assemble into higher order structures and stabilize mineral prenucleation clusters, and organize them into parallel arrays of linear chains, yielding the formation of crystallite bundles [102]. There is a hypothesis that the higher order structures of the self-assembled ameloblastin (or most likely its N-terminal moiety) contribute to the oriented growth of

Dentin forms a support for hard tissues of a tooth. It covers both crown and root structures and is responsible for its shape. The formation of dentin is closely related to bone. Both bone and dentin are composed of a collagenous matrix and a mineral phase with hydroxyapatite plate-like crystals. Mineralization of dentin and bone extracellular matrix is initiated with the aid of matrix vesicles and later involves secretion of families of a specialized matrix protein [105]. In contrast to bones, dentin is not remodeled and does not participate in the regulation of calcium and phosphate metabolism. The tooth formation takes place throughout the lifetime of a tooth. Unlike enamel, dentin mineral content increases with age due to continuous mineral deposition either as physiological secondary dentin or as tertiary dentin

Dentin is composed of a collagen I, which accounts for about 92% of organic components. In addition to collagen, there is also a group of noncollagen proteins, which include dentin-specific phosphoryls (DPPs) and dentin sialoproteins (DSP), sialic acid, osteopontin, dentin matrix proteins (DMP1, DMP2, DMP3), BSP, acidic bone-75 glycoprotein, osteonectin, and proteins rich in γ-carboxyglutamic acid. Among organic components should also be mentioned proteoglycans, growth factors, phospholipids, and enzymes [106, 107]. Inorganic dentin components make up to about 70% of the tissue mass, making the dentin harder than the bone. The mass of a single hydroxyapatite crystal is about 10 times greater than in bone, but many

times less than in enamel, and its size is about 35 × 10 × 100 nm [108].

Cementum protects the dental root dentin with a very thin layer. In many respects, it is very unique: it is not vascularized and is not innervated, it does not undergo constant remodeling like a bone, but it grows all the time. Cellular and acellular cement are distinguished based on the presence of cementocytes in its structure. The structure and composition of cement resembles bone tissue. Inorganic constituents make up to about 65% of tissue mass and consist mainly of

Cementum provides contact between roots of the teeth and periodontium ligaments [111]. hrCEMP1 (recombinant human cementum protein 1) is an isolated form of cementum human β-sheet protein. It has been showed that hrCEMP1 forms clusters of 6.5 nm diameter [111]. hrCEMP1 is an inducer of specific

**60**

The matrix of bone consists of type I collagen molecule self-assembled into a triple helix consisting of two α1 and one α2 chains (ca. 300 nm in length and 1.5 nm in diameter) and hydroxyapatite (Ca10(PO4)6(OH)2) nanocrystals (plateshaped, 1.5–4 nm in thickness) deposited along collagen fibrils [112, 113]. The collagen fiber does not undergo spontaneous mineralization in the presence of phosphate and calcium ions [20], and the involvement of noncollagen proteins is necessary. The majority of these proteins belong to the SIBLING family described above [27].

Bones and dentin are characterized by a similar composition and the mechanism of their formation [112]. Their organic matrix consists of type I collagen and the mineral matrix built of hydroxyapatite. Osteoblasts and odontoblasts, cells involved in osteogenesis and dentinogenesis, first secrete a nonmineralized matrix—a bone osteoid, and in case of dentin—predentin and then with fibers of collagen I form matrix for biomineralization [112]. Calcium and phosphate ions are dislocated from the vascular network into the mineralization matrix. Osteoid and predentin contain numerous noncollagenous proteins called NCPs. On the basis of mutation experiments and suppression of NCP genes, it is suggested that they stimulate the nucleation and growth of hydroxyapatite crystals [112].

Bones and dentin differ significantly. Bone is a dynamic tissue because it is constantly remodeled, while dentin is a rather static tissue [112]. Osteoblasts produce matrix components as a result of controlling growth factors to form a bone. Hydroxyapatite crystals grow above vesicles, thus creating mineralization centers. Bones have a lot of vessels, and they store calcium ions. The reason for bone resorption is the action of lysosomal enzymes produced by osteoclasts and lowering the pH in extracellular bone matrix.

Osteoclasts are responsible for the attachment, bone degradation, and subsequent tissue resorption. It was found that this is possible due to the strong polarization of osteoclasts. They have both frontal and posterior abdominal polarization. On the underside of the osteoclasts, there are structures responsible for the degradation of the mineral surface. The adhesion zone is located in the sealing zone [114]—a ring made of actin and podosomes in which extracellular resorption pit is segregated and is used to retain heavy substances (minerals) formed during bone resorption near the bone surface [43, 114]. Podosomes are dynamic structures built of actin. They perform important functions, including adhesion, destruction of the bone and its matrix, and recognition of the appropriate medium [115]. Podosomes contain a large number of V-ATPase molecules that pump protons and secrete proteases, which promote bone degradation [114].

#### **Acknowledgements**

This work was supported by the National Science Center (Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity subsidy from the Polish Ministry of Science and High Education for the Faculty of Chemistry of Wroclaw University of Science and Technology.

### **Conflict of interest**

Authors declare no conflict of interest.

#### **Author details**

Marta Kalka, Anna Zoglowek, Andrzej Ożyhar and Piotr Dobryczycki\* Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wrocław, Poland

\*Address all correspondence to: piotr.dobryszycki@pwr.edu.pl

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**63**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

> [10] Jain A, Karadag A, Fohr B, Fisher LW, Fedarko NS. Three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) enhance factor H's cofactor activity enabling MCP-like cellular evasion of complement-mediated attack. The Journal of Biological Chemistry.

2002;**277**:13700-13708

[11] Wojtas M, Dobryszycki P, Ożyhar A. Intrinsically disordered proteins in biomineralization. In: Advanced Topics in Biomineralization. UK: IntechOpen; 2012. pp. 3-32. DOI: 10.5772/31121

[12] Uversky VN, Gillespie JR, Fink AL. Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins. 2000;**41**:415-427

[13] Romero P, Obradovic Z, Li X, Garner EC, Brown CJ, Dunker AK. Sequence complexity of disordered protein. Proteins. 2001;**42**:38-48

Multifunctional ECM proteins in bone and teeth. Experimental Cell Research.

[16] Brodsky B, Persikov AV. Molecular structure of the collagen triple helix. Advances in Protein Chemistry.

[17] Van der Rest M, Garrone R. Collagen family of proteins. The FASEB Journal.

[18] Olszta MJ, Cheng X, Jee SS, Kumar R, Kim Y, Kaufman MJ, et al. Bone structure and formation, a new perspective. Materials Science and Engineering R. 2007;**58**:77-116

[15] Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. In: The Extracellular Matrix of Animals. Available from: https://www.ncbi.nlm. nih.gov/books/NBK26810/. 4th ed. New York: Garland Science; 2002

[14] Ravindran S, George A.

2014;**325**(2):148-154

2005;**70**:301-339

1991;**13**:2814-2823

[1] Lowenstam HA, Weiner S. On Biomineralization. New York: Oxford

University Press; 1989. 324 p

[2] Dorozhkin SV. Calcium orthophosphates occurrence, properties, biomineralization, pathological calcification and biomimetic applications. Biomatter.

[3] Dorozhkin SV. Calcium

orthophosphates (CaPO4): Occurrence and properties. Progress in Biomaterials.

[4] Grynpas MD, Omelon S. Transient precursor strategy or very small biological apatite crystals? Bone.

[5] Young RA. Biological apatite vs hydroxyapatite at the atomic level. Clinical Orthopaedics and Related

Research. 1975;**113**:249-262

[6] Wopenka B, Pasteris JD. A mineralogical perspective on the apatite in bone. Materials Science and Engineering. 2005;**C 25**:131-143

M, Różycka M, Dobryszycki P. Biomineralization-precision of shape, structure and properties controlled by proteins. Postepy Biochemii.

[8] Marin F, Luquet G. Unusually acidic proteins in biomineralization. In: Handbook of Biomineralization: Biological Aspects and Structure Formation. USA: Wiley; 2008. DOI: 10.1002/9783527619443.ch16

[9] Gorski JP. Acidic phosphoproteins from bone matrix: A structural rationalization of their role in biomineralization. Calcified Tissue International. 1992;**50**:391-396

2015;**61**:364-380

[7] Hołubowicz R, Porębska A, Poznar

**References**

2011;**1**:121-164

2016;**5**:9-70

2007;**4**:162-164

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

#### **References**

*Contemporary Topics about Phosphorus in Biology and Materials*

Authors declare no conflict of interest.

**Conflict of interest**

**62**

**Author details**

and Technology, Wrocław, Poland

provided the original work is properly cited.

Marta Kalka, Anna Zoglowek, Andrzej Ożyhar and Piotr Dobryczycki\*

\*Address all correspondence to: piotr.dobryszycki@pwr.edu.pl

Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] Lowenstam HA, Weiner S. On Biomineralization. New York: Oxford University Press; 1989. 324 p

[2] Dorozhkin SV. Calcium orthophosphates occurrence, properties, biomineralization, pathological calcification and biomimetic applications. Biomatter. 2011;**1**:121-164

[3] Dorozhkin SV. Calcium orthophosphates (CaPO4): Occurrence and properties. Progress in Biomaterials. 2016;**5**:9-70

[4] Grynpas MD, Omelon S. Transient precursor strategy or very small biological apatite crystals? Bone. 2007;**4**:162-164

[5] Young RA. Biological apatite vs hydroxyapatite at the atomic level. Clinical Orthopaedics and Related Research. 1975;**113**:249-262

[6] Wopenka B, Pasteris JD. A mineralogical perspective on the apatite in bone. Materials Science and Engineering. 2005;**C 25**:131-143

[7] Hołubowicz R, Porębska A, Poznar M, Różycka M, Dobryszycki P. Biomineralization-precision of shape, structure and properties controlled by proteins. Postepy Biochemii. 2015;**61**:364-380

[8] Marin F, Luquet G. Unusually acidic proteins in biomineralization. In: Handbook of Biomineralization: Biological Aspects and Structure Formation. USA: Wiley; 2008. DOI: 10.1002/9783527619443.ch16

[9] Gorski JP. Acidic phosphoproteins from bone matrix: A structural rationalization of their role in biomineralization. Calcified Tissue International. 1992;**50**:391-396

[10] Jain A, Karadag A, Fohr B, Fisher LW, Fedarko NS. Three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) enhance factor H's cofactor activity enabling MCP-like cellular evasion of complement-mediated attack. The Journal of Biological Chemistry. 2002;**277**:13700-13708

[11] Wojtas M, Dobryszycki P, Ożyhar A. Intrinsically disordered proteins in biomineralization. In: Advanced Topics in Biomineralization. UK: IntechOpen; 2012. pp. 3-32. DOI: 10.5772/31121

[12] Uversky VN, Gillespie JR, Fink AL. Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins. 2000;**41**:415-427

[13] Romero P, Obradovic Z, Li X, Garner EC, Brown CJ, Dunker AK. Sequence complexity of disordered protein. Proteins. 2001;**42**:38-48

[14] Ravindran S, George A. Multifunctional ECM proteins in bone and teeth. Experimental Cell Research. 2014;**325**(2):148-154

[15] Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. In: The Extracellular Matrix of Animals. Available from: https://www.ncbi.nlm. nih.gov/books/NBK26810/. 4th ed. New York: Garland Science; 2002

[16] Brodsky B, Persikov AV. Molecular structure of the collagen triple helix. Advances in Protein Chemistry. 2005;**70**:301-339

[17] Van der Rest M, Garrone R. Collagen family of proteins. The FASEB Journal. 1991;**13**:2814-2823

[18] Olszta MJ, Cheng X, Jee SS, Kumar R, Kim Y, Kaufman MJ, et al. Bone structure and formation, a new perspective. Materials Science and Engineering R. 2007;**58**:77-116

[19] Knott L, Bailey AJ. Collagen crosslinks in mineralizing tissues, a review of their chemistry, function, and clinical relevance. Bone. 1998;**22**:181-187

[20] Nudelman F, Lausch AJ, Sommerdijk NA, Sone ED. In vitro models of collagen biomineralization. Journal of Structural Biology. 2013;**183**:258-269

[21] Boskey AL. Biomineralization: An overview. Connective Tissue Research. 2003;**44**:5-9

[22] Wright PE, Dyson HJ. Intrinsically unstructured proteins, re-assessing the protein structure-function paradigm. Journal of Molecular Biology. 1999;**293**:321-331

[23] Tompa P. Intrinsically unstructured proteins. Trends in Biochemical Sciences. 2002;**27**:527-533

[24] Iakoucheva LM, Radivojac P, Brown CJ, O'Connor TR, Sikes JG, Obradovic Z, et al. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Research. 2004;**32**:1037-1049

[25] Kalmar L, Homola D, Varga D, Tompa P. Structural disorder in proteins brings order to crystal growth in biomineralization. Bone. 2012;**51**:528-534

[26] Boskey AL, Villarreal-Ramirez E. Intrinsically disordered proteins and biomineralization. Matrix Biology. 2016;**52-54**:43-59

[27] Staines KA, MacRae VE, Farquharson C. The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling. Journal of Endocrinology. 2012;**214**:241-255

[28] Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS. Flexible structures of SIBLING proteins,

bone sialoprotein, and osteopontin. Biochemical and Biophysical Research Communications. 2001;**280**:460-465

[29] Rowe PS, Kumagai Y, Gutierrez G, Garrett IR, Blacher R, Rosen D, et al. MEPE has the properties of an osteoblastic phosphatonin and minhibin. Bone. 2004;**34**:303-319

[30] David V, Martin AC, Hedge AM, Drezner MK, Rowe PS. ASARM peptides: PHEX-dependent, independent regulation of serum phosphate. American Journal of Physiology. Renal Physiology. 2010;**300**:F783-F791

[31] Qin C, Baba O, Butler WT. Posttranslational modification of SIBLING proteins and their roles in osteogenesis and dentinogenesis. Critical Reviews in Oral Biology and Medicine. 2004;**15**:126-136

[32] Icer MA, Gezmen-Karadag M. The multiple functions and mechanisms of osteopontin. Clinical Biochemistry. 2018;**59**:17-24

[33] Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT. Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (SPP-1 or osteopontin) gene expression. Proceedings of the National Academy of Sciences of the United States of America. 1990;**87**:9995-9999

[34] Reinholt FP, Hultenby K, Oldberg A, Heinegård D. Osteopontin—A possible anchor of osteoclasts to bone. Proceedings of the National Academy of Sciences. 1990;**87**:4473-4475

[35] Sodek J, Ganss B, McKee MD. Osteopontin. Critical Reviews in Oral Biology and Medicine. 2000;**11**:279-303

**65**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

[36] Giachelli CM, Steitz S. Osteopontin: A versatile regulator of inflammation and biomineralization. Matrix Biology.

Biophysical Research Communications.

[44] Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. Expression of bone sialoprotein (BSP) in developing human tissues. Calcified Tissue International. 1991;**49**:421-426

[45] Tye CE, Rattray KR, Warner KJ, Gordon JA, Sodek J, Hunter GK, et al. Delineation of the hydroxyapatitenucleating domains of bone

sialoprotein. The Journal of Biological Chemistry. 2003;**278**:7949-7955

[46] Tye CE, Hunter GK, Goldberg HA. Identification of the type I collagen-binding domain of bone sialoprotein and characterization of the mechanism of interaction. The Journal of Biological Chemistry.

[47] Wuttke M, Muller S, Nitsche DP, Paulsson M, Hanisch FG, Maurer P. Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding. The Journal of Biological Chemistry. 2001;**276**:36839-36848

[48] Ganss B, Kim RH, Sodek J. Bone sialoprotein. Critical Reviews in Oral Biology and Medicine. 1999;**10**:79-98

[49] Malaval L, Wade-Gueye NM, Boudiffa M, Fei J, Zirngibl R, et al. Bonesialoprotein plays a functional role in bone formation and osteoclastogenesis. The Journal of Experimental Medicine.

[50] Bouleftour W, Boudiffa M, Wade-Gueye NM, Bouët G, Cardelli M, Laroche N, et al. Skeletal development of mice lacking bone sialoprotein (BSP)—Impairment of long bone growth and progressive establishment of high trabecular bone mass. PLoS One.

2008;**205**:1145-1153

2014;**9**:e95144

2005;**280**:13487-13492

2012;**419**:333-338

[37] McKee M, Nanci A. Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: Ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microscopy Research and Technique.

[38] Chellaiah M, Hruska K. The integrin αVβ3 and CD44 regulate the actions of osteopontin on osteoclast motility. Calcified Tissue International.

[39] Boskey AL, Maresca M, Ullrich W, Doty SB, Butler WT, Prince CW. Osteopontin-hydroxyapatite interactions in vitro: Inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone and Mineral.

[40] Hunter GK. Role of osteopontin in modulation of hydroxyapatite formation. Calcified Tissue International. 2013;**93**:348-354

[41] Boskey AL, Spevak L, Paschalis E, Doty SB, McKee MD. Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone. Calcified Tissue International.

[42] Addison WN, Masica DL, Gray JJ, McKee MD. Phosphorylationdependent inhibition of mineralization by osteopontin ASARM peptides is regulated by PHEX cleavage. Journal of Bone and Mineral Research.

[43] Boskey AL, Christensen B, Taleb H,

Sorensen ES. Post-translational modification of osteopontin: Effects on in vitro hydroxyapatite formation and growth. Biochemical and

2000;**19**:615-622

1996;**33**:141-164

2002;**72**:97-205

1993;**22**:147-159

2002;**71**:145-154

2010;**25**:695-705

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

*Contemporary Topics about Phosphorus in Biology and Materials*

bone sialoprotein, and osteopontin. Biochemical and Biophysical Research Communications. 2001;**280**:460-465

[29] Rowe PS, Kumagai Y, Gutierrez G, Garrett IR, Blacher R, Rosen D, et al. MEPE has the properties of an osteoblastic phosphatonin and minhibin. Bone. 2004;**34**:303-319

[30] David V, Martin AC, Hedge AM, Drezner MK, Rowe PS. ASARM peptides: PHEX-dependent, independent regulation of serum phosphate. American Journal of Physiology. Renal Physiology.

[31] Qin C, Baba O, Butler WT. Posttranslational modification of SIBLING proteins and their roles in osteogenesis and dentinogenesis. Critical Reviews in Oral Biology and Medicine.

[32] Icer MA, Gezmen-Karadag M. The multiple functions and mechanisms of osteopontin. Clinical Biochemistry.

[33] Noda M, Vogel RL, Craig AM, Prahl

1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (SPP-1 or osteopontin) gene expression. Proceedings of the National Academy of Sciences of the United States of America. 1990;**87**:9995-9999

[34] Reinholt FP, Hultenby K, Oldberg A, Heinegård D. Osteopontin—A possible anchor of osteoclasts to bone. Proceedings of the National Academy of

Sciences. 1990;**87**:4473-4475

[35] Sodek J, Ganss B, McKee MD. Osteopontin. Critical Reviews in Oral Biology and Medicine.

2000;**11**:279-303

J, DeLuca HF, Denhardt DT. Identification of a DNA sequence responsible for binding of the

2010;**300**:F783-F791

2004;**15**:126-136

2018;**59**:17-24

[19] Knott L, Bailey AJ. Collagen crosslinks in mineralizing tissues, a review of their chemistry, function, and clinical relevance. Bone. 1998;**22**:181-187

[20] Nudelman F, Lausch AJ, Sommerdijk NA, Sone ED. In vitro models of collagen biomineralization.

Journal of Structural Biology.

[21] Boskey AL. Biomineralization: An overview. Connective Tissue Research.

[22] Wright PE, Dyson HJ. Intrinsically unstructured proteins, re-assessing the protein structure-function

paradigm. Journal of Molecular Biology.

[23] Tompa P. Intrinsically unstructured

proteins. Trends in Biochemical Sciences. 2002;**27**:527-533

[24] Iakoucheva LM, Radivojac P, Brown CJ, O'Connor TR, Sikes JG, Obradovic Z, et al. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Research. 2004;**32**:1037-1049

[25] Kalmar L, Homola D, Varga D, Tompa P. Structural disorder in proteins brings order to crystal growth in biomineralization. Bone.

[26] Boskey AL, Villarreal-Ramirez E. Intrinsically disordered proteins and biomineralization. Matrix Biology.

2013;**183**:258-269

1999;**293**:321-331

2012;**51**:528-534

2016;**52-54**:43-59

2012;**214**:241-255

[27] Staines KA, MacRae VE, Farquharson C. The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling. Journal of Endocrinology.

[28] Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS. Flexible structures of SIBLING proteins,

2003;**44**:5-9

**64**

[36] Giachelli CM, Steitz S. Osteopontin: A versatile regulator of inflammation and biomineralization. Matrix Biology. 2000;**19**:615-622

[37] McKee M, Nanci A. Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: Ultrastructural distribution and implications for mineralized tissue formation, turnover, and repair. Microscopy Research and Technique. 1996;**33**:141-164

[38] Chellaiah M, Hruska K. The integrin αVβ3 and CD44 regulate the actions of osteopontin on osteoclast motility. Calcified Tissue International. 2002;**72**:97-205

[39] Boskey AL, Maresca M, Ullrich W, Doty SB, Butler WT, Prince CW. Osteopontin-hydroxyapatite interactions in vitro: Inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone and Mineral. 1993;**22**:147-159

[40] Hunter GK. Role of osteopontin in modulation of hydroxyapatite formation. Calcified Tissue International. 2013;**93**:348-354

[41] Boskey AL, Spevak L, Paschalis E, Doty SB, McKee MD. Osteopontin deficiency increases mineral content and mineral crystallinity in mouse bone. Calcified Tissue International. 2002;**71**:145-154

[42] Addison WN, Masica DL, Gray JJ, McKee MD. Phosphorylationdependent inhibition of mineralization by osteopontin ASARM peptides is regulated by PHEX cleavage. Journal of Bone and Mineral Research. 2010;**25**:695-705

[43] Boskey AL, Christensen B, Taleb H, Sorensen ES. Post-translational modification of osteopontin: Effects on in vitro hydroxyapatite formation and growth. Biochemical and

Biophysical Research Communications. 2012;**419**:333-338

[44] Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. Expression of bone sialoprotein (BSP) in developing human tissues. Calcified Tissue International. 1991;**49**:421-426

[45] Tye CE, Rattray KR, Warner KJ, Gordon JA, Sodek J, Hunter GK, et al. Delineation of the hydroxyapatitenucleating domains of bone sialoprotein. The Journal of Biological Chemistry. 2003;**278**:7949-7955

[46] Tye CE, Hunter GK, Goldberg HA. Identification of the type I collagen-binding domain of bone sialoprotein and characterization of the mechanism of interaction. The Journal of Biological Chemistry. 2005;**280**:13487-13492

[47] Wuttke M, Muller S, Nitsche DP, Paulsson M, Hanisch FG, Maurer P. Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding. The Journal of Biological Chemistry. 2001;**276**:36839-36848

[48] Ganss B, Kim RH, Sodek J. Bone sialoprotein. Critical Reviews in Oral Biology and Medicine. 1999;**10**:79-98

[49] Malaval L, Wade-Gueye NM, Boudiffa M, Fei J, Zirngibl R, et al. Bonesialoprotein plays a functional role in bone formation and osteoclastogenesis. The Journal of Experimental Medicine. 2008;**205**:1145-1153

[50] Bouleftour W, Boudiffa M, Wade-Gueye NM, Bouët G, Cardelli M, Laroche N, et al. Skeletal development of mice lacking bone sialoprotein (BSP)—Impairment of long bone growth and progressive establishment of high trabecular bone mass. PLoS One. 2014;**9**:e95144

[51] Valverde P, Zhang J, Fix A, Zhu J, Ma W, Tu Q, et al. Overexpression of bone sialoprotein leads to an uncoupling of bone formation and bone resorption in mice. Journal of Bone and Mineral Research. 2008;**23**:1775-1788

[52] Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences. 1993;**90**:8562-8565

[53] Baht GS, Hunter GK, Goldberg HA. Bone sialoprotein-collagen interactionpromotes hydroxyapatite nucleation. Matrix Biology. 2008;**27**:600-608

[54] George A, Veis A. Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition. Chemical Reviews. 2008;**108**:4670-4693

[55] Baht GS, O'Young J, Borovina A, Chen H, Tye CE, Karttunen M, et al. Phosphorylation of Ser136 is critical for potent bone sialoproteinmediated nucleation of hydroxyapatite crystals. The Biochemical Journal. 2010;**428**:385-395

[56] Foster BL, Ao M, Willoughby C, Soenjaya Y, Holm E, Lukashova L, et al. Mineralization defects in cementum and craniofacial bone from loss of bone sialoprotein. Bone. 2015;**78**:150-164

[57] Nampei A, Hashimoto J, Hayashida K, Tsuboi H, Shi K, Tsuji I, et al. Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone. Journal of Bone and Mineral Metabolism. 2004;**22**:176-184

[58] Gowen LC, Petersen DN, Mansolf AL, Qi H, Stock JL, et al. Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. Journal of Biological Chemistry. 2003;**278**:1998-2007

[59] Quarles LD. FGF23, PHEX, and MEPE regulation of phosphate homeostasis and skeletal mineralization. American Journal of Physiology. Endocrinology and Metabolism. 2003;**285**:1-9

[60] Dobbie H, Unwin RJ, Faria NJ, Shirley DG. Matrix extracellular phosphoglycoprotein causes phosphaturia in rats by inhibiting tubular phosphate reabsorption. Nephrology, Dialysis, Transplantation. 2008;**23**:730-733

[61] Addison WN, Nakano Y, Loisel T, Crine P, McKee MD. MEPE-ASARM peptides control extracellular matrix mineralization by binding to hydroxyapatite: An inhibition regulated by PHEX cleavage of ASARM. Journal of Bone and Mineral Research. 2008;**23**:1638-1649

[62] Hayashibara T, Hiraga T, Yi B, Nomizu M, Kumagai Y, Nishimura R, et al. A synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo. Journal of Bone and Mineral Research. 2004;**19**:455-462

[63] George A, Sabsay B, Simonian PA, Veis A. Characterization of a novel dentin matrix acidic phosphoprotein, implications for induction of biomineralization. Journal of Biological Chemistry. 1993;**268**:12624-12630

[64] MacDougall M, Gu TT, Luan X, Simmons D, Chen J. Identification of a novel isoform of mouse dentin matrix protein 1: Spatial expression in mineralized tissues. Journal of Bone and Mineral Research. 1998;**13**:422-431

[65] Sun Y, Chen L, Ma S, Zhou J, Zhang H, Feng JQ, et al. Roles of DMP1 processing in osteogenesis, dentinogenesis and chondrogenesis. Cells, Tissues, Organs. 2011;**194**:199-204

[66] Gericke A, Qin C, Sun Y, Redfern R, Redfern D, Fujimoto Y, et al. Different

**67**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

> [74] Porębska A, Ożyhar A, Dobryszycki P. Intrinsically disordered recombinant

influences the in vitro crystallization of CaCO3. Protein Science. 2015;**24**(Suppl 1):137-138. DOI: 10.1002/pro.2823

[75] Qin C, Brunn JC, Cadena E, Ridall A, Tsujigiwa H, Nagatsuka H, et al. The expression of dentin sialophosphoprotein gene in bone. Journal of Dental Research.

[76] Baba O, Qin C, Brunn JC, Jones JE, Wygant JN, BW MI, et al. Detection of dentin sialoprotein in rat periodontium. European Journal of Oral Sciences.

[77] Kim JW, Hu JCC, Lee JI, et al. Mutational hot spot in the DSPP gene causing dentinogenesis imperfecta type II. Human Genetics. 2005;**116**:186-191

[78] Verdelis K, Ling Y, Sreenath T, Haruyama N, MacDougall M, Van der Meulen MC, et al. DSPP effects on in vivo bone mineralization. Bone.

[79] Yamakoshi Y, Simmer JP. Structural

features, processing mechanism and gene splice variants of dentin sialophosphoprotein. Japanese Dental Science Review. 2018;**54**:183-196

[80] Yamakoshi Y, Hu JC, Fukae M, Zhang H, Simmer JP. Dentin glycoprotein: The protein in the middle of the dentin sialophosphoprotein chimera. The Journal of Biological Chemistry. 2005;**280**:17472-17479

[81] Yamakoshi Y, Lu Y, Hu JC, Kim JW, Iwata T, Kobayashi K, et al. Porcine dentin sialophosphoprotein: Length polymorphisms, glycosylation, phosphorylation, and stability. The Journal of Biological Chemistry.

[82] He G, Ramachandran A, Dahl T, George S, Schultz D, Cookson D, et al.

2008;**283**:14835-14844

57K fragment of human DMP1

2002;**81**:392-394

2004;**112**:163-170

2008;**43**:983-990

forms of DMP1 play distinct roles in mineralization. Journal of Dental

[67] He G, Dahl T, Veis A, George A. Dentin matrix protein 1 initiates hydroxyapatite formation in vitro. Connective Tissue Research.

[68] Tartaix PH, Doulaverakis M, George A, Fisher LW, Butler WT, Qin C, et al. In vitro effects of dentin matrix protein-1 on hydroxyapatite formation provide insights into in vivo functions. Journal of Biological Chemistry.

[69] Qin C, Brunn JC, Cook RG, Orkiszewski RS, Malone JP, Veis A, et al. Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites. Journal of Biological Chemistry.

[70] Kulkarni GV, Chen B, Malone JP, Sampath Narayanan A, George A. Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1. Archives of Oral Biology.

[71] Rowe PS. The wrickkened pathways of FGF23, MEPE and PHEX. Critical Reviews in Oral Biology and Medicine.

[72] Gajjeraman S, Narayanan K, Hao J, Qin C, George A. Matrix macromolecules in hard tissues

control the nucleation and hierarchical assembly of hydroxyapatite. The Journal of Biological Chemistry.

[73] Lu Y, Qin C, Xie Y, Bonewald LF, Feng JQ. Studies of the DMP1 57-kDa functional domain both in vivo and in vitro. Cells, Tissues, Organs.

Research. 2010;**89**:355-359

2003;**44**:240-245

2004;**279**:18115-18120

2003;**278**:34700-34708

2000;**45**:475-484

2004;**15**:264-281

2007;**282**:1193-1204

2008;**189**:175-185

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

forms of DMP1 play distinct roles in mineralization. Journal of Dental Research. 2010;**89**:355-359

*Contemporary Topics about Phosphorus in Biology and Materials*

[59] Quarles LD. FGF23, PHEX, and MEPE regulation of phosphate homeostasis and skeletal mineralization.

American Journal of Physiology. Endocrinology and Metabolism.

[60] Dobbie H, Unwin RJ, Faria NJ, Shirley DG. Matrix extracellular phosphoglycoprotein causes phosphaturia in rats by inhibiting tubular phosphate reabsorption. Nephrology, Dialysis, Transplantation.

[61] Addison WN, Nakano Y, Loisel T, Crine P, McKee MD. MEPE-ASARM peptides control extracellular matrix mineralization by binding to hydroxyapatite: An inhibition regulated by PHEX cleavage of ASARM. Journal of Bone and Mineral Research.

[62] Hayashibara T, Hiraga T, Yi B, Nomizu M, Kumagai Y, Nishimura R, et al. A synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo. Journal of Bone and Mineral Research.

[63] George A, Sabsay B, Simonian PA, Veis A. Characterization of a novel dentin matrix acidic phosphoprotein,

biomineralization. Journal of Biological Chemistry. 1993;**268**:12624-12630

[64] MacDougall M, Gu TT, Luan X, Simmons D, Chen J. Identification of a novel isoform of mouse dentin matrix protein 1: Spatial expression in mineralized tissues. Journal of Bone and Mineral Research. 1998;**13**:422-431

[65] Sun Y, Chen L, Ma S, Zhou J, Zhang H, Feng JQ, et al. Roles of DMP1 processing in osteogenesis, dentinogenesis and chondrogenesis. Cells, Tissues, Organs. 2011;**194**:199-204

[66] Gericke A, Qin C, Sun Y, Redfern R, Redfern D, Fujimoto Y, et al. Different

implications for induction of

2003;**285**:1-9

2008;**23**:730-733

2008;**23**:1638-1649

2004;**19**:455-462

[51] Valverde P, Zhang J, Fix A, Zhu J, Ma W, Tu Q, et al. Overexpression of bone sialoprotein leads to an uncoupling of bone formation and bone resorption in mice. Journal of Bone and Mineral

Research. 2008;**23**:1775-1788

[52] Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proceedings of the National Academy of Sciences.

Bone sialoprotein-collagen

nucleation. Matrix Biology.

[53] Baht GS, Hunter GK, Goldberg HA.

interactionpromotes hydroxyapatite

[54] George A, Veis A. Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition. Chemical Reviews.

[55] Baht GS, O'Young J, Borovina A, Chen H, Tye CE, Karttunen M, et al. Phosphorylation of Ser136 is critical for potent bone sialoproteinmediated nucleation of hydroxyapatite crystals. The Biochemical Journal.

[56] Foster BL, Ao M, Willoughby C, Soenjaya Y, Holm E, Lukashova L, et al. Mineralization defects in cementum and craniofacial bone from loss of bone sialoprotein. Bone. 2015;**78**:150-164

[57] Nampei A, Hashimoto J, Hayashida K, Tsuboi H, Shi K, Tsuji I, et al. Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone. Journal of Bone and Mineral Metabolism.

[58] Gowen LC, Petersen DN, Mansolf AL, Qi H, Stock JL, et al. Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. Journal of Biological Chemistry.

1993;**90**:8562-8565

2008;**27**:600-608

2008;**108**:4670-4693

2010;**428**:385-395

2004;**22**:176-184

2003;**278**:1998-2007

**66**

[67] He G, Dahl T, Veis A, George A. Dentin matrix protein 1 initiates hydroxyapatite formation in vitro. Connective Tissue Research. 2003;**44**:240-245

[68] Tartaix PH, Doulaverakis M, George A, Fisher LW, Butler WT, Qin C, et al. In vitro effects of dentin matrix protein-1 on hydroxyapatite formation provide insights into in vivo functions. Journal of Biological Chemistry. 2004;**279**:18115-18120

[69] Qin C, Brunn JC, Cook RG, Orkiszewski RS, Malone JP, Veis A, et al. Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites. Journal of Biological Chemistry. 2003;**278**:34700-34708

[70] Kulkarni GV, Chen B, Malone JP, Sampath Narayanan A, George A. Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1. Archives of Oral Biology. 2000;**45**:475-484

[71] Rowe PS. The wrickkened pathways of FGF23, MEPE and PHEX. Critical Reviews in Oral Biology and Medicine. 2004;**15**:264-281

[72] Gajjeraman S, Narayanan K, Hao J, Qin C, George A. Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite. The Journal of Biological Chemistry. 2007;**282**:1193-1204

[73] Lu Y, Qin C, Xie Y, Bonewald LF, Feng JQ. Studies of the DMP1 57-kDa functional domain both in vivo and in vitro. Cells, Tissues, Organs. 2008;**189**:175-185

[74] Porębska A, Ożyhar A, Dobryszycki P. Intrinsically disordered recombinant 57K fragment of human DMP1 influences the in vitro crystallization of CaCO3. Protein Science. 2015;**24**(Suppl 1):137-138. DOI: 10.1002/pro.2823

[75] Qin C, Brunn JC, Cadena E, Ridall A, Tsujigiwa H, Nagatsuka H, et al. The expression of dentin sialophosphoprotein gene in bone. Journal of Dental Research. 2002;**81**:392-394

[76] Baba O, Qin C, Brunn JC, Jones JE, Wygant JN, BW MI, et al. Detection of dentin sialoprotein in rat periodontium. European Journal of Oral Sciences. 2004;**112**:163-170

[77] Kim JW, Hu JCC, Lee JI, et al. Mutational hot spot in the DSPP gene causing dentinogenesis imperfecta type II. Human Genetics. 2005;**116**:186-191

[78] Verdelis K, Ling Y, Sreenath T, Haruyama N, MacDougall M, Van der Meulen MC, et al. DSPP effects on in vivo bone mineralization. Bone. 2008;**43**:983-990

[79] Yamakoshi Y, Simmer JP. Structural features, processing mechanism and gene splice variants of dentin sialophosphoprotein. Japanese Dental Science Review. 2018;**54**:183-196

[80] Yamakoshi Y, Hu JC, Fukae M, Zhang H, Simmer JP. Dentin glycoprotein: The protein in the middle of the dentin sialophosphoprotein chimera. The Journal of Biological Chemistry. 2005;**280**:17472-17479

[81] Yamakoshi Y, Lu Y, Hu JC, Kim JW, Iwata T, Kobayashi K, et al. Porcine dentin sialophosphoprotein: Length polymorphisms, glycosylation, phosphorylation, and stability. The Journal of Biological Chemistry. 2008;**283**:14835-14844

[82] He G, Ramachandran A, Dahl T, George S, Schultz D, Cookson D, et al. Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization. The Journal of Biological Chemistry. 2005;**280**:33109-33114

[83] Boskey AL, Maresca M, Doty S, Sabsay B, Veis A. Concentration dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone and Mineral. 1990;**11**:55-65

[84] Stetler-Stevenson WG, Veis A. Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcified Tissue International. 1986;**38**:135-141

[85] Traub W, Jodaikin A, Arad T, Veis A, Sabsay B. Dentin phosphophoryn binding to collagen fibrils. Matrix. 1992;**12**:197-201

[86] Prasad M, Butler WT, Qin C. Dentin sialophosphoprotein in biomineralization. Connective Tissue Research. 2010;**51**:404-417

[87] Butler WT. Dentin matrix proteins. European Journal of Oral Sciences. 1998;**106**(Suppl 1):204-210

[88] Butler WT, Brunn JC, Qin C. Dentin extracellular matrix (ECM) proteins: Comparison to bone ECM and contribution to dynamics of dentinogenesis. Connective Tissue Research. 2003;**44**(Suppl 1):171-178

[89] Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: Structure, composition and mineralization. The role of dentin ECM in dentin formation and mineralization. Frontiers in Bioscience. 2011;**3**:711-735

[90] Moradian-Oldak J. Proteinmediated enamel mineralization. Frontiers in bioscience (Landmark edition). 2012;**17**:1996-2023

[91] Ye L, Le TQ, Zhu L, Butcher K, Schneider RA, Li W, et al. Amelogenins in human developing and mature dental pulp. Journal of Dental Research. 2006;**85**:814-818

[92] Delak K, Harcup C, Lakshminarayanan R, Sun Z, Fan YJ, Moradian-Oldak J, et al. The tooth enamel protein, porcine amelogenin, is an intrinsically disordered protein with an extended molecular configuration in the monomeric form. Biochemistry. 2009;**48**:2272-2281

[93] Paine ML, Luo W, Zhu DH, Bringas PJ, Snead ML. Functional domains for amelogenin revealed by compound genetic defects. Journal of Bone and Mineral Research. 2003;**18**:466-472

[94] Nanci A, Zalzal S, Lavoie P, Kunikata M, Chen W, Krebsbach PH, et al. Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors. The Journal of Histochemistry and Cytochemistry. 1998;**46**:911-934

[95] Krebsbach PH, Lee SK, Matsuki Y, Kozak CA, Yamada KM, Yamada Y. Full length sequence, localization, and chromosomal mapping of ameloblastin: A novel tooth-specific gene. The Journal of Biological Chemistry. 1996;**271**:4431-4435

[96] Fukumoto S, Kiba T, Hall B, Iehara N, Nakamura T, Longenecker G, et al. Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts. The Journal of Cell Biology. 2004;**167**:973-983

[97] Vymetal J, Slaby I, Spahr A, Vondrasek J, Lyngstadaas SP. Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two-domain intrinsically unstructured calcium-binding protein. European Journal of Oral Sciences. 2008;**116**:124-134

**69**

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

> P, et al. Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif. Proceedings of the National Academy of Sciences.

[105] MacDougall MJ, Javed A. Dentin

[106] Orsini G, Ruggeri A, Mazzoni A, Nato F, Manzoli L, Putignano A, et al. A review of the nature, role, and function of dentin non-collagenous proteins. Part 1: Proteoglycans and glycoproteins. Endodontic Topics.

[107] Niu LN, Zhang W, Pashley DH, Breschi L, Mao J, Chen JH, et al.

Dental Materials. 2014;**30**:77-96

Medicine. 2003;**14**:13-29

2000;**1997**(13):41-75

Biomimetic remineralization of dentin.

[108] Kinney JH, Marshall SJ, Marshall GW. The mechanical properties of human dentin: A critical review and re-evaluation of the dental literature. Critical Reviews in Oral Biology and

[109] Bosshardt DD, Selvig KA. Dental cementum: The dynamic tissue covering of the root. Periodontology 2000.

[110] Saygin NE, Giannobile WV, Somerman MJ. Molecular and cell biology of cementum. Periodontology

[111] Villarreal-Ramírez E, Moreno A, Mas-Oliva J, Chávez-Pacheco LJ, Narayanan AS, Gil-Chavarría I, et al. Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization. Biochemical and Biophysical Research Communications. 2009;**384**:49-54

2000. 2000;**2000**(24):73-98

and bone: Similar collagenous mineralized tissues. In: Bronner F, Farach-Carson M, Roach H, editors. Bone and Development. Topics in Bone Biology. Vol. 6. London: Springer; 2010

2017;**114**:E1641-E1650

2009;**21**:1-18

[98] Mazumder P, Prajapati S, Lokappa SB, Gallon V, Moradian-Oldak J. Analysis of co-assembly and co-localization of ameloblastin and amelogenin. Frontiers in Physiology.

[99] Mazumder P, Prajapati S, Bapat R, Moradian-Oldak J. Amelogenin ameloblastin spatial interaction around maturing enamel rods. Journal of Dental

Research. 2016;**95**(9):1042-1048

[100] Uchida T, Tanabe T, Fukae M, Shimizu M, Yamada M, Miake K, et al. Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of the pig and rat. Histochemistry. 1991;**96**:129-138

[101] Uchida T, Murakami C, Dohi N, Wakida K, Satoda T, Takahashi O. Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using regionspecific antibodies. The Journal of Histochemistry and Cytochemistry.

[102] Fang PA, Conway JF, Margolis HC, Simmer JP, Beniash E. Hierarchical self assembly of amelogenin and the regulation of biomineralization at the nanoscale. Proceedings of the National Academy of Sciences of the United States of America.

1997;**45**:1329-1340

2011;**108**:14097-14102

2013;**288**:22333-22345

[103] Wald T et al. Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5. The Journal of Biological Chemistry.

[104] Wald T, Spoutil F, Osickova A, Prochazkova M, Benada O, Kasparek

2014;**5**:274

*Proteins in Calcium Phosphates Biomineralization DOI: http://dx.doi.org/10.5772/intechopen.86718*

*Contemporary Topics about Phosphorus in Biology and Materials*

in human developing and mature dental pulp. Journal of Dental Research.

Lakshminarayanan R, Sun Z, Fan YJ, Moradian-Oldak J, et al. The tooth enamel protein, porcine amelogenin, is an intrinsically disordered protein with an extended molecular configuration in the monomeric form. Biochemistry.

[93] Paine ML, Luo W, Zhu DH, Bringas PJ, Snead ML. Functional domains for amelogenin revealed by compound genetic defects. Journal of Bone and Mineral Research. 2003;**18**:466-472

[94] Nanci A, Zalzal S, Lavoie P, Kunikata M, Chen W, Krebsbach PH, et al. Comparative immunochemical analyses of the developmental expression and distribution of ameloblastin and amelogenin in rat incisors. The Journal of Histochemistry and Cytochemistry. 1998;**46**:911-934

[95] Krebsbach PH, Lee SK, Matsuki Y, Kozak CA, Yamada KM, Yamada Y. Full length sequence, localization, and chromosomal mapping of ameloblastin:

[96] Fukumoto S, Kiba T, Hall B, Iehara N, Nakamura T, Longenecker G, et al. Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.

A novel tooth-specific gene. The Journal of Biological Chemistry.

The Journal of Cell Biology.

[97] Vymetal J, Slaby I, Spahr A, Vondrasek J, Lyngstadaas SP.

Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two-domain intrinsically unstructured calcium-binding protein. European Journal of Oral Sciences.

2004;**167**:973-983

2008;**116**:124-134

1996;**271**:4431-4435

2006;**85**:814-818

2009;**48**:2272-2281

[92] Delak K, Harcup C,

Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization. The Journal of Biological Chemistry.

[83] Boskey AL, Maresca M, Doty S, Sabsay B, Veis A. Concentration dependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone and Mineral. 1990;**11**:55-65

[84] Stetler-Stevenson WG, Veis A. Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. Calcified Tissue International. 1986;**38**:135-141

[85] Traub W, Jodaikin A, Arad T, Veis A, Sabsay B. Dentin phosphophoryn binding to collagen fibrils. Matrix.

[86] Prasad M, Butler WT, Qin C. Dentin sialophosphoprotein in biomineralization. Connective Tissue

[87] Butler WT. Dentin matrix proteins. European Journal of Oral Sciences.

Research. 2010;**51**:404-417

1998;**106**(Suppl 1):204-210

[88] Butler WT, Brunn JC, Qin C. Dentin extracellular matrix (ECM) proteins: Comparison to bone ECM and contribution to dynamics of dentinogenesis. Connective Tissue Research. 2003;**44**(Suppl 1):171-178

[89] Goldberg M, Kulkarni AB, Young M, Boskey A. Dentin: Structure, composition and mineralization. The role of dentin ECM in dentin formation and mineralization. Frontiers in Bioscience. 2011;**3**:711-735

[90] Moradian-Oldak J. Proteinmediated enamel mineralization. Frontiers in bioscience (Landmark edition). 2012;**17**:1996-2023

[91] Ye L, Le TQ, Zhu L, Butcher K, Schneider RA, Li W, et al. Amelogenins

1992;**12**:197-201

2005;**280**:33109-33114

**68**

[98] Mazumder P, Prajapati S, Lokappa SB, Gallon V, Moradian-Oldak J. Analysis of co-assembly and co-localization of ameloblastin and amelogenin. Frontiers in Physiology. 2014;**5**:274

[99] Mazumder P, Prajapati S, Bapat R, Moradian-Oldak J. Amelogenin ameloblastin spatial interaction around maturing enamel rods. Journal of Dental Research. 2016;**95**(9):1042-1048

[100] Uchida T, Tanabe T, Fukae M, Shimizu M, Yamada M, Miake K, et al. Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of the pig and rat. Histochemistry. 1991;**96**:129-138

[101] Uchida T, Murakami C, Dohi N, Wakida K, Satoda T, Takahashi O. Synthesis, secretion, degradation, and fate of ameloblastin during the matrix formation stage of the rat incisor as shown by immunocytochemistry and immunochemistry using regionspecific antibodies. The Journal of Histochemistry and Cytochemistry. 1997;**45**:1329-1340

[102] Fang PA, Conway JF, Margolis HC, Simmer JP, Beniash E. Hierarchical self assembly of amelogenin and the regulation of biomineralization at the nanoscale. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**:14097-14102

[103] Wald T et al. Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5. The Journal of Biological Chemistry. 2013;**288**:22333-22345

[104] Wald T, Spoutil F, Osickova A, Prochazkova M, Benada O, Kasparek P, et al. Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif. Proceedings of the National Academy of Sciences. 2017;**114**:E1641-E1650

[105] MacDougall MJ, Javed A. Dentin and bone: Similar collagenous mineralized tissues. In: Bronner F, Farach-Carson M, Roach H, editors. Bone and Development. Topics in Bone Biology. Vol. 6. London: Springer; 2010

[106] Orsini G, Ruggeri A, Mazzoni A, Nato F, Manzoli L, Putignano A, et al. A review of the nature, role, and function of dentin non-collagenous proteins. Part 1: Proteoglycans and glycoproteins. Endodontic Topics. 2009;**21**:1-18

[107] Niu LN, Zhang W, Pashley DH, Breschi L, Mao J, Chen JH, et al. Biomimetic remineralization of dentin. Dental Materials. 2014;**30**:77-96

[108] Kinney JH, Marshall SJ, Marshall GW. The mechanical properties of human dentin: A critical review and re-evaluation of the dental literature. Critical Reviews in Oral Biology and Medicine. 2003;**14**:13-29

[109] Bosshardt DD, Selvig KA. Dental cementum: The dynamic tissue covering of the root. Periodontology 2000. 2000;**1997**(13):41-75

[110] Saygin NE, Giannobile WV, Somerman MJ. Molecular and cell biology of cementum. Periodontology 2000. 2000;**2000**(24):73-98

[111] Villarreal-Ramírez E, Moreno A, Mas-Oliva J, Chávez-Pacheco LJ, Narayanan AS, Gil-Chavarría I, et al. Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization. Biochemical and Biophysical Research Communications. 2009;**384**:49-54

[112] Stock SR. The mineral–collagen interface in bone. Calcified Tissue International. 2015;**97**:262-280

[113] Weiner S, Wagner HD. The material bone: Structure-mechanical function relations. Annual Review of Materials Science. 1998;**28**:271-298

[114] Akisaka T, Yoshida A. Ultrastructural analysis of apatitedegrading capability of extended invasive podosomes in resorbing osteoclasts. Micron. 2016;**88**:37-47

[115] Veillat V, Spuul P, Daubon T, Egaña I, Kramer I, Génot E. Podosomes: Multipurpose organelles? The International Journal of Biochemistry & Cell Biology. 2015;**65**:52-60

**71**

plasma

**1. Introduction**

**Chapter 5**

**Abstract**

Inorganic Polyphosphates Are

Important for Cell Survival

and Motility of Human Skin

*Cynthia M. Simbulan-Rosenthal, Bonnie C. Carney,* 

potent modulator of the wound healing process remain to be elucidated.

**Keywords:** inorganic polyphosphate, wound healing, keratinocytes, platelet-rich

PolyP is a simple prebiotic molecule that varies in chain length between three and several thousand inorganic phosphates linked by phosphoanhydride bonds (**Figure 1A**). It is continuously synthesized from ATP or GTP and degraded by cellular enzymes in bacteria [1–5] and eukaryotes, yet its pleiotropic functions remain

*Anirudh Gaur, Manish Moghe, Elliott Crooke,* 

Wound Healing

Keratinocytes and Play a Role in

*Lauren T. Moffatt, Jeffrey W. Shupp and Dean S. Rosenthal*

Inorganic polyphosphate (polyP) is a simple ancient polymer of linear chains of orthophosphate residues linked by high energy phospho-anhydride bonds ubiquitously found in all organisms. Despite its structural simplicity, it plays diverse functional roles. polyP is involved in myriad of processes including serving as microbial phosphagens, buffer against alkalis, Ca2+ storage, metal-chelating agents, pathogen virulence, cell viability and proliferation, structural component and chemical chaperones, and in the microbial stress response. In mammalian cells, polyP has been implicated in blood coagulation, inflammation, bone differentiation, cell bioenergetics, signal transduction, Ca2+-signaling, neuronal excitability, as a protein-stabilizing scaffold, and in wound healing, among others. This chapter will discuss (1) polyP metabolism and roles of polyP in prokaryotic and eukaryotic cells, (2) the contribution of polyP to survival, cell proliferation, and motility involved in wound healing in human skin keratinocytes, (3) the use of polyP-containing platelet-rich plasma (PRP) to promote wound healing in acute and chronic wounds, including burns, and (4) the use of polyP-containing PRP in excisional wound models to promote faster healing. While polyP shows promise as a therapeutic agent to accelerate healing for acute and chronic wounds, the molecular mechanisms as a

#### **Chapter 5**

*Contemporary Topics about Phosphorus in Biology and Materials*

[112] Stock SR. The mineral–collagen interface in bone. Calcified Tissue International. 2015;**97**:262-280

[113] Weiner S, Wagner HD. The material bone: Structure-mechanical function relations. Annual Review of Materials Science. 1998;**28**:271-298

[114] Akisaka T, Yoshida A.

Cell Biology. 2015;**65**:52-60

Ultrastructural analysis of apatitedegrading capability of extended invasive podosomes in resorbing osteoclasts. Micron. 2016;**88**:37-47

[115] Veillat V, Spuul P, Daubon T, Egaña I, Kramer I, Génot E. Podosomes: Multipurpose organelles? The

International Journal of Biochemistry &

**70**

## Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin Keratinocytes and Play a Role in Wound Healing

*Cynthia M. Simbulan-Rosenthal, Bonnie C. Carney, Anirudh Gaur, Manish Moghe, Elliott Crooke, Lauren T. Moffatt, Jeffrey W. Shupp and Dean S. Rosenthal*

#### **Abstract**

Inorganic polyphosphate (polyP) is a simple ancient polymer of linear chains of orthophosphate residues linked by high energy phospho-anhydride bonds ubiquitously found in all organisms. Despite its structural simplicity, it plays diverse functional roles. polyP is involved in myriad of processes including serving as microbial phosphagens, buffer against alkalis, Ca2+ storage, metal-chelating agents, pathogen virulence, cell viability and proliferation, structural component and chemical chaperones, and in the microbial stress response. In mammalian cells, polyP has been implicated in blood coagulation, inflammation, bone differentiation, cell bioenergetics, signal transduction, Ca2+-signaling, neuronal excitability, as a protein-stabilizing scaffold, and in wound healing, among others. This chapter will discuss (1) polyP metabolism and roles of polyP in prokaryotic and eukaryotic cells, (2) the contribution of polyP to survival, cell proliferation, and motility involved in wound healing in human skin keratinocytes, (3) the use of polyP-containing platelet-rich plasma (PRP) to promote wound healing in acute and chronic wounds, including burns, and (4) the use of polyP-containing PRP in excisional wound models to promote faster healing. While polyP shows promise as a therapeutic agent to accelerate healing for acute and chronic wounds, the molecular mechanisms as a potent modulator of the wound healing process remain to be elucidated.

**Keywords:** inorganic polyphosphate, wound healing, keratinocytes, platelet-rich plasma

#### **1. Introduction**

PolyP is a simple prebiotic molecule that varies in chain length between three and several thousand inorganic phosphates linked by phosphoanhydride bonds (**Figure 1A**). It is continuously synthesized from ATP or GTP and degraded by cellular enzymes in bacteria [1–5] and eukaryotes, yet its pleiotropic functions remain

#### **Figure 1.**

*(A) Schematic of linear structure of polyP, and its synthesis by polyphosphate kinase (PPK) and degradation by exopolyphosphatases (PPX1) or endopolyphosphatases (PPN). Constitutive (B and D) or Tet-inducible PPX1 (C and E) expression slows wound healing in cultured human keratinocytes (modified with permission from [33]).*

to be clarified. In bacteria, but not mammalian cells, the enzymes that catalyze these activities have been identified [6–10]. polyP is synthesized following osmotic, oxidative, UVB, or other cellular stresses, and augments bacterial survival [11–16]; four roles have been proposed: an energy source, chelation of metal ions, storage of phosphate and response to cellular stresses. Necessary for bacterial survival, polyP is implicated in essential biological processes in prokaryotes including stress response, motility, biofilm formation [17–22], virulence, sporulation, and quorumsensing [21–23]. Enzymes involved in polyP synthesis and degradation have been isolated and characterized in bacteria and other lower eukaryotes, and maintain tight control of polyP levels, as might be expected for a polymer controlling vital biological processes.

#### **1.1 PolyP in prokaryotes**

Drawing from the cellular ATP pool, bacterial polyphosphate kinase (PPK1) catalyzes the reversible transfer of the terminal γ-phosphate of ATP to polyP [24], whereas PPK2, expressed in other prokaryotes, transfers the terminal inorganic phosphate (Pi) from polyP to GDP to form GTP [4, 25, 26]. In contrast, the exopolyphosphatase PPX1 hydrolyzes polyP into phosphate monomers, thus maintaining phosphate homeostasis [5]. PPK levels and activity are tightly regulated, maintaining steady-state polyP concentrations in the bacterial cytosol at low micromolar levels, even in mutant strains deficient in PPX [27]. PolyP synthesis is

**73**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

other investigators have used to specifically inhibit the function of polyP.

Not surprisingly, *ppk* mutants are extremely sensitive to environmental stresses [13, 31, 32, 40, 41], and exhibit reduced motility, virulence, and biofilm production [42]. *ppk* gene expression is regulated by σ38, a transcriptional regulator for late stationary phase genes [43] and polyP, in turn, amplifies its own synthesis by inducing transcription of the gene encoding σ38 (RpoS) [29, 41, 42]. In response to oxidative or heat stress, polyP synthesis is regulated at a transcriptional and/or post-translational level, as PPK synthesis and levels are altered by antisense RNA that target *ppk* mRNA transcripts [44] as well as transient inactivation of PPX by stress-sensitive regulators that allow polyP levels to remain high until normal condi-

While polyP is found in eukaryotes from protists to mammalian cells [11], the mechanism of polyP synthesis remains largely unknown for most eukaryotic organisms [1, 45], except for *S. cerevisiae*, where the vacuolar transporter chaperone 4 (VTC4) synthesizes polyP from ATP and then transports the polymer into vacuoles [46, 47]. Vacuolar polyP maintains phosphate homeostasis by appropriating phosphate during growth in phosphate-rich conditions [46], and releases phosphate

There is no sequence or structural homology between the polyP-synthesizing enzymes PPK1, PPK2, or VTC4, and homologues have yet to be found in higher eukaryotes [1]. While phylogenetic analysis of the prokaryotic branch reveals no clear homologues of *E. coli* PPK in a large number of polyP-synthesizing species [49], a few enzymes have been identified that use polyP as phosphate donor in reactions that can be reversed in the presence of excess substrate *in vitro* [49, 50]. In the absence of a polyP-synthesizing enzyme, polyP may be synthesized by the mitochondrial proton-motive force [51], in a complex process involving intact mitochondrial membranes [52]. Decreased polyP production resulting from depolarization of the mitochondrial membrane [52, 53] suggests that this may be a spontaneous process that does not need catalysis. Alternatively, inositol phosphates have also been implicated in polyP metabolism, since polyP levels are diminished in cells lacking the enzyme that synthesizes highly phosphorylated inositols [54–57]. Similar to polyP synthesis, polyP-degrading enzymes, such as yeast PPX1, have been found in lower eukaryotes, while mammalian polyP-specific degradation enzymes are mostly uncharacterized. However, h-prune, which regulates cell migration, also acts as a exopolyphosphatase for short-chain polyP *in vitro* [58, 59]. Subcellular fractionation, immunofluorescent staining, and biochemical quantification reveal that polyP is localized to the nucleus, cell membrane, cytoplasm, and

during the cell cycle to provide precursors for DNA replication [48].

upregulated during nutrient deprivation [28, 29], or during osmotic [28], acidic pH [30], oxidative [31], or heat [32] stresses, potentially depleting cellular ATP pools by converting millimolar levels of ATP to long polyP chains (>1000 Pi) [31]. polyP levels are measured using enzyme-based assays that employ ppk to generate ATP from ADP, using luciferase as a reporter [33]. In another assay, cells are labeled with 32Pi; polyP is isolated and hydrolyzed with PPX1, and thin-layer chromatography or phosphoimage analysis is performed [17]. Toluidine blue binding assays are effective for different chain-lengths but are relatively insensitive. Recently, a rapid and simple method has been described [34, 35].31P-NMR Spectroscopy is an effective and accurate method for measuring polyP in intact cells [36]. Electron ionization mass spectrometry [37], cryoelectron tomography and spectroscopy imaging [38] have also been employed. Protein affinity labeling *in vivo* uses the affinity of a recombinant polyP-binding domain of *E. coli* PPX1 (PPXbd) [39], which we and

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

tions are restored [45].

**1.2 PolyP in eukaryotes**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

upregulated during nutrient deprivation [28, 29], or during osmotic [28], acidic pH [30], oxidative [31], or heat [32] stresses, potentially depleting cellular ATP pools by converting millimolar levels of ATP to long polyP chains (>1000 Pi) [31]. polyP levels are measured using enzyme-based assays that employ ppk to generate ATP from ADP, using luciferase as a reporter [33]. In another assay, cells are labeled with 32Pi; polyP is isolated and hydrolyzed with PPX1, and thin-layer chromatography or phosphoimage analysis is performed [17]. Toluidine blue binding assays are effective for different chain-lengths but are relatively insensitive. Recently, a rapid and simple method has been described [34, 35].31P-NMR Spectroscopy is an effective and accurate method for measuring polyP in intact cells [36]. Electron ionization mass spectrometry [37], cryoelectron tomography and spectroscopy imaging [38] have also been employed. Protein affinity labeling *in vivo* uses the affinity of a recombinant polyP-binding domain of *E. coli* PPX1 (PPXbd) [39], which we and other investigators have used to specifically inhibit the function of polyP.

Not surprisingly, *ppk* mutants are extremely sensitive to environmental stresses [13, 31, 32, 40, 41], and exhibit reduced motility, virulence, and biofilm production [42]. *ppk* gene expression is regulated by σ38, a transcriptional regulator for late stationary phase genes [43] and polyP, in turn, amplifies its own synthesis by inducing transcription of the gene encoding σ38 (RpoS) [29, 41, 42]. In response to oxidative or heat stress, polyP synthesis is regulated at a transcriptional and/or post-translational level, as PPK synthesis and levels are altered by antisense RNA that target *ppk* mRNA transcripts [44] as well as transient inactivation of PPX by stress-sensitive regulators that allow polyP levels to remain high until normal conditions are restored [45].

#### **1.2 PolyP in eukaryotes**

*Contemporary Topics about Phosphorus in Biology and Materials*

to be clarified. In bacteria, but not mammalian cells, the enzymes that catalyze these activities have been identified [6–10]. polyP is synthesized following osmotic, oxidative, UVB, or other cellular stresses, and augments bacterial survival [11–16]; four roles have been proposed: an energy source, chelation of metal ions, storage of phosphate and response to cellular stresses. Necessary for bacterial survival, polyP is implicated in essential biological processes in prokaryotes including stress response, motility, biofilm formation [17–22], virulence, sporulation, and quorumsensing [21–23]. Enzymes involved in polyP synthesis and degradation have been isolated and characterized in bacteria and other lower eukaryotes, and maintain tight control of polyP levels, as might be expected for a polymer controlling vital

*(A) Schematic of linear structure of polyP, and its synthesis by polyphosphate kinase (PPK) and degradation by exopolyphosphatases (PPX1) or endopolyphosphatases (PPN). Constitutive (B and D) or Tet-inducible PPX1 (C and E) expression slows wound healing in cultured human keratinocytes (modified with permission* 

Drawing from the cellular ATP pool, bacterial polyphosphate kinase (PPK1) catalyzes the reversible transfer of the terminal γ-phosphate of ATP to polyP [24], whereas PPK2, expressed in other prokaryotes, transfers the terminal inorganic phosphate (Pi) from polyP to GDP to form GTP [4, 25, 26]. In contrast, the exopolyphosphatase PPX1 hydrolyzes polyP into phosphate monomers, thus maintaining phosphate homeostasis [5]. PPK levels and activity are tightly regulated, maintaining steady-state polyP concentrations in the bacterial cytosol at low micromolar levels, even in mutant strains deficient in PPX [27]. PolyP synthesis is

**72**

biological processes.

**Figure 1.**

*from [33]).*

**1.1 PolyP in prokaryotes**

While polyP is found in eukaryotes from protists to mammalian cells [11], the mechanism of polyP synthesis remains largely unknown for most eukaryotic organisms [1, 45], except for *S. cerevisiae*, where the vacuolar transporter chaperone 4 (VTC4) synthesizes polyP from ATP and then transports the polymer into vacuoles [46, 47]. Vacuolar polyP maintains phosphate homeostasis by appropriating phosphate during growth in phosphate-rich conditions [46], and releases phosphate during the cell cycle to provide precursors for DNA replication [48].

There is no sequence or structural homology between the polyP-synthesizing enzymes PPK1, PPK2, or VTC4, and homologues have yet to be found in higher eukaryotes [1]. While phylogenetic analysis of the prokaryotic branch reveals no clear homologues of *E. coli* PPK in a large number of polyP-synthesizing species [49], a few enzymes have been identified that use polyP as phosphate donor in reactions that can be reversed in the presence of excess substrate *in vitro* [49, 50]. In the absence of a polyP-synthesizing enzyme, polyP may be synthesized by the mitochondrial proton-motive force [51], in a complex process involving intact mitochondrial membranes [52]. Decreased polyP production resulting from depolarization of the mitochondrial membrane [52, 53] suggests that this may be a spontaneous process that does not need catalysis. Alternatively, inositol phosphates have also been implicated in polyP metabolism, since polyP levels are diminished in cells lacking the enzyme that synthesizes highly phosphorylated inositols [54–57]. Similar to polyP synthesis, polyP-degrading enzymes, such as yeast PPX1, have been found in lower eukaryotes, while mammalian polyP-specific degradation enzymes are mostly uncharacterized. However, h-prune, which regulates cell migration, also acts as a exopolyphosphatase for short-chain polyP *in vitro* [58, 59].

Subcellular fractionation, immunofluorescent staining, and biochemical quantification reveal that polyP is localized to the nucleus, cell membrane, cytoplasm, and intracellular organelles in mammalian cells. It is specifically enriched in nucleoli, acidocalcisomes (organelles rich in protons, calcium (Ca2+), and phosphorus), and mitochondria [11, 60–63]. In the brain, astrocytes secrete polyP, which is taken up by neurons, indicating both intra- and extracellular localization [64, 65]. Similar to vesicular packaging of ATP, astrocytes release polyP *via* exocytosis from vesicular nucleotide transporter (VNUT)-containing vesicles [65]. A putative G proteincoupled receptor in *D. discoideum* mediates cell surface binding of extracellular polyP, which as a signaling molecule, elicits differential effects on cell-substratum adhesion and cytoskeletal F-actin levels [66].

Eukaryotic polyP levels are in the 20–100 μM range (expressed as Pi concentration), with chain lengths ranging from 50 to 800 Pi residues (rat tissues) [67], averaging ~80 Pi residues in human platelets [68] to 200 Pi residues in yeast [67], compared to bacterial polyP, which can range up to thousands of Pi units long [53]; however, up to 130 mm medium-sized polyP chains are stored in dense granules in thrombocytes and mast cells [62, 68, 69]. Brain tissue exhibits among the highest polyP levels (~100 µm), which drop with age and neurodegenerative disease [11, 70–72], consistent with the role of polyP in stabilizing protein unfolding intermediates as amyloid-like precursors [32]. High levels of polyphosphate are also found in osteoblast matrix vesicles, the initial sites of bone mineral formation [73]. PolyP concentrations and chain lengths are dynamic, and depend on growth conditions of cells; for example in *Plasmodia*, polyP has an average chain length of 100 Pi, which is degraded to 10 Pi during sporulation [67].

Studies on the myriad roles of polyP in higher eukaryotes have recently gained momentum. PolyP is directly or indirectly involved in diverse cell processes, including control of cell bioenergetics, signal transduction, activation of the mitochondrial permeability transition pore (mPTP), Ca2+-signaling [74, 75], and maintenance of the mitochondrial membrane potential [74]. Associated with mPTP [74] and voltage-gated channels, polyP regulates neuronal excitability [76] and astroglial signaling [64]. About 39% of intracellular polyP pools in astrocytes are in mitochondria [77], playing a role in bioenergetics [52, 77] and Ca2+-handling [74, 78, 79]. As a signaling molecule, polyP released from astrocytes can mediate the physiological response to brain hypoxia [65].

In addition to its role as a gliotransmitter in the autonomic nervous system [64], the polymer also interacts with a variety of proteins, such as mammalian target of rapamycin (mTOR), fibroblast growth factor (FGF)-2, TRPM8, integrin β1, and glycosomal and ribosomal proteins and enzymes, consequently modulating cell survival and cell growth [80–86]. A fascinating finding of several recent studies is that polyP can covalently and non-enzymatically modify a small number of specific proteins in yeast [81] and humans containing lysine residues located in poly-acidic, serine, and lysine-rich (PASK) motifs, some of which are involved in ribosome biogenesis [87].

PolyP is involved in mTOR signaling, cell proliferation, and apoptosis [74, 80], and stimulates the mTOR pathway [80] at concentrations normally found in mammalian cells (0.15–1.5 mM); [11], suggesting a role for the polymer in mammalian cell proliferation. By promoting release of translation initiation factor eIF4E, mTOR stimulates initiation of translation, particularly proteins involved in cell growth and proliferation. PolyP also enhances the mitogenic activity of FGF-2 by promoting its binding to cell surface receptors [83]. PolyP appears to regulate apoptosis by inducing activation of caspase-3 in human plasma cells [88]. This polyanion also chelates metals, such as manganese and cadmium, blocking metalinduced cell damage [89, 90].

Other roles for polyP in mammalian cells include coagulation *via* activation of blood clotting factor XII [69], inflammation [91] as well as Ca2+ chelation for bone

**75**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

mineralization and osteogenic differentiation [92]. The polymer also contributes to pro-inflammatory responses upon release from mast cells [62]. Finally, serving as a stabilizing scaffold for protein-folding intermediates, polyP was recently shown to work as a protein-like chaperone protecting cells against stress-induced protein

**2. The contribution of polyP to cell survival, proliferation, and motility** 

Whereas candidates for mammalian polyP metabolism have been shown to exhibit additional enzymatic activities [59], more specific polyphosphatases have been identified in lower eukaryotes including yeast, trypanosomes, and *Dictyostelium*. We therefore used exopolyphosphatase derived from *S. cerevisiae* (ScPPX1) to target intracellular polyP in human skin keratinocytes, an obvious choice for UV resistance, motility, and wound healing. The functions of polyP in the response of keratinocytes to UVB or wounding was studied by expressing ScPPX1, which selectively breaks down endogenous inorganic polyP, and not phosphoproteins, DNA, RNA, or nucleotide mono-, di-, or triphosphates [6]. Cells depleted of intracellular polyP by ScPPX1 expression exhibited increased sensitivity to UVB *via* enhanced apoptosis, and impaired wound healing [33]. Human keratinocytes stably expressing constitutive HisPPX1 or tetracycline (Tet)-inducible HisPPX1 were used to deplete cells of endogenous polyP, and study its role in wound healing assays performed on confluent monolayers, mimicking cell re-epithelialization during wound healing *in vivo*. RT-PCR and immunoblot analysis confirmed PPX1 expression in stable HisPPX1-expressing cells or in the presence of Tet (**Figure 1B** and **C**). Scratch gaps demonstrate marked attenuation of wound healing following constitu-

Since keratinocyte proliferation and migration are crucial to re-epithelialization

during wound healing, the contribution of polyP to cell growth and motility involved in wound healing was next determined. Vector control cells exhibited significantly higher rates of cell growth as well as BrdU incorporation into newly synthesized DNA in cells at the wound edge, compared with polyP-depleted HisPPX1-expressing cells (**Figure 2**). Further, real time monitoring and measurement of cell motility performed in an xCelligence impedance-based system revealed significant decreases in cell motility in polyP-depleted keratinocytes (**Figure 3**). These results demonstrate that polyP depletion by either constitutive or inducible expression of PPX1 retards the rate of wound healing in human skin keratinocytes, by decreasing cell proliferation and motility. To determine if the loss of endogenous polyP can be supplemented with exogenous extracellular polyP, ScPPX1-expressing cells were grown in the presence of different concentrations of polyP, or with polyPrich platelet lysate (next section). Exogenously added polyP was found to accelerate wound healing in human keratinocytes in polyP dose-response experiments on confluent monolayers of keratinocytes subjected to scratch wound healing assays

Inorganic polyP shows promise in different phases of wound healing, including hemostasis and re-epithelialization, as polyP is a normal component of different cells that play a role in this process, including platelets, dermal fibroblasts, and keratinocytes. The use of polyP as a therapeutic for acute and chronic wounding has begun to garner interest, in part because of experiments elucidating its role in wound healing [33], as well as in hemostasis [69, 94–99]. We have recently shown a role for polyP in the response to UV survival, cell motility, and wound healing [33]. Addition of exogenous polyP increased the rate of wound healing in standard

**involved in wound healing in skin keratinocytes**

tive HisPPX1 or Tet-induced expression (**Figure 1D** and **E**).

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

scratch wound assays *in vitro* [33].

aggregation [93].

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

mineralization and osteogenic differentiation [92]. The polymer also contributes to pro-inflammatory responses upon release from mast cells [62]. Finally, serving as a stabilizing scaffold for protein-folding intermediates, polyP was recently shown to work as a protein-like chaperone protecting cells against stress-induced protein aggregation [93].

#### **2. The contribution of polyP to cell survival, proliferation, and motility involved in wound healing in skin keratinocytes**

Inorganic polyP shows promise in different phases of wound healing, including hemostasis and re-epithelialization, as polyP is a normal component of different cells that play a role in this process, including platelets, dermal fibroblasts, and keratinocytes. The use of polyP as a therapeutic for acute and chronic wounding has begun to garner interest, in part because of experiments elucidating its role in wound healing [33], as well as in hemostasis [69, 94–99]. We have recently shown a role for polyP in the response to UV survival, cell motility, and wound healing [33]. Addition of exogenous polyP increased the rate of wound healing in standard scratch wound assays *in vitro* [33].

Whereas candidates for mammalian polyP metabolism have been shown to exhibit additional enzymatic activities [59], more specific polyphosphatases have been identified in lower eukaryotes including yeast, trypanosomes, and *Dictyostelium*. We therefore used exopolyphosphatase derived from *S. cerevisiae* (ScPPX1) to target intracellular polyP in human skin keratinocytes, an obvious choice for UV resistance, motility, and wound healing. The functions of polyP in the response of keratinocytes to UVB or wounding was studied by expressing ScPPX1, which selectively breaks down endogenous inorganic polyP, and not phosphoproteins, DNA, RNA, or nucleotide mono-, di-, or triphosphates [6]. Cells depleted of intracellular polyP by ScPPX1 expression exhibited increased sensitivity to UVB *via* enhanced apoptosis, and impaired wound healing [33]. Human keratinocytes stably expressing constitutive HisPPX1 or tetracycline (Tet)-inducible HisPPX1 were used to deplete cells of endogenous polyP, and study its role in wound healing assays performed on confluent monolayers, mimicking cell re-epithelialization during wound healing *in vivo*. RT-PCR and immunoblot analysis confirmed PPX1 expression in stable HisPPX1-expressing cells or in the presence of Tet (**Figure 1B** and **C**). Scratch gaps demonstrate marked attenuation of wound healing following constitutive HisPPX1 or Tet-induced expression (**Figure 1D** and **E**).

Since keratinocyte proliferation and migration are crucial to re-epithelialization during wound healing, the contribution of polyP to cell growth and motility involved in wound healing was next determined. Vector control cells exhibited significantly higher rates of cell growth as well as BrdU incorporation into newly synthesized DNA in cells at the wound edge, compared with polyP-depleted HisPPX1-expressing cells (**Figure 2**). Further, real time monitoring and measurement of cell motility performed in an xCelligence impedance-based system revealed significant decreases in cell motility in polyP-depleted keratinocytes (**Figure 3**). These results demonstrate that polyP depletion by either constitutive or inducible expression of PPX1 retards the rate of wound healing in human skin keratinocytes, by decreasing cell proliferation and motility. To determine if the loss of endogenous polyP can be supplemented with exogenous extracellular polyP, ScPPX1-expressing cells were grown in the presence of different concentrations of polyP, or with polyPrich platelet lysate (next section). Exogenously added polyP was found to accelerate wound healing in human keratinocytes in polyP dose-response experiments on confluent monolayers of keratinocytes subjected to scratch wound healing assays

*Contemporary Topics about Phosphorus in Biology and Materials*

adhesion and cytoskeletal F-actin levels [66].

is degraded to 10 Pi during sporulation [67].

physiological response to brain hypoxia [65].

intracellular organelles in mammalian cells. It is specifically enriched in nucleoli, acidocalcisomes (organelles rich in protons, calcium (Ca2+), and phosphorus), and mitochondria [11, 60–63]. In the brain, astrocytes secrete polyP, which is taken up by neurons, indicating both intra- and extracellular localization [64, 65]. Similar to vesicular packaging of ATP, astrocytes release polyP *via* exocytosis from vesicular nucleotide transporter (VNUT)-containing vesicles [65]. A putative G proteincoupled receptor in *D. discoideum* mediates cell surface binding of extracellular polyP, which as a signaling molecule, elicits differential effects on cell-substratum

Eukaryotic polyP levels are in the 20–100 μM range (expressed as Pi concentration), with chain lengths ranging from 50 to 800 Pi residues (rat tissues) [67], averaging ~80 Pi residues in human platelets [68] to 200 Pi residues in yeast [67], compared to bacterial polyP, which can range up to thousands of Pi units long [53]; however, up to 130 mm medium-sized polyP chains are stored in dense granules in thrombocytes and mast cells [62, 68, 69]. Brain tissue exhibits among the highest polyP levels (~100 µm), which drop with age and neurodegenerative disease [11, 70–72], consistent with the role of polyP in stabilizing protein unfolding intermediates as amyloid-like precursors [32]. High levels of polyphosphate are also found in osteoblast matrix vesicles, the initial sites of bone mineral formation [73]. PolyP concentrations and chain lengths are dynamic, and depend on growth conditions of cells; for example in *Plasmodia*, polyP has an average chain length of 100 Pi, which

Studies on the myriad roles of polyP in higher eukaryotes have recently gained

In addition to its role as a gliotransmitter in the autonomic nervous system [64], the polymer also interacts with a variety of proteins, such as mammalian target of rapamycin (mTOR), fibroblast growth factor (FGF)-2, TRPM8, integrin β1, and glycosomal and ribosomal proteins and enzymes, consequently modulating cell survival and cell growth [80–86]. A fascinating finding of several recent studies is that polyP can covalently and non-enzymatically modify a small number of specific proteins in yeast [81] and humans containing lysine residues located in poly-acidic, serine, and lysine-rich (PASK) motifs, some of which are involved in ribosome

PolyP is involved in mTOR signaling, cell proliferation, and apoptosis [74, 80], and stimulates the mTOR pathway [80] at concentrations normally found in mammalian cells (0.15–1.5 mM); [11], suggesting a role for the polymer in mammalian cell proliferation. By promoting release of translation initiation factor eIF4E, mTOR stimulates initiation of translation, particularly proteins involved in cell growth and proliferation. PolyP also enhances the mitogenic activity of FGF-2 by promoting its binding to cell surface receptors [83]. PolyP appears to regulate apoptosis by inducing activation of caspase-3 in human plasma cells [88]. This polyanion also chelates metals, such as manganese and cadmium, blocking metal-

Other roles for polyP in mammalian cells include coagulation *via* activation of blood clotting factor XII [69], inflammation [91] as well as Ca2+ chelation for bone

momentum. PolyP is directly or indirectly involved in diverse cell processes, including control of cell bioenergetics, signal transduction, activation of the mitochondrial permeability transition pore (mPTP), Ca2+-signaling [74, 75], and maintenance of the mitochondrial membrane potential [74]. Associated with mPTP [74] and voltage-gated channels, polyP regulates neuronal excitability [76] and astroglial signaling [64]. About 39% of intracellular polyP pools in astrocytes are in mitochondria [77], playing a role in bioenergetics [52, 77] and Ca2+-handling [74, 78, 79]. As a signaling molecule, polyP released from astrocytes can mediate the

**74**

biogenesis [87].

induced cell damage [89, 90].

#### **Figure 2.**

*Constitutive* PPX1 *expression decreases growth rate of keratinocytes. Viable cell counts were performed over 11 days, and growth curves plotted for HisPPX1-expressing cells compared to vector cells (top). HisPPX1 and vector controls were subjected to scratch assays and proliferation was measured by in situ BrdU incorporation in cells at the wound edge (middle and bottom; modified with permission from [33]).*

#### **Figure 3.**

*Real-time monitoring and measurement of cell motility was performed in an xCelligence impedance-based system. Total number of cells attached to the bottom chamber were measured every 15 min over 24 hours, and are shown as technical duplicates, with assays repeated twice (left). Percentage of cell migration at the 24-hour time-point based on 100,000 cells plated at the top chamber (right; modified with permission from [33]).*

**77**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

(**Figure 4**). Interestingly, both intracellular and extracellular polyP dose-depend-

*PolyP dose-dependently accelerates wound healing in cultured human keratinocyte, using polyP at 1 μM (left)* 

**3. The use of polyP-containing PRP to promote wound healing in acute** 

Wound healing is a highly coordinated process involving biochemical and physiological interplay of keratinocytes and fibroblasts to restore skin integrity. Platelets are components of blood responsible for blood clotting and wound healing. Although the importance of platelets in wound healing has been extensively studied, the bioactive substance playing a major role in skin re-epithelialization during wound healing is still unclear. PRP has been shown to support the survival and proliferation of human keratinocytes [100], and is currently used as therapeutic for both acute and chronic wounds (for review see [101]). Discovery of the release of growth factors triggered an interest in using PRP for wound healing, and platelet lysates have been examined as a replacement for using fetal bovine serum in cell cultures, which may contain contaminants such as prions, or elicit an unwanted immune response in patients. Platelet-rich lysates derived from platelets are a by-product of blood preparation, and are thus inexpensive. Most studies focused on lysates for mesenchymal stromal cell culture for cell therapy, in which platelets are

In recent years, PRP has gained traction in many different specialties including in dermatology where it is used to treat acne [102], scarring [103], and alopecia [104, 105], in regenerative medicine where it is used to treat acute and chronic injuries to bone and cartilage [106, 107], in orthopedics and sports medicine where it is used to treat rotator cuff tears, osteoarthritis of the knee, hamstring injuries, and Achilles tendinopathy [108–110], in dentistry where it is used during tooth extractions, periodontal surgery, and dental implant surgery [111, 112], and more recently in wound healing to promote enhanced healing [113–118]. PRP is an autologous blood product generated from multiple rounds of centrifugation that serve to concentrate the number of platelets in plasma. PRP contains high concentrations of growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor beta (TGFβ) compared to plasma and whole blood [119, 120]. It also contains higher levels of pro- and anti-inflammatory cytokines that promote enhanced healing. Lastly, PRP is known to contain inorganic polyP which is continually synthesized from ATP or

The role of polyP secreted by platelets and present in PRP on cell proliferation and wound healing was investigated in human HaCaT keratinocytes co-transduced

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

ently increased the rate of wound healing *in vitro*.

*or 10 μM (right; modified with permission from [33]).*

**Figure 4.**

**and chronic wounds, including burns**

activated by thrombin and CaCl2, or by freeze-thaw.

GTP, and is degraded by cellular enzymes in bacteria and eukaryotes.

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

**Figure 4.** *PolyP dose-dependently accelerates wound healing in cultured human keratinocyte, using polyP at 1 μM (left) or 10 μM (right; modified with permission from [33]).*

(**Figure 4**). Interestingly, both intracellular and extracellular polyP dose-dependently increased the rate of wound healing *in vitro*.

#### **3. The use of polyP-containing PRP to promote wound healing in acute and chronic wounds, including burns**

Wound healing is a highly coordinated process involving biochemical and physiological interplay of keratinocytes and fibroblasts to restore skin integrity. Platelets are components of blood responsible for blood clotting and wound healing. Although the importance of platelets in wound healing has been extensively studied, the bioactive substance playing a major role in skin re-epithelialization during wound healing is still unclear. PRP has been shown to support the survival and proliferation of human keratinocytes [100], and is currently used as therapeutic for both acute and chronic wounds (for review see [101]). Discovery of the release of growth factors triggered an interest in using PRP for wound healing, and platelet lysates have been examined as a replacement for using fetal bovine serum in cell cultures, which may contain contaminants such as prions, or elicit an unwanted immune response in patients. Platelet-rich lysates derived from platelets are a by-product of blood preparation, and are thus inexpensive. Most studies focused on lysates for mesenchymal stromal cell culture for cell therapy, in which platelets are activated by thrombin and CaCl2, or by freeze-thaw.

In recent years, PRP has gained traction in many different specialties including in dermatology where it is used to treat acne [102], scarring [103], and alopecia [104, 105], in regenerative medicine where it is used to treat acute and chronic injuries to bone and cartilage [106, 107], in orthopedics and sports medicine where it is used to treat rotator cuff tears, osteoarthritis of the knee, hamstring injuries, and Achilles tendinopathy [108–110], in dentistry where it is used during tooth extractions, periodontal surgery, and dental implant surgery [111, 112], and more recently in wound healing to promote enhanced healing [113–118]. PRP is an autologous blood product generated from multiple rounds of centrifugation that serve to concentrate the number of platelets in plasma. PRP contains high concentrations of growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor beta (TGFβ) compared to plasma and whole blood [119, 120]. It also contains higher levels of pro- and anti-inflammatory cytokines that promote enhanced healing. Lastly, PRP is known to contain inorganic polyP which is continually synthesized from ATP or GTP, and is degraded by cellular enzymes in bacteria and eukaryotes.

The role of polyP secreted by platelets and present in PRP on cell proliferation and wound healing was investigated in human HaCaT keratinocytes co-transduced

*Contemporary Topics about Phosphorus in Biology and Materials*

**76**

**Figure 3.**

**Figure 2.**

*Real-time monitoring and measurement of cell motility was performed in an xCelligence impedance-based system. Total number of cells attached to the bottom chamber were measured every 15 min over 24 hours, and are shown as technical duplicates, with assays repeated twice (left). Percentage of cell migration at the 24-hour time-point based on 100,000 cells plated at the top chamber (right; modified with permission from [33]).*

*Constitutive* PPX1 *expression decreases growth rate of keratinocytes. Viable cell counts were performed over 11 days, and growth curves plotted for HisPPX1-expressing cells compared to vector cells (top). HisPPX1 and vector controls were subjected to scratch assays and proliferation was measured by in situ BrdU incorporation in* 

*cells at the wound edge (middle and bottom; modified with permission from [33]).*

with either ScPPX1 or vector control, along with DsRed or GFP, respectively, as fluorescent markers in order to visualize and track cells that have reduced or normal levels of polyP. Cells stably expressing fluorescent-tagged DsRed-PPX1 or GFPempty vector were incubated with platelet lysate (4%) supplemented with or without exogenous pure polyP (1 μM). In both vector-GFP control and polyP-depleted PPX1-DsRed cells treated with polyP, platelet lysate, or platelet lysate + polyP, cell growth curves revealed a significant increase in cell proliferation compared to untreated controls (data to be published elsewhere). PolyP quantification in platelet lysates using a micromolar polyP assay kit showed that a 4% platelet lysate contains ~8 μM polyP, which was within the range used for exogenously added polyP. This assay measures increase in fluorescence intensity (emission 550 nm, excitation 415 nm) of a PPD dye upon binding to polyP.

Cell migration/scratch assays were performed on PPX1-DsRed or vector-GFP control keratinocytes to assess the effects on wound healing and cell motility. Fluorescent pictures were taken at 10 min intervals for 36 hours using an EVOS FL time-lapse imaging system, and gap closure was quantified by Image J. In both GFP-vector cells and PPX1-expressing cells, the rate of wound closure in the scratch assays were significantly increased when cells were incubated either with platelet lysate alone, polyP alone, or both (data to be published elsewhere). These results together indicate that exogenous polyP, delivered either purified or from plateletenriched plasma, can accelerate wound healing.

To assess whether the increased rate of wound healing is attributable to polyP in platelet lysates, specific polyP inhibitors (polyP-binding protein PPXbd or UHRA-9, a kind gift from Dr. James Morrissey) were utilized in wound healing assays. PPXbd, a recombinant polyP-binding domain of *E. coli* exopolyphosphatase, binds to platelet-derived polyP and blocks FXI activation, thrombin and fibrin generation, and consequently, inhibiting polyP procoagulant activity [99]. Interestingly, the enhanced rates of wound healing in vector control or polyP-depleted ScPPXexpressing cells induced by supplementation with exogenous extracellular polyP from pure polyP or in platelet lysates, was completely reversed by addition of the polyP inhibitors PPXbd or UHRA-9 (data to be published elsewhere). PolyP secreted by platelets and present in platelet lysate or PRP may therefore play an essential role in re-epithelialization during wound healing.

#### **3.1 Chronic wounds**

The acceleration of wound healing is of paramount importance in the setting of acute and chronic wounds, as well as burn wounds. Open chronic wounds are a significant cause of additional morbidity in patient populations that already have a plethora of comorbidities [121]. Significant improvements in complete healing were reported in chronic wounds treated with PRP compared to no topical treatments in a 2011 systematic review and meta-analysis on the use of PRP in acute and chronic wounds [101]. Another review of PubMed and Cochrane databases found significant benefit of PRP for diabetic chronic wounds, specifically in wounds unresponsive to standard of care treatment options [113]. A third systematic review of nine randomized controlled clinical trials (RCT) suggested that well-designed high-powered RCTs are needed to demonstrate increased wound healing with PRP treatment [122].

Treatment of 56 patients with diabetic foot ulcers with twice weekly applications of PRP resulted in complete healing in 86% of patients in the treated groups vs. only 68% in the control group [123]. Animal models using exosomes derived from PRP for full thickness skin wounds in a diabetic rat model also showed increased healing, as well as increased fibroblast proliferation and migration [124]. Platelet-rich fibrin also improved diabetic animal skin wound healing [125]. Overall, while there is no

**79**

showed improved outcomes [135].

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

consensus on this treatment modality in chronic wounds, it is becoming widely used, and many trials seek to understand its potential beneficial effects. Improvements in open wound area have been shown in a number of animal and clinical studies.

Meta-analysis of rodent and non-rodent studies using a systematic review conducted under preferred reported items for systematic review of interventions (PRISMA) guidelines indicated that the treatment of wounds with PRP resulted in reduction of open wound area [126]. In addition to its role in wound healing, PRP reduced complications such as wound infection, exudate (mass of cells and fluid that seeps out of a wound), drainage, and hematoma formation [101]. PRP and PRP with keratinocyte and fibroblast cells were shown to increase re-epithelialization at 7–14 days post-injury in mouse models, compared to non-treated controls [127]. In full thickness porcine wounds treated with the secreted proteins of PRP, wound re-epithelialization and collagen deposition were significantly increased in treated animals vs. saline controls [128]. Thus, PRP may improve wound healing in acute surgical wounds by secreting growth factors that support local microenvironments that promotes healing [129]. PRP's effectiveness has also been shown in bone grafting, cartilage regeneration, and non-cutaneous surgical procedures. The impact of PRP on normal and damaged (derived from chronic ulcers or irradiated) fibroblasts have been described [130]. In addition, despite the lack of reproducibility of platelet concentrations due to differences in manufacturer-specific protocols for PRP preparation and differences in treatment methodologies, PRP has been shown to affect fibroblast proliferation and migration in a number of *in vitro* studies. As with chronic wounds, it is unclear why some studies, but not others, show a beneficial effect of PRP treatment.

PRP has been used as a topical treatment to accelerate wound healing in burn wounds, however, like in chronic and acute wounds, its use is still debated due to conflicting results [114]. Some papers recommend its use [115, 117, 118, 131–135], while others have shown non-significant changes in outcomes after treatment with PRP, and advise caution in using it in a wide-spread manner [114, 136–138]. A review of PRP for burns concluded that PRP may be useful in regeneration of dermal structures, increasing graft-take, and increasing re-epithelialization, but recommended further research on characterization of the mechanisms by which PRP can improve burn wound healing, donor site healing, and scar outcomes [139]. The use of side-by-side treatment of a split thickness skin graft (STSG) donor site with standard treatment or with PRP showed complete re-epithelialization in the PRP-treated side at day 11 *vs.* day 13 for the control. Histological samples taken from these healing wounds, and by H and E staining revealed increased epidermal thickness in PRP-treated wounds, as well as a significant increase in the number of blood vessels. After platelet concentrate in conjunction with STSG was used for deep burns, monitoring of viscoelastic properties of the resultant scars over 12 months revealed that the skin's return to normal viscoelastic properties was accelerated in burns treated with PRP compared to controls [133]. Compared to historic institutional standard of care controls, treatment of deep partial thickness (DPT) burns using PRP applied with the autograft during skin grafting, pain scores, inflammation, pruritis (itchiness), cosmesis of the scar, and perfusion all

Animal models of burn injury in rats treated with topical PRP or control showed that PRP treatment resulted in increased hydroxyproline, decreased inflammatory

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

**3.2 Acute wounds**

**3.3 Burns**

consensus on this treatment modality in chronic wounds, it is becoming widely used, and many trials seek to understand its potential beneficial effects. Improvements in open wound area have been shown in a number of animal and clinical studies.

#### **3.2 Acute wounds**

*Contemporary Topics about Phosphorus in Biology and Materials*

415 nm) of a PPD dye upon binding to polyP.

enriched plasma, can accelerate wound healing.

essential role in re-epithelialization during wound healing.

**3.1 Chronic wounds**

with either ScPPX1 or vector control, along with DsRed or GFP, respectively, as fluorescent markers in order to visualize and track cells that have reduced or normal levels of polyP. Cells stably expressing fluorescent-tagged DsRed-PPX1 or GFPempty vector were incubated with platelet lysate (4%) supplemented with or without exogenous pure polyP (1 μM). In both vector-GFP control and polyP-depleted PPX1-DsRed cells treated with polyP, platelet lysate, or platelet lysate + polyP, cell growth curves revealed a significant increase in cell proliferation compared to untreated controls (data to be published elsewhere). PolyP quantification in platelet lysates using a micromolar polyP assay kit showed that a 4% platelet lysate contains ~8 μM polyP, which was within the range used for exogenously added polyP. This assay measures increase in fluorescence intensity (emission 550 nm, excitation

Cell migration/scratch assays were performed on PPX1-DsRed or vector-GFP control keratinocytes to assess the effects on wound healing and cell motility. Fluorescent pictures were taken at 10 min intervals for 36 hours using an EVOS FL time-lapse imaging system, and gap closure was quantified by Image J. In both GFP-vector cells and PPX1-expressing cells, the rate of wound closure in the scratch assays were significantly increased when cells were incubated either with platelet lysate alone, polyP alone, or both (data to be published elsewhere). These results together indicate that exogenous polyP, delivered either purified or from platelet-

To assess whether the increased rate of wound healing is attributable to polyP in platelet lysates, specific polyP inhibitors (polyP-binding protein PPXbd or UHRA-9, a kind gift from Dr. James Morrissey) were utilized in wound healing assays. PPXbd, a recombinant polyP-binding domain of *E. coli* exopolyphosphatase, binds to platelet-derived polyP and blocks FXI activation, thrombin and fibrin generation, and consequently, inhibiting polyP procoagulant activity [99]. Interestingly, the enhanced rates of wound healing in vector control or polyP-depleted ScPPXexpressing cells induced by supplementation with exogenous extracellular polyP from pure polyP or in platelet lysates, was completely reversed by addition of the polyP inhibitors PPXbd or UHRA-9 (data to be published elsewhere). PolyP secreted by platelets and present in platelet lysate or PRP may therefore play an

The acceleration of wound healing is of paramount importance in the setting of acute and chronic wounds, as well as burn wounds. Open chronic wounds are a significant cause of additional morbidity in patient populations that already have a plethora of comorbidities [121]. Significant improvements in complete healing were reported in chronic wounds treated with PRP compared to no topical treatments in a 2011 systematic review and meta-analysis on the use of PRP in acute and chronic wounds [101]. Another review of PubMed and Cochrane databases found significant benefit of PRP for diabetic chronic wounds, specifically in wounds unresponsive to standard of care treatment options [113]. A third systematic review of nine randomized controlled clinical trials (RCT) suggested that well-designed high-powered RCTs are needed to demonstrate increased wound healing with PRP treatment [122]. Treatment of 56 patients with diabetic foot ulcers with twice weekly applications of PRP resulted in complete healing in 86% of patients in the treated groups vs. only 68% in the control group [123]. Animal models using exosomes derived from PRP for full thickness skin wounds in a diabetic rat model also showed increased healing, as well as increased fibroblast proliferation and migration [124]. Platelet-rich fibrin also improved diabetic animal skin wound healing [125]. Overall, while there is no

**78**

Meta-analysis of rodent and non-rodent studies using a systematic review conducted under preferred reported items for systematic review of interventions (PRISMA) guidelines indicated that the treatment of wounds with PRP resulted in reduction of open wound area [126]. In addition to its role in wound healing, PRP reduced complications such as wound infection, exudate (mass of cells and fluid that seeps out of a wound), drainage, and hematoma formation [101]. PRP and PRP with keratinocyte and fibroblast cells were shown to increase re-epithelialization at 7–14 days post-injury in mouse models, compared to non-treated controls [127]. In full thickness porcine wounds treated with the secreted proteins of PRP, wound re-epithelialization and collagen deposition were significantly increased in treated animals vs. saline controls [128]. Thus, PRP may improve wound healing in acute surgical wounds by secreting growth factors that support local microenvironments that promotes healing [129]. PRP's effectiveness has also been shown in bone grafting, cartilage regeneration, and non-cutaneous surgical procedures. The impact of PRP on normal and damaged (derived from chronic ulcers or irradiated) fibroblasts have been described [130]. In addition, despite the lack of reproducibility of platelet concentrations due to differences in manufacturer-specific protocols for PRP preparation and differences in treatment methodologies, PRP has been shown to affect fibroblast proliferation and migration in a number of *in vitro* studies. As with chronic wounds, it is unclear why some studies, but not others, show a beneficial effect of PRP treatment.

#### **3.3 Burns**

PRP has been used as a topical treatment to accelerate wound healing in burn wounds, however, like in chronic and acute wounds, its use is still debated due to conflicting results [114]. Some papers recommend its use [115, 117, 118, 131–135], while others have shown non-significant changes in outcomes after treatment with PRP, and advise caution in using it in a wide-spread manner [114, 136–138]. A review of PRP for burns concluded that PRP may be useful in regeneration of dermal structures, increasing graft-take, and increasing re-epithelialization, but recommended further research on characterization of the mechanisms by which PRP can improve burn wound healing, donor site healing, and scar outcomes [139].

The use of side-by-side treatment of a split thickness skin graft (STSG) donor site with standard treatment or with PRP showed complete re-epithelialization in the PRP-treated side at day 11 *vs.* day 13 for the control. Histological samples taken from these healing wounds, and by H and E staining revealed increased epidermal thickness in PRP-treated wounds, as well as a significant increase in the number of blood vessels. After platelet concentrate in conjunction with STSG was used for deep burns, monitoring of viscoelastic properties of the resultant scars over 12 months revealed that the skin's return to normal viscoelastic properties was accelerated in burns treated with PRP compared to controls [133]. Compared to historic institutional standard of care controls, treatment of deep partial thickness (DPT) burns using PRP applied with the autograft during skin grafting, pain scores, inflammation, pruritis (itchiness), cosmesis of the scar, and perfusion all showed improved outcomes [135].

Animal models of burn injury in rats treated with topical PRP or control showed that PRP treatment resulted in increased hydroxyproline, decreased inflammatory

cells infiltration, but no difference in fibroblast collagen production or angiogenesis [134]. In a rat animal model of DPT burns, PRP was effective in increasing % wound closure, but showed little effectiveness in the full thickness injury group. PRP treatment resulted in increased neo-epidermal thickness at day 21, as well as decreases in CD31, 68, and 163, TGFβ1, MMP2, and MPO+ cells indicating an increased resolution of inflammation [118]. PRP injection in burn wound scars in a rat animal model of burn injury also showed pain-associated markers to be decreased with treatment [132]. While positive healing was observed in most models, in a recent study using a swine model of burn, tangential excision, and grafting with or without PRP, PRP showed similar effect on re-epithelialization and scarring in full thickness wounds compared to control wounds [136].

In a randomized clinical trial from 2018, 27 patients with DPT burns that did not get autografted were treated with lyophilized PRP powder, and showed a significant increased percent wound closure at 3 weeks in the treated group compared to control. Additionally, the infection rate in the PRP group was 26%, while 33% of control patients had postoperative infections [115]. PRP's concentrated secretion of growth factors may include basic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor (PDGF), insulin-like growth factor, transforming growth factor β (TGFβ1), and vascular endothelial growth factor (VEGF) as probable mechanisms by which it can accelerate healing [139]. However, quantification of growth factors TGFβ1, PDGF-AA, and VEGF in a cohort of five burn patients compared to five healthy volunteers showed comparable levels of growth factors in the PRP from burn patients and health volunteers. Thus, there may be an additional factor in PRP that is possibly altered in burn patients (or patients with other pathologies such as diabetes or other conditions that would lead them to have surgical procedures yielding acute wounds) that may contribute to its success in treating some wounds and failure in others. Due to the effect burns have on the pathophysiology of blood coagulopathy [140–142] and capillary endotheliopathy [143, 144] after injury, it is reasonable to assume that platelets from burn patients may have differing levels of polyP. Associating this data with what is known about polyP, its ubiquitous presence in all prokaryotic and eukaryotic organisms, and its role in the response to cellular stress, it was hypothesized that polyP may contribute to wound healing.

#### **4. PolyP and polyP-containing platelet rich plasma accelerates re-epithelialization** *in vitro* **and** *in vivo*

A HaCaT keratinocyte polyP-depleted cell line and vector control was used in growth curves and scratch assays to evaluate polyP, platelet lysate, or combined treatment to accelerate wound healing *in vitro*. PolyP-containing PRP was also evaluated as a treatment in a splinted model of excisional wounding *in vivo*. Exogenous polyP was also spiked into PRP to assess its role. Treatment with the polyP-containing treatments increased cell growth and attenuated open wound area *in vitro* (p < 0.001). Addition of a polyP inhibitor abrogated these effects (p < 0.0001). PRP-treated wounds re-epithelialized faster compared to untreated wounds when analyzed at Days 3 and 5 (n = 6 wounds, p < 0.05). Re-epithelialization was further enhanced by exogenous polyP addition to PRP as evidenced by elongated epithelial tongues (**Figure 5**) in the low and high dose PRP + polyP treatment groups compared to PRP alone (n = 8 wounds, p < 0.05; data to be published elsewhere). Due to its autologous nature, PRP serves as a safe and efficacious option for accelerating wound healing, and may be enhanced by the exogenous addition of polyP.

**81**

**Figure 5.**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

**4.1 PRP-treated wounds heal faster than controls** *in vivo*

*was measured with Image J and quantified.*

were taken at days 3, 4, 5, 6, or 7. By day 7, wounds were mostly closed.

PRP was generated based on a previously published protocol [145]. Briefly, whole blood was collected, and centrifuged to create platelet-poor plasma (PPP) and a pellet of platelets. PPP was then removed and the platelets were resuspended and activated with thrombin and calcium chloride (CaCl2) to form a "biobandage"-like gel. Whole blood, packed RBCS, and PRP were stained with Wright and Giemsa stains to confirm PRP platelet concentration. A murine model of full thickness excisional wound healing was used where 6 mm punch biopsies were created on dorsal flanks of C57BL/6 mice [146, 147]. Wounds were splinted to encourage healing by re-epithelialization as opposed to contracture. They were subsequently treated with PRP, or no treatment was applied. Tegaderm dressing was applied and kept in place for 3 days. Pictures

*Untreated or treated wounds were excised on day 5 and fixed in formalin, paraffin embedded, sectioned, and stained with H&E. Sections were imaged, and composited to create an image with areas of normal skin on both sides with the epithelial tongue (orange arrow) protruding from each side of normal skin. The epithelial gap is demarcated by the black arrow where no epithelium is present. Scale bar = 200 μm. Representative histology of untreated (A), PRP only treated (B), PRP + 10 μM polyP-treated wounds are shown. Epithelial tongue length* 

In a second experiment, wounded mice were divided into four treatment groups: untreated, PRP only, PRP + 10 μM polyP (low dose) and PRP + 100 μM polyP (high dose). Doses of polyP were calculated by examining historical data from the literature on platelet levels, as well as polyP concentrations in platelets. The mean platelet count for C57BL/6 mice is 9.85 ± 1.40 × 1011 platelets/L, however, because there are no reliable reports of polyP concentrations per platelet in mice, human platelet counts and polyP concentrations were extrapolated for this experiment [148]. Human platelet concentrations range from 1.5 to 4.5 × 1011 platelets/L [149]. It is also known that platelets contain 0.74 ± 0.08 μmol polyP/1 × 1011 platelets [150]. Therefore, whole blood should contain between 1 and 3 μM polyP, and PRP, which contains at least 3-fold higher levels of platelets compared to whole blood, should contain 3–9 μM of polyP. We added ~10 μM in the low dose group, and a 10× concentration compared to the low dose group for the high dose group (100 μM). Lyophilized polyP was reconstituted in dH2O to 1 M. PolyP took up 10% of the total treatment volume of PRP. Treatments were applied on Day 0, and *Tegaderm*™ dressings stayed in place through day 3. On day 3 and 4, dressings were removed, and pictures were taken. On day 5, dressings were removed, pictures were taken, and wounds were excised

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

#### **Figure 5.**

*Contemporary Topics about Phosphorus in Biology and Materials*

in full thickness wounds compared to control wounds [136].

**4. PolyP and polyP-containing platelet rich plasma accelerates** 

A HaCaT keratinocyte polyP-depleted cell line and vector control was used in growth curves and scratch assays to evaluate polyP, platelet lysate, or combined treatment to accelerate wound healing *in vitro*. PolyP-containing PRP was also evaluated as a treatment in a splinted model of excisional wounding *in vivo*. Exogenous polyP was also spiked into PRP to assess its role. Treatment with the polyP-containing treatments increased cell growth and attenuated open wound area *in vitro* (p < 0.001). Addition of a polyP inhibitor abrogated these effects (p < 0.0001). PRP-treated wounds re-epithelialized faster compared to untreated wounds when analyzed at Days 3 and 5 (n = 6 wounds, p < 0.05). Re-epithelialization was further enhanced by exogenous polyP addition to PRP as evidenced by elongated epithelial tongues (**Figure 5**) in the low and high dose PRP + polyP treatment groups compared to PRP alone (n = 8 wounds, p < 0.05; data to be published elsewhere). Due to its autologous nature, PRP serves as a safe and efficacious option for accelerating wound healing, and may be enhanced by

**re-epithelialization** *in vitro* **and** *in vivo*

cells infiltration, but no difference in fibroblast collagen production or angiogenesis [134]. In a rat animal model of DPT burns, PRP was effective in increasing % wound closure, but showed little effectiveness in the full thickness injury group. PRP treatment resulted in increased neo-epidermal thickness at day 21, as well as decreases in CD31, 68, and 163, TGFβ1, MMP2, and MPO+ cells indicating an increased resolution of inflammation [118]. PRP injection in burn wound scars in a rat animal model of burn injury also showed pain-associated markers to be decreased with treatment [132]. While positive healing was observed in most models, in a recent study using a swine model of burn, tangential excision, and grafting with or without PRP, PRP showed similar effect on re-epithelialization and scarring

In a randomized clinical trial from 2018, 27 patients with DPT burns that did not get autografted were treated with lyophilized PRP powder, and showed a significant increased percent wound closure at 3 weeks in the treated group compared to control. Additionally, the infection rate in the PRP group was 26%, while 33% of control patients had postoperative infections [115]. PRP's concentrated secretion of growth factors may include basic fibroblast growth factor, epidermal growth factor, platelet-derived growth factor (PDGF), insulin-like growth factor, transforming growth factor β (TGFβ1), and vascular endothelial growth factor (VEGF) as probable mechanisms by which it can accelerate healing [139]. However, quantification of growth factors TGFβ1, PDGF-AA, and VEGF in a cohort of five burn patients compared to five healthy volunteers showed comparable levels of growth factors in the PRP from burn patients and health volunteers. Thus, there may be an additional factor in PRP that is possibly altered in burn patients (or patients with other pathologies such as diabetes or other conditions that would lead them to have surgical procedures yielding acute wounds) that may contribute to its success in treating some wounds and failure in others. Due to the effect burns have on the pathophysiology of blood coagulopathy [140–142] and capillary endotheliopathy [143, 144] after injury, it is reasonable to assume that platelets from burn patients may have differing levels of polyP. Associating this data with what is known about polyP, its ubiquitous presence in all prokaryotic and eukaryotic organisms, and its role in the response to cellular stress, it was hypothesized that polyP may contribute

**80**

the exogenous addition of polyP.

to wound healing.

*Untreated or treated wounds were excised on day 5 and fixed in formalin, paraffin embedded, sectioned, and stained with H&E. Sections were imaged, and composited to create an image with areas of normal skin on both sides with the epithelial tongue (orange arrow) protruding from each side of normal skin. The epithelial gap is demarcated by the black arrow where no epithelium is present. Scale bar = 200 μm. Representative histology of untreated (A), PRP only treated (B), PRP + 10 μM polyP-treated wounds are shown. Epithelial tongue length was measured with Image J and quantified.*

#### **4.1 PRP-treated wounds heal faster than controls** *in vivo*

PRP was generated based on a previously published protocol [145]. Briefly, whole blood was collected, and centrifuged to create platelet-poor plasma (PPP) and a pellet of platelets. PPP was then removed and the platelets were resuspended and activated with thrombin and calcium chloride (CaCl2) to form a "biobandage"-like gel. Whole blood, packed RBCS, and PRP were stained with Wright and Giemsa stains to confirm PRP platelet concentration. A murine model of full thickness excisional wound healing was used where 6 mm punch biopsies were created on dorsal flanks of C57BL/6 mice [146, 147]. Wounds were splinted to encourage healing by re-epithelialization as opposed to contracture. They were subsequently treated with PRP, or no treatment was applied. Tegaderm dressing was applied and kept in place for 3 days. Pictures were taken at days 3, 4, 5, 6, or 7. By day 7, wounds were mostly closed.

In a second experiment, wounded mice were divided into four treatment groups: untreated, PRP only, PRP + 10 μM polyP (low dose) and PRP + 100 μM polyP (high dose). Doses of polyP were calculated by examining historical data from the literature on platelet levels, as well as polyP concentrations in platelets. The mean platelet count for C57BL/6 mice is 9.85 ± 1.40 × 1011 platelets/L, however, because there are no reliable reports of polyP concentrations per platelet in mice, human platelet counts and polyP concentrations were extrapolated for this experiment [148]. Human platelet concentrations range from 1.5 to 4.5 × 1011 platelets/L [149]. It is also known that platelets contain 0.74 ± 0.08 μmol polyP/1 × 1011 platelets [150]. Therefore, whole blood should contain between 1 and 3 μM polyP, and PRP, which contains at least 3-fold higher levels of platelets compared to whole blood, should contain 3–9 μM of polyP. We added ~10 μM in the low dose group, and a 10× concentration compared to the low dose group for the high dose group (100 μM). Lyophilized polyP was reconstituted in dH2O to 1 M. PolyP took up 10% of the total treatment volume of PRP.

Treatments were applied on Day 0, and *Tegaderm*™ dressings stayed in place through day 3. On day 3 and 4, dressings were removed, and pictures were taken. On day 5, dressings were removed, pictures were taken, and wounds were excised with underlying fascia and were sewn into histological cassettes to retain wound orientation. These samples were then paraffin embedded and H&E stained. Sections were imaged, and epithelial tongue length was measured using Image J. Epithelial tongues were defined as new epithelium if there was no uninjured dermis underneath the epithelium.

Compared to whole blood, PRP contained a higher concentration of platelets (data to be published elsewhere). The splinted wound model was used to shift the healing towards re-epithelialization instead of the normal contraction observed in mice. PRP application was easily applied as a "bio-bandage" gel-like liquid (data to be published elsewhere). The 6 mm punch biopsies allowed for the creation of similar wound size between animal groups at day 0 (0.33 cm2 ± 0.13 vs. 0.39 ± 0.15 cm2 , p = n.s.; data to be published elsewhere). At days 3 (0.09 ± 0.06 vs. 0.23 ± 0.13) and 5 (0.12 ± 0.07 vs. 0.25 ± 0.12) PRP-treated wounds had significantly smaller open wound areas compared to control animals (n = 6, p < 0.05). At days 6 and 7, this difference leveled off.

#### **4.2 Exogenous spiking of PRP with polyP further accelerates healing** *in vivo*

To further investigate the potential role of polyP in PRP, wounds were treated with PRP or with PRP with low or high dose-spiked polyP. Untreated wounds were largely open by day 5, while PRP treated wounds were smaller and contained newly formed epithelium (data to be published elsewhere). Spiking with low or high dose polyP further stimulated epithelialization, and wounds were smaller with increasing doses. By histomorphometric analysis, epithelial tongues can be seen by H&E staining. In untreated wounds, these tongues are small and shallow. PRP treatment results in a more proliferative epithelium that is thicker and longer than untreated samples. Spiking with low and high dose polyP creates longer epithelial tongues). Epithelial tongue measurement by Image J shows a significant decrease in tongue length in untreated and PRP only treated vs. PRP + high dose polyP (737.38 ± 121.21 and 925.55 ± 214.17 vs. 1186.91 ± 255.06 μM, n = 8, p < 0.0001, p < 0.05). PRP + high dose polyP-treated wounds also had significantly longer epithelial tongues compared to PRP + low dose polyP (n = 8, p < 0.05). PRP contains polyP at a concentration near 5 μM. The exogenous addition of polyP to the PRP promoted keratinocyte growth and proliferation, as is evidence by the increased epithelial tongue length with increasing doses of polyP administration.

#### **5. Conclusion**

PolyP plays key roles in essential biological processes in bacteria, and its increasing importance in eukaryotes is becoming apparent, including its participation in blood coagulation and wound healing. Recent advances in measurement and localization of polyP, along with our growing understanding of polyP metabolism and its interaction with specific proteins allows us to begin to analyze mechanisms responsible for cell-specific roles of polyP. Our ability to regulate polyP in eukaryotic cells opens possibilities for therapeutic intervention. Future work related to wound healing should be aimed at investigating the specific roles of intra- and extracellular polyP in keratinocytes, as well as the potential importance of polyP in other skin cells, including dermal fibroblasts, as these cells make up the majority of the skin. As polyP is also secreted by activated platelets and is important for normal blood clotting, the application of polyP-containing PRP as a biologic dressing may positively contribute to wound healing. We have completed two clinical trials using platelet-rich plasma for wound healing, a phenomenon that may be explained by

**83**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

the presence of polyP. PolyP levels and chain lengths should also be quantified in healthy and pathologic conditions in order to assess appropriate levels when treat-

Authors are grateful for the technical assistance of Sixian Song in the scratch gap assays, and Dr. James Morrissey (University of Michigan Medical School) for the generous gift of polyP, and its inhibitors PPXbd and UHRA-9, used in their studies. This work was partially supported by the NIH STTR grant 1R41ES026908 (to DSR), and the office of the Dean of Research, Georgetown University School of Medicine.

, Bonnie C. Carney1,2, Anirudh Gaur1

, Lauren T. Moffatt1,2, Jeffrey W. Shupp1,2,3,4

,

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

ing acute or chronic wounds in the future.

The authors have no conflicts of interest to declare.

**Acknowledgements**

**Conflict of interest**

**Author details**

Manish Moghe1

and Dean S. Rosenthal1

Washington, DC, USA

Washington, DC, USA

Cynthia M. Simbulan-Rosenthal1

Institute, Washington, DC, USA

, Elliott Crooke1

\*

1 Department of Biochemistry and Molecular and Cellular Biology, Georgetown University School of Medicine, Washington, DC, USA

4 Department of Surgery, Georgetown University School of Medicine,

\*Address all correspondence to: rosenthd@georgetown.edu

provided the original work is properly cited.

2 Firefighters' Burn and Surgical Research Laboratory, MedStar Health Research

3 The Burn Center, Department of Surgery, MedStar Washington Hospital Center,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

the presence of polyP. PolyP levels and chain lengths should also be quantified in healthy and pathologic conditions in order to assess appropriate levels when treating acute or chronic wounds in the future.

### **Acknowledgements**

*Contemporary Topics about Phosphorus in Biology and Materials*

lar wound size between animal groups at day 0 (0.33 cm<sup>2</sup>

with increasing doses of polyP administration.

dermis underneath the epithelium.

difference leveled off.

**5. Conclusion**

with underlying fascia and were sewn into histological cassettes to retain wound orientation. These samples were then paraffin embedded and H&E stained. Sections were imaged, and epithelial tongue length was measured using Image J. Epithelial tongues were defined as new epithelium if there was no uninjured

Compared to whole blood, PRP contained a higher concentration of platelets (data to be published elsewhere). The splinted wound model was used to shift the healing towards re-epithelialization instead of the normal contraction observed in mice. PRP application was easily applied as a "bio-bandage" gel-like liquid (data to be published elsewhere). The 6 mm punch biopsies allowed for the creation of simi-

p = n.s.; data to be published elsewhere). At days 3 (0.09 ± 0.06 vs. 0.23 ± 0.13) and 5 (0.12 ± 0.07 vs. 0.25 ± 0.12) PRP-treated wounds had significantly smaller open wound areas compared to control animals (n = 6, p < 0.05). At days 6 and 7, this

**4.2 Exogenous spiking of PRP with polyP further accelerates healing** *in vivo*

To further investigate the potential role of polyP in PRP, wounds were treated with PRP or with PRP with low or high dose-spiked polyP. Untreated wounds were largely open by day 5, while PRP treated wounds were smaller and contained newly formed epithelium (data to be published elsewhere). Spiking with low or high dose polyP further stimulated epithelialization, and wounds were smaller with increasing doses. By histomorphometric analysis, epithelial tongues can be seen by H&E staining. In untreated wounds, these tongues are small and shallow. PRP treatment results in a more proliferative epithelium that is thicker and longer than untreated samples. Spiking with low and high dose polyP creates longer epithelial tongues). Epithelial tongue measurement by Image J shows a significant decrease in tongue length in untreated and PRP only treated vs. PRP + high dose polyP (737.38 ± 121.21 and 925.55 ± 214.17 vs. 1186.91 ± 255.06 μM, n = 8, p < 0.0001, p < 0.05). PRP + high dose polyP-treated wounds also had significantly longer epithelial tongues compared to PRP + low dose polyP (n = 8, p < 0.05). PRP contains polyP at a concentration near 5 μM. The exogenous addition of polyP to the PRP promoted keratinocyte growth and proliferation, as is evidence by the increased epithelial tongue length

PolyP plays key roles in essential biological processes in bacteria, and its increas-

ing importance in eukaryotes is becoming apparent, including its participation in blood coagulation and wound healing. Recent advances in measurement and localization of polyP, along with our growing understanding of polyP metabolism and its interaction with specific proteins allows us to begin to analyze mechanisms responsible for cell-specific roles of polyP. Our ability to regulate polyP in eukaryotic cells opens possibilities for therapeutic intervention. Future work related to wound healing should be aimed at investigating the specific roles of intra- and extracellular polyP in keratinocytes, as well as the potential importance of polyP in other skin cells, including dermal fibroblasts, as these cells make up the majority of the skin. As polyP is also secreted by activated platelets and is important for normal blood clotting, the application of polyP-containing PRP as a biologic dressing may positively contribute to wound healing. We have completed two clinical trials using platelet-rich plasma for wound healing, a phenomenon that may be explained by

± 0.13 vs. 0.39 ± 0.15 cm2

,

**82**

Authors are grateful for the technical assistance of Sixian Song in the scratch gap assays, and Dr. James Morrissey (University of Michigan Medical School) for the generous gift of polyP, and its inhibitors PPXbd and UHRA-9, used in their studies. This work was partially supported by the NIH STTR grant 1R41ES026908 (to DSR), and the office of the Dean of Research, Georgetown University School of Medicine.

### **Conflict of interest**

The authors have no conflicts of interest to declare.

### **Author details**

Cynthia M. Simbulan-Rosenthal1 , Bonnie C. Carney1,2, Anirudh Gaur1 , Manish Moghe1 , Elliott Crooke1 , Lauren T. Moffatt1,2, Jeffrey W. Shupp1,2,3,4 and Dean S. Rosenthal1 \*

1 Department of Biochemistry and Molecular and Cellular Biology, Georgetown University School of Medicine, Washington, DC, USA

2 Firefighters' Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington, DC, USA

3 The Burn Center, Department of Surgery, MedStar Washington Hospital Center, Washington, DC, USA

4 Department of Surgery, Georgetown University School of Medicine, Washington, DC, USA

\*Address all correspondence to: rosenthd@georgetown.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Rao NN, Gomez-Garcia MR, Kornberg A. Inorganic polyphosphate: Essential for growth and survival. Annual Review of Biochemistry. 2009;**78**:605-647. DOI: 10.1146/annurev. biochem.77.083007.093039

[2] Brown MR, Kornberg A. The long and short of it: Polyphosphate PPK and bacterial survival. Trends in Biochemical Sciences. 2008;**33**(6):284- 290. DOI: 10.1016/j.tibs.2008.04.005

[3] Gomez-Garcia MR, Kornberg A. Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(45):15876-15880. DOI: 10.1073/pnas.0406923101

[4] Zhang H, Ishige K, Kornberg A. A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(26):16678-16683. DOI: 10.1073/pnas.262655199

[5] Akiyama M, Crooke E, Kornberg A. An exopolyphosphatase of *Escherichia coli*. The enzyme and its ppx gene in a polyphosphate operon. The Journal of Biological Chemistry. 1993;**268**(1):633-639

[6] Wurst H, Kornberg A. A soluble exopolyphosphatase of *Saccharomyces cerevisiae*. Purification and characterization. The Journal of Biological Chemistry. 1994;**269**(15):10996-11001

[7] Wurst H, Shiba T, Kornberg A. The gene for a major exopolyphosphatase of *Saccharomyces cerevisiae*. Journal of Bacteriology. 1995;**177**(4):898-906

[8] Lichko LP, Kulakovskaya TV, Kulaev IS. Inorganic polyphosphate and

exopolyphosphatase in the nuclei of *Saccharomyces cerevisiae*: Dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Yeast. 2006;**23**(10):735-740. DOI: 10.1002/ yea.1391

[9] Sethuraman A, Rao NN, Kornberg A. The endopolyphosphatase gene: Essential in *Saccharomyces cerevisiae*. Proceedings of the National Academy of Sciences of the United States of America. 2001;**98**(15):8542-8547. DOI: 10.1073/pnas.151269398

[10] Luginbuehl E et al. The exopolyphosphatase TbrPPX1 of *Trypanosoma brucei*. BMC Microbiology. 2011;**11**:4. DOI: 10.1186/1471-2180-11-4

[11] Kumble KD, Kornberg A. Inorganic polyphosphate in mammalian cells and tissues. The Journal of Biological Chemistry. 1995;**270**(11):5818-5822

[12] Rao NN, Kornberg A. Inorganic polyphosphate regulates responses of *Escherichia coli* to nutritional stringencies, environmental stresses and survival in the stationary phase. Progress in Molecular and Subcellular Biology. 1999;**23**:183-195

[13] Crooke E et al. Genetically altered levels of inorganic polyphosphate in *Escherichia coli*. The Journal of Biological Chemistry. 1994;**269**(9):6290-6295

[14] Alcantara C et al. Accumulation of polyphosphate in *Lactobacillus* spp. and its involvement in stress resistance. Applied and Environmental Microbiology. 2014;**80**(5):1650-1659. DOI: 10.1128/AEM.03997-13

[15] Nikel PI et al. Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in *Pseudomonas putida* KT2440. Microbial Cell Factories. 2013;**12**:50. DOI: 10.1186/1475-2859-12-50

**85**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

survival and virulence factors in *Shigella* and *Salmonella* spp. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(11):7675-7680. DOI: 10.1073/

[24] Ahn K, Kornberg A. Polyphosphate kinase from *Escherichia coli*. Purification and demonstration of a phosphoenzyme intermediate. The Journal of Biological Chemistry. 1990;**265**(20):11734-11739

[25] Ishige K, Zhang H, Kornberg A. Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(26):16684- 16688. DOI: 10.1073/pnas.262655299

[26] Nocek B et al. Polyphosphatedependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(46):17730-17735.

DOI: 10.1073/pnas.0807563105

[27] Rudat AK et al. Mutations in *Escherichia coli* polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels. Journal of Bacteriology. 2018;**200**(6):pii: e00697-17.

[28] Ault-Riche D et al. Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in *Escherichia* 

DOI: 10.1128/JB.00697-17

*coli*. Journal of Bacteriology. 1998;**180**(7):1841-1847

[29] Shiba T et al. Inorganic polyphosphate and the induction of rpoS expression. Proceedings of the National Academy of Sciences of the United States of America.

1997;**94**(21):11210-11215

[30] Mullan A, Quinn JP, McGrath JW. Enhanced phosphate uptake and polyphosphate accumulation in

pnas.112210499

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

[16] Singh R et al. Polyphosphate deficiency in *Mycobacterium tuberculosis* is associated with enhanced drug susceptibility and impaired growth in Guinea pigs. Journal of Bacteriology. 2013;**195**(12):2839-2851. DOI: 10.1128/

[17] Rao NN, Liu S, Kornberg A. Inorganic polyphosphate in *Escherichia coli*: The phosphate regulon and the stringent response. Journal of Bacteriology. 1998;**180**(8):2186-2193

[18] Rao NN, Kornberg A. Inorganic polyphosphate supports resistance and survival of stationary-phase *Escherichia coli*. Journal of Bacteriology.

[19] Rashid MH, Kornberg A. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of *Pseudomonas aeruginosa*. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(9):4885-4890. DOI: 10.1073/

[20] Rashid MH, Rao NN, Kornberg A. Inorganic polyphosphate is required for motility of bacterial pathogens. Journal of Bacteriology.

[21] Shi X, Rao NN, Kornberg A. Inorganic polyphosphate in *Bacillus cereus*: Motility, biofilm formation, and sporulation. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(49):17061- 17065. DOI: 10.1073/pnas.0407787101

[22] Rashid MH et al. Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of *Pseudomonas aeruginosa*. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(17):9636-9641. DOI:

10.1073/pnas.170283397

[23] Kim KS et al. Inorganic

polyphosphate is essential for long-term

1996;**178**(5):1394-1400

pnas.060030097

2000;**182**(1):225-227

JB.00038-13

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

[16] Singh R et al. Polyphosphate deficiency in *Mycobacterium tuberculosis* is associated with enhanced drug susceptibility and impaired growth in Guinea pigs. Journal of Bacteriology. 2013;**195**(12):2839-2851. DOI: 10.1128/ JB.00038-13

[17] Rao NN, Liu S, Kornberg A. Inorganic polyphosphate in *Escherichia coli*: The phosphate regulon and the stringent response. Journal of Bacteriology. 1998;**180**(8):2186-2193

[18] Rao NN, Kornberg A. Inorganic polyphosphate supports resistance and survival of stationary-phase *Escherichia coli*. Journal of Bacteriology. 1996;**178**(5):1394-1400

[19] Rashid MH, Kornberg A. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of *Pseudomonas aeruginosa*. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(9):4885-4890. DOI: 10.1073/ pnas.060030097

[20] Rashid MH, Rao NN, Kornberg A. Inorganic polyphosphate is required for motility of bacterial pathogens. Journal of Bacteriology. 2000;**182**(1):225-227

[21] Shi X, Rao NN, Kornberg A. Inorganic polyphosphate in *Bacillus cereus*: Motility, biofilm formation, and sporulation. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(49):17061- 17065. DOI: 10.1073/pnas.0407787101

[22] Rashid MH et al. Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of *Pseudomonas aeruginosa*. Proceedings of the National Academy of Sciences of the United States of America. 2000;**97**(17):9636-9641. DOI: 10.1073/pnas.170283397

[23] Kim KS et al. Inorganic polyphosphate is essential for long-term survival and virulence factors in *Shigella* and *Salmonella* spp. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(11):7675-7680. DOI: 10.1073/ pnas.112210499

[24] Ahn K, Kornberg A. Polyphosphate kinase from *Escherichia coli*. Purification and demonstration of a phosphoenzyme intermediate. The Journal of Biological Chemistry. 1990;**265**(20):11734-11739

[25] Ishige K, Zhang H, Kornberg A. Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(26):16684- 16688. DOI: 10.1073/pnas.262655299

[26] Nocek B et al. Polyphosphatedependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(46):17730-17735. DOI: 10.1073/pnas.0807563105

[27] Rudat AK et al. Mutations in *Escherichia coli* polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels. Journal of Bacteriology. 2018;**200**(6):pii: e00697-17. DOI: 10.1128/JB.00697-17

[28] Ault-Riche D et al. Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in *Escherichia coli*. Journal of Bacteriology. 1998;**180**(7):1841-1847

[29] Shiba T et al. Inorganic polyphosphate and the induction of rpoS expression. Proceedings of the National Academy of Sciences of the United States of America. 1997;**94**(21):11210-11215

[30] Mullan A, Quinn JP, McGrath JW. Enhanced phosphate uptake and polyphosphate accumulation in

**84**

*Contemporary Topics about Phosphorus in Biology and Materials*

exopolyphosphatase in the nuclei of *Saccharomyces cerevisiae*: Dependence on the growth phase and inactivation of the PPX1 and PPN1 genes. Yeast. 2006;**23**(10):735-740. DOI: 10.1002/

[9] Sethuraman A, Rao NN, Kornberg A.

*Trypanosoma brucei*. BMC Microbiology. 2011;**11**:4. DOI: 10.1186/1471-2180-11-4

[11] Kumble KD, Kornberg A. Inorganic polyphosphate in mammalian cells and tissues. The Journal of Biological Chemistry. 1995;**270**(11):5818-5822

[12] Rao NN, Kornberg A. Inorganic polyphosphate regulates responses of *Escherichia coli* to nutritional stringencies, environmental stresses and survival in the stationary phase. Progress in Molecular and Subcellular

[13] Crooke E et al. Genetically altered levels of inorganic polyphosphate in *Escherichia coli*. The Journal of Biological Chemistry. 1994;**269**(9):6290-6295

[14] Alcantara C et al. Accumulation of polyphosphate in *Lactobacillus* spp. and its involvement in stress resistance. Applied and Environmental Microbiology. 2014;**80**(5):1650-1659.

DOI: 10.1128/AEM.03997-13

[15] Nikel PI et al. Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in *Pseudomonas putida* KT2440. Microbial

Cell Factories. 2013;**12**:50. DOI: 10.1186/1475-2859-12-50

Biology. 1999;**23**:183-195

The endopolyphosphatase gene: Essential in *Saccharomyces cerevisiae*. Proceedings of the National Academy of Sciences of the United States of America. 2001;**98**(15):8542-8547. DOI:

10.1073/pnas.151269398

[10] Luginbuehl E et al. The exopolyphosphatase TbrPPX1 of

yea.1391

**References**

[1] Rao NN, Gomez-Garcia MR, Kornberg A. Inorganic polyphosphate: Essential for growth and survival. Annual Review of Biochemistry. 2009;**78**:605-647. DOI: 10.1146/annurev.

biochem.77.083007.093039

[2] Brown MR, Kornberg A. The long and short of it: Polyphosphate PPK and bacterial survival. Trends in Biochemical Sciences. 2008;**33**(6):284- 290. DOI: 10.1016/j.tibs.2008.04.005

[3] Gomez-Garcia MR, Kornberg A. Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(45):15876-15880. DOI: 10.1073/pnas.0406923101

[4] Zhang H, Ishige K, Kornberg A. A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2002;**99**(26):16678-16683. DOI:

[5] Akiyama M, Crooke E, Kornberg A. An exopolyphosphatase of *Escherichia coli*. The enzyme and its ppx gene in a polyphosphate operon. The Journal of Biological Chemistry.

[7] Wurst H, Shiba T, Kornberg A. The gene for a major exopolyphosphatase of *Saccharomyces cerevisiae*. Journal of Bacteriology. 1995;**177**(4):898-906

[8] Lichko LP, Kulakovskaya TV, Kulaev IS. Inorganic polyphosphate and

10.1073/pnas.262655199

1993;**268**(1):633-639

[6] Wurst H, Kornberg A. A soluble exopolyphosphatase of *Saccharomyces cerevisiae*. Purification

and characterization. The Journal of Biological Chemistry. 1994;**269**(15):10996-11001

*Burkholderia cepacia* grown under low pH conditions. Microbial Ecology. 2002;**44**(1):69-77. DOI: 10.1007/ s00248-002-3004-x

[31] Gray MJ et al. Polyphosphate is a primordial chaperone. Molecular Cell. 2014;**53**(5):689-699. DOI: 10.1016/j. molcel.2014.01.012

[32] Yoo NG et al. Polyphosphate stabilizes protein unfolding intermediates as soluble amyloid-like oligomers. Journal of Molecular Biology. 2018;**430**(21):4195-4208. DOI: 10.1016/j. jmb.2018.08.016

[33] Simbulan-Rosenthal CM et al. Inorganic polyphosphates are important for cell survival and motility of human skin keratinocytes. Experimental Dermatology. 2015;**24**(8):636-639. DOI: 10.1111/exd.12729

[34] Werner TP, Amrhein N, Freimoser FM. Novel method for the quantification of inorganic polyphosphate (iPoP) in *Saccharomyces cerevisiae* shows dependence of iPoP content on the growth phase. Archives of Microbiology. 2005;**184**(2):129-136. DOI: 10.1007/s00203-005-0031-2

[35] Freimoser FM et al. Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary metabolism. Genome Biology. 2006;**7**(11):R109. DOI: 10.1186/ gb-2006-7-11-r109

[36] Zakrzewska J, Zizic M, Zivic M. The effect of anoxia on PolyP content of *Phycomyces blakesleeanus* mycelium studied by 31P NMR spectroscopy. Annals of the New York Academy of Sciences. 2005;**1048**:482-486. DOI: 10.1196/annals.1342.073

[37] Choi BK, Hercules DM, Houalla M. Characterization of polyphosphates by electrospray mass spectrometry. Analytical Chemistry. 2000;**72**(20):5087-5091

[38] Comolli LR, Kundmann M, Downing KH. Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging. Journal of Microscopy. 2006;**223**(Pt 1):40-52. DOI: 10.1111/j.1365-2818.2006.01597.x

[39] Saito K et al. Direct labeling of polyphosphate at the ultrastructural level in *Saccharomyces cerevisiae* by using the affinity of the polyphosphate binding domain of *Escherichia coli* exopolyphosphatase. Applied and Environmental Microbiology. 2005;**71**(10):5692-5701. DOI: 10.1128/ AEM.71.10.5692-5701.2005

[40] Groitl B et al. *Pseudomonas aeruginosa* defense systems against microbicidal oxidants. Molecular Microbiology. 2017;**106**(3):335-350. DOI: 10.1111/mmi.13768

[41] Dahl JU et al. The antiinflammatory drug mesalamine targets bacterial polyphosphate accumulation. Nature Microbiology. 2017;**2**:16267. DOI: 10.1038/nmicrobiol.2016.267

[42] Fraley CD et al. A polyphosphate kinase 1 (ppk1) mutant of *Pseudomonas aeruginosa* exhibits multiple ultrastructural and functional defects. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(9):3526-3531. DOI: 10.1073/pnas.0609733104

[43] Maciag A et al. In vitro transcription profiling of the sigmaS subunit of bacterial RNA polymerase: Re-definition of the sigmaS regulon and identification of sigmaS-specific promoter sequence elements. Nucleic Acids Research. 2011;**39**(13):5338-5355. DOI: 10.1093/nar/gkr129

[44] Silby MW, Nicoll JS, Levy SB. Regulation of polyphosphate kinase production by antisense RNA in *Pseudomonas fluorescens* Pf0-1. Applied and Environmental Microbiology.

**87**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

[52] Pavlov E et al. Inorganic polyphosphate and energy metabolism in mammalian cells. The Journal of Biological Chemistry. 2010;**285**(13):9420-9428. DOI: 10.1074/

[53] Kornberg A, Rao NN, Ault-Riche D. Inorganic polyphosphate: A molecule of many functions. Annual Review of Biochemistry. 1999;**68**:89-125. DOI: 10.1146/annurev.biochem.68.1.89

[54] Lonetti A et al. Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases. The Journal of Biological Chemistry. 2011;**286**(37):31966-31974. DOI:

[55] Cordeiro CD, Saiardi A, Docampo

hexakisphosphate kinase 1 maintains hemostasis in mice by regulating platelet polyphosphate levels. Blood. 2013;**122**(8):1478-1486. DOI: 10.1182/

[57] Hou Q et al. Inhibition of IP6K1 suppresses neutrophil-mediated pulmonary damage in bacterial pneumonia. Science Translational Medicine. 2018;**10**(435):pii: eaal4045. DOI: 10.1126/scitranslmed.aal4045

[58] Carotenuto M et al. H-prune through GSK-3beta interaction sustains canonical WNT/beta-catenin signaling enhancing cancer progression in NSCLC. Oncotarget. 2014;**5**(14):5736- 5749. DOI: 10.18632/oncotarget.2169

[59] Tammenkoski M et al. Human metastasis regulator protein H-prune is a short-chain exopolyphosphatase.

R. The inositol pyrophosphate synthesis pathway in *Trypanosoma brucei* is linked to polyphosphate synthesis in acidocalcisomes. Molecular Microbiology. 2017;**106**(2):319-333.

10.1074/jbc.M111.266320

DOI: 10.1111/mmi.13766

[56] Ghosh S et al. Inositol

blood-2013-01-481549

jbc.M109.013011

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

2012;**78**(12):4533-4537. DOI: 10.1128/

Polyphosphate kinase as a nucleoside diphosphate kinase in *Escherichia coli* and *Pseudomonas aeruginosa*. Proceedings of the National Academy of Sciences of the United States of America. 1997;**94**(2):439-442

[46] Ogawa N, DeRisi J, Brown PO. New components of a system for phosphate accumulation and polyphosphate metabolism in *Saccharomyces cerevisiae* revealed by genomic expression analysis. Molecular Biology of the Cell. 2000;**11**(12):4309-4321. DOI: 10.1091/

[47] Hothorn M et al. Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase. Science. 2009;**324**(5926):513-516. DOI: 10.1126/

[48] Bru S et al. Polyphosphate is involved in cell cycle progression and genomic stability in *Saccharomyces cerevisiae*. Molecular Microbiology. 2016;**101**(3):367-

[49] Wang L et al. Distribution patterns of polyphosphate metabolism pathway and its relationships with bacterial durability and virulence. Frontiers in Microbiology. 2018;**9**:782. DOI: 10.3389/

[50] Nahalka J, Patoprsty V. Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3). Organic & Biomolecular Chemistry. 2009;**7**(9):1778-1780. DOI:

[51] Pestov NA, Kulakovskaya TV, Kulaev IS. Inorganic polyphosphate in mitochondria of *Saccharomyces cerevisiae* at phosphate limitation and phosphate excess. FEMS Yeast Research. 2004;**4**(6):643-648. DOI: 10.1016/j.

380. DOI: 10.1111/mmi.13396

[45] Kuroda A, Kornberg A.

AEM.07836-11

mbc.11.12.4309

science.1168120

fmicb.2018.00782

10.1039/b822549b

femsyr.2003.12.008

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

2012;**78**(12):4533-4537. DOI: 10.1128/ AEM.07836-11

*Contemporary Topics about Phosphorus in Biology and Materials*

[38] Comolli LR, Kundmann M, Downing KH. Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging. Journal of Microscopy. 2006;**223**(Pt 1):40-52. DOI:

10.1111/j.1365-2818.2006.01597.x

binding domain of *Escherichia coli* exopolyphosphatase. Applied and Environmental Microbiology. 2005;**71**(10):5692-5701. DOI: 10.1128/

AEM.71.10.5692-5701.2005

DOI: 10.1111/mmi.13768

[41] Dahl JU et al. The anti-

10.1038/nmicrobiol.2016.267

*aeruginosa* exhibits multiple

10.1073/pnas.0609733104

DOI: 10.1093/nar/gkr129

profiling of the sigmaS subunit of bacterial RNA polymerase: Re-definition of the sigmaS regulon and identification of sigmaS-specific promoter sequence elements. Nucleic Acids Research. 2011;**39**(13):5338-5355.

[44] Silby MW, Nicoll JS, Levy SB. Regulation of polyphosphate kinase production by antisense RNA in *Pseudomonas fluorescens* Pf0-1. Applied and Environmental Microbiology.

inflammatory drug mesalamine targets bacterial polyphosphate accumulation. Nature Microbiology. 2017;**2**:16267. DOI:

[42] Fraley CD et al. A polyphosphate kinase 1 (ppk1) mutant of *Pseudomonas* 

ultrastructural and functional defects. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(9):3526-3531. DOI:

[43] Maciag A et al. In vitro transcription

[40] Groitl B et al. *Pseudomonas aeruginosa* defense systems against microbicidal oxidants. Molecular Microbiology. 2017;**106**(3):335-350.

[39] Saito K et al. Direct labeling of polyphosphate at the ultrastructural level in *Saccharomyces cerevisiae* by using the affinity of the polyphosphate

*Burkholderia cepacia* grown under low pH conditions. Microbial Ecology. 2002;**44**(1):69-77. DOI: 10.1007/

[31] Gray MJ et al. Polyphosphate is a primordial chaperone. Molecular Cell. 2014;**53**(5):689-699. DOI: 10.1016/j.

[32] Yoo NG et al. Polyphosphate stabilizes protein unfolding

intermediates as soluble amyloid-like oligomers. Journal of Molecular Biology. 2018;**430**(21):4195-4208. DOI: 10.1016/j.

[33] Simbulan-Rosenthal CM et al. Inorganic polyphosphates are important for cell survival and motility of human skin keratinocytes. Experimental Dermatology. 2015;**24**(8):636-639. DOI:

s00248-002-3004-x

molcel.2014.01.012

jmb.2018.08.016

10.1111/exd.12729

[34] Werner TP, Amrhein N, Freimoser FM. Novel method for the quantification of inorganic

polyphosphate (iPoP) in *Saccharomyces cerevisiae* shows dependence of iPoP content on the growth phase. Archives of Microbiology. 2005;**184**(2):129-136. DOI: 10.1007/s00203-005-0031-2

[35] Freimoser FM et al. Systematic screening of polyphosphate (poly P) levels in yeast mutant cells reveals strong interdependence with primary

[36] Zakrzewska J, Zizic M, Zivic M. The effect of anoxia on PolyP content of *Phycomyces blakesleeanus* mycelium studied by 31P NMR spectroscopy. Annals of the New York Academy of Sciences. 2005;**1048**:482-486. DOI:

metabolism. Genome Biology. 2006;**7**(11):R109. DOI: 10.1186/

gb-2006-7-11-r109

10.1196/annals.1342.073

2000;**72**(20):5087-5091

[37] Choi BK, Hercules DM, Houalla M. Characterization of polyphosphates by electrospray mass spectrometry. Analytical Chemistry.

**86**

[45] Kuroda A, Kornberg A. Polyphosphate kinase as a nucleoside diphosphate kinase in *Escherichia coli* and *Pseudomonas aeruginosa*. Proceedings of the National Academy of Sciences of the United States of America. 1997;**94**(2):439-442

[46] Ogawa N, DeRisi J, Brown PO. New components of a system for phosphate accumulation and polyphosphate metabolism in *Saccharomyces cerevisiae* revealed by genomic expression analysis. Molecular Biology of the Cell. 2000;**11**(12):4309-4321. DOI: 10.1091/ mbc.11.12.4309

[47] Hothorn M et al. Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase. Science. 2009;**324**(5926):513-516. DOI: 10.1126/ science.1168120

[48] Bru S et al. Polyphosphate is involved in cell cycle progression and genomic stability in *Saccharomyces cerevisiae*. Molecular Microbiology. 2016;**101**(3):367- 380. DOI: 10.1111/mmi.13396

[49] Wang L et al. Distribution patterns of polyphosphate metabolism pathway and its relationships with bacterial durability and virulence. Frontiers in Microbiology. 2018;**9**:782. DOI: 10.3389/ fmicb.2018.00782

[50] Nahalka J, Patoprsty V. Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3). Organic & Biomolecular Chemistry. 2009;**7**(9):1778-1780. DOI: 10.1039/b822549b

[51] Pestov NA, Kulakovskaya TV, Kulaev IS. Inorganic polyphosphate in mitochondria of *Saccharomyces cerevisiae* at phosphate limitation and phosphate excess. FEMS Yeast Research. 2004;**4**(6):643-648. DOI: 10.1016/j. femsyr.2003.12.008

[52] Pavlov E et al. Inorganic polyphosphate and energy metabolism in mammalian cells. The Journal of Biological Chemistry. 2010;**285**(13):9420-9428. DOI: 10.1074/ jbc.M109.013011

[53] Kornberg A, Rao NN, Ault-Riche D. Inorganic polyphosphate: A molecule of many functions. Annual Review of Biochemistry. 1999;**68**:89-125. DOI: 10.1146/annurev.biochem.68.1.89

[54] Lonetti A et al. Identification of an evolutionarily conserved family of inorganic polyphosphate endopolyphosphatases. The Journal of Biological Chemistry. 2011;**286**(37):31966-31974. DOI: 10.1074/jbc.M111.266320

[55] Cordeiro CD, Saiardi A, Docampo R. The inositol pyrophosphate synthesis pathway in *Trypanosoma brucei* is linked to polyphosphate synthesis in acidocalcisomes. Molecular Microbiology. 2017;**106**(2):319-333. DOI: 10.1111/mmi.13766

[56] Ghosh S et al. Inositol hexakisphosphate kinase 1 maintains hemostasis in mice by regulating platelet polyphosphate levels. Blood. 2013;**122**(8):1478-1486. DOI: 10.1182/ blood-2013-01-481549

[57] Hou Q et al. Inhibition of IP6K1 suppresses neutrophil-mediated pulmonary damage in bacterial pneumonia. Science Translational Medicine. 2018;**10**(435):pii: eaal4045. DOI: 10.1126/scitranslmed.aal4045

[58] Carotenuto M et al. H-prune through GSK-3beta interaction sustains canonical WNT/beta-catenin signaling enhancing cancer progression in NSCLC. Oncotarget. 2014;**5**(14):5736- 5749. DOI: 10.18632/oncotarget.2169

[59] Tammenkoski M et al. Human metastasis regulator protein H-prune is a short-chain exopolyphosphatase.

Biochemistry. 2008;**47**(36):9707-9713. DOI: 10.1021/bi8010847

[60] Aschar-Sobbi R et al. High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach. Journal of Fluorescence. 2008;**18**(5):859-866. DOI: 10.1007/s10895-008-0315-4

[61] Jimenez-Nunez MD et al. Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription. Haematologica. 2012;**97**(8):1264-1271. DOI: 10.3324/ haematol.2011.051409

[62] Moreno-Sanchez D et al. Polyphosphate is a novel proinflammatory regulator of mast cells and is located in acidocalcisomes. The Journal of Biological Chemistry. 2012;**287**(34):28435- 28444. DOI: 10.1074/jbc.M112.385823

[63] Angelova PR et al. In situ investigation of mammalian inorganic polyphosphate localization using novel selective fluorescent probes JC-D7 and JC-D8. ACS Chemical Biology. 2014;**9**(9):2101-2110. DOI: 10.1021/cb5000696

[64] Holmstrom KM et al. Signalling properties of inorganic polyphosphate in the mammalian brain. Nature Communications. 2013;**4**:1362. DOI: 10.1038/ncomms2364

[65] Angelova PR et al. Signal transduction in astrocytes: Localization and release of inorganic polyphosphate. Glia. 2018;**66**(10):2126-2136. DOI: 10.1002/glia.23466

[66] Suess PM, Tang Y, Gomer RH. The putative G protein-coupled receptor GrlD mediates extracellular polyphosphate sensing in Dictyostelium discoideum. Molecular Biology of the Cell. 2019;**30**(9):1118-1128. DOI: 10.1091/mbc.E18-10-0686

[67] Chen KY. Study of polyphosphate metabolism in intact cells by 31-P nuclear magnetic resonance

spectroscopy. Progress in Molecular and Subcellular Biology. 1999;**23**:253-273

[68] Ruiz FA et al. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. The Journal of Biological Chemistry. 2004;**279**(43):44250-44257. DOI: 10.1074/jbc.M406261200

[69] Muller F et al. Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell. 2009;**139**(6):1143-1156. DOI: 10.1016/j.cell.2009.11.001

[70] Gabel NW, Thomas V. Evidence for the occurrence and distribution of inorganic polyphosphates in vertebrate tissues. Journal of Neurochemistry. 1971;**18**(7):1229-1242

[71] Cremers CM et al. Polyphosphate: A conserved modifier of amyloidogenic processes. Molecular Cell. 2016;**63**(5):768-780. DOI: 10.1016/j. molcel.2016.07.016

[72] Lorenz B et al. Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis. Biochimica et Biophysica Acta. 1997;**1335**(1-2):51-60. DOI: 10.1016/ S0304-4165(96)00121-3

[73] Li L et al. Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization. Biochimica et Biophysica Acta: General Subjects. 2019;**1863**(1):199-209. DOI: 10.1016/j. bbagen.2018.10.003

[74] Abramov AY et al. Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(46):18091-18096. DOI: 10.1073/pnas.0708959104

**89**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

2015;**58**(1):71-82. DOI: 10.1016/j.

[82] Zakharian E et al. Inorganic polyphosphate modulates TRPM8 channels. PLoS ONE. 2009;**4**(4):e5404. DOI: 10.1371/journal.pone.0005404

[83] Shiba T et al. Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate. The Journal of Biological Chemistry. 2003;**278**(29):26788-26792. DOI:

[84] Segawa S et al. Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway. PLoS ONE. 2011;**6**(8):e23278. DOI: 10.1371/

10.1074/jbc.M303468200

journal.pone.0023278

DOI: 10.1111/mmi.14131

10.1021/acschembio.8b00357

celrep.2018.02.104

[87] Bentley-DeSousa A et al. A screen for candidate targets of lysine polyphosphorylation uncovers a conserved network implicated in ribosome biogenesis. Cell Reports. 2018;**22**(13):3427-3439. DOI: 10.1016/j.

[88] Hernandez-Ruiz L et al. Inorganic polyphosphate and specific induction of apoptosis in human plasma cells. Haematologica. 2006;**91**(9):1180-1186

[89] Trilisenko L, Kulakovskaya E, Kulakovskaya T. The cadmium tolerance in *Saccharomyces cerevisiae* depends on inorganic polyphosphate. Journal of

[85] Negreiros RS et al. Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids. Molecular Microbiology. 2018;**110**(6):973-994.

[86] Azevedo C et al. Screening a protein Array with synthetic biotinylated inorganic polyphosphate to define the human PolyP-ome. ACS Chemical Biology. 2018;**13**(8):1958-1963. DOI:

molcel.2015.02.010

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

[75] Seidlmayer LK et al. Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes. The Journal of General Physiology. 2012;**139**(5):321-331. DOI: 10.1085/

[76] Stotz SC et al. Inorganic polyphosphate regulates neuronal excitability through modulation of voltage-gated channels.

10.1186/1756-6606-7-42

[77] Angelova PR et al. Role of

Molecular Brain. 2014;**7**(1):42. DOI:

inorganic polyphosphate in mammalian cells: From signal transduction and mitochondrial metabolism to cell death. Biochemical Society Transactions. 2016;**44**(1):40-45. DOI: 10.1042/

[78] Baev AY, Negoda A, Abramov AY. Modulation of mitochondrial ion transport by inorganic polyphosphate:

[79] Elustondo PA et al. Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate. Cell Death Discovery. 2016;**2**:16070. DOI:

polyphosphate stimulates mammalian

proliferation of mammary cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 2003;**100**(20):11249-11254. DOI: 10.1073/pnas.1534805100

[81] Azevedo C, Livermore T, Saiardi A. Protein polyphosphorylation of lysine residues by inorganic polyphosphate. Molecular Cell.

Essential role in mitochondrial permeability transition pore. Journal of Bioenergetics and Biomembranes. 2017;**49**(1):49-55. DOI: 10.1007/

10.1038/cddiscovery.2016.70

[80] Wang L et al. Inorganic

TOR, a kinase involved in the

jgp.201210788

BST20150223

s10863-016-9650-3

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

[75] Seidlmayer LK et al. Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes. The Journal of General Physiology. 2012;**139**(5):321-331. DOI: 10.1085/ jgp.201210788

*Contemporary Topics about Phosphorus in Biology and Materials*

spectroscopy. Progress in Molecular and Subcellular Biology. 1999;**23**:253-273

[68] Ruiz FA et al. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. The Journal of Biological Chemistry. 2004;**279**(43):44250-44257. DOI:

10.1074/jbc.M406261200

[69] Muller F et al. Platelet

10.1016/j.cell.2009.11.001

1971;**18**(7):1229-1242

molcel.2016.07.016

processes. Molecular Cell.

polyphosphates are proinflammatory and procoagulant mediators in vivo. Cell. 2009;**139**(6):1143-1156. DOI:

[70] Gabel NW, Thomas V. Evidence for the occurrence and distribution of inorganic polyphosphates in vertebrate tissues. Journal of Neurochemistry.

[71] Cremers CM et al. Polyphosphate: A conserved modifier of amyloidogenic

2016;**63**(5):768-780. DOI: 10.1016/j.

metabolism of inorganic polyphosphate

[72] Lorenz B et al. Changes in

in rat tissues and human cells during development and apoptosis. Biochimica et Biophysica Acta. 1997;**1335**(1-2):51-60. DOI: 10.1016/

S0304-4165(96)00121-3

[73] Li L et al. Long-chain

bbagen.2018.10.003

polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization. Biochimica et Biophysica Acta: General Subjects. 2019;**1863**(1):199-209. DOI: 10.1016/j.

[74] Abramov AY et al. Targeted polyphosphatase expression alters mitochondrial metabolism and

inhibits calcium-dependent cell death. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**(46):18091-18096. DOI: 10.1073/pnas.0708959104

Biochemistry. 2008;**47**(36):9707-9713.

sensitivity, quantitative measurements

[61] Jimenez-Nunez MD et al. Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription. Haematologica. 2012;**97**(8):1264-1271. DOI: 10.3324/

inflammatory regulator of mast cells and is located in acidocalcisomes. The Journal of Biological Chemistry. 2012;**287**(34):28435- 28444. DOI: 10.1074/jbc.M112.385823

[63] Angelova PR et al. In situ investigation of mammalian inorganic polyphosphate localization using novel selective

fluorescent probes JC-D7 and JC-D8. ACS Chemical Biology. 2014;**9**(9):2101-2110.

[64] Holmstrom KM et al. Signalling properties of inorganic polyphosphate in the mammalian brain. Nature Communications. 2013;**4**:1362. DOI:

transduction in astrocytes: Localization and release of inorganic polyphosphate. Glia. 2018;**66**(10):2126-2136. DOI:

[66] Suess PM, Tang Y, Gomer RH. The putative G protein-coupled receptor GrlD mediates extracellular polyphosphate sensing in Dictyostelium discoideum. Molecular Biology of the Cell. 2019;**30**(9):1118-1128. DOI:

[67] Chen KY. Study of polyphosphate

10.1091/mbc.E18-10-0686

metabolism in intact cells by 31-P nuclear magnetic resonance

DOI: 10.1021/bi8010847

[60] Aschar-Sobbi R et al. High

of polyphosphate using a new DAPI-based approach. Journal of Fluorescence. 2008;**18**(5):859-866. DOI:

10.1007/s10895-008-0315-4

haematol.2011.051409

DOI: 10.1021/cb5000696

10.1038/ncomms2364

10.1002/glia.23466

[65] Angelova PR et al. Signal

[62] Moreno-Sanchez D et al. Polyphosphate is a novel pro-

**88**

[76] Stotz SC et al. Inorganic polyphosphate regulates neuronal excitability through modulation of voltage-gated channels. Molecular Brain. 2014;**7**(1):42. DOI: 10.1186/1756-6606-7-42

[77] Angelova PR et al. Role of inorganic polyphosphate in mammalian cells: From signal transduction and mitochondrial metabolism to cell death. Biochemical Society Transactions. 2016;**44**(1):40-45. DOI: 10.1042/ BST20150223

[78] Baev AY, Negoda A, Abramov AY. Modulation of mitochondrial ion transport by inorganic polyphosphate: Essential role in mitochondrial permeability transition pore. Journal of Bioenergetics and Biomembranes. 2017;**49**(1):49-55. DOI: 10.1007/ s10863-016-9650-3

[79] Elustondo PA et al. Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate. Cell Death Discovery. 2016;**2**:16070. DOI: 10.1038/cddiscovery.2016.70

[80] Wang L et al. Inorganic polyphosphate stimulates mammalian TOR, a kinase involved in the proliferation of mammary cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 2003;**100**(20):11249-11254. DOI: 10.1073/pnas.1534805100

[81] Azevedo C, Livermore T, Saiardi A. Protein polyphosphorylation of lysine residues by inorganic polyphosphate. Molecular Cell.

2015;**58**(1):71-82. DOI: 10.1016/j. molcel.2015.02.010

[82] Zakharian E et al. Inorganic polyphosphate modulates TRPM8 channels. PLoS ONE. 2009;**4**(4):e5404. DOI: 10.1371/journal.pone.0005404

[83] Shiba T et al. Modulation of mitogenic activity of fibroblast growth factors by inorganic polyphosphate. The Journal of Biological Chemistry. 2003;**278**(29):26788-26792. DOI: 10.1074/jbc.M303468200

[84] Segawa S et al. Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway. PLoS ONE. 2011;**6**(8):e23278. DOI: 10.1371/ journal.pone.0023278

[85] Negreiros RS et al. Inorganic polyphosphate interacts with nucleolar and glycosomal proteins in trypanosomatids. Molecular Microbiology. 2018;**110**(6):973-994. DOI: 10.1111/mmi.14131

[86] Azevedo C et al. Screening a protein Array with synthetic biotinylated inorganic polyphosphate to define the human PolyP-ome. ACS Chemical Biology. 2018;**13**(8):1958-1963. DOI: 10.1021/acschembio.8b00357

[87] Bentley-DeSousa A et al. A screen for candidate targets of lysine polyphosphorylation uncovers a conserved network implicated in ribosome biogenesis. Cell Reports. 2018;**22**(13):3427-3439. DOI: 10.1016/j. celrep.2018.02.104

[88] Hernandez-Ruiz L et al. Inorganic polyphosphate and specific induction of apoptosis in human plasma cells. Haematologica. 2006;**91**(9):1180-1186

[89] Trilisenko L, Kulakovskaya E, Kulakovskaya T. The cadmium tolerance in *Saccharomyces cerevisiae* depends on inorganic polyphosphate. Journal of

Basic Microbiology. 2017;**57**(11):982- 986. DOI: 10.1002/jobm.201700257

[90] Andreeva N et al. Adaptation of *Saccharomyces cerevisiae* to toxic manganese concentration triggers changes in inorganic polyphosphates. FEMS Yeast Research. 2013;**13**(5):463-470. DOI: 10.1111/1567-1364.12049

[91] Dinarvand P et al. Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor. Blood. 2014;**123**(6):935-945. DOI: 10.1182/blood-2013-09-529602

[92] Tsutsumi K et al. Morphogenetic study on the maturation of osteoblastic cell as induced by inorganic polyphosphate. PLoS ONE. 2014;**9**(2):e86834. DOI: 10.1371/journal.pone.0086834

[93] Xie L, Jakob U. Inorganic polyphosphate, a multifunctional polyanionic protein scaffold. The Journal of Biological Chemistry. 2019;**294**(6):2180-2190. DOI: 10.1074/ jbc.REV118.002808

[94] Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011;**118**(26):6963-6970. DOI: 10.1182/blood-2011-07-368811

[95] Smith SA et al. Inhibition of polyphosphate as a novel strategy for preventing thrombosis and inflammation. Blood. 2012;**120**(26):5103-5110. DOI: 10.1182/ blood-2012-07-444935

[96] Smith SA et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood. 2010;**116**(20):4353-4359. DOI: 10.1182/blood-2010-01-266791

[97] Smith SA, Morrissey JH. Polyphosphate: A new player in the field of hemostasis. Current Opinion in Hematology. 2014;**21**(5):388-394. DOI: 10.1097/MOH.0000000000000069

[98] Szymusiak M et al. Colloidal confinement of polyphosphate on gold nanoparticles robustly activates the contact pathway of blood coagulation. Bioconjugate Chemistry. 2016;**27**(1):102-109. DOI: 10.1021/acs. bioconjchem.5b00524

[99] Zhu S et al. FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow. Blood. 2015;**126**(12):1494-1502. DOI: 10.1182/blood-2015-04-641472

[100] Baik SY et al. Effects of platelet lysate preparations on the proliferation of HaCaT cells. Annals of Laboratory Medicine. 2014;**34**(1):43-50. DOI: 10.3343/alm.2014.34.1.43

[101] Carter MJ, Fylling CP, Parnell LK. Use of platelet rich plasma gel on wound healing: A systematic review and meta-analysis. Eplasty. 2011;**11**:e38

[102] Porwal S, Chahar YS, Singh PK. A comparative study of combined dermaroller and platelet-rich plasma versus dermaroller alone in acne scars and assessment of quality of life before and after treatment. Indian Journal of Dermatology. 2018;**63**(5):403-408. DOI: 10.4103/ijd.IJD\_118\_17

[103] Alser OH, Goutos I. The evidence behind the use of platelet-rich plasma (PRP) in scar management: A literature review. Scars, Burns & Healing. 2018;**4**:2059513118808773. DOI: 10.1177/2059513118808773

[104] Garg S, Manchanda S. Plateletrich plasma-an 'Elixir' for treatment of alopecia: Personal experience on 117 patients with review of literature. Stem Cell Investigation. 2017;**4**:64. DOI: 10.21037/sci.2017.06.07

[105] Alves R, Grimalt R. Doubleblind, placebo-controlled pilot study

**91**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

concise review and outlook on future possibilities. International Journal of Oral Science. 2017;**9**(1):1-9. DOI:

[112] Agrawal AA. Evolution, current status and advances in application of platelet concentrate in periodontics and implantology. World Journal of Clinical Cases. 2017;**5**(5):159-171. DOI:

10.1038/ijos.2017.1

10.12998/wjcc.v5.i5.159

10.1111/wrr.12317

wrr.12266

[113] Picard F et al. The growing evidence for the use of platelet-rich plasma on diabetic chronic wounds: A review and a proposal for a new standard care. Wound Repair and Regeneration. 2015;**23**(5):638-643. DOI:

[114] Picard F et al. Should we use platelet-rich plasma as an adjunct therapy to treat "acute wounds," "burns," and "laser therapies": A review and a proposal of a quality criteria checklist for further studies. Wound Repair and Regeneration. 2015;**23**(2):163-170. DOI: 10.1111/

[115] Yeung CY et al. Efficacy of lyophilised platelet-rich plasma

powder on healing rate in patients with deep second degree burn injury: A prospective double-blind randomized clinical trial. Annals of Plastic Surgery. 2018;**80**(2S Suppl 1):S66-S69. DOI: 10.1097/SAP.0000000000001328

[116] Hara T et al. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway. Journal of Artificial Organs. 2016;**19**(4):372-377. DOI: 10.1007/

[117] Kakudo N et al. Platelet-rich plasma promotes epithelialization and angiogenesis in a splitthickness skin graft donor site. Medical Molecular Morphology. 2011;**44**(4):233-236. DOI:

10.1007/s00795-010-0532-1

s10047-016-0913-x

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

on the use of platelet-rich plasma in women with female androgenetic alopecia. Dermatologic Surgery. 2018;**44**(1):132-133. DOI: 10.1097/

[106] Liou JJ et al. Effect of platelet-rich plasma on chondrogenic differentiation of adipose- and bone marrow-derived mesenchymal stem cells. Tissue Engineering. Part A. 2018;**24**(19- 20):1432-1443. DOI: 10.1089/ten.

[107] Xie X, Zhang C, Tuan RS. Biology of platelet-rich plasma and its clinical application in cartilage repair. Arthritis Research & Therapy. 2014;**16**(1):204.

[108] Chiavaras MM et al. IMpact of Platelet Rich plasma OVer alternative therapies in patients with lateral epicondylitis (IMPROVE): Protocol for a multicenter randomized controlled study: A multicenter, randomized trial comparing autologous platelet-rich plasma, autologous whole blood, dry needle tendon fenestration, and physical therapy exercises alone on pain and quality of life in patients with lateral epicondylitis. Academic Radiology. 2014;**21**(9):1144-1155. DOI: 10.1016/j.

[109] Hussain N, Johal H, Bhandari M. An evidence-based evaluation on the use of platelet rich plasma in orthopedics: A review of the literature. SICOT J. 2017;**3**:57. DOI: 10.1051/

[110] Bansal H et al. Intra-articular injection in the knee of adipose

derived stromal cells (stromal vascular fraction) and platelet rich plasma for osteoarthritis. Journal of Translational Medicine. 2017;**15**(1):141. DOI: 10.1186/

[111] Huang Y et al. Platelet-rich plasma for regeneration of neural feedback pathways around dental implants: A

DSS.0000000000001197

tea.2018.0065

DOI: 10.1186/ar4493

acra.2014.05.003

sicotj/2017036

s12967-017-1242-4

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

on the use of platelet-rich plasma in women with female androgenetic alopecia. Dermatologic Surgery. 2018;**44**(1):132-133. DOI: 10.1097/ DSS.0000000000001197

*Contemporary Topics about Phosphorus in Biology and Materials*

Hematology. 2014;**21**(5):388-394. DOI: 10.1097/MOH.0000000000000069

coagulation. Bioconjugate Chemistry. 2016;**27**(1):102-109. DOI: 10.1021/acs.

[99] Zhu S et al. FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow. Blood. 2015;**126**(12):1494-1502. DOI: 10.1182/blood-2015-04-641472

[100] Baik SY et al. Effects of platelet lysate preparations on the proliferation of HaCaT cells. Annals of Laboratory Medicine. 2014;**34**(1):43-50. DOI:

[101] Carter MJ, Fylling CP, Parnell LK. Use of platelet rich plasma gel on wound healing: A systematic review and meta-analysis. Eplasty. 2011;**11**:e38

[102] Porwal S, Chahar YS, Singh PK. A comparative study of combined dermaroller and platelet-rich plasma versus dermaroller alone in acne scars and assessment of quality of life before and after treatment. Indian Journal of Dermatology. 2018;**63**(5):403-408. DOI:

[103] Alser OH, Goutos I. The evidence behind the use of platelet-rich plasma (PRP) in scar management: A literature

review. Scars, Burns & Healing. 2018;**4**:2059513118808773. DOI: 10.1177/2059513118808773

[104] Garg S, Manchanda S. Plateletrich plasma-an 'Elixir' for treatment of alopecia: Personal experience on 117 patients with review of literature. Stem Cell Investigation. 2017;**4**:64. DOI:

[105] Alves R, Grimalt R. Doubleblind, placebo-controlled pilot study

10.3343/alm.2014.34.1.43

10.4103/ijd.IJD\_118\_17

10.21037/sci.2017.06.07

[98] Szymusiak M et al. Colloidal confinement of polyphosphate on gold nanoparticles robustly activates

the contact pathway of blood

bioconjchem.5b00524

Basic Microbiology. 2017;**57**(11):982- 986. DOI: 10.1002/jobm.201700257

manganese concentration triggers changes in inorganic polyphosphates. FEMS Yeast Research. 2013;**13**(5):463-470. DOI:

[91] Dinarvand P et al. Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor. Blood. 2014;**123**(6):935-945. DOI: 10.1182/blood-2013-09-529602

[92] Tsutsumi K et al. Morphogenetic study on the maturation of osteoblastic cell as induced by inorganic polyphosphate. PLoS ONE. 2014;**9**(2):e86834. DOI: 10.1371/journal.pone.0086834

[93] Xie L, Jakob U. Inorganic polyphosphate, a multifunctional polyanionic protein scaffold. The Journal of Biological Chemistry. 2019;**294**(6):2180-2190. DOI: 10.1074/

[94] Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011;**118**(26):6963-6970. DOI: 10.1182/blood-2011-07-368811

[95] Smith SA et al. Inhibition of polyphosphate as a novel strategy for preventing thrombosis

2012;**120**(26):5103-5110. DOI: 10.1182/

[96] Smith SA et al. Polyphosphate exerts differential effects on blood clotting, depending on polymer size. Blood. 2010;**116**(20):4353-4359. DOI: 10.1182/blood-2010-01-266791

[97] Smith SA, Morrissey JH. Polyphosphate: A new player in the field of hemostasis. Current Opinion in

and inflammation. Blood.

blood-2012-07-444935

jbc.REV118.002808

[90] Andreeva N et al. Adaptation of *Saccharomyces cerevisiae* to toxic

10.1111/1567-1364.12049

**90**

[106] Liou JJ et al. Effect of platelet-rich plasma on chondrogenic differentiation of adipose- and bone marrow-derived mesenchymal stem cells. Tissue Engineering. Part A. 2018;**24**(19- 20):1432-1443. DOI: 10.1089/ten. tea.2018.0065

[107] Xie X, Zhang C, Tuan RS. Biology of platelet-rich plasma and its clinical application in cartilage repair. Arthritis Research & Therapy. 2014;**16**(1):204. DOI: 10.1186/ar4493

[108] Chiavaras MM et al. IMpact of Platelet Rich plasma OVer alternative therapies in patients with lateral epicondylitis (IMPROVE): Protocol for a multicenter randomized controlled study: A multicenter, randomized trial comparing autologous platelet-rich plasma, autologous whole blood, dry needle tendon fenestration, and physical therapy exercises alone on pain and quality of life in patients with lateral epicondylitis. Academic Radiology. 2014;**21**(9):1144-1155. DOI: 10.1016/j. acra.2014.05.003

[109] Hussain N, Johal H, Bhandari M. An evidence-based evaluation on the use of platelet rich plasma in orthopedics: A review of the literature. SICOT J. 2017;**3**:57. DOI: 10.1051/ sicotj/2017036

[110] Bansal H et al. Intra-articular injection in the knee of adipose derived stromal cells (stromal vascular fraction) and platelet rich plasma for osteoarthritis. Journal of Translational Medicine. 2017;**15**(1):141. DOI: 10.1186/ s12967-017-1242-4

[111] Huang Y et al. Platelet-rich plasma for regeneration of neural feedback pathways around dental implants: A

concise review and outlook on future possibilities. International Journal of Oral Science. 2017;**9**(1):1-9. DOI: 10.1038/ijos.2017.1

[112] Agrawal AA. Evolution, current status and advances in application of platelet concentrate in periodontics and implantology. World Journal of Clinical Cases. 2017;**5**(5):159-171. DOI: 10.12998/wjcc.v5.i5.159

[113] Picard F et al. The growing evidence for the use of platelet-rich plasma on diabetic chronic wounds: A review and a proposal for a new standard care. Wound Repair and Regeneration. 2015;**23**(5):638-643. DOI: 10.1111/wrr.12317

[114] Picard F et al. Should we use platelet-rich plasma as an adjunct therapy to treat "acute wounds," "burns," and "laser therapies": A review and a proposal of a quality criteria checklist for further studies. Wound Repair and Regeneration. 2015;**23**(2):163-170. DOI: 10.1111/ wrr.12266

[115] Yeung CY et al. Efficacy of lyophilised platelet-rich plasma powder on healing rate in patients with deep second degree burn injury: A prospective double-blind randomized clinical trial. Annals of Plastic Surgery. 2018;**80**(2S Suppl 1):S66-S69. DOI: 10.1097/SAP.0000000000001328

[116] Hara T et al. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway. Journal of Artificial Organs. 2016;**19**(4):372-377. DOI: 10.1007/ s10047-016-0913-x

[117] Kakudo N et al. Platelet-rich plasma promotes epithelialization and angiogenesis in a splitthickness skin graft donor site. Medical Molecular Morphology. 2011;**44**(4):233-236. DOI: 10.1007/s00795-010-0532-1

[118] Venter NG et al. Use of platelet-rich plasma in deep second- and thirddegree burns. Burns. 2016;**42**(4):807- 814. DOI: 10.1016/j.burns.2016.01.002

[119] Alves R, Grimalt R. A review of platelet-rich plasma: History, biology, mechanism of action, and classification. Skin Appendage Disorders. 2018;**4**(1):18-24. DOI: 10.1159/000477353

[120] Arnoczky SP, Sheibani-Rad S. The basic science of platelet-rich plasma (PRP): What clinicians need to know. Sports Medicine and Arthroscopy Review. 2013;**21**(4):180-185. DOI: 10.1097/JSA.0b013e3182999712

[121] Martinengo L et al. Prevalence of chronic wounds in the general population: Systematic review and meta-analysis of observational studies. Annals of Epidemiology. 2019;**29**:8-15. DOI: 10.1016/j.annepidem.2018.10.005

[122] Brick N. Autologous platelet-rich plasma for treating chronic wounds. The American Journal of Nursing. 2013;**113**(8):54. DOI: 10.1097/01. NAJ.0000432965.18634.85

[123] Ahmed M et al. Platelet-rich plasma for the treatment of clean diabetic foot ulcers. Annals of Vascular Surgery. 2017;**38**:206-211. DOI: 10.1016/j.avsg.2016.04.023

[124] Guo SC et al. Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model. Theranostics. 2017;**7**(1):81-96. DOI: 10.7150/thno.16803

[125] Ding Y et al. Platelet-rich fibrin accelerates skin wound healing in diabetic mice. Annals of Plastic Surgery. 2017;**79**(3):e15-e19. DOI: 10.1097/ SAP.0000000000001091

[126] Tambella AM et al. Platelet-rich plasma to treat experimentally-induced skin wounds in animals: A systematic

review and meta-analysis. PLoS ONE. 2018;**13**(1):e0191093. DOI: 10.1371/ journal.pone.0191093

[127] Law JX et al. Platelet-rich plasma with keratinocytes and fibroblasts enhance healing of full-thickness wounds. Journal of Tissue Viability. 2017;**26**(3):208-215. DOI: 10.1016/j. jtv.2017.05.003

[128] Long DW et al. Controlled delivery of platelet-derived proteins enhances porcine wound healing. Journal of Controlled Release. 2017;**253**:73-81. DOI: 10.1016/j.jconrel.2017.03.021

[129] Fernandez-Moure JS et al. Platelet-rich plasma: A biomimetic approach to enhancement of surgical wound healing. The Journal of Surgical Research. 2017;**207**:33-44. DOI: 10.1016/j.jss.2016.08.063

[130] Devereaux J et al. Effects of platelet-rich plasma and platelet-poor plasma on human dermal fibroblasts. Maturitas. 2018;**117**:34-44. DOI: 10.1016/j.maturitas.2018.09.001

[131] Maciel FB et al. Scanning electron microscopy and microbiological evaluation of equine burn wound repair after platelet-rich plasma gel treatment. Burns. 2012;**38**(7):1058-1065. DOI: 10.1016/j.burns.2012.02.029

[132] Huang SH et al. Platelet-rich plasma injection in burn scar areas alleviates neuropathic scar pain. International Journal of Medical Sciences. 2018;**15**(3):238-247. DOI: 10.7150/ijms.22563

[133] Klosova H et al. Objective evaluation of the effect of autologous platelet concentrate on post-operative scarring in deep burns. Burns. 2013;**39**(6):1263-1276. DOI: 10.1016/j.burns.2013.01.020

[134] Ozcelik U et al. Effect of topical platelet-rich plasma on burn healing after partial-thickness burn

**93**

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin…*

jth.13283

2018;**31**(1):17-22

2016;**14**(5):865-874. DOI: 10.1111/

[143] Luker JN et al. Shedding of the endothelial glycocalyx is quantitatively proportional to burn injury severity. Annals of Burns and Fire Disasters.

[144] Vigiola Cruz M et al. Plasma ameliorates endothelial dysfunction in burn injury. The Journal of Surgical Research. 2019;**233**:459-466. DOI:

[145] Messora MR, Nagata MJH, Furlaneto FAC, Dornelles RCM, Bomfim SRM, Deliberador TM, et al. A standardized research protocol for plateletrich plasma (PRP) preparation in rats. Revista Brasileira De Saude Ocupacional. 2011;**8**(3):299-304

[146] Galiano RD et al. Quantitative and reproducible murine model of excisional wound healing. Wound Repair and Regeneration. 2004;**12**(4):485-492. DOI:

10.1111/j.1067-1927.2004.12404.x

[148] Barrios M et al. Comparative hemostatic parameters in BALB/c, C57BL/6 and C3H/He mice. Thrombosis Research. 2009;**124**(3):338-343. DOI: 10.1016/j.thromres.2008.11.001

[149] Giacomini A et al. Platelet count and parameters determined by the Bayer ADVIA 120 in reference subjects and patients. Clinical and Laboratory Haematology. 2001;**23**(3):181-186

[150] Morrissey JH, Choi SH, Smith SA. Polyphosphate: An ancient molecule that links platelets,

blood-2012-03-306605

coagulation, and inflammation. Blood. 2012;**119**(25):5972-5979. DOI: 10.1182/

[147] Wang X et al. The mouse excisional wound splinting model, including applications for stem cell transplantation. Nature Protocols. 2013;**8**(2):302-309. DOI: 10.1038/

nprot.2013.002

10.1016/j.jss.2018.08.027

*DOI: http://dx.doi.org/10.5772/intechopen.87183*

injury. Medical Science Monitor.

[135] Prochazka V et al. Addition of platelet concentrate to dermo-epidermal skin graft in deep burn trauma reduces scarring and need for revision surgeries. Biomedical Papers of the Medical Faculty of the University Palacky, Olomouc, Czech Republic. 2014;**158**(2):242-258.

[136] Singer AJ et al. The effects of platelet rich plasma on healing of full thickness burns in swine. Burns. 2018;**44**(6):1543-1550. DOI: 10.1016/j.

[137] Marck RE, Middelkoop E,

Breederveld RS. Considerations on the use of platelet-rich plasma, specifically for burn treatment. Journal of Burn Care & Research. 2014;**35**(3):219-227. DOI: 10.1097/BCR.0b013e31829b334e

[138] Marck RE et al. The application of platelet-rich plasma in the treatment of deep dermal burns: A randomized, double-blind, intra-patient controlled study. Wound Repair and Regeneration. 2016;**24**(4):712-720. DOI: 10.1111/

[139] Pallua N, Wolter T, Markowicz M. Platelet-rich plasma in burns. Burns. 2010;**36**(1):4-8. DOI: 10.1016/j.

[140] Tejiram S et al. In-depth analysis of clotting dynamics in burn patients. The Journal of Surgical Research. 2016;**202**(2):341-351. DOI: 10.1016/j.

[141] Shupp JW et al. Analysis of factor XIa, factor IXa and tissue factor activity in burn patients. Burns. 2018;**44**(2):436- 444. DOI: 10.1016/j.burns.2017.08.003

[142] Glas GJ, Levi M, Schultz MJ. Coagulopathy and its management in patients with severe burns. Journal of Thrombosis and Haemostasis.

2016;**22**:1903-1909

DOI: 10.5507/bp.2013.070

burns.2018.04.021

wrr.12443

burns.2009.05.002

jss.2016.01.006

*Inorganic Polyphosphates Are Important for Cell Survival and Motility of Human Skin… DOI: http://dx.doi.org/10.5772/intechopen.87183*

injury. Medical Science Monitor. 2016;**22**:1903-1909

*Contemporary Topics about Phosphorus in Biology and Materials*

review and meta-analysis. PLoS ONE. 2018;**13**(1):e0191093. DOI: 10.1371/

[127] Law JX et al. Platelet-rich plasma with keratinocytes and fibroblasts enhance healing of full-thickness wounds. Journal of Tissue Viability. 2017;**26**(3):208-215. DOI: 10.1016/j.

[128] Long DW et al. Controlled delivery of platelet-derived proteins enhances porcine wound healing. Journal of Controlled Release. 2017;**253**:73-81. DOI: 10.1016/j.jconrel.2017.03.021

[129] Fernandez-Moure JS et al. Platelet-rich plasma: A biomimetic approach to enhancement of surgical wound healing. The Journal of Surgical

Research. 2017;**207**:33-44. DOI:

[130] Devereaux J et al. Effects of platelet-rich plasma and platelet-poor plasma on human dermal fibroblasts. Maturitas. 2018;**117**:34-44. DOI: 10.1016/j.maturitas.2018.09.001

[131] Maciel FB et al. Scanning electron microscopy and microbiological

evaluation of equine burn wound repair after platelet-rich plasma gel treatment. Burns. 2012;**38**(7):1058-1065. DOI: 10.1016/j.burns.2012.02.029

[133] Klosova H et al. Objective evaluation of the effect of autologous platelet concentrate on post-operative scarring in deep burns. Burns. 2013;**39**(6):1263-1276.

DOI: 10.1016/j.burns.2013.01.020

[134] Ozcelik U et al. Effect of topical platelet-rich plasma on burn healing after partial-thickness burn

[132] Huang SH et al. Platelet-rich plasma injection in burn scar areas alleviates neuropathic scar pain. International Journal of Medical Sciences. 2018;**15**(3):238-247. DOI:

10.7150/ijms.22563

10.1016/j.jss.2016.08.063

journal.pone.0191093

jtv.2017.05.003

[118] Venter NG et al. Use of platelet-rich plasma in deep second- and thirddegree burns. Burns. 2016;**42**(4):807- 814. DOI: 10.1016/j.burns.2016.01.002

[119] Alves R, Grimalt R. A review of platelet-rich plasma: History, biology, mechanism of action, and classification. Skin Appendage Disorders. 2018;**4**(1):18-24. DOI:

[120] Arnoczky SP, Sheibani-Rad S. The basic science of platelet-rich plasma (PRP): What clinicians need to know. Sports Medicine and Arthroscopy Review. 2013;**21**(4):180-185. DOI: 10.1097/JSA.0b013e3182999712

[121] Martinengo L et al. Prevalence of chronic wounds in the general population: Systematic review and meta-analysis of observational studies. Annals of Epidemiology. 2019;**29**:8-15. DOI: 10.1016/j.annepidem.2018.10.005

[122] Brick N. Autologous platelet-rich plasma for treating chronic wounds. The American Journal of Nursing. 2013;**113**(8):54. DOI: 10.1097/01. NAJ.0000432965.18634.85

[123] Ahmed M et al. Platelet-rich plasma for the treatment of clean diabetic foot ulcers. Annals of Vascular

Surgery. 2017;**38**:206-211. DOI: 10.1016/j.avsg.2016.04.023

[124] Guo SC et al. Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model. Theranostics.

2017;**7**(1):81-96. DOI: 10.7150/thno.16803

[125] Ding Y et al. Platelet-rich fibrin accelerates skin wound healing in diabetic mice. Annals of Plastic Surgery. 2017;**79**(3):e15-e19. DOI: 10.1097/

[126] Tambella AM et al. Platelet-rich plasma to treat experimentally-induced skin wounds in animals: A systematic

SAP.0000000000001091

10.1159/000477353

**92**

[135] Prochazka V et al. Addition of platelet concentrate to dermo-epidermal skin graft in deep burn trauma reduces scarring and need for revision surgeries. Biomedical Papers of the Medical Faculty of the University Palacky, Olomouc, Czech Republic. 2014;**158**(2):242-258. DOI: 10.5507/bp.2013.070

[136] Singer AJ et al. The effects of platelet rich plasma on healing of full thickness burns in swine. Burns. 2018;**44**(6):1543-1550. DOI: 10.1016/j. burns.2018.04.021

[137] Marck RE, Middelkoop E, Breederveld RS. Considerations on the use of platelet-rich plasma, specifically for burn treatment. Journal of Burn Care & Research. 2014;**35**(3):219-227. DOI: 10.1097/BCR.0b013e31829b334e

[138] Marck RE et al. The application of platelet-rich plasma in the treatment of deep dermal burns: A randomized, double-blind, intra-patient controlled study. Wound Repair and Regeneration. 2016;**24**(4):712-720. DOI: 10.1111/ wrr.12443

[139] Pallua N, Wolter T, Markowicz M. Platelet-rich plasma in burns. Burns. 2010;**36**(1):4-8. DOI: 10.1016/j. burns.2009.05.002

[140] Tejiram S et al. In-depth analysis of clotting dynamics in burn patients. The Journal of Surgical Research. 2016;**202**(2):341-351. DOI: 10.1016/j. jss.2016.01.006

[141] Shupp JW et al. Analysis of factor XIa, factor IXa and tissue factor activity in burn patients. Burns. 2018;**44**(2):436- 444. DOI: 10.1016/j.burns.2017.08.003

[142] Glas GJ, Levi M, Schultz MJ. Coagulopathy and its management in patients with severe burns. Journal of Thrombosis and Haemostasis.

2016;**14**(5):865-874. DOI: 10.1111/ jth.13283

[143] Luker JN et al. Shedding of the endothelial glycocalyx is quantitatively proportional to burn injury severity. Annals of Burns and Fire Disasters. 2018;**31**(1):17-22

[144] Vigiola Cruz M et al. Plasma ameliorates endothelial dysfunction in burn injury. The Journal of Surgical Research. 2019;**233**:459-466. DOI: 10.1016/j.jss.2018.08.027

[145] Messora MR, Nagata MJH, Furlaneto FAC, Dornelles RCM, Bomfim SRM, Deliberador TM, et al. A standardized research protocol for plateletrich plasma (PRP) preparation in rats. Revista Brasileira De Saude Ocupacional. 2011;**8**(3):299-304

[146] Galiano RD et al. Quantitative and reproducible murine model of excisional wound healing. Wound Repair and Regeneration. 2004;**12**(4):485-492. DOI: 10.1111/j.1067-1927.2004.12404.x

[147] Wang X et al. The mouse excisional wound splinting model, including applications for stem cell transplantation. Nature Protocols. 2013;**8**(2):302-309. DOI: 10.1038/ nprot.2013.002

[148] Barrios M et al. Comparative hemostatic parameters in BALB/c, C57BL/6 and C3H/He mice. Thrombosis Research. 2009;**124**(3):338-343. DOI: 10.1016/j.thromres.2008.11.001

[149] Giacomini A et al. Platelet count and parameters determined by the Bayer ADVIA 120 in reference subjects and patients. Clinical and Laboratory Haematology. 2001;**23**(3):181-186

[150] Morrissey JH, Choi SH, Smith SA. Polyphosphate: An ancient molecule that links platelets, coagulation, and inflammation. Blood. 2012;**119**(25):5972-5979. DOI: 10.1182/ blood-2012-03-306605

**95**

**Figure 1.**

*acids.*

**Chapter 6**

**Abstract**

*Paweł Kafarski*

antibiotics, 31P NMR, genome mining

**1. Introduction**

Phosphonates: Their Natural

Occurrence and Physiological Role

The first natural compound containing carbon-to-phosphorus bond—ciliatine was discovered 60 years ago, and for four decades, phosphonates were considered simply as a biological curiosity. Finding the importance of these compounds in biogeochemical phosphorus cycling, their role in methane production, as well as discovery of numerous phosphonates and phosphonopeptides of promising antibacterial and antifungal activities has stimulated the development of studies on this class of compounds, especially on their metabolism and biochemistry. These studies are driven by the use of 31P NMR and by a clever combination of genomics and innovative chemistry by using the method of selective labeling of metabolites. These studies revealed unusual and interesting chemistry of these compounds.

**Keywords:** C—P bond, phosphonates, ciliatine, phosphonopeptides, mimetics,

Phosphonates are organophosphorus compounds characterized by a stable carbon-to-phosphorus (C—P) bond, which usually resists biochemical, thermal, and photochemical decomposition. The first phosphonate (compound **1**, **Figure 1**), being an analog of β-alanine and taurine, was isolated in 1959 from ciliated protozoa in the rumen of sheep [1]. That was the cause why its discoverers—M. Horiguchi

*Ciliatine (2-aminoethylphosphonic acids) and its derivatives found in lipids, glycans, glycoproteins, and bile* 

#### **Chapter 6**

## Phosphonates: Their Natural Occurrence and Physiological Role

*Paweł Kafarski*

#### **Abstract**

The first natural compound containing carbon-to-phosphorus bond—ciliatine was discovered 60 years ago, and for four decades, phosphonates were considered simply as a biological curiosity. Finding the importance of these compounds in biogeochemical phosphorus cycling, their role in methane production, as well as discovery of numerous phosphonates and phosphonopeptides of promising antibacterial and antifungal activities has stimulated the development of studies on this class of compounds, especially on their metabolism and biochemistry. These studies are driven by the use of 31P NMR and by a clever combination of genomics and innovative chemistry by using the method of selective labeling of metabolites. These studies revealed unusual and interesting chemistry of these compounds.

**Keywords:** C—P bond, phosphonates, ciliatine, phosphonopeptides, mimetics, antibiotics, 31P NMR, genome mining

#### **1. Introduction**

Phosphonates are organophosphorus compounds characterized by a stable carbon-to-phosphorus (C—P) bond, which usually resists biochemical, thermal, and photochemical decomposition. The first phosphonate (compound **1**, **Figure 1**), being an analog of β-alanine and taurine, was isolated in 1959 from ciliated protozoa in the rumen of sheep [1]. That was the cause why its discoverers—M. Horiguchi

#### **Figure 1.**

*Ciliatine (2-aminoethylphosphonic acids) and its derivatives found in lipids, glycans, glycoproteins, and bile acids.*

and M. Kandatsu, named it ciliatine. This amino acid was then considered as a possible marker of the content of protozoa in sheep rumen, which appeared further to be misleading. For many years, natural compounds containing the C—P bond had been considered as curiosity being only scarcely studied. This is not the case in science currently because of their involvement in the global phosphorus cycle and in oceanic methane production. Some aspects of their occurrence, environmental role, biochemistry, and biological functions have been reviewed [2–5]. This chapter will concentrate on discussion of chemical diversity of the naturally occurring phosphonates and on the indication of open problems, which have not yet been solved.

#### **2. Occurrence of carbon-to-phosphorus bond**

The discovery of ciliatine stimulated intensive studies on the distribution of phosphonates in nature. Despite the fact that early studies were hampered by the lack of simple and sensitive methods for the identification of the presence of carbon-to-phosphorus bond in natural samples, it was found to exist in protozoa, bacteria, coelenterates, and mollusks [6–11]. Presumably, the unbreakable record is held by the snail *Helisoma* sp. freshly laid eggs, which contain over 95% of total phosphorus in phosphonate form [12]. Upon embryonic development, phosphonate is converted into phosphoric acid and subsequently incorporated into cellular constituents. It is believed that the physiological role of incorporation of phosphonates into the lipid fraction might function as a means to protect the eggs against predators, because they are presumably not able to disrupt and digest such membranes.

The advent of 31P NMR for the analysis of tissue extracts, body fluids, and later—whole cells provided an effective tool for tracking the forms of phosphorus and its interchanges during organism development and growth. Quite paradoxically, the availability of 31P NMR was accompanied with a significant decrease in the number of papers dealing with distribution of phosphonates in various species. Applications of this simple technique enabled the determination of the presence of C—P bond in bacteria and bacterial communities [13, 14], cyanobacteria [15], sponges [16], higher fungi [17, 18], or even human specimens [19]. However, these studies did not explain if phosphonates are synthesized *de novo* or are introduced to these organisms by cohabiting organisms or diet. On the other hand, phosphonate xenobiotics are quite massively released into environment [20], and various organisms might use them, or products of their decomposition, as building blocks of more complex structures.

Next, gene-based methods for assessing the abundance and identity of biological phosphonate producers were applied. This approach based on knowledge regarding C—P compound biosynthesis. Thus, with a single exception [21], all the known phosphonates are derived from phosphoenolpyruvate by isomerization to phosphonopyruvate in a reaction catalyzed by the phosphoenolpyruvate mutase, followed by its fast utilization because the reaction of formation of the C—P bond is thermodynamically unfavorable (see **Figure 3**). Most common, decarboxylation of phosphonopyruvate by phosphonopyruvate decarboxylase to produce phosphonoacetaldehyde is the next, irreversible step [3, 22–24]. Mining in genome databases for genes related to these two enzymes, as well as their homologs, enabled to determine that 10–15% of bacterial species are able to produce phosphonates [23–25].

Discovery that phosphonates form around 10% of dissolved and particulate phosphorus in the oceans [15, 25, 26] brought the increasing recognition of the importance of these compounds in biogeochemical phosphorus cycling and an awareness of the interdependence between the global phosphorus cycle and those of the other biologically significant elements [27, 28]. It is important because

**97**

studies [42].

*Phosphonates: Their Natural Occurrence and Physiological Role*

a resident of the oxygen-rich regions of the open oceans [31, 32].

by finding several phosphonic acids in Murchison meteorite [39].

**3. Ciliatine (AEP, 2-aminoethylphosphonic acid)**

cation/characterization are difficult and cumbersome.

phosphorus availability has been shown to be a key determinant of marine phytoplankton productivity [15]. Phosphonates are mostly concentrated in dissolved organic phosphorus (DOP), an integral and dynamic part of the marine organic matter pool. The composition of the DOP pool is complex and largely unknown, but phosphonates account for one third of its high molecular weight fraction. Thus, they seem to be an important resource of this element for aquatic organisms; however, the understanding of their utilization by eukaryotic phytoplankton is severely limited [29, 30]. They most likely occur in a form of polysaccharides esterified with methylphosphonate (compound **2**, **Figure 1**) and 2-hydroxyethylphosphonate (compound **3**, **Figure 1**). These compounds have been mainly found in *Nitrosopumilus maritimus*, one of the most abundant organisms on the planet and

Up to 4% of the methane on Earth comes from the oxygen-rich waters through the cleavage of the highly unreactive carbon-to-phosphorus bond in methyl phosphonate [32]. The production of methylphosphonic acid (MPn) by cyanobacteria or marine archaea related to *N. maritimus* and its subsequent decomposition by phosphate-starved bacterioplankton may partially explain the production of methane in oceanic and lake surfaces [33–35]. The concentration of methane in the upper ocean being above equilibrium with the atmosphere is known as the oceanic

Some researchers believe that phosphonates are a form of relic of evolution. Being of slightly lower formal oxidation state, they might predominate in prebiotic reductive conditions [37]. This assumption, although debatable, finds some support

Ciliatine (compound **1**) is the most ubiquitous phosphonate present in lower organisms and occurs in remarkably high amounts. It is either presented in a free, unbound form being a common intermediate in numerous phosphonate biosynthetic pathways or incorporated into lipids and glycans. It is not surprising if considering that ciliatine is a formal analog of common component of lipids—phosphoethanolamine (compound **4**). Most of the studies on natural occurrence of ciliatine and its lipids had been published in 1960–1990 and are comprehensively reviewed [2–5]. Only single paper was published after this period. As shown in **Figure 1,** its methylated forms, namely *N*-methyl, *N*,*N*-dimethyl-, and *N*,*N*,*N*-trimethylciliatine (compounds **5**, **6,** and **7**), were also found in lipid fractions of some organisms albeit in significantly smaller quantities. Compound **7** is an analog of the most common component of lipids—phosphocholine (compound **8**). The presence of an unusual aminophosphonate—(R)-2-amino-1-hydroxyethylphosphonic acid (compound **9**) and its acetyl derivative has been determined in lipid fractions of *Bacteriovorax stolpii* [40, 41]. Its configuration was elegantly determined by a combination of chemical synthesis and biochemical

Lipids containing aminophosphonates are called phosphonolipids. There are two classes of these compounds—glycerophosphonolipids and sphingophosphonolipids (representative structures are shown in **Figure 2**). They have been isolated from numerous organisms including humans, mammals (sheep, goats, and rats), egg yolk, fish, insects, sea anemones, sponges, numerous species of freshwater and marine mollusks, seeds of plants, protozoa, and bacteria [3, 43–46]. Usually they are a small fraction of the total lipids present, and their isolation and exact identifi-

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

methane paradox [36, 38].

*Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

*Contemporary Topics about Phosphorus in Biology and Materials*

**2. Occurrence of carbon-to-phosphorus bond**

and M. Kandatsu, named it ciliatine. This amino acid was then considered as a possible marker of the content of protozoa in sheep rumen, which appeared further to be misleading. For many years, natural compounds containing the C—P bond had been considered as curiosity being only scarcely studied. This is not the case in science currently because of their involvement in the global phosphorus cycle and in oceanic methane production. Some aspects of their occurrence, environmental role, biochemistry, and biological functions have been reviewed [2–5]. This chapter will concentrate on discussion of chemical diversity of the naturally occurring phosphonates and on the indication of open problems, which have not yet been solved.

The discovery of ciliatine stimulated intensive studies on the distribution of phosphonates in nature. Despite the fact that early studies were hampered by the lack of simple and sensitive methods for the identification of the presence of carbon-to-phosphorus bond in natural samples, it was found to exist in protozoa, bacteria, coelenterates, and mollusks [6–11]. Presumably, the unbreakable record is held by the snail *Helisoma* sp. freshly laid eggs, which contain over 95% of total phosphorus in phosphonate form [12]. Upon embryonic development, phosphonate is converted into phosphoric acid and subsequently incorporated into cellular constituents. It is believed that the physiological role of incorporation of phosphonates into the lipid fraction might function as a means to protect the eggs against predators, because they are presumably not able to disrupt and digest such membranes. The advent of 31P NMR for the analysis of tissue extracts, body fluids, and later—whole cells provided an effective tool for tracking the forms of phosphorus and its interchanges during organism development and growth. Quite paradoxically, the availability of 31P NMR was accompanied with a significant decrease in the number of papers dealing with distribution of phosphonates in various species. Applications of this simple technique enabled the determination of the presence of C—P bond in bacteria and bacterial communities [13, 14], cyanobacteria [15], sponges [16], higher fungi [17, 18], or even human specimens [19]. However, these studies did not explain if phosphonates are synthesized *de novo* or are introduced to these organisms by cohabiting organisms or diet. On the other hand, phosphonate xenobiotics are quite massively released into environment [20], and various organisms might use them, or products of their decomposition, as building blocks of

Next, gene-based methods for assessing the abundance and identity of biological phosphonate producers were applied. This approach based on knowledge regarding C—P compound biosynthesis. Thus, with a single exception [21], all the known phosphonates are derived from phosphoenolpyruvate by isomerization to phosphonopyruvate in a reaction catalyzed by the phosphoenolpyruvate mutase, followed by its fast utilization because the reaction of formation of the C—P bond is thermodynamically unfavorable (see **Figure 3**). Most common, decarboxylation of phosphonopyruvate by phosphonopyruvate decarboxylase to produce phosphonoacetaldehyde is the next, irreversible step [3, 22–24]. Mining in genome databases for genes related to these two enzymes, as well as their homologs, enabled to determine that 10–15% of bacterial species are able to produce phosphonates [23–25]. Discovery that phosphonates form around 10% of dissolved and particulate phosphorus in the oceans [15, 25, 26] brought the increasing recognition of the importance of these compounds in biogeochemical phosphorus cycling and an awareness of the interdependence between the global phosphorus cycle and those of the other biologically significant elements [27, 28]. It is important because

**96**

more complex structures.

phosphorus availability has been shown to be a key determinant of marine phytoplankton productivity [15]. Phosphonates are mostly concentrated in dissolved organic phosphorus (DOP), an integral and dynamic part of the marine organic matter pool. The composition of the DOP pool is complex and largely unknown, but phosphonates account for one third of its high molecular weight fraction. Thus, they seem to be an important resource of this element for aquatic organisms; however, the understanding of their utilization by eukaryotic phytoplankton is severely limited [29, 30]. They most likely occur in a form of polysaccharides esterified with methylphosphonate (compound **2**, **Figure 1**) and 2-hydroxyethylphosphonate (compound **3**, **Figure 1**). These compounds have been mainly found in *Nitrosopumilus maritimus*, one of the most abundant organisms on the planet and a resident of the oxygen-rich regions of the open oceans [31, 32].

Up to 4% of the methane on Earth comes from the oxygen-rich waters through the cleavage of the highly unreactive carbon-to-phosphorus bond in methyl phosphonate [32]. The production of methylphosphonic acid (MPn) by cyanobacteria or marine archaea related to *N. maritimus* and its subsequent decomposition by phosphate-starved bacterioplankton may partially explain the production of methane in oceanic and lake surfaces [33–35]. The concentration of methane in the upper ocean being above equilibrium with the atmosphere is known as the oceanic methane paradox [36, 38].

Some researchers believe that phosphonates are a form of relic of evolution. Being of slightly lower formal oxidation state, they might predominate in prebiotic reductive conditions [37]. This assumption, although debatable, finds some support by finding several phosphonic acids in Murchison meteorite [39].

#### **3. Ciliatine (AEP, 2-aminoethylphosphonic acid)**

Ciliatine (compound **1**) is the most ubiquitous phosphonate present in lower organisms and occurs in remarkably high amounts. It is either presented in a free, unbound form being a common intermediate in numerous phosphonate biosynthetic pathways or incorporated into lipids and glycans. It is not surprising if considering that ciliatine is a formal analog of common component of lipids—phosphoethanolamine (compound **4**). Most of the studies on natural occurrence of ciliatine and its lipids had been published in 1960–1990 and are comprehensively reviewed [2–5]. Only single paper was published after this period. As shown in **Figure 1,** its methylated forms, namely *N*-methyl, *N*,*N*-dimethyl-, and *N*,*N*,*N*-trimethylciliatine (compounds **5**, **6,** and **7**), were also found in lipid fractions of some organisms albeit in significantly smaller quantities. Compound **7** is an analog of the most common component of lipids—phosphocholine (compound **8**). The presence of an unusual aminophosphonate—(R)-2-amino-1-hydroxyethylphosphonic acid (compound **9**) and its acetyl derivative has been determined in lipid fractions of *Bacteriovorax stolpii* [40, 41]. Its configuration was elegantly determined by a combination of chemical synthesis and biochemical studies [42].

Lipids containing aminophosphonates are called phosphonolipids. There are two classes of these compounds—glycerophosphonolipids and sphingophosphonolipids (representative structures are shown in **Figure 2**). They have been isolated from numerous organisms including humans, mammals (sheep, goats, and rats), egg yolk, fish, insects, sea anemones, sponges, numerous species of freshwater and marine mollusks, seeds of plants, protozoa, and bacteria [3, 43–46]. Usually they are a small fraction of the total lipids present, and their isolation and exact identification/characterization are difficult and cumbersome.

The physiologic function of phosphonolipids is still unknown, and the suggested protecting role against predators resulting from their stability toward hydrolysis by lipases and phosphatases has not been proved so far. Moreover, the distribution and abundance of phosphonolipids among organisms vary with species, tissue, or cellular location. For example, vertebrates have sphingophosphonolipids as components of nervous tissue sphingomyelin, while invertebrates frequently contain high levels of these lipids as outer membrane components.

Whereas phosphate is a common modification of polysaccharides, there are only a few examples of polysaccharides containing phosphonate moieties. Their characterization/identification was made possible as well as substantially accelerated by the development of glycomics [47]. Ciliatine and compound **9** have been found to be bound to the sugar moieties of variable glycans (see **Figure 2** for schematic structures). Their occurrence was documented in fractions of glycocerebrosides (lipids) of many lower marine phyla [2, 12, 48], bacterial exopolysaccharides (secreted polysugars into the environment), and outer membrane components

**99**

**Figure 3**.

pounds related to ciliatine.

*Phosphonates: Their Natural Occurrence and Physiological Role*

[49–51] and glycoproteins deriving from marine snails, common jellyfish and locust [51–54]. Genome scanning led to the identification of methylphosphonic acid (compound **2**) in the exopolysaccharide of the marine archeon *N. maritimus*. Its function is not known, but it is ultimately a major source of methane production by

Similarly as in the case of phosphonolipids, the physiological role of phosphonoglycans is not known and thus awaits determination. This might be important in the context that the glycans are essential molecules being well known to enable adaptive response to environmental changes [55]. The speculative roles of phosphonoglycans include cell-cell signaling or their action as phosphorus reservoirs in the environments of low phosphate concentration. The second assumption might be supported by the conservation of phosphonolipids at the expense of phosphodiesters in starved conditions by the oyster *Crassostrea virginica* [56]. Other possibility is demonstrated by the fact that *Bacteroides fragilis*, a part of the normal microbiota of the human colon, produces a capsular polysaccharide complex containing ciliatine, which is directly involved in abscess formation in animal models when bacteria are

It is also important to mention that the phosphonic analog of taurocholic acid was found in the gall bladders of cows [58]; however, this finding may require

Biosynthesis of phosphonates starts from rearrangement of phosphoenolpyruvate (compound **10**) into phosphonopyruvate (compound **11**), a reaction catalyzed by phosphonoenolpyruvate mutase. In this equilibrium process, the thermodynamics favors phosphonoenolpyruvate by a factor of at least 500. Thus, phosphonopyruvate has to be rapidly converted into metabolically useful compounds, most favorably in the irreversible reactions. Consequently, it is a key substrate in the synthesis of ciliatine (compound **1**), phosphonoalalnine (compound **12**), 2-hydroxyethylphosphonic acid (compound **3**), phosphonoacetaldehyde (compound **13**), phosphonomethylmalic acid (compound **14**), and 2-keto-4-hydroxy-5-phosphonopentanoic acid (compound **15**). Most of the enzymes involved in the production of these compounds have been isolated and characterized and comprehensively reviewed [2, 59]. The metabolic relationships between these compounds and their precursor role in the synthesis of phosphonate antibiotics are shown in

Low-molecular antibiotics such as fosfomycin (compound **17**) [60], fosfonochlorin (compound **18** produced by several strains of *Fusarium* and *Talaromyces flavus*) [61], nitrilaphos and hydroxynitrilaphos (compounds **19** and **20** found in cultivating media of *Streptomyces*) [62]**,** and herbicidal phosphonothrixin (compound **21**, produced by *Saccharothrix*) [63] might be also considered as low-molecular com-

Only one of them—*fosfomycin* (also known as *Monuril*, *Monurol,* or *Monural*), produced by *Pseudomonas* and *Streptomyces,* has found limited use as therapeutic agent to cure urinary tract infections and diabetic foot [60]. It is an active site directed covalent inactivator of muramyl ligase A, the first enzyme of peptidoglycan synthesis, and causes disruption of bacterial cell wall. Unfortunately, bacteria adapted to be able to open the epoxy ring functionality of fosfomycin, thus resulting in the compound deactivation/degradation of this antibiotic and in the microorganism ability to readily develop drug resistance [64]. Quite interestingly, pathways for the biosynthesis of fosfomycin in *Streptomyces* and *Pseudomonas* are different

**4. Low-molecular phosphonates metabolically related to ciliatine**

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

displaced into the bloodstream [57].

additional confirmation.

the oceans [32].

*Contemporary Topics about Phosphorus in Biology and Materials*

levels of these lipids as outer membrane components.

The physiologic function of phosphonolipids is still unknown, and the suggested protecting role against predators resulting from their stability toward hydrolysis by lipases and phosphatases has not been proved so far. Moreover, the distribution and abundance of phosphonolipids among organisms vary with species, tissue, or cellular location. For example, vertebrates have sphingophosphonolipids as components of nervous tissue sphingomyelin, while invertebrates frequently contain high

Whereas phosphate is a common modification of polysaccharides, there are only a few examples of polysaccharides containing phosphonate moieties. Their characterization/identification was made possible as well as substantially accelerated by the development of glycomics [47]. Ciliatine and compound **9** have been found to be bound to the sugar moieties of variable glycans (see **Figure 2** for schematic structures). Their occurrence was documented in fractions of glycocerebrosides (lipids) of many lower marine phyla [2, 12, 48], bacterial exopolysaccharides (secreted polysugars into the environment), and outer membrane components

**98**

**Figure 2.**

*Representative structures of phosphonolipids, phosphonoglycans, and phosphonosteroid.*

[49–51] and glycoproteins deriving from marine snails, common jellyfish and locust [51–54]. Genome scanning led to the identification of methylphosphonic acid (compound **2**) in the exopolysaccharide of the marine archeon *N. maritimus*. Its function is not known, but it is ultimately a major source of methane production by the oceans [32].

Similarly as in the case of phosphonolipids, the physiological role of phosphonoglycans is not known and thus awaits determination. This might be important in the context that the glycans are essential molecules being well known to enable adaptive response to environmental changes [55]. The speculative roles of phosphonoglycans include cell-cell signaling or their action as phosphorus reservoirs in the environments of low phosphate concentration. The second assumption might be supported by the conservation of phosphonolipids at the expense of phosphodiesters in starved conditions by the oyster *Crassostrea virginica* [56]. Other possibility is demonstrated by the fact that *Bacteroides fragilis*, a part of the normal microbiota of the human colon, produces a capsular polysaccharide complex containing ciliatine, which is directly involved in abscess formation in animal models when bacteria are displaced into the bloodstream [57].

It is also important to mention that the phosphonic analog of taurocholic acid was found in the gall bladders of cows [58]; however, this finding may require additional confirmation.

#### **4. Low-molecular phosphonates metabolically related to ciliatine**

Biosynthesis of phosphonates starts from rearrangement of phosphoenolpyruvate (compound **10**) into phosphonopyruvate (compound **11**), a reaction catalyzed by phosphonoenolpyruvate mutase. In this equilibrium process, the thermodynamics favors phosphonoenolpyruvate by a factor of at least 500. Thus, phosphonopyruvate has to be rapidly converted into metabolically useful compounds, most favorably in the irreversible reactions. Consequently, it is a key substrate in the synthesis of ciliatine (compound **1**), phosphonoalalnine (compound **12**), 2-hydroxyethylphosphonic acid (compound **3**), phosphonoacetaldehyde (compound **13**), phosphonomethylmalic acid (compound **14**), and 2-keto-4-hydroxy-5-phosphonopentanoic acid (compound **15**). Most of the enzymes involved in the production of these compounds have been isolated and characterized and comprehensively reviewed [2, 59]. The metabolic relationships between these compounds and their precursor role in the synthesis of phosphonate antibiotics are shown in **Figure 3**.

Low-molecular antibiotics such as fosfomycin (compound **17**) [60], fosfonochlorin (compound **18** produced by several strains of *Fusarium* and *Talaromyces flavus*) [61], nitrilaphos and hydroxynitrilaphos (compounds **19** and **20** found in cultivating media of *Streptomyces*) [62]**,** and herbicidal phosphonothrixin (compound **21**, produced by *Saccharothrix*) [63] might be also considered as low-molecular compounds related to ciliatine.

Only one of them—*fosfomycin* (also known as *Monuril*, *Monurol,* or *Monural*), produced by *Pseudomonas* and *Streptomyces,* has found limited use as therapeutic agent to cure urinary tract infections and diabetic foot [60]. It is an active site directed covalent inactivator of muramyl ligase A, the first enzyme of peptidoglycan synthesis, and causes disruption of bacterial cell wall. Unfortunately, bacteria adapted to be able to open the epoxy ring functionality of fosfomycin, thus resulting in the compound deactivation/degradation of this antibiotic and in the microorganism ability to readily develop drug resistance [64]. Quite interestingly, pathways for the biosynthesis of fosfomycin in *Streptomyces* and *Pseudomonas* are different

#### *Contemporary Topics about Phosphorus in Biology and Materials*

(see **Figure 3**). This shows that synthesis of natural phosphonates does not have to be normalized; many metabolic pathways are still yet to be discovered.

The separate class is aminophosphonate antibacterial antibiotics possessing an amino group in the gamma position in relation to the phosphonic functional group, namely fosmidomycin (compound **22**), and its derivatives denoted as FR900098 (compound **23**), FR-33289 (compound **24**), and FR32863 (compound **25**), originally isolated from culture broths of *Streptomyces* as well as cyclic SF2312 (compound **26**) isolated from *Micromonospora* sp. [65–67]. Their structures are shown in **Figure 4**.

Fosmidomycin and its homologs are potent inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis being active against a broad range of enterobacteria,

**Figure 3.**

*Metabolic relationship between naturally occurring phosphonates.*

**101**

in bialaphos metabolism.

action is shown schematically in **Figure 6**.

*Phosphonates: Their Natural Occurrence and Physiological Role*

agents (for representative structure, see **Figure 4**) [68].

but not against Gram-positive organisms or anaerobes. More importantly, they are blocking the development of isoprenoids in the parasite apicoplast, and thus, structurally modified fosmidomycin derivatives are considered as promising antimalarial

*Aphanizomenon flos-aquae* is a cyanobacterium that grows in eutrophic Balgavies Loch in Scotland. From its water blooms, a novel biosurfactant of lipidic character—2-acyloxyethylphosphonate (compound **27**) was isolated; however, its ecologi-

Two unusual placotylene A esters [69] of ciliatine (phosphoiodyn A, compound **28**) and its phosphate congener—phosphoethanolamine (phosphoiodyn B) were isolated from a Korean marine sponge *Placospongia* sp. [70]. Phosphoiodyn A was found to exhibit a potent agonistic activity on human peroxisome proliferatoractivated receptor delta (hPPARδ), which is thought to function as an integrator of transcriptional repression and nuclear receptor signaling [16, 71]. This compound, as well as its analogs, demonstrates significant neuroprotective activity in an *in vitro* cellular model indicating that such phosphonates may be an effective novel scaffold for the design of therapeutics for the treatment of neurodegenerative disorders [71].

Half of the century after the discovery of ciliatine witnessed a slow progress in the isolation and identification of natural compounds containing the C—P bond with most of them being antibacterials. The majority of these compounds appeared to be peptides containing C-terminal phosphonic acids and mostly differ by their *N*-terminal peptide structure. They have drawn attention not only because of their bioactivity but also because of unusual and interesting chemistry associated with the biosynthesis and biodegradation of these molecules. Structures of antibiotic

Bialaphos (compound **29**, [72]) was isolated from as the first such an antibiotic from the culture filtrates of *Streptomyces viridochromogenes* and *Streptomyces hygroscopicus* [72–74]. Further studies indicated that its antibacterial activity is a result of active transport of the peptide across bacterial membrane followed by hydrolysis of the peptide and release of terminal phosphonate—phosphinothricin, which inhibits glutamine synthetase. This enzyme converts glutamic acid and ammonia into glutamine; this reaction is an important step of the nitrogen metabolism in bacteria and plants [75]. That activity of phosphinothricin resulted in its introduction to agriculture as a popular herbicide, and it is sold as ammonium salt under the name glufosinate. Its application causes accumulation of ammonia in plants and consequently plant death [76]. It is worth to notice that bialaphos also exerts herbicidal activity and was applied in Japan [77]. Its activity relays on hydrolysis of bialaphos in plant tissues and release of herbicidal phosphinothricin. Further studies on bialaphos resulted in isolation of tetrapeptide trialaphos (compound **30**) [78] and phosalacine (compound **31**) [79] both of the same mechanism of action. Finally, studies on biosynthesis of this compound resulted in the identification of its desmethyl analog **32**, which is an intermediate

The antibacterial activity of bialaphos is typical for all the phosphonopeptides. Peptide parts of these antibiotics usually function as a targeting unit. Thus, the peptides are efficiently transported through bacterial (or fungal) membranes and after hydrolysis release phosphonic acid, which exerts its toxic action by inhibiting parasite vital enzymes—in this case glutamine synthetase. This mechanism of

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

cal function remains to be evaluated [69].

**5. Phosphonopeptide antibiotics**

phosphonopeptides are shown in **Figure 5**.

**Figure 4.** *Antibiotics structurally related to fosmidomycin.*

*Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

*Contemporary Topics about Phosphorus in Biology and Materials*

**Figure 4**.

(see **Figure 3**). This shows that synthesis of natural phosphonates does not have to

The separate class is aminophosphonate antibacterial antibiotics possessing an amino group in the gamma position in relation to the phosphonic functional group, namely fosmidomycin (compound **22**), and its derivatives denoted as FR900098 (compound **23**), FR-33289 (compound **24**), and FR32863 (compound **25**), originally isolated from culture broths of *Streptomyces* as well as cyclic SF2312 (compound **26**) isolated from *Micromonospora* sp. [65–67]. Their structures are shown in

Fosmidomycin and its homologs are potent inhibitors of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, an essential enzyme of the non-mevalonate pathway of isoprenoid biosynthesis being active against a broad range of enterobacteria,

be normalized; many metabolic pathways are still yet to be discovered.

**100**

**Figure 4.**

**Figure 3.**

*Antibiotics structurally related to fosmidomycin.*

*Metabolic relationship between naturally occurring phosphonates.*

but not against Gram-positive organisms or anaerobes. More importantly, they are blocking the development of isoprenoids in the parasite apicoplast, and thus, structurally modified fosmidomycin derivatives are considered as promising antimalarial agents (for representative structure, see **Figure 4**) [68].

*Aphanizomenon flos-aquae* is a cyanobacterium that grows in eutrophic Balgavies Loch in Scotland. From its water blooms, a novel biosurfactant of lipidic character—2-acyloxyethylphosphonate (compound **27**) was isolated; however, its ecological function remains to be evaluated [69].

Two unusual placotylene A esters [69] of ciliatine (phosphoiodyn A, compound **28**) and its phosphate congener—phosphoethanolamine (phosphoiodyn B) were isolated from a Korean marine sponge *Placospongia* sp. [70]. Phosphoiodyn A was found to exhibit a potent agonistic activity on human peroxisome proliferatoractivated receptor delta (hPPARδ), which is thought to function as an integrator of transcriptional repression and nuclear receptor signaling [16, 71]. This compound, as well as its analogs, demonstrates significant neuroprotective activity in an *in vitro* cellular model indicating that such phosphonates may be an effective novel scaffold for the design of therapeutics for the treatment of neurodegenerative disorders [71].

#### **5. Phosphonopeptide antibiotics**

Half of the century after the discovery of ciliatine witnessed a slow progress in the isolation and identification of natural compounds containing the C—P bond with most of them being antibacterials. The majority of these compounds appeared to be peptides containing C-terminal phosphonic acids and mostly differ by their *N*-terminal peptide structure. They have drawn attention not only because of their bioactivity but also because of unusual and interesting chemistry associated with the biosynthesis and biodegradation of these molecules. Structures of antibiotic phosphonopeptides are shown in **Figure 5**.

Bialaphos (compound **29**, [72]) was isolated from as the first such an antibiotic from the culture filtrates of *Streptomyces viridochromogenes* and *Streptomyces hygroscopicus* [72–74]. Further studies indicated that its antibacterial activity is a result of active transport of the peptide across bacterial membrane followed by hydrolysis of the peptide and release of terminal phosphonate—phosphinothricin, which inhibits glutamine synthetase. This enzyme converts glutamic acid and ammonia into glutamine; this reaction is an important step of the nitrogen metabolism in bacteria and plants [75]. That activity of phosphinothricin resulted in its introduction to agriculture as a popular herbicide, and it is sold as ammonium salt under the name glufosinate. Its application causes accumulation of ammonia in plants and consequently plant death [76]. It is worth to notice that bialaphos also exerts herbicidal activity and was applied in Japan [77]. Its activity relays on hydrolysis of bialaphos in plant tissues and release of herbicidal phosphinothricin. Further studies on bialaphos resulted in isolation of tetrapeptide trialaphos (compound **30**) [78] and phosalacine (compound **31**) [79] both of the same mechanism of action. Finally, studies on biosynthesis of this compound resulted in the identification of its desmethyl analog **32**, which is an intermediate in bialaphos metabolism.

The antibacterial activity of bialaphos is typical for all the phosphonopeptides. Peptide parts of these antibiotics usually function as a targeting unit. Thus, the peptides are efficiently transported through bacterial (or fungal) membranes and after hydrolysis release phosphonic acid, which exerts its toxic action by inhibiting parasite vital enzymes—in this case glutamine synthetase. This mechanism of action is shown schematically in **Figure 6**.

The following years brought the discovery of a family of antibiotics called rhizocticins (compounds **33–36**) [80, 81], plumbemycins (compounds **37** and **38**) [81–83], and phosacetamycin (compound **39**) [84], first isolated as secondary metabolites of *Bacillus subtilis* on the basis of their antifungal activity and were

**103**

pound **42**) [91].

*Phosphonates: Their Natural Occurrence and Physiological Role*

later found as products of *Streptomyces plumbeus*. They form a library of di- and tripeptides containing C-terminal (*Z*)-*L*-2-amino-5-phosphono-3-pentenoic acid, a mimetic of phosphonothreonine, which is the substrate for threonine synthetase. Thus, after the release from the peptide aminophosphonate acts as a potent inhibi-

Dehydrophos (compound **40**) was first isolated from the broth of *Streptomyces luridus* as a broad-spectrum antibiotic affective in chicken model of *Salmonella* infection [86]. The history of determination of its structure is rather long and led to three propositions of which the last one appeared to be reasonable and compelling. It is a dehydrophosphonopeptide, which, after the cleavage of the peptide bond, provides an analog of dehydroalanine, which is then converted into methyl acetylphosphonate (compound **41**, an analog of pyruvic acid), which is strongly antibacterial by acting most likely as antimetabolite of pyruvate (**Figure 6**) [87]. Thus, it was considered as a lead compound for the design of novel antibacterial agents [88]. The non-typical and innovative is the application of its biosynthetic enzymes for obtaining new antibacterial phosphonopeptides [89]. Recently, the role of nonribosomal peptidyl transferase DhpH in the formation of peptide bond in dehydrophos was studied in detail using phosphonic analog of alanine and various

Phosphonopeptides have very limited utility in human medicine because they are readily hydrolyzed in body fluids and released aminophosphonic acids that are not able to cross bacterial or fungal cell barriers and to exert antibiotic action.

Published in 2015 work of Metcalf and van der Donk brought a significant breakthrough in studies on naturally occurring phosphonate antibiotics. By a clever combination of the mining of the genome of 10,000 of actinomycetes and selective labeling of phosphonate metabolites, they rediscovered a large number of old phosphonates and discovered 19 new compounds [24]. This opened a genetic approach in natural phosphonate chemistry and biochemistry, especially enabling the identification of metabolic pathways leading to this class of compounds. An important and instructive example here is an activation of gene cluster from *Streptomyces* sp. NRRL F-525 and its reengineering in *Streptomyces lividans*, which resulted in the isolation of *O*-phosphonoacetic acid serine (com-

One of the examples of rediscovered compounds is fosfazinomycins A and B (compounds **43** and **44**), identified 30 years after their original isolation from *Streptomyces lavendofoliae* and *Streptomyces unzenensis* [92, 93]. They are a very specific since they contain an exotic structural feature, which is the hydrazide linkage between the carboxylic acid of peptidyl arginine and the phosphonic acid. Fosfazinomycin was also found further in one of 210 substances present in 42 actinomycetes associated with the Baltic sponge *Halichondria panacea* [94].

The genetic approach also enabled the isolation and characterization of novel of *Streptomyces* peptidomimetics such as argolaphos A and B (compounds **45** and **46**) and valinophos (compound **47**) [24]. Similar approach was used for the isolation of phosphonocystoximate and its hydroxylated derivative (compounds **48** and **49**) [24]. Detailed NMR studies on their biosynthesis, which starts from ciliatine and its analog—compound **9**, enabled to confirm the presence of intermediates such as mixtures of the (E)- and (Z)-isomers of corresponding oximes (compounds **50** and **51**), substrates for the synthesis of phosphonocystoximate and its hydroxylated derivative [95]. They are formed by the action of specific flavin-dependent, oxime-forming N-oxidases. These oxidases are also able to convert the oximes **50** and **51** into corresponding nitroethylphosphonates (compounds **52** and **53**) [96]. Structures of these intermediates and side products are depicted in **Figure 7**.

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

amino acid-tRNAs as substrates [90].

Additionally, they are being readily excreted through urine.

tor of this enzyme [85].

#### *Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

*Contemporary Topics about Phosphorus in Biology and Materials*

**102**

**Figure 6.**

*Representative mechanism of action of phosphonopeptides.*

**Figure 5.**

*Phosphonopeptide antibiotics.*

The following years brought the discovery of a family of antibiotics called rhizocticins (compounds **33–36**) [80, 81], plumbemycins (compounds **37** and **38**) [81–83], and phosacetamycin (compound **39**) [84], first isolated as secondary metabolites of *Bacillus subtilis* on the basis of their antifungal activity and were

later found as products of *Streptomyces plumbeus*. They form a library of di- and tripeptides containing C-terminal (*Z*)-*L*-2-amino-5-phosphono-3-pentenoic acid, a mimetic of phosphonothreonine, which is the substrate for threonine synthetase. Thus, after the release from the peptide aminophosphonate acts as a potent inhibitor of this enzyme [85].

Dehydrophos (compound **40**) was first isolated from the broth of *Streptomyces luridus* as a broad-spectrum antibiotic affective in chicken model of *Salmonella* infection [86]. The history of determination of its structure is rather long and led to three propositions of which the last one appeared to be reasonable and compelling. It is a dehydrophosphonopeptide, which, after the cleavage of the peptide bond, provides an analog of dehydroalanine, which is then converted into methyl acetylphosphonate (compound **41**, an analog of pyruvic acid), which is strongly antibacterial by acting most likely as antimetabolite of pyruvate (**Figure 6**) [87]. Thus, it was considered as a lead compound for the design of novel antibacterial agents [88]. The non-typical and innovative is the application of its biosynthetic enzymes for obtaining new antibacterial phosphonopeptides [89]. Recently, the role of nonribosomal peptidyl transferase DhpH in the formation of peptide bond in dehydrophos was studied in detail using phosphonic analog of alanine and various amino acid-tRNAs as substrates [90].

Phosphonopeptides have very limited utility in human medicine because they are readily hydrolyzed in body fluids and released aminophosphonic acids that are not able to cross bacterial or fungal cell barriers and to exert antibiotic action. Additionally, they are being readily excreted through urine.

Published in 2015 work of Metcalf and van der Donk brought a significant breakthrough in studies on naturally occurring phosphonate antibiotics. By a clever combination of the mining of the genome of 10,000 of actinomycetes and selective labeling of phosphonate metabolites, they rediscovered a large number of old phosphonates and discovered 19 new compounds [24]. This opened a genetic approach in natural phosphonate chemistry and biochemistry, especially enabling the identification of metabolic pathways leading to this class of compounds. An important and instructive example here is an activation of gene cluster from *Streptomyces* sp. NRRL F-525 and its reengineering in *Streptomyces lividans*, which resulted in the isolation of *O*-phosphonoacetic acid serine (compound **42**) [91].

One of the examples of rediscovered compounds is fosfazinomycins A and B (compounds **43** and **44**), identified 30 years after their original isolation from *Streptomyces lavendofoliae* and *Streptomyces unzenensis* [92, 93]. They are a very specific since they contain an exotic structural feature, which is the hydrazide linkage between the carboxylic acid of peptidyl arginine and the phosphonic acid. Fosfazinomycin was also found further in one of 210 substances present in 42 actinomycetes associated with the Baltic sponge *Halichondria panacea* [94].

The genetic approach also enabled the isolation and characterization of novel of *Streptomyces* peptidomimetics such as argolaphos A and B (compounds **45** and **46**) and valinophos (compound **47**) [24]. Similar approach was used for the isolation of phosphonocystoximate and its hydroxylated derivative (compounds **48** and **49**) [24]. Detailed NMR studies on their biosynthesis, which starts from ciliatine and its analog—compound **9**, enabled to confirm the presence of intermediates such as mixtures of the (E)- and (Z)-isomers of corresponding oximes (compounds **50** and **51**), substrates for the synthesis of phosphonocystoximate and its hydroxylated derivative [95]. They are formed by the action of specific flavin-dependent, oxime-forming N-oxidases. These oxidases are also able to convert the oximes **50** and **51** into corresponding nitroethylphosphonates (compounds **52** and **53**) [96]. Structures of these intermediates and side products are depicted in **Figure 7**.

#### **Figure 7.**

*Intermediates and side products in the synthesis of phosphonocystoximate.*

A separate group of phosphonic peptidomimetics is compounds denoted as K-26, K4, and I5B2 (compounds **54**, **55,** and **56,** respectively) [21, 97–99], a small family of bacterial secondary metabolites, tripeptides terminated by an unusual phosphonate analog of tyrosine (see **Figure 6**). They are produced by three different actinomycetales and act as potent inhibitors of human angiotensin-I converting enzyme selectively targeting the eukaryotic family of the enzyme [100, 101]. These compounds derived from *L*-tyrosine, which suggests the existence of novel and not discovered yet reaction of carbon-to-phosphorus bond formation [21, 102].

#### **6. Conclusions**

Natural phosphonates might be considered as simple analogs of phosphate esters and/or carboxylic acids. The inherent stability of the C—P bond causes that they often display promising activities as enzyme inhibitors and therefore might be considered as drugs or agrochemicals. Moreover, the wide use of xenobiotics containing carbon-to-phosphorus bond has led to the spread of these compounds in the environment, which may result in their incorporation into variable metabolic pathways. All of this stimulate interest in these, still somewhat exotic, compounds. The development of 31P NMR and genomics supplemented by biochemical studies resulted in the development of new detection technologies, which enormously speed out the discovery of novel naturally occurring phosphonates, identification of their metabolic pathways (both biosynthesis and degradation), and their use as lead compounds for the design of new promising medicines. With the exception of the identification of antibacterial and antifungal antibiotics, these studies are not accompanied, however, with the determination of physiologic importance of these compounds.

#### **Acknowledgements**

This work was supported by statuary grants of Wrocław University of Science and Technology and National Science Centre, Poland (grant 2016/21/B/ ST5/00115).

**105**

**Author details**

Paweł Kafarski

Science and Technology, Wrocław, Poland

provided the original work is properly cited.

\*Address all correspondence to: pawel.kafarski@pwr.edu.pl

*Phosphonates: Their Natural Occurrence and Physiological Role*

I declare that there is no conflict of interest that might have any bearing on

Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

**Conflict of interest**

research reported in this work.

*Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

### **Conflict of interest**

*Contemporary Topics about Phosphorus in Biology and Materials*

*Intermediates and side products in the synthesis of phosphonocystoximate.*

A separate group of phosphonic peptidomimetics is compounds denoted as K-26, K4, and I5B2 (compounds **54**, **55,** and **56,** respectively) [21, 97–99], a small family of bacterial secondary metabolites, tripeptides terminated by an unusual phosphonate analog of tyrosine (see **Figure 6**). They are produced by three different actinomycetales and act as potent inhibitors of human angiotensin-I converting enzyme selectively targeting the eukaryotic family of the enzyme [100, 101]. These compounds derived from *L*-tyrosine, which suggests the existence of novel and not

discovered yet reaction of carbon-to-phosphorus bond formation [21, 102].

Natural phosphonates might be considered as simple analogs of phosphate esters and/or carboxylic acids. The inherent stability of the C—P bond causes that they often display promising activities as enzyme inhibitors and therefore might be considered as drugs or agrochemicals. Moreover, the wide use of xenobiotics containing carbon-to-phosphorus bond has led to the spread of these compounds in the environment, which may result in their incorporation into variable metabolic pathways. All of this stimulate interest in these, still somewhat exotic, compounds. The development of 31P NMR and genomics supplemented by biochemical studies resulted in the development of new detection technologies, which enormously speed out the discovery of novel naturally occurring phosphonates, identification of their metabolic pathways (both biosynthesis and degradation), and their use as lead compounds for the design of new promising medicines. With the exception of the identification of antibacterial and antifungal antibiotics, these studies are not accompanied, however, with the determination of physiologic importance of these

This work was supported by statuary grants of Wrocław University of Science and Technology and National Science Centre, Poland (grant 2016/21/B/

**104**

compounds.

ST5/00115).

**Acknowledgements**

**6. Conclusions**

**Figure 7.**

I declare that there is no conflict of interest that might have any bearing on research reported in this work.

### **Author details**

Paweł Kafarski

Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology, Wrocław, Poland

\*Address all correspondence to: pawel.kafarski@pwr.edu.pl

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Horiguchi M, Kandatsu M. Isolation of 2-aminoethane phosphonic acid from rumen protozoa. Nature. 1959;**184**:901-902. DOI: 10.1038/184901b0

[2] Horsman GP, Zechel DL. Phosphonate biochemistry. Chemical Reviews. 2017;**117**:5704-5783. DOI: 10.1021/acs.chemrev.6b00536

[3] Ju K-S, Doroghazi JR, Metcalf WW. Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways. Journal of Industrial Microbiology & Biotechnology. 2014;**421**:345-356. DOI: 10.1007/s10295-013-1375-2

[4] Mastalerz P, Kafarski P. Naturally occurring aminophsophonic and aminophosphinic acids. In: Kukhar VP, Hudson HR, editors. Aminophosphonic and Aminophosphinic Acids. Chichester: Wiley; 2000. pp. 1-31

[5] Petkowski JJ, Bains W, Seager S. Natural products containing "rare" organophosphorus functional groups. Molecules. 2019;**24**:866. DOI: 10.3390/ molecules24050866

[6] Kitterdge JS, Roberts E. A carbonphosphorus bond in nature. Science. 1969;**164**:37-42. DOI: 10.1126/ science.164.3875.37

[7] Horiguchi M. Occurrence, identification and properties of phosphonic and phosphinic acids. In: Hori T, Horiguchi M, Hayashi M, editors. Biochemistry of Natural C-P Compounds. Shiga: Japanese Association for Research on the Biochemistry of C-P Compounds; 1984. pp. 24-52

[8] Tamari M, Kametaka M. Isolation and identification of ciliatine (2-aminoethylphosphonic acid) from phospholipids of the oyster, *Crassostrea gigas*. Agricultural and Biological

Chemistry. 1972;**36**:1147-1152. DOI: 10.1271/bbb1961.36.1147

[9] Kittredge JS, Roberts E, Simonsen DG. The occurrence of free 2-aminoethylphosphonic acid in the aea anemone, *Anthopleura elegantissima*. Biochemistry. 1962;**1**:624-628. DOI: 10.1021/bi00910a013

[10] Liang CR, Rosenberg H. On the distribution and biosynthesis of 2-aminoethylphosphonate in two terrestrial molluscs. Comparative Biochemistry and Physiology. 1968;**25**:673-681. DOI: 10.1016/0010-406X(68)90377-0

[11] Kariotoglou DM, Mastronicolis SK. Sphingophosphonolipid molecular species from edible mollusks and a jellyfish. Comparative Biochemistry and Physiology. B. 2003;**136**:27-44. DOI: 10.1016/S1096-4959(03)00168-4

[12] Miceli MV, Henderson TO, Myers TC. Alkylphosphonic acid distribution in the planorbid snail *Helisoma* sp. Comparative Biochemistry and Physiology. B. 1987;**2**:603-611. DOI: 10.1016/0305-0491(87)90351-8

[13] Jayasimhulu K, Hunt SM, Kaneshiro ES. Detection and identification of *Bacteriovorax stolpii* UKi2 sphingophosphonol;ipid molecular species. Journal of the American Society for Mass Spectrometry. 2007;**18**:394- 403. DOI: 10.1016/j.jasms.2006.10.014

[14] Turner BL, Baxter R, Mathieu N, Sjögersten S, Whitton B. Phosphorus compounds in subartic Fennoscandian soils at the mountain birch (*Betula pubescens* )-Tundra ecotone. Soil Biology and Biochemistry. 2004;**36**:815-823. DOI: 10.1016/j.soilbio.2004.01.011

[15] Dyhrman ST, Benitez-Nelson CR, Orchard ED, Haley ST, Pellechia PJ. A microbial source of phosphonates

**107**

*Phosphonates: Their Natural Occurrence and Physiological Role*

enzymology. Current Opinion in Chemical Biology. 2013;**19**:580-588. DOI: 10.1016/j.cbpa.2013.06.018

[23] Villarreal-Chiu JF, Quinn JP,

2012;**3**:art.19. DOI: 10.3389/

10.1073/pnas.1315107110

pbio.0040383

[25] Scott KM, Sievert SM, Abril FN, Ball LA, Barrett CJ, Blake RA, et al. The genome of deep-see vent chemolithoautitroph *Thiomicrospira crunogena* XCL-2. PLoS Biology. 2006;**4**:e383. DOI: 10.1371/journal.

[26] Whitney LP, Lomas LW.

[27] Chin JP, McGrath JW, Quinn JP. Microbial transformations in phosphonate biosynthesis and catabolism, and their importance in nutrient cycling. Current Opinion in Chemical Biology. 2016;**31**:50-57. DOI:

DOI: 10.1002/lol2.10100

10.1016/j.cbpa.2016.01.010

Villanueva M, Jetten MSM. Metagenomic analysis of nitrogen and methane cycling in the Arabian Sea oxygen minimum zone. PeerJ. 2016;**4**:e1924. DOI: 10.7717/peerj.1924

[28] Lüke C, Speeth DR, Kox MAR,

[29] Born DA, Ulrich EC, Ju K-S, Peck S, van der Donk WA, Drennan CL. Structural basis for

Phosphonate utilization by eukaryotic phytoplankton. Limnology and Oceanography Letters. 2019;**4**:18-24.

[24] Yu JX, Doroghazi JR, Janga SC, Zhang JK, Circello B, Grifiinj BM, et al. Diversity and abundance of phosphonate biosynthetic genes in nature. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:20759-20764. DOI:

fmicb.2012.00019

McGrath JW. The genes and enzymes of phosphonate metabolism by bacteria, and their distribution in the marine environment. Frontiers in Microbiology.

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

[16] Kim H, Chin J, Choi H, Baek K, Lee TG, Park SE, et al. Phosphoiodyns A and B, unique phosphorus-containing iodinated polyacetylenes from a

Korean sponge *Placospongia* sp. Organic Letters. 2013;**15**:100-103. DOI: 10.1021/

[17] Koukol O, Novák F, Hrabal R. Composition of the organic phosphorus fraction in basidiocarps of saprotrophic and mycorrhizal fungi. Soil Biology and Biochemistry. 2008;**40**:2464-2467. DOI: 10.1016/j.

[18] Maciejczyk E, Wieczorek D, Zwyrzykowska A, Halama M, Jasicka-Misiak I, Kafarski P. Phosphorus profile of basidomycetes. Phosphorus, Sulfur and Silicon and the Related Elements. 2015;**190**:763-768. DOI:

10.1080/10426507.2014.99

[19] Glonek T, Henderson TO,

Hilderbrand RL, Myers TC. Biological phosphonates: Determination by phosphorus-31 nuclear magnetic resonance. Science. 1970;**169**:172-174. DOI: 10.1126/science.169.3941.192

[20] Studnik H, Liebsch S, Forlani G, Wieczorek D, Kafarski P, Lipok J. Aminopolyphosphonates— Chemical features and practical uses, environmental durability and biodegradation. New Biotechnology.

[21] Ntai I, Manier ML, Hachey DL, Bachmann BO. Biosynthetic origins of C-P bond containing tripeptide K-26. Organic Letters. 2005;**7**:2763-2765. DOI:

[22] Peck SC, van der Donk WA. Phosphonate biosynthesis and

catabolism: A treasure trove of unusual

2015;**32**:1-6. DOI: 10.1016/j.

nbt.2014.06.007

10.1021/ol051091d

soilbio.2008.04.021

in oligotrophic marine systems. Nature Geoscience. 2009;**2**:699.

DOI: 10.1038/ngeo639

ol3031318

*Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

in oligotrophic marine systems. Nature Geoscience. 2009;**2**:699. DOI: 10.1038/ngeo639

[16] Kim H, Chin J, Choi H, Baek K, Lee TG, Park SE, et al. Phosphoiodyns A and B, unique phosphorus-containing iodinated polyacetylenes from a Korean sponge *Placospongia* sp. Organic Letters. 2013;**15**:100-103. DOI: 10.1021/ ol3031318

[17] Koukol O, Novák F, Hrabal R. Composition of the organic phosphorus fraction in basidiocarps of saprotrophic and mycorrhizal fungi. Soil Biology and Biochemistry. 2008;**40**:2464-2467. DOI: 10.1016/j. soilbio.2008.04.021

[18] Maciejczyk E, Wieczorek D, Zwyrzykowska A, Halama M, Jasicka-Misiak I, Kafarski P. Phosphorus profile of basidomycetes. Phosphorus, Sulfur and Silicon and the Related Elements. 2015;**190**:763-768. DOI: 10.1080/10426507.2014.99

[19] Glonek T, Henderson TO, Hilderbrand RL, Myers TC. Biological phosphonates: Determination by phosphorus-31 nuclear magnetic resonance. Science. 1970;**169**:172-174. DOI: 10.1126/science.169.3941.192

[20] Studnik H, Liebsch S, Forlani G, Wieczorek D, Kafarski P, Lipok J. Aminopolyphosphonates— Chemical features and practical uses, environmental durability and biodegradation. New Biotechnology. 2015;**32**:1-6. DOI: 10.1016/j. nbt.2014.06.007

[21] Ntai I, Manier ML, Hachey DL, Bachmann BO. Biosynthetic origins of C-P bond containing tripeptide K-26. Organic Letters. 2005;**7**:2763-2765. DOI: 10.1021/ol051091d

[22] Peck SC, van der Donk WA. Phosphonate biosynthesis and catabolism: A treasure trove of unusual enzymology. Current Opinion in Chemical Biology. 2013;**19**:580-588. DOI: 10.1016/j.cbpa.2013.06.018

[23] Villarreal-Chiu JF, Quinn JP, McGrath JW. The genes and enzymes of phosphonate metabolism by bacteria, and their distribution in the marine environment. Frontiers in Microbiology. 2012;**3**:art.19. DOI: 10.3389/ fmicb.2012.00019

[24] Yu JX, Doroghazi JR, Janga SC, Zhang JK, Circello B, Grifiinj BM, et al. Diversity and abundance of phosphonate biosynthetic genes in nature. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:20759-20764. DOI: 10.1073/pnas.1315107110

[25] Scott KM, Sievert SM, Abril FN, Ball LA, Barrett CJ, Blake RA, et al. The genome of deep-see vent chemolithoautitroph *Thiomicrospira crunogena* XCL-2. PLoS Biology. 2006;**4**:e383. DOI: 10.1371/journal. pbio.0040383

[26] Whitney LP, Lomas LW. Phosphonate utilization by eukaryotic phytoplankton. Limnology and Oceanography Letters. 2019;**4**:18-24. DOI: 10.1002/lol2.10100

[27] Chin JP, McGrath JW, Quinn JP. Microbial transformations in phosphonate biosynthesis and catabolism, and their importance in nutrient cycling. Current Opinion in Chemical Biology. 2016;**31**:50-57. DOI: 10.1016/j.cbpa.2016.01.010

[28] Lüke C, Speeth DR, Kox MAR, Villanueva M, Jetten MSM. Metagenomic analysis of nitrogen and methane cycling in the Arabian Sea oxygen minimum zone. PeerJ. 2016;**4**:e1924. DOI: 10.7717/peerj.1924

[29] Born DA, Ulrich EC, Ju K-S, Peck S, van der Donk WA, Drennan CL. Structural basis for

**106**

*Contemporary Topics about Phosphorus in Biology and Materials*

Chemistry. 1972;**36**:1147-1152. DOI:

[9] Kittredge JS, Roberts E, Simonsen

2-aminoethylphosphonic acid in the aea anemone, *Anthopleura elegantissima*. Biochemistry. 1962;**1**:624-628. DOI:

10.1271/bbb1961.36.1147

DG. The occurrence of free

[10] Liang CR, Rosenberg H. On the distribution and biosynthesis of 2-aminoethylphosphonate in two terrestrial molluscs. Comparative Biochemistry and Physiology. 1968;**25**:673-681. DOI: 10.1016/0010-406X(68)90377-0

[11] Kariotoglou DM, Mastronicolis SK. Sphingophosphonolipid molecular species from edible mollusks and a jellyfish. Comparative Biochemistry and Physiology. B. 2003;**136**:27-44. DOI: 10.1016/S1096-4959(03)00168-4

[12] Miceli MV, Henderson TO, Myers TC. Alkylphosphonic acid distribution in the planorbid snail *Helisoma* sp. Comparative Biochemistry and Physiology. B. 1987;**2**:603-611. DOI: 10.1016/0305-0491(87)90351-8

[13] Jayasimhulu K, Hunt SM, Kaneshiro

[14] Turner BL, Baxter R, Mathieu N, Sjögersten S, Whitton B. Phosphorus compounds in subartic Fennoscandian soils at the mountain birch (*Betula pubescens* )-Tundra ecotone. Soil Biology and Biochemistry. 2004;**36**:815-823. DOI: 10.1016/j.soilbio.2004.01.011

[15] Dyhrman ST, Benitez-Nelson CR, Orchard ED, Haley ST, Pellechia PJ. A microbial source of phosphonates

ES. Detection and identification of *Bacteriovorax stolpii* UKi2 sphingophosphonol;ipid molecular species. Journal of the American Society for Mass Spectrometry. 2007;**18**:394- 403. DOI: 10.1016/j.jasms.2006.10.014

10.1021/bi00910a013

[1] Horiguchi M, Kandatsu M. Isolation

Phosphonate biochemistry. Chemical Reviews. 2017;**117**:5704-5783. DOI: 10.1021/acs.chemrev.6b00536

[3] Ju K-S, Doroghazi JR, Metcalf WW. Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways. Journal of Industrial Microbiology & Biotechnology. 2014;**421**:345-356. DOI:

10.1007/s10295-013-1375-2

and Aminophosphinic Acids. Chichester: Wiley; 2000. pp. 1-31

molecules24050866

science.164.3875.37

[7] Horiguchi M. Occurrence, identification and properties of phosphonic and phosphinic acids. In: Hori T, Horiguchi M, Hayashi M, editors. Biochemistry of Natural C-P Compounds. Shiga: Japanese Association for Research on the Biochemistry of C-P

Compounds; 1984. pp. 24-52

and identification of ciliatine

[8] Tamari M, Kametaka M. Isolation

(2-aminoethylphosphonic acid) from phospholipids of the oyster, *Crassostrea gigas*. Agricultural and Biological

[5] Petkowski JJ, Bains W, Seager S. Natural products containing "rare" organophosphorus functional groups. Molecules. 2019;**24**:866. DOI: 10.3390/

[6] Kitterdge JS, Roberts E. A carbonphosphorus bond in nature. Science. 1969;**164**:37-42. DOI: 10.1126/

[4] Mastalerz P, Kafarski P. Naturally occurring aminophsophonic and aminophosphinic acids. In: Kukhar VP, Hudson HR, editors. Aminophosphonic

of 2-aminoethane phosphonic acid from rumen protozoa. Nature. 1959;**184**:901-902. DOI:

[2] Horsman GP, Zechel DL.

10.1038/184901b0

**References**

methylphosphonate biosynthesis. Science. 2017;**358**:1336-1339. DOI: 10.1126/science.aao3435

[30] Nowack B. Environmental chemistry of phosphonates. Water Resources. 2003;**37**:2533-2546. DOI: 10.1016/S0043-1354(03)00079-4

[31] Benitez-Nelson CR, O'Neill L, Kolowith LC, Pellecia P, Thunell R. Phosphonates and particulate organic phosphorus cycling in an anoxic marine basin. Limnology and Oceanography. 2004;**49**:1593-1604. DOI: 10.4319/ lo.2004.49.5.1593

[32] Metcalf WW, Griffin BM, Cicchillo RM, Gao J, Chandra Janga S, Cooke HA, et al. Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean. Science. 2012;**337**:1104-1107. DOI: 10.1126/ science.1219875

[33] del Valle DA, Karl DM. Aerobic production of methane from dissolved water-column methylphosphonate and sinking particles in the North Pacific subtropical gyre. Aquatic Microbial Ecology. 2014;**73**:93-105. DOI: 10.3354/ ame01714

[34] Bižić-Ionescu M, Klintzsch T, Ionescu D, Hindiyeh MY, Günthel M, Muro-Pastor AM, et al. Widespread methane formation by Cyanobacteria in aquatic and terrestrial ecosystems. BioRxiv. DOI: 10.1101/398958

[35] Beversdorf LJ, White AE, Björkman KM, Letelier RM, Karl DM. Phosphonate metabolism of *Trichodesmium* IMS101 and the production of greenhouse gases. Limnology and Oceanography. 2010;**55**:1768-1778. DOI: 10.4319/ lo.2010.55.4.1768

[36] Repeta DJ, Ferrón S, Sosa OA, Johnson CG, Repeta LD, Acke M, et al. Marine methane paradox explained by bacterial degradation of dissolved

organic matter. Nature Geoscience. 2016;**9**:884-887. DOI: 10.1038/ ngeo2837

[37] McGrath JW, Chin JP, Quinn JP. Organophosphonate reveals: New insights into microbial metabolism of ancient molecules. Nature Reviews. Microbiology. 2013;**11**:411-419. DOI: 10.1038/nrmicro3011

[38] Schwartz AW. Phosphorus in prebiotic chemistry. Philosophical Transactions of the Royal Society B. 2006;**361**:1743-1749. DOI: 10.1098/ rstb.2006.1901

[39] Cooper GW, Onwo WM, Cronin JR. Alkyl phosphonic acids and sulfonic acids in the Murchison meteorite. Geochimica et Cosmochimica Acta. 1992;**56**:4109-4115. DOI: 10.1016/0016-7037(92)90023-C

[40] Jayasimhulu K, Hunt SM, Kaneshiro ES, Watanabe Y, Giner J-L. Detection and identification of *Bacteriovorax stolpii* UKi2 sphingophosphonolipid molecular species. Journal of the American Society for Mass Spectrometry. 2007;**18**:394-403. DOI: 10.1016/j.jasms.2006.10.014

[41] Watanabe Y, Nakayima N, Hoshino T, Jayasimhulu K, Brooks EE, Kaneshiro ES. A novel sphingophosphonolipid head group 1-hydroxy-2-aminoethyl phosphonate in *Bdellovibrio stolpii*. Lipids. 2001;**36**:513-519. DOI: 10.1007/ s11745-001-0751-3

[42] Pallitsch K, Happl B, Stieger C. Determination of the absolute configuration of (−)-hydroxynitrilaphos and related biosynthetic questions. Chemistry-A European Journal. 2017;**23**:15655-15665. DOI: 10.1002/chem.201702904

[43] Moschidis MC. Phosphonolipids. Progress in Lipid Research. 1984;**23**:223-246. DOI: 10.1016/0163-7827(84)90012-2

**109**

*Phosphonates: Their Natural Occurrence and Physiological Role*

1992;**31**:4081-4089. DOI: 10.1021/

[51] Young NM, Foote SJ, Wakarchuk WW. Review of phosphocholine substituents on bacterial pathogen plycans: Synthesis, structures and interactions with host proteins. Molecular Immunology. 2013;**56**:563- 573. DOI: 10.1016/j.molimm.2013.05.237

[52] Urai M, Nakamura T, Uzawa J, Baba T, Taniguchi K, Seki H, et al. Structural analysis of *O*-glycans of mucin from jellyfish (*Aurelia aurita*) containing 2-aminoethylphosphonate. Carbohydrate Research. 2009;**344**:2182- 2187. DOI: 10.1016/j.carres.2009.08.001

[53] Hård K, Van Doorn JM, Thomas-Oates JE, Kamerling JP, Van der Horst DJ. Structure of the Asn-linked oligosaccharides of Apolipophorin III from the insect *Locusta migratoria*. Carbohydratelinked 2-aminoethylphosphonate as a constituent of a glycoprotein. Biochemistry. 1993;**32**:766-775. DOI:

10.1021/bi00054a005

M115.051573

fgene.2014.00145

BF02533632

[54] Eckmair B, Jin C, Abed-Avandi D, Paschinger K. Multistep fractionation

zwitterionic and anionic modifications of the N- and O-glycans of a marine snail. Molecular & Cellular Proteomics. 2016;**15**:573-597. DOI: 10.1074/mcp.

[56] Swift ML. Phosphono-lipid content of the oyster, *Crassostrea virginica*, in three physiological conditions. Lipids. 1977;**12**:449-451. DOI: 10.1007/

[57] Onderdonk AB, Kasper DL, Cisneros RL, Bartlett JG. The capsular

and mass spectrometry reveal

[55] Lauc G, Krištić J, Zoldoš V. Glycans—The third revolution in evolution. Frontiers in Genetics. 2014;**5**:art.145. DOI: 10.3389/

bi00131a026

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

[44] Muralidhar P, Radhika P, Krishna N, Venkata Rao D, Bheemasankara Rao CH. Sphingolipids from marine organisms: A review. Natural Product

[45] Satake M, Miyamoto E. A group of glycosphingolipids found in an invertebrate: Their structures and biological significance. Proceedings of the Japan Academy, Series B. 2012;**88**:509-517. DOI: 10.2183/

[46] Mukhamedova KS, Glushenkova

[47] Paschinger K, Wilson IB. Analysis of zwitterionic and anionic *N*-linked glycans from invertebrates and protists by mass spectrometry. Glycoconjugate Journal. 2016;**33**:273-283. DOI: 10.1007/

[48] Korn ED, Dearborn DG, Fales HM, Sokolowski EA. Phosphonoglycan. A major polysaccharide constituent of the amoeba plasma membrane contains 2-aminoethylphosphonic acid and 1-hydroxy-2-aminoethylphosphonic acid. The Journal of Biological Chemistry. 1973;**248**:2257-2259

[49] Vinogradov E, Egbosimba EE, Perry MB, Lam JS, Forsberg CW. Structural analysis of the

lacking cellulolytic ruminal bacterium *Fibrobacter succinogenes*. European Journal of Biochemistry.

2001;**268**:3566-3576. DOI: 10.1046/j.1432-1327.2001.02264.x

elucidation of two capsular polysaccharides from one strain of *Bacteroides fragilis* using high-resolution NMR spectroscopy. Biochemistry.

carbohydrate components of the outer membrane of the lipopolysaccharide-

[50] Baumann H, Tzianabos AO, Brisson JR, Kasper DL, Jennings HJ. Structural

AI. Natural phosphonolipids. Chemistry of Natural Compound.

2000;**36**:329-341. DOI: 10.1023/A:1002804409503

s10719-016-9650-x

Sciences. 2009;**9**:117-142

pjab.88.509

*Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

[44] Muralidhar P, Radhika P, Krishna N, Venkata Rao D, Bheemasankara Rao CH. Sphingolipids from marine organisms: A review. Natural Product Sciences. 2009;**9**:117-142

*Contemporary Topics about Phosphorus in Biology and Materials*

organic matter. Nature Geoscience. 2016;**9**:884-887. DOI: 10.1038/

[37] McGrath JW, Chin JP, Quinn JP. Organophosphonate reveals: New insights into microbial metabolism of ancient molecules. Nature Reviews. Microbiology. 2013;**11**:411-419. DOI:

[38] Schwartz AW. Phosphorus in prebiotic chemistry. Philosophical Transactions of the Royal Society B. 2006;**361**:1743-1749. DOI: 10.1098/

[39] Cooper GW, Onwo WM, Cronin JR. Alkyl phosphonic acids and sulfonic acids in the Murchison meteorite. Geochimica et Cosmochimica Acta. 1992;**56**:4109-4115. DOI: 10.1016/0016-7037(92)90023-C

[40] Jayasimhulu K, Hunt SM, Kaneshiro ES, Watanabe Y, Giner J-L. Detection and identification of *Bacteriovorax stolpii* UKi2 sphingophosphonolipid molecular species. Journal of the American Society for Mass Spectrometry. 2007;**18**:394-403. DOI:

[41] Watanabe Y, Nakayima N, Hoshino T, Jayasimhulu K, Brooks EE, Kaneshiro ES. A novel sphingophosphonolipid head group 1-hydroxy-2-aminoethyl phosphonate in *Bdellovibrio stolpii*. Lipids. 2001;**36**:513-519. DOI: 10.1007/

10.1016/j.jasms.2006.10.014

s11745-001-0751-3

[42] Pallitsch K, Happl B, Stieger C. Determination of the absolute configuration of (−)-hydroxynitrilaphos and related biosynthetic questions. Chemistry-A European Journal. 2017;**23**:15655-15665.

DOI: 10.1002/chem.201702904

10.1016/0163-7827(84)90012-2

Progress in Lipid Research. 1984;**23**:223-246. DOI:

[43] Moschidis MC. Phosphonolipids.

ngeo2837

10.1038/nrmicro3011

rstb.2006.1901

methylphosphonate biosynthesis. Science. 2017;**358**:1336-1339. DOI:

[30] Nowack B. Environmental chemistry of phosphonates. Water Resources. 2003;**37**:2533-2546. DOI: 10.1016/S0043-1354(03)00079-4

[31] Benitez-Nelson CR, O'Neill L, Kolowith LC, Pellecia P, Thunell R. Phosphonates and particulate organic phosphorus cycling in an anoxic marine basin. Limnology and Oceanography. 2004;**49**:1593-1604. DOI: 10.4319/

[32] Metcalf WW, Griffin BM, Cicchillo RM, Gao J, Chandra Janga S, Cooke HA, et al. Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean. Science. 2012;**337**:1104-1107. DOI: 10.1126/

[33] del Valle DA, Karl DM. Aerobic production of methane from dissolved water-column methylphosphonate and sinking particles in the North Pacific subtropical gyre. Aquatic Microbial Ecology. 2014;**73**:93-105. DOI: 10.3354/

[34] Bižić-Ionescu M, Klintzsch T, Ionescu D, Hindiyeh MY, Günthel M, Muro-Pastor AM, et al. Widespread methane formation by Cyanobacteria in aquatic and terrestrial ecosystems.

BioRxiv. DOI: 10.1101/398958

KM, Letelier RM, Karl DM. Phosphonate metabolism of *Trichodesmium* IMS101 and the production of greenhouse gases. Limnology and Oceanography. 2010;**55**:1768-1778. DOI: 10.4319/

lo.2010.55.4.1768

[35] Beversdorf LJ, White AE, Björkman

[36] Repeta DJ, Ferrón S, Sosa OA, Johnson CG, Repeta LD, Acke M, et al. Marine methane paradox explained by bacterial degradation of dissolved

10.1126/science.aao3435

lo.2004.49.5.1593

science.1219875

ame01714

**108**

[45] Satake M, Miyamoto E. A group of glycosphingolipids found in an invertebrate: Their structures and biological significance. Proceedings of the Japan Academy, Series B. 2012;**88**:509-517. DOI: 10.2183/ pjab.88.509

[46] Mukhamedova KS, Glushenkova AI. Natural phosphonolipids. Chemistry of Natural Compound. 2000;**36**:329-341. DOI: 10.1023/A:1002804409503

[47] Paschinger K, Wilson IB. Analysis of zwitterionic and anionic *N*-linked glycans from invertebrates and protists by mass spectrometry. Glycoconjugate Journal. 2016;**33**:273-283. DOI: 10.1007/ s10719-016-9650-x

[48] Korn ED, Dearborn DG, Fales HM, Sokolowski EA. Phosphonoglycan. A major polysaccharide constituent of the amoeba plasma membrane contains 2-aminoethylphosphonic acid and 1-hydroxy-2-aminoethylphosphonic acid. The Journal of Biological Chemistry. 1973;**248**:2257-2259

[49] Vinogradov E, Egbosimba EE, Perry MB, Lam JS, Forsberg CW. Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharidelacking cellulolytic ruminal bacterium *Fibrobacter succinogenes*. European Journal of Biochemistry. 2001;**268**:3566-3576. DOI: 10.1046/j.1432-1327.2001.02264.x

[50] Baumann H, Tzianabos AO, Brisson JR, Kasper DL, Jennings HJ. Structural elucidation of two capsular polysaccharides from one strain of *Bacteroides fragilis* using high-resolution NMR spectroscopy. Biochemistry.

1992;**31**:4081-4089. DOI: 10.1021/ bi00131a026

[51] Young NM, Foote SJ, Wakarchuk WW. Review of phosphocholine substituents on bacterial pathogen plycans: Synthesis, structures and interactions with host proteins. Molecular Immunology. 2013;**56**:563- 573. DOI: 10.1016/j.molimm.2013.05.237

[52] Urai M, Nakamura T, Uzawa J, Baba T, Taniguchi K, Seki H, et al. Structural analysis of *O*-glycans of mucin from jellyfish (*Aurelia aurita*) containing 2-aminoethylphosphonate. Carbohydrate Research. 2009;**344**:2182- 2187. DOI: 10.1016/j.carres.2009.08.001

[53] Hård K, Van Doorn JM, Thomas-Oates JE, Kamerling JP, Van der Horst DJ. Structure of the Asn-linked oligosaccharides of Apolipophorin III from the insect *Locusta migratoria*. Carbohydratelinked 2-aminoethylphosphonate as a constituent of a glycoprotein. Biochemistry. 1993;**32**:766-775. DOI: 10.1021/bi00054a005

[54] Eckmair B, Jin C, Abed-Avandi D, Paschinger K. Multistep fractionation and mass spectrometry reveal zwitterionic and anionic modifications of the N- and O-glycans of a marine snail. Molecular & Cellular Proteomics. 2016;**15**:573-597. DOI: 10.1074/mcp. M115.051573

[55] Lauc G, Krištić J, Zoldoš V. Glycans—The third revolution in evolution. Frontiers in Genetics. 2014;**5**:art.145. DOI: 10.3389/ fgene.2014.00145

[56] Swift ML. Phosphono-lipid content of the oyster, *Crassostrea virginica*, in three physiological conditions. Lipids. 1977;**12**:449-451. DOI: 10.1007/ BF02533632

[57] Onderdonk AB, Kasper DL, Cisneros RL, Bartlett JG. The capsular polysaccharide of *Bacteroides fragilis* as a virulence factor: Comparison of the pathogenic potential of encapsulated and unencapsulated strains. The Journal of Infectious Diseases. 1977;**136**:82-89. DOI: 10.1093/infdis/136.1.82

[58] Tamari M, Ogawa M, Kametaka M. A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile. Journal of Biochemistry. 1976;**80**:371- 377. DOI: 10.1093/oxfordjournals. jbchem.a131286

[59] Peck SC, van der Donk W. Phosphonate biosynthesis and catabolism: A treasure trove for unusual enzymology. Current Opinion in Chemical Biology. 2013;**17**:580-588. DOI: 10.1016/j.chpa.2013.06.018

[60] Falagas ME, Vouloumanou K, Samonis G, Vardakas KZ. Fosfomycin. Clinical Microbiology Reviews. 2016;**29**:321-347. DOI: 10.1128/ CMR.00068-15

[61] Takeuchi M, Nakajima M, Ogita T, Inukai K, Kodama K, Furuya K, et al. Fosfonochlorin, a new antibiotic with spheroplast forming activity. Journal of Antibiotics. 1989;**42**:198-205. DOI: 10.7164/antibiotics.42.198

[62] Cioni JP, Doroghazi JR, Ju JR, Yu K-S, Evans BS, Lee J, et al. Cyanohydrin phosphonate natural products from *Streptomycin regenesis*. Journal of Natural Products. 2014;**7**:243-249. DOI: 10.1021/ np400722m

[63] Takahashi E, Kimura T, Nakamura K, Arahira M, IIda M. Phosphonothrixin, a novel herbicidal antibiotic produced by Saccharothrix sp. ST-888. I. Taxonomy, fermentation, isolation and biological properties. Journal of Antibiotics (Tokyo). 1995;**48**:1124-1129. DOI: 10.7164/ antibiotics.48.1124

[64] Silver LL. Fosfomycin: Mechanism and resistance. Cold Spring

Harbor Perspectives in Medicine. 2017;**7**:a025262. DOI: 10.1101/ cshperspect.a025262

[65] Iguchi E, Okuhara M, Koshaka M, Aoki H, Imanaka H. Studies on new phosphonic acid antibiotics. II. Taxonomic studies on producing organisms of the phosphonic acid and related compounds. Journal of Antibiotics. 1980;**33**:19-23. DOI: 10.7164/antibiotics.33.18

[66] Ohba K, Sato Y, Sasaki T, Sezaki M. Studies on a new phosphonic acid antibiotic SF2312.II isolation, physicochemical properties and structure. Science Reports. 1986;**25**:18-22

[67] Okuhara M, Kuroda Y, Goto T, Okamoto M, Terano H, Kohsaka M, et al. Studies on a new phosphonic acid antibiotic III. Isolation and characterisation of FRFR-31564, FR-32863 and FR-33289. Journal of Antibiotics. 1980;**33**:24-28. DOI: 10.7164/antibiotics.33.24

[68] Edwards RL, Brothers RC, Wang X, Maron MI, Ziniel PD, Tsang PS, et al. MEPicides: Potent antimalarial prodrugs targeting isoprenoid biosynthesis. Scientific Reports. 2017;**7**:art.8400

[69] Kaya K, Morrison LF, Codd GA, Metcalf JS, Sano T, Takagi H, et al. A novel biosurfactant, 2-Acyloxyethylphosphonate, isolated from Waterblooms of *Aphanizomenon flos-aquae*. Molecules. 2006;**11**:539-548. DOI: 10.3390/11070539

[70] Kim H, Kim K-J, Jeon J-T, Kim S-H, Won DH, Choi H, et al. Placotylene A, an inhibitor of the receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation, from a Korean sponge *Placospongia* sp. Molecules. 2014;**12**:2054-2065. DOI: 10.3390/ d12042054

[71] Kinarivala N, Suh JH, Botros M, Webb P, Trippier PC. Pharmacophore

**111**

*Phosphonates: Their Natural Occurrence and Physiological Role*

structure and biological activity of trialaphos. Agricultural and Biological Chemistry. 1991;**55**:1133-1134. DOI: 10.1080/00021369.1991.10870694

[79] Omura S, Murata M, Hanaki H, Hinotozawa K, Oiwa R, Tanaka H. Phosalacine, a new herbicidal

DOI: 10.7164/antibiotics.37.829

jlac.198819880707

bmc.2013.06.064

bbb1961.41.573

antibiotic containing phosphinothricin. Fermentation, isolation, biological activity and mechanism of action. The Journal of Antibiotics. 1984;**37**:829-835.

[80] Rapp C, Jung G, Kugler M, Loeffler W. Rhizocticins—New phosphonooligopeptides with antifungal

activity. European Journal of Organic Chemistry. 1988:655-661. DOI: 10.1002/

[81] Gahungu M, Arguelles-Arias A, Fickers P, Zervosen A, Joris B, Damblon C, et al. Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A. Bioorganic & Medicinal Chemistry. 2013;**21**:4958-4967. DOI: 10.1016/j.

[82] Park BK, Hirota A, Sakai H. Studies on new antimetabolite produced by microorganism. III. Structure of plumbemycin A and B, antagonists of L-threonine from *Streptomyces plumbeus*. Agricultural and Biological Chemistry.

1977;**41**:573579. DOI: 10.1271/

[83] Borisova SA, Circello BT, Zhang JK, van der Donk WW, Metcalf WW. Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by *Bacillus subtilis* ATCC6633. Chemistry & Biology. 2010;**17**:28-37. DOI: 10.1016/j.chembiol.2009.11.017

[84] Evans BS, Zhao C, Gao J, Evans CM, Ju K-S, Doroghazi JR, et al. Discovery of the antibiotic phosacetamycin via a new mass spectrometry-based method for phosphonic acid detection. ACS

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

elucidation of phosphoiodyn A—Potent and selective peroxisome proliferatoractivated receptor β/δ agonists with neuroprotective activity. Bioorganic & Medicinal Chemistry Letters. 2016;**26**:1889-1893. DOI: 10.1016/j.

[72] Ogawa Y, Tsuruoka T, Inouye S, Niida T. Studies on a new antibiotic SF-1293. II. Chemical structure of antibiotic SF-1293. Science Reports.

[73] Bayer E, Gugel KH, Hägele K, Hagenmaier H, Jessipow S, König WA, et al. Metabolic products of microorganisms. 98. Phosphinothricin and phosphinothricyl-alanyl-alanine. Helvetica Chimica Acta. 1972;**55**:224- 239. DOI: 10.1002/hlca.19720550126

[74] Blodgett JAV, Zhang JK, Yu X, Metcalf WW. Conserved biosynthetic pathways for phosalacine, bialaphos and newly discovered phosphonic acid natural products. The Journal of Antibiotics. 2016;**69**:15-25. DOI:

[75] Wild A, Ziegler C. The effect of bialaphos on ammonium-assimilation and photosynthesis I. Effect on the enzymes of ammonium-assimilation. Zeitschrift für Naturforschung. 1984;**44**:97-102. DOI: 10.1515/

[76] Devkota P, Johnson WG. Glufosinate efficacy as influenced by carrier water pH, hardness, foliar fertilizer, and ammonium sulfate. Weed Technology. 2016;**30**:848-859. DOI: 10.1614/

bmcl.2016.03.028

1973;**13**:42-48

10.1038/ja.2015.77

znc-1989-1-217

WT-D-16-00053.1

jpestics.11.297

[77] Tachibana T, Kaneko T. Development of an herbicide, bialaphos. Journal of Pest Science. 1986;**11**:297-304. DOI: 10.1584/

[78] Kato H, Nagayama K, Abe H, Kobayashi R, Ishihara E. Isolation,

#### *Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

elucidation of phosphoiodyn A—Potent and selective peroxisome proliferatoractivated receptor β/δ agonists with neuroprotective activity. Bioorganic & Medicinal Chemistry Letters. 2016;**26**:1889-1893. DOI: 10.1016/j. bmcl.2016.03.028

*Contemporary Topics about Phosphorus in Biology and Materials*

Harbor Perspectives in Medicine. 2017;**7**:a025262. DOI: 10.1101/

[65] Iguchi E, Okuhara M, Koshaka M, Aoki H, Imanaka H. Studies on new phosphonic acid antibiotics. II. Taxonomic studies on producing organisms of the phosphonic acid and related compounds. Journal of Antibiotics. 1980;**33**:19-23. DOI:

[66] Ohba K, Sato Y, Sasaki T, Sezaki M. Studies on a new phosphonic acid antibiotic SF2312.II isolation, physicochemical properties and structure. Science Reports. 1986;**25**:18-22

[67] Okuhara M, Kuroda Y, Goto T, Okamoto M, Terano H, Kohsaka M, et al. Studies on a new phosphonic acid antibiotic III. Isolation and characterisation of FRFR-31564, FR-32863 and FR-33289. Journal of Antibiotics. 1980;**33**:24-28. DOI:

[68] Edwards RL, Brothers RC, Wang X, Maron MI, Ziniel PD, Tsang PS, et al. MEPicides: Potent antimalarial prodrugs targeting isoprenoid biosynthesis. Scientific Reports. 2017;**7**:art.8400

[69] Kaya K, Morrison LF, Codd GA, Metcalf JS, Sano T, Takagi H, et al. A novel biosurfactant,

2-Acyloxyethylphosphonate, isolated from Waterblooms of *Aphanizomenon flos-aquae*. Molecules. 2006;**11**:539-548.

[70] Kim H, Kim K-J, Jeon J-T, Kim S-H, Won DH, Choi H, et al. Placotylene A, an inhibitor of the receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation, from a Korean sponge *Placospongia* sp. Molecules. 2014;**12**:2054-2065. DOI: 10.3390/

[71] Kinarivala N, Suh JH, Botros M, Webb P, Trippier PC. Pharmacophore

cshperspect.a025262

10.7164/antibiotics.33.18

10.7164/antibiotics.33.24

DOI: 10.3390/11070539

d12042054

polysaccharide of *Bacteroides fragilis* as a virulence factor: Comparison of the pathogenic potential of encapsulated and unencapsulated strains. The Journal of Infectious Diseases. 1977;**136**:82-89.

[58] Tamari M, Ogawa M, Kametaka M. A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile. Journal of Biochemistry. 1976;**80**:371- 377. DOI: 10.1093/oxfordjournals.

catabolism: A treasure trove for unusual enzymology. Current Opinion in Chemical Biology. 2013;**17**:580-588. DOI: 10.1016/j.chpa.2013.06.018

DOI: 10.1093/infdis/136.1.82

[59] Peck SC, van der Donk W. Phosphonate biosynthesis and

[60] Falagas ME, Vouloumanou K, Samonis G, Vardakas KZ. Fosfomycin. Clinical Microbiology Reviews. 2016;**29**:321-347. DOI: 10.1128/

[61] Takeuchi M, Nakajima M, Ogita T, Inukai K, Kodama K, Furuya K, et al. Fosfonochlorin, a new antibiotic with spheroplast forming activity. Journal of Antibiotics. 1989;**42**:198-205. DOI:

[62] Cioni JP, Doroghazi JR, Ju JR, Yu K-S, Evans BS, Lee J, et al. Cyanohydrin phosphonate natural products from *Streptomycin regenesis*. Journal of Natural Products. 2014;**7**:243-249. DOI: 10.1021/

[63] Takahashi E, Kimura T, Nakamura

Phosphonothrixin, a novel herbicidal antibiotic produced by Saccharothrix sp. ST-888. I. Taxonomy, fermentation, isolation and biological properties. Journal of Antibiotics (Tokyo). 1995;**48**:1124-1129. DOI: 10.7164/

[64] Silver LL. Fosfomycin: Mechanism

and resistance. Cold Spring

jbchem.a131286

CMR.00068-15

np400722m

K, Arahira M, IIda M.

antibiotics.48.1124

10.7164/antibiotics.42.198

**110**

[72] Ogawa Y, Tsuruoka T, Inouye S, Niida T. Studies on a new antibiotic SF-1293. II. Chemical structure of antibiotic SF-1293. Science Reports. 1973;**13**:42-48

[73] Bayer E, Gugel KH, Hägele K, Hagenmaier H, Jessipow S, König WA, et al. Metabolic products of microorganisms. 98. Phosphinothricin and phosphinothricyl-alanyl-alanine. Helvetica Chimica Acta. 1972;**55**:224- 239. DOI: 10.1002/hlca.19720550126

[74] Blodgett JAV, Zhang JK, Yu X, Metcalf WW. Conserved biosynthetic pathways for phosalacine, bialaphos and newly discovered phosphonic acid natural products. The Journal of Antibiotics. 2016;**69**:15-25. DOI: 10.1038/ja.2015.77

[75] Wild A, Ziegler C. The effect of bialaphos on ammonium-assimilation and photosynthesis I. Effect on the enzymes of ammonium-assimilation. Zeitschrift für Naturforschung. 1984;**44**:97-102. DOI: 10.1515/ znc-1989-1-217

[76] Devkota P, Johnson WG. Glufosinate efficacy as influenced by carrier water pH, hardness, foliar fertilizer, and ammonium sulfate. Weed Technology. 2016;**30**:848-859. DOI: 10.1614/ WT-D-16-00053.1

[77] Tachibana T, Kaneko T. Development of an herbicide, bialaphos. Journal of Pest Science. 1986;**11**:297-304. DOI: 10.1584/ jpestics.11.297

[78] Kato H, Nagayama K, Abe H, Kobayashi R, Ishihara E. Isolation, structure and biological activity of trialaphos. Agricultural and Biological Chemistry. 1991;**55**:1133-1134. DOI: 10.1080/00021369.1991.10870694

[79] Omura S, Murata M, Hanaki H, Hinotozawa K, Oiwa R, Tanaka H. Phosalacine, a new herbicidal antibiotic containing phosphinothricin. Fermentation, isolation, biological activity and mechanism of action. The Journal of Antibiotics. 1984;**37**:829-835. DOI: 10.7164/antibiotics.37.829

[80] Rapp C, Jung G, Kugler M, Loeffler W. Rhizocticins—New phosphonooligopeptides with antifungal activity. European Journal of Organic Chemistry. 1988:655-661. DOI: 10.1002/ jlac.198819880707

[81] Gahungu M, Arguelles-Arias A, Fickers P, Zervosen A, Joris B, Damblon C, et al. Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A. Bioorganic & Medicinal Chemistry. 2013;**21**:4958-4967. DOI: 10.1016/j. bmc.2013.06.064

[82] Park BK, Hirota A, Sakai H. Studies on new antimetabolite produced by microorganism. III. Structure of plumbemycin A and B, antagonists of L-threonine from *Streptomyces plumbeus*. Agricultural and Biological Chemistry. 1977;**41**:573579. DOI: 10.1271/ bbb1961.41.573

[83] Borisova SA, Circello BT, Zhang JK, van der Donk WW, Metcalf WW. Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by *Bacillus subtilis* ATCC6633. Chemistry & Biology. 2010;**17**:28-37. DOI: 10.1016/j.chembiol.2009.11.017

[84] Evans BS, Zhao C, Gao J, Evans CM, Ju K-S, Doroghazi JR, et al. Discovery of the antibiotic phosacetamycin via a new mass spectrometry-based method for phosphonic acid detection. ACS

Chemical Biology. 2013;**8**:908-913. DOI: 10.1021/cb400102t

[85] Kugler M, Loeffler W, Rapp C, Kern A, Jung G. Rhizocticin A, an antifungal phosphono-oligopeptide of *Bacillus subtilis* ATCC 6633: Biological properties. Archives of Microbiology. 1990;**153**:276-281. DOI: 10.1007/ BF00249082

[86] Johnson RD, Kastner RM, Larsen SH, Ose EE. Antibiotic A53868 and process for production thereof. U.S. patent 4,482,488; 1988

[87] Circello BT, Miller CG, Lee J-H, van der Donk WA, Metcalf WW. The antibiotic dehydrophos is converted to a toxic pyruvate analog by peptide bond cleavage in *Salmonella enterica*. Antimicrobial Agents and Chemotherapy. 2011;**55**:3357-3362. DOI: 10.1128/AAC.01483-10

[88] Jiménez-Andreu MM, Quintana AL, Aínsa JA, Sayago FJ, Cativiela C. Synthesis and biological activity of dehydrophos derivatives. Organic & Biomolecular Chemistry. 2019;**5**:1097- 1112. DOI: 10.1039/c8ob03079k

[89] Bougioukou DJ, Ting CP, Peck SC, Mukherjee S, van der Donk WA. Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin. Organic & Biomolecular Chemistry. 2019;**4**:822-829. DOI: 10.1039/C8OB02860E

[90] Bougioukou DJ, Mukherjee S, van der Donk WA. Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:10952-11057. DOI: 10.1073/ pnas.1303568110

[91] Freestone TS, Ju K-S, Wang B, Zhao H. Discovery of a phosphonoacetic acid derived natural product by pathway refactoring. ACS Synthetic

Biology. 2017;**6**:217-223. DOI: 10.1021/ acssynbio.6b00299

[92] Gunji S, Arima K, Beppu T. Screening of antifungal antibiotics according to activities inducing morphological abnormalities. Agricultural and Biological Chemistry. 1983;**41**:2016-2069. DOI: 10.1080/00021369.1983.10865911

[93] Gao J, Ju K-S, Yu X, Velásques JE, Mukherjee S, Lee J, et al. Use of phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster. Angewandte Chemie, International Edition. 2014;**53**:1334-1337. DOI: 10.1002/ anie.201308363

[94] Schneemann I, Nagel K, Labes A, Wiese J, Imhoff JF. Comprehensivei of marine *Actinobacteria* associated with the sponge *Halichondria panacea*. Applied and Environmental Microbiology. 2010;**76**:3702-3714. DOI: 10.1128/AEM.00780-10

[95] Goettge MN, Cioni JP, Ju K-S, Pallitsch K, Metcalf WW. PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in phosphonocystoximic acid biosynthesis in *Streptomyces*. The Journal of Biological Chemistry. 2018;**293**:6859-6868. DOI: 10.1074/jbc.RA118.001721

[96] Pallitsch K, Kalina T, Stanković T. Synthetic phosphonic acids as potent tools to study phosphonate enzymology. Synlett. 2019;**30**:770-776. DOI: 10.1055/s-0037-1611460

[97] Yamato M, Koguchi T, Okachi R, Yamada K, Nakayama K, Kase H, et al. K-26, a novel inhibitor of angiotensin I converting enzyme produced cy an *Actinomycete* K-26. The Journal of Antibiotics. 1986;**39**:44-52. DOI: 10.7164/antibiotics.39.44

[98] Hirayama N, Kasai M, Hirata K. Structure and conformation of a novel

**113**

*Phosphonates: Their Natural Occurrence and Physiological Role*

*DOI: http://dx.doi.org/10.5772/intechopen.87155*

inhibitor of angiotensin I converting enzyme—A tripeptide containing phosphonic acid. International Journal of Peptide and Protein Research. 1991;**38**:20-24. DOI: 10.1111/j.1399-

[99] Kido Y, Hamakado T, Anno M, Miyagawa E, Motoki Y, Wakamiya T, et al. Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by *Actinomadura* sp. The Journal of Antibiotics. 1984;**37**:965- 969. DOI: 10.7164/antibiotics.37.965

[100] Kramer GJ, Mohd A, Schwager LSU, Masuyer G, Acharya KR, Sturrock ED, et al. Interkingdom pharmacology of angiotensin-I converting enzyme inhibitor phosphonates produced by *Actinomycetes*. ACS Medicinal Chemistry Letters. 2014;**5**:346-351. DOI: 10.1021/

[101] Masuyer G, Cozier GE, Kramer GJ, Bachmann BO, Acharya KR. Crystal structure of a peptidyl-dipeptidase K-26-DCP from *Actinomycetein* complex with its natural inhibitor. The FEBS Journal. 2016;**283**:4357-4369. DOI:

[102] Ntai I, Phelan VV, Bachmann BO. Phosphonopeptide K-26 biosynthetic intermediates in

*Astrosporangium hypotensionis*. Chemical Communications. 2006:4518-4520. DOI:

3011.1991.tb01404.x

ml4004588

10.1111/febs.13928

10.1039/b611768f

*Phosphonates: Their Natural Occurrence and Physiological Role DOI: http://dx.doi.org/10.5772/intechopen.87155*

inhibitor of angiotensin I converting enzyme—A tripeptide containing phosphonic acid. International Journal of Peptide and Protein Research. 1991;**38**:20-24. DOI: 10.1111/j.1399- 3011.1991.tb01404.x

*Contemporary Topics about Phosphorus in Biology and Materials*

Biology. 2017;**6**:217-223. DOI: 10.1021/

T. Screening of antifungal antibiotics according to activities inducing morphological abnormalities. Agricultural and Biological

Chemistry. 1983;**41**:2016-2069. DOI: 10.1080/00021369.1983.10865911

[93] Gao J, Ju K-S, Yu X, Velásques JE, Mukherjee S, Lee J, et al. Use of phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster. Angewandte

Chemie, International Edition. 2014;**53**:1334-1337. DOI: 10.1002/

[94] Schneemann I, Nagel K, Labes A, Wiese J, Imhoff JF. Comprehensivei of marine *Actinobacteria* associated with the sponge *Halichondria* 

*panacea*. Applied and Environmental Microbiology. 2010;**76**:3702-3714. DOI:

[95] Goettge MN, Cioni JP, Ju K-S, Pallitsch K, Metcalf WW. PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in

phosphonocystoximic acid biosynthesis in *Streptomyces*. The Journal of Biological Chemistry. 2018;**293**:6859-6868. DOI:

[96] Pallitsch K, Kalina T, Stanković T. Synthetic phosphonic acids as potent tools to study phosphonate enzymology.

[97] Yamato M, Koguchi T, Okachi R, Yamada K, Nakayama K, Kase H, et al. K-26, a novel inhibitor of angiotensin I converting enzyme produced cy an *Actinomycete* K-26. The Journal of Antibiotics. 1986;**39**:44-52. DOI:

[98] Hirayama N, Kasai M, Hirata K. Structure and conformation of a novel

Synlett. 2019;**30**:770-776. DOI: 10.1055/s-0037-1611460

10.7164/antibiotics.39.44

10.1128/AEM.00780-10

10.1074/jbc.RA118.001721

anie.201308363

[92] Gunji S, Arima K, Beppu

acssynbio.6b00299

Chemical Biology. 2013;**8**:908-913. DOI:

[85] Kugler M, Loeffler W, Rapp C, Kern A, Jung G. Rhizocticin A, an antifungal phosphono-oligopeptide of *Bacillus subtilis* ATCC 6633: Biological properties. Archives of Microbiology. 1990;**153**:276-281. DOI: 10.1007/

[86] Johnson RD, Kastner RM, Larsen SH, Ose EE. Antibiotic A53868 and process for production thereof. U.S.

[87] Circello BT, Miller CG, Lee J-H, van der Donk WA, Metcalf WW. The antibiotic dehydrophos is converted to a toxic pyruvate analog by peptide bond cleavage in *Salmonella enterica*. Antimicrobial Agents and Chemotherapy. 2011;**55**:3357-3362. DOI:

[88] Jiménez-Andreu MM, Quintana AL,

[89] Bougioukou DJ, Ting CP, Peck SC, Mukherjee S, van der Donk WA. Use of the dehydrophos biosynthetic enzymes to prepare antimicrobial analogs of alaphosphin. Organic & Biomolecular Chemistry. 2019;**4**:822-829. DOI:

[90] Bougioukou DJ, Mukherjee S, van der Donk WA. Revisiting the biosynthesis of dehydrophos reveals a tRNA-dependent pathway. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:10952-11057. DOI: 10.1073/

[91] Freestone TS, Ju K-S, Wang B, Zhao H. Discovery of a phosphonoacetic acid derived natural product by pathway refactoring. ACS Synthetic

Aínsa JA, Sayago FJ, Cativiela C. Synthesis and biological activity of dehydrophos derivatives. Organic & Biomolecular Chemistry. 2019;**5**:1097- 1112. DOI: 10.1039/c8ob03079k

10.1021/cb400102t

BF00249082

patent 4,482,488; 1988

10.1128/AAC.01483-10

10.1039/C8OB02860E

pnas.1303568110

**112**

[99] Kido Y, Hamakado T, Anno M, Miyagawa E, Motoki Y, Wakamiya T, et al. Isolation and characterization of I5B2, a new phosphorus containing inhibitor of angiotensin I converting enzyme produced by *Actinomadura* sp. The Journal of Antibiotics. 1984;**37**:965- 969. DOI: 10.7164/antibiotics.37.965

[100] Kramer GJ, Mohd A, Schwager LSU, Masuyer G, Acharya KR, Sturrock ED, et al. Interkingdom pharmacology of angiotensin-I converting enzyme inhibitor phosphonates produced by *Actinomycetes*. ACS Medicinal Chemistry Letters. 2014;**5**:346-351. DOI: 10.1021/ ml4004588

[101] Masuyer G, Cozier GE, Kramer GJ, Bachmann BO, Acharya KR. Crystal structure of a peptidyl-dipeptidase K-26-DCP from *Actinomycetein* complex with its natural inhibitor. The FEBS Journal. 2016;**283**:4357-4369. DOI: 10.1111/febs.13928

[102] Ntai I, Phelan VV, Bachmann BO. Phosphonopeptide K-26 biosynthetic intermediates in *Astrosporangium hypotensionis*. Chemical Communications. 2006:4518-4520. DOI: 10.1039/b611768f

**115**

Section 3

Phosphates in Biomaterials

Section 3
