**3. Bone-like calcium phosphates**

Bone and teeth are the hardest human tissues. Bone provides support and protection to organs. When skeletal system is damaged, an immediate fix is required to avoid any complications, physiological function and mobility impair and even death.

An ideal bone substitute should be biomechanically stable, able to resorb as natural bone within an appropriate time frame while new bone regenerate, exhibit osteoconductive (interconnected porous scaffold onto which bone cells can attach, migrate, differentiate, and grow new bone tissue). osteogenic and osteoinductive properties (ability to stimulate differentiation of a progenitor cells toward an osteoblast lineage) and provide a favorable environment for invading blood vessels and bone forming cell [43].

When it comes to bone substitution, autogenous bone is typically considered as the gold standard for bone defect regeneration since it is living tissue and contains osteogenic cells, still involves harvesting bone from one part of the patient's body and putting it in a damaged bone area. It was and still the method of choice in reconstructing bone either for dental or orthopedic applications. It provides perfect biocompatibility along with the body's own growth factors and structural proteins. Because of limited supply, the need of a second surgery associated with site morbidity and infection risks, negative effect on the mechanics, autograft is not always possible or the best option.

Calcium phosphate (CaP) is the main constituent of inorganic phase of natural bone and teeth and it play essential roles in our daily lives. Damaged calcified natural tissues would be best repaired with something similar. CaP biomaterials are the most legitimate candidates when it comes to regenerate bone. They have been extensively used for decade with great success in orthopedic and dental fields [44]. CaP bone substitutes materials are safe and efficient. They are biocompatible with bone tissues. When implanted they have the particularity to go over the same biological osteoclastic resorption and new bone regeneration processes as the natural bone. They are highly bio-similar to the inorganic phase of autologous bone tissue. Their resorbability and solubility depend in general in their ratio Ca/P (**Table 4**) empirical formulations were proposed to describe the mineral composition of natural bone [45]. The chemical formula of Calcium Phosphate materials eq. (1) is shown below:

$$\left(\text{Ca}\_{8,31,7}\left(\text{PO}\_4\right)\_{4,3}\left(\text{HPO}\_4\text{ or }\text{CO}\_3\right)\_{1,7}\left(\text{OH}\text{or }\text{"}^{1/2}\text{CO}\_3\right)\_{0,31,7}\right) \tag{1}$$

**83**

**Table 5.**

lism, etc).

Dicalcium phosphate

dihydrate

**Table 4.**

*Chitosan Based Biocomposites for Hard Tissue Engineering*

The composition and crystallinity of bone tissues depends on many parameters (location: cortical, cancellous, dental enamel, dentine, the age, biological metabo-

*Chemical composition of calcified hard tissues vs. stochiometric synthetic Hfydroxyapatite (HA).*

**% Element Enamel Dentine Bone HA** Ca 37,6 40,3 36,6 39 P 18,3 18,6 17,1 18,5 CO2 3,0 4,8 4,8 / Na 0,7 0,1 1,0 / K 0,05 0,07 0,07 / Mg 0,2 1,1 0,6 / Sr 0,03 0,04 0,05 / Cl 0,4 0,27 0,1 / F 0,01 0,07 0,1 / Ratio Ca/P 1,59 1,67 1,65 1,67 Crystallinity good low low good

Many studies have reported development of CaP products that would be used as potential bone substitutes. In the **Table 5**, a list of the main most popular products

One of the furthermost interesting CaP biomaterials are the osteoconductive biphasic calcium phosphate (BCP) containing Hydroxyapatite (HA) and Beta TCP. These two phases have different resorption rates. HA (less soluble) will provide short- and long-term physical stability to the bone defect and scaffold for bone ingrowth, whereas Beta TCP (more resorbable) will provide locally Ca and phosphate ions to regenerate new bone and activate the osteogenesis process [46]. To enhance the physical, physiological and/or therapeutical properties, CaP biomaterials could be easily assorted with polymers, drug, proteins, Growth Factors, cells,

CaP biomaterials are relatively easy to make osteoconducteurs by different methods, to mimic the trabecular structure of natural bone (**Figures 3** and **4**).

**CaP Biomaterial Formula; Abbreviation Ca/P Solubility at** 

Octocalcium phosphate CasH2(PO4)6·5H2O;OCP 1.33 8.1 Hydroxyapatites Ca10(PO4)(OH)2*; HA* 1.67 9.4 α-Tricalcium phosphate α-Ca3(PO4)2; α-TCP 1.50 2.5 β-Tricalcium phosphate β-Ca3(PO4)2; β-TCP 1.50 0.5

BCP

Tetracalcium phosphate Ca4(PO4)2O; TTCP 2.00 0.7

CaHPO4·2H2O; DCPD 1.00 88

**25C mg/L)**

1.50–1.67 0.3–0.5

used in the development or formulation of CaP biomaterials.

blood cells, bone marrow and even autologous bone tissue.

Biphasic Calcium phosphate xβ-Ca3(PO4)2 + yCa10(PO4)6(OH)2;

*Short list of calcium phosphates with biological interest.*

*DOI: http://dx.doi.org/10.5772/intechopen.98468*

Actually, mineral bone composition is more versatile, it has many other minor chemical elements such: Mg, Sr., Si, F, Na, and others (**Table 4**).


#### *Chitosan Based Biocomposites for Hard Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.98468*

#### **Table 4.**

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

inorganic biomaterial similar to the calcified phase of natural bone [42].

icry of natural bone tissue.

and bone forming cell [43].

even death.

**3. Bone-like calcium phosphates**

have reported some studies that have been performed with chitosan polymer or its derivatives to treat bone defects. Despite the good biological properties of chitosan formulations developed till now for hard tissues, the poor mechanical properties and lack of certain bioactivity proper to bone tissue such as osteoconduction, chitosan and derivatives are so far not the best clinical choice to treat bone defects. Researchers have tried and are still trying to overwhelm the shortfalls. The most hopeful ones are those that combine chitosan-based formulations and synthetic

In the next section, we will review some of interesting options related to bone substitutes' candidates that could be used along with chitosan to achieve a biomim-

Bone and teeth are the hardest human tissues. Bone provides support and protection to organs. When skeletal system is damaged, an immediate fix is required

An ideal bone substitute should be biomechanically stable, able to resorb as natural bone within an appropriate time frame while new bone regenerate, exhibit osteoconductive (interconnected porous scaffold onto which bone cells can attach, migrate, differentiate, and grow new bone tissue). osteogenic and osteoinductive properties (ability to stimulate differentiation of a progenitor cells toward an osteoblast lineage) and provide a favorable environment for invading blood vessels

When it comes to bone substitution, autogenous bone is typically considered as the gold standard for bone defect regeneration since it is living tissue and contains osteogenic cells, still involves harvesting bone from one part of the patient's body and putting it in a damaged bone area. It was and still the method of choice in reconstructing bone either for dental or orthopedic applications. It provides perfect biocompatibility along with the body's own growth factors and structural proteins. Because of limited supply, the need of a second surgery associated with site morbidity and infection risks, negative effect on the mechanics, autograft is not always possible or the best option. Calcium phosphate (CaP) is the main constituent of inorganic phase of natural bone and teeth and it play essential roles in our daily lives. Damaged calcified natural tissues would be best repaired with something similar. CaP biomaterials are the most legitimate candidates when it comes to regenerate bone. They have been extensively used for decade with great success in orthopedic and dental fields [44]. CaP bone substitutes materials are safe and efficient. They are biocompatible with bone tissues. When implanted they have the particularity to go over the same biological osteoclastic resorption and new bone regeneration processes as the natural bone. They are highly bio-similar to the inorganic phase of autologous bone tissue. Their resorbability and solubility depend in general in their ratio Ca/P (**Table 4**) empirical formulations were proposed to describe the mineral composition of natural bone [45]. The chemical formula of Calcium Phosphate materials eq. (1) is shown below:

> ( ) ( ) ( ) 1/2 8,31,7 4 4,3 4 3 <sup>3</sup> 1.7 0,31.7

Actually, mineral bone composition is more versatile, it has many other minor

chemical elements such: Mg, Sr., Si, F, Na, and others (**Table 4**).

Ca PO HPO orCO OH or CO (1)

to avoid any complications, physiological function and mobility impair and

**82**

*Chemical composition of calcified hard tissues vs. stochiometric synthetic Hfydroxyapatite (HA).*

The composition and crystallinity of bone tissues depends on many parameters (location: cortical, cancellous, dental enamel, dentine, the age, biological metabolism, etc).

Many studies have reported development of CaP products that would be used as potential bone substitutes. In the **Table 5**, a list of the main most popular products used in the development or formulation of CaP biomaterials.

One of the furthermost interesting CaP biomaterials are the osteoconductive biphasic calcium phosphate (BCP) containing Hydroxyapatite (HA) and Beta TCP. These two phases have different resorption rates. HA (less soluble) will provide short- and long-term physical stability to the bone defect and scaffold for bone ingrowth, whereas Beta TCP (more resorbable) will provide locally Ca and phosphate ions to regenerate new bone and activate the osteogenesis process [46]. To enhance the physical, physiological and/or therapeutical properties, CaP biomaterials could be easily assorted with polymers, drug, proteins, Growth Factors, cells, blood cells, bone marrow and even autologous bone tissue.

CaP biomaterials are relatively easy to make osteoconducteurs by different methods, to mimic the trabecular structure of natural bone (**Figures 3** and **4**).


#### **Table 5.**

*Short list of calcium phosphates with biological interest.*

#### **Figure 3.**

*Optical microscopic pictures (x8) of natural cancellous bone (left) and synthetic osteoconductive bioceramics (BCP 50–50%) (right, porosity >70%, Biomatcan).*

#### **Figure 4.**

*Histological picture of Osteoconductive bone graft implanted in rabbit tibia bone after 12 weeks. Pores are filled with new bone (Osteoconduction). BV: Blood vessels, NB: New bone, I: Implant (x50) (Biomatcan).*

New developed formulations were found to have some outstanding properties similar to biological growth factor in autologous bone such bone morphogenic proteins (BMPs). They are osteoinductive. The osteoinduction is trigged either by the addition of chemical elements such silicates ions (Actifuse bone graft, by Baxter) or by tailored sub-micron surface topography and porosity [47] that has the capability to induce bone formation in ectopic or heterotopic location such in muscle or under skin. (**Figures 5** and **6**). The mechanism through which a Ca-P graft mediates an osteoinduction in the host bed is still an active subject of research.

This approach has more benefit. It is less expensive and safer than the BMPs therapy that has limitations (e.g.: not recommended in bone joints or small bones, serious complication, and side effects (cancer, unpredictable ectopic bone growth, neurological impairment, fertility problem …) [48].

A study conducted by Van Dijk et al., showed that in spine fusing in ovine model, formulation of osteoinductive submicron surface topography of BCP bone graft (Magnetos) outperforms Bioglass and monophasic Tricalcium phosphate CaP bioceramics (Vitoss) mixed with Bioglass. The induced bone growth was found similar when using autologous bone (**Figure 5**) [48]. Unlike the other natural substitutes, there is no risks of incompatibility, allergy, or transmission of diseases.

**85**

**Figure 5.**

**Figure 6.**

*Chitosan Based Biocomposites for Hard Tissue Engineering*

We can confirm that synthetic CaP biomaterials are safe and a reliable alternative for autograft or allograft. With a history of safety and effectiveness in clinical both human and animal health, they are gaining more attention and started to be

*Ct-scan of heterotopic implantation of Osteoinductive BCP (50–50%) in mice model, noticeable increase* 

*Posterolateral fusion on ovine model: Histomorphometry diagrams of bone performed on low-magnification micrographs of histologic sections. Data are presented as area%, in mean and SD.* ★*, significantly different from BG and TCP/BG (P < 0.001). (P < 0.005) and TCP/BG. AB: Autograft bone; BCP < μm, biphasic calcium phosphate with submicron topography; BG, bioglass; TCP, tricalcium phosphate. [47].*

Many researchers have worked on development of biocomposites containing CS [49–55] and CaP biomaterials. If the biological properties were improved in some cases, the mechanical properties still not comparable to natural bone. In this section we are going to report some testing and results on the developed biocomposites:

considered the new gold standard in bone regeneration therapy.

**4. Biocomposites: Chitosan-CaP bioceramics**

*(10.6%) of implant size after 40 days. (Biomatcan).*

*DOI: http://dx.doi.org/10.5772/intechopen.98468*

*Chitosan Based Biocomposites for Hard Tissue Engineering DOI: http://dx.doi.org/10.5772/intechopen.98468*

#### **Figure 5.**

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

New developed formulations were found to have some outstanding properties similar to biological growth factor in autologous bone such bone morphogenic proteins (BMPs). They are osteoinductive. The osteoinduction is trigged either by the addition of chemical elements such silicates ions (Actifuse bone graft, by Baxter) or by tailored sub-micron surface topography and porosity [47] that has the capability to induce bone formation in ectopic or heterotopic location such in muscle or under skin. (**Figures 5** and **6**). The mechanism through which a Ca-P graft mediates an

*Histological picture of Osteoconductive bone graft implanted in rabbit tibia bone after 12 weeks. Pores are filled* 

*with new bone (Osteoconduction). BV: Blood vessels, NB: New bone, I: Implant (x50) (Biomatcan).*

*Optical microscopic pictures (x8) of natural cancellous bone (left) and synthetic osteoconductive bioceramics* 

This approach has more benefit. It is less expensive and safer than the BMPs therapy that has limitations (e.g.: not recommended in bone joints or small bones, serious complication, and side effects (cancer, unpredictable ectopic bone growth,

A study conducted by Van Dijk et al., showed that in spine fusing in ovine model, formulation of osteoinductive submicron surface topography of BCP bone graft (Magnetos) outperforms Bioglass and monophasic Tricalcium phosphate CaP bioceramics (Vitoss) mixed with Bioglass. The induced bone growth was found similar when using autologous bone (**Figure 5**) [48]. Unlike the other natural substitutes, there is no risks of incompatibility, allergy, or transmission of

osteoinduction in the host bed is still an active subject of research.

neurological impairment, fertility problem …) [48].

**84**

diseases.

**Figure 3.**

**Figure 4.**

*(BCP 50–50%) (right, porosity >70%, Biomatcan).*

*Posterolateral fusion on ovine model: Histomorphometry diagrams of bone performed on low-magnification micrographs of histologic sections. Data are presented as area%, in mean and SD.* ★*, significantly different from BG and TCP/BG (P < 0.001). (P < 0.005) and TCP/BG. AB: Autograft bone; BCP < μm, biphasic calcium phosphate with submicron topography; BG, bioglass; TCP, tricalcium phosphate. [47].*

#### **Figure 6.**

*Ct-scan of heterotopic implantation of Osteoinductive BCP (50–50%) in mice model, noticeable increase (10.6%) of implant size after 40 days. (Biomatcan).*

We can confirm that synthetic CaP biomaterials are safe and a reliable alternative for autograft or allograft. With a history of safety and effectiveness in clinical both human and animal health, they are gaining more attention and started to be considered the new gold standard in bone regeneration therapy.

## **4. Biocomposites: Chitosan-CaP bioceramics**

Many researchers have worked on development of biocomposites containing CS [49–55] and CaP biomaterials. If the biological properties were improved in some cases, the mechanical properties still not comparable to natural bone. In this section we are going to report some testing and results on the developed biocomposites:

An injectable bone graft formulation and hardening injectable bone cements. The mechanical properties were evaluated in both of cases.
