**3. Platelet-Rich Plasma**

#### **3.1 Platelets as a source of bioactive molecules**

PRP is an autologous biological therapy framed in the regenerative medicine whose basic principle is to obtain a fraction of blood plasma that contains platelets at a higher concentration than in the blood. From the pharmacological perspective, it is very difficult to define it since the PRP presents a large number and variety of active substances, even often antagonistic. Its therapeutic potential lies both in the biomolecules present in the plasma and in the platelet and its content that is the core element of this therapy.

The platelets are produced by the megakaryocytes of the bone marrow, which migrate to the endothelial barrier after maturation and project their prolongations releasing into the bloodstream the proplatelets or precursors that will generate the platelets [28]. Platelets are discoid and anucleated blood elements with a diameter of 2–3 μm; their blood concentration is 150.000–400.000 platelets/μL with a life span of 7 to 10 days. Platelets are limited by an external plasma membrane that contains a large network of receptors that trigger intracellular signals that allow platelets to perform their numerous functions. Among them glycoprotein Ib (GPiB) and glycoprotein VI (GPVI) receptors can be found, which are involved in functions related to homeostasis, the main function of platelets. GPIb and GPVI bind to von Willebrand factor (VWF) and collagen when there is a discontinuity in the endothelial barrier that exposes the extracellular matrix. These interactions cause conformational changes in platelets and allow them to bind to fibrinogen, tissues, and other platelets to form the thrombus that will participate in tissue repair. In addition, this platelet activation also causes the release of their internal content that has regenerative abilities and justifies the use of PRP.

The internal content of platelets is stored in different granules called dense granules, α-granules, and lysosomes. The material present in these granules may have been synthesized by the original megakaryocyte as well as captured by platelets by endocytosis. The α-granules are those that have a higher content of active biomolecules related to tissue repair. Hundreds of these molecules have been identified, including adhesive proteins, fibrinolytic and coagulation factors, antimicrobial molecules, cytokines, and growth factors, among others. These last two groups of molecules participate in tissue repair and regeneration processes such as angiogenesis, chemotaxis, migration, or cell proliferation [29]. When platelets are activated, not only these molecules are released but also other elements such as platelet microparticles, which are involved in anti-inflammatory processes, or exosomes. Exosomes are small vesicles of 100–400 nm that carry several proteins in addition to other biomolecules as genetic material. Although not much is known about these platelet exosomes, it has been found that they are very important in cellular communication [30].

The activation of the PRP platelets causes the release of platelet content related to tissue repair to the outside, and it joins to the circulating biomolecules in the plasma. Thus, the levels of many growth factors will depend on the platelet concentration of

**73**

*Isolation, Activation, and Mechanism of Action of Platelet-Rich Plasma and Its Applications...*

the PRP. Among these platelet growth factors, there is platelet-derived growth factor (PDGF), which is a potent chemotactic for several cell types and has an important effect on tissue repair over tissues such as cartilage and meniscus. Another growth factor with a large presence in platelets is TGF-β, whose effects are varied and can be of different nature depending on the molecules and cells with which it interacts. It influences early responses in tissue repair, on the differentiation processes of mesenchymal stem cells, and on the maintenance of cartilage and subchondral bone. Other regulatory factors in tissue repair are VEGF, epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) with key roles in cell migration, proliferation, differentiation, or angiogenesis. In addition, circulating molecules such as insulin-like growth factor type I (IGF-I) or hepatocyte growth factor (HGF) have also crucial importance in the effect of PRP; they are growth factor enhancers of regeneration processes as well as modulators of inflammatory processes [31]. Therefore, PRP is a cocktail of thousands of biomolecules from plasma and platelets that regulate hemostasis, coagulation, tissue repair and regeneration, inflammation, cellular behavior, or defense against microorganisms, among other biological processes. All this therapeutic potential depends largely on its composition, which may vary according to the method used to obtain it. As a result, there is

As stated previously, the PRP obtaining technique is used to achieve a fraction of blood plasma with higher levels of platelets than blood. The first step consists in the collection of a small volume of peripheral blood from the patient using tubes with anticoagulant—to prevent blood clotting. Different types of anticoagulants can be used such as sodium citrate and ethylenediaminetetraacetic acid (EDTA), which chelate calcium and prevent the coagulation cascade, or heparin that inhibits thrombin. However, sodium citrate is the most recommended anticoagulant since it ensures a better preservation of platelets [32]. It also causes less secretion of microvesicles that are the result of platelet activation, which is increased when

After blood collection, a centrifugation process is performed, whose force and time vary according to the methodology and, hence, the PRP formulation to be obtained. Centrifugation has to generate sufficient force to create a gradient that separates the blood into different fractions but without damaging its components (**Figure 3**). These centrifugations can be single or double, with a centrifugal force of between 350 and 2000 g and a centrifugation time of 3 to 15 minutes depending on the method used. Thus, the blood is divided into a lower fraction of red blood cells, a thin layer of leukocytes or buffy coat, and finally the plasma fraction with platelets, which gradually decrease their number in the uppermost areas. This last layer will constitute the PRP, and depending on the centrifugation process, the number of platelets may vary. However, a higher number of platelets are not strictly linked to an improved effect of PRP. In fact, several studies have reported that an excessive concentration of platelets may have inhibitory effects on cell proliferation or differentiation in populations such as tenocytes or adipose tissue-derived stem cells. Thus, the optimal platelet concentration for an optimized function is considered

*DOI: http://dx.doi.org/10.5772/intechopen.90543*

a wide variety of PRP products as will be explained below.

EDTA and heparin are used as blood anticoagulants [33].

*3.2.2 Blood fractionation and Platelet-Rich Plasma obtention*

two- to threefold compared to blood levels [34, 35].

**3.2 Obtaining process**

*3.2.1 Blood collection*

*Isolation, Activation, and Mechanism of Action of Platelet-Rich Plasma and Its Applications... DOI: http://dx.doi.org/10.5772/intechopen.90543*

the PRP. Among these platelet growth factors, there is platelet-derived growth factor (PDGF), which is a potent chemotactic for several cell types and has an important effect on tissue repair over tissues such as cartilage and meniscus. Another growth factor with a large presence in platelets is TGF-β, whose effects are varied and can be of different nature depending on the molecules and cells with which it interacts. It influences early responses in tissue repair, on the differentiation processes of mesenchymal stem cells, and on the maintenance of cartilage and subchondral bone. Other regulatory factors in tissue repair are VEGF, epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) with key roles in cell migration, proliferation, differentiation, or angiogenesis. In addition, circulating molecules such as insulin-like growth factor type I (IGF-I) or hepatocyte growth factor (HGF) have also crucial importance in the effect of PRP; they are growth factor enhancers of regeneration processes as well as modulators of inflammatory processes [31].

Therefore, PRP is a cocktail of thousands of biomolecules from plasma and platelets that regulate hemostasis, coagulation, tissue repair and regeneration, inflammation, cellular behavior, or defense against microorganisms, among other biological processes. All this therapeutic potential depends largely on its composition, which may vary according to the method used to obtain it. As a result, there is a wide variety of PRP products as will be explained below.

#### **3.2 Obtaining process**

#### *3.2.1 Blood collection*

*Regenerative Medicine*

**3. Platelet-Rich Plasma**

element of this therapy.

disorganized collagen fibers [26]. Finally, muscle tissue is also affected by inflammation produced in joint degeneration, showing fibrosis, collagen depositions, and muscle wasting [27]. Considering all this, it is clear that joint degeneration is not a sole cartilage disease. Instead, it affects all the elements present in the joint, and, therefore, it should be clinically tackled taking into consideration all of them in

PRP is an autologous biological therapy framed in the regenerative medicine whose basic principle is to obtain a fraction of blood plasma that contains platelets at a higher concentration than in the blood. From the pharmacological perspective, it is very difficult to define it since the PRP presents a large number and variety of active substances, even often antagonistic. Its therapeutic potential lies both in the biomolecules present in the plasma and in the platelet and its content that is the core

The platelets are produced by the megakaryocytes of the bone marrow, which migrate to the endothelial barrier after maturation and project their prolongations releasing into the bloodstream the proplatelets or precursors that will generate the platelets [28]. Platelets are discoid and anucleated blood elements with a diameter of 2–3 μm; their blood concentration is 150.000–400.000 platelets/μL with a life span of 7 to 10 days. Platelets are limited by an external plasma membrane that contains a large network of receptors that trigger intracellular signals that allow platelets to perform their numerous functions. Among them glycoprotein Ib (GPiB) and glycoprotein VI (GPVI) receptors can be found, which are involved in functions related to homeostasis, the main function of platelets. GPIb and GPVI bind to von Willebrand factor (VWF) and collagen when there is a discontinuity in the endothelial barrier that exposes the extracellular matrix. These interactions cause conformational changes in platelets and allow them to bind to fibrinogen, tissues, and other platelets to form the thrombus that will participate in tissue repair. In addition, this platelet activation also causes the release of their internal content that

The internal content of platelets is stored in different granules called dense granules, α-granules, and lysosomes. The material present in these granules may have been synthesized by the original megakaryocyte as well as captured by platelets by endocytosis. The α-granules are those that have a higher content of active biomolecules related to tissue repair. Hundreds of these molecules have been identified, including adhesive proteins, fibrinolytic and coagulation factors, antimicrobial molecules, cytokines, and growth factors, among others. These last two groups of molecules participate in tissue repair and regeneration processes such as angiogenesis, chemotaxis, migration, or cell proliferation [29]. When platelets are activated, not only these molecules are released but also other elements such as platelet microparticles, which are involved in anti-inflammatory processes, or exosomes. Exosomes are small vesicles of 100–400 nm that carry several proteins in addition to other biomolecules as genetic material. Although not much is known about these platelet exosomes, it has been found that they are very important in

The activation of the PRP platelets causes the release of platelet content related to tissue repair to the outside, and it joins to the circulating biomolecules in the plasma. Thus, the levels of many growth factors will depend on the platelet concentration of

order to reverse or slow down the degenerative progression.

**3.1 Platelets as a source of bioactive molecules**

has regenerative abilities and justifies the use of PRP.

**72**

cellular communication [30].

As stated previously, the PRP obtaining technique is used to achieve a fraction of blood plasma with higher levels of platelets than blood. The first step consists in the collection of a small volume of peripheral blood from the patient using tubes with anticoagulant—to prevent blood clotting. Different types of anticoagulants can be used such as sodium citrate and ethylenediaminetetraacetic acid (EDTA), which chelate calcium and prevent the coagulation cascade, or heparin that inhibits thrombin. However, sodium citrate is the most recommended anticoagulant since it ensures a better preservation of platelets [32]. It also causes less secretion of microvesicles that are the result of platelet activation, which is increased when EDTA and heparin are used as blood anticoagulants [33].

#### *3.2.2 Blood fractionation and Platelet-Rich Plasma obtention*

After blood collection, a centrifugation process is performed, whose force and time vary according to the methodology and, hence, the PRP formulation to be obtained. Centrifugation has to generate sufficient force to create a gradient that separates the blood into different fractions but without damaging its components (**Figure 3**). These centrifugations can be single or double, with a centrifugal force of between 350 and 2000 g and a centrifugation time of 3 to 15 minutes depending on the method used. Thus, the blood is divided into a lower fraction of red blood cells, a thin layer of leukocytes or buffy coat, and finally the plasma fraction with platelets, which gradually decrease their number in the uppermost areas. This last layer will constitute the PRP, and depending on the centrifugation process, the number of platelets may vary. However, a higher number of platelets are not strictly linked to an improved effect of PRP. In fact, several studies have reported that an excessive concentration of platelets may have inhibitory effects on cell proliferation or differentiation in populations such as tenocytes or adipose tissue-derived stem cells. Thus, the optimal platelet concentration for an optimized function is considered two- to threefold compared to blood levels [34, 35].

#### **Figure 3.**

*Obtaining Platelet-Rich Plasma. After blood fractionation, the platelet-enriched plasma fraction is obtained. The activation of this fraction causes the release of the platelet content that together with the plasma molecules constitutes the effector biomolecules of the PRP. It also generates the polymerization of the fibrinogen that will create a network of fibrin where these biomolecules will be trapped, and that will be released progressively.*

When separating the PRP from the rest of the blood fractions, there is the option to include or not the leukocyte layer, thus obtaining different PRP products, which will be detailed below. Although in some musculoskeletal disorders the use of Leukocyte-Rich PRP (LR-PRP) need further research, there is an increasingly broad consensus by which the use of leukocyte-poor PRP (LP-PRP) preparations is recommended for joint degeneration [36]. The inclusion of leukocytes in the PRP generated pro-inflammatory molecules that had negative effects on cell proliferation and chondrogenic differentiation as well as a worse regeneration of articular cartilage [37]. However, the fraction of red blood cells must be discarded in order to avoid the presence of erythrocytes in the PRP. The presence of erythrocytes in the PRP entails their own degradation processes such as hemolysis and eryptosis. As a result, products that promote inflammation and cellular stress are generated, which would hinder the beneficial action of PRP [38].

#### *3.2.3 Platelet-Rich Plasma activation*

The last step in the process to obtain PRP is the activation of platelets, through which its platelet content not only is released but also triggers the polymerization of fibrinogen in a fibrin mesh that traps the molecules. Thus, a controlled release system that delivers the molecules as it degrades is obtained. Activation can be exogenous either by physical methods such as freeze–thaw cycles or by the addition of certain substances (calcium chloride, thrombin). Some methods propose endogenous activation in which PRP is administered without prior activation and platelets are physiologically activated inside the body [39]. However, the use of exogenous activation allows a more versatile PRP, and depending on the time that has elapsed since the activation, different formulations are achieved at the point of care. The addition of calcium chloride as an activation method avoids the use of

**75**

*Isolation, Activation, and Mechanism of Action of Platelet-Rich Plasma and Its Applications...*

exogenous biological elements such as thrombin. It also prevents local hypocalcemia that can be caused by the calcium-chelating anticoagulants previously used in blood collection to prepare the PRP. Thus, PRP can be used as an injectable liquid formulation immediately after activation or as a fibrin membrane-clot minutes after adding the activator. In this case, and due to its consistency, this formulation can be used as a biological and autologous scaffold in surgical interventions that

As mentioned above, many variables may be involved in the obtaining process. It is not the intention of this chapter to delve into the large number of PRP types that exist both in the market and in the scientific literature. However, it is important to mention the variables that condition not only the type of PRP and therefore the

The three main variables that condition the obtaining of PRP, namely, number of platelets, presence or not of leukocytes, and activation, generate many different products under the PRP term which are necessary to differentiate. Not only a wide variety of products have emerged but also several classification systems that have attempted to clarify the inconsistency that accompanies the term PRP. Initially, the main difference was the presence or not of leukocytes. In the first classification of Dohan et al., PRPs could be distinguished in leukocyte-poor PRP and Leukocyte-Rich PRP, besides contemplating the fibrin presence [41]. Subsequently, Mishra [42] and DeLong [43] took into consideration the number of platelets and the activation of PRP. In the following classifications, the presence of erythrocytes [44] was also mentioned, and in recent years aspects such as recovery efficiency or centrifugation and application methods were addressed [45, 46], trying to classify as much as

As if that were not enough, new denominations are being coined in products derived from blood but that share the fundamental principles of PRP. This is the case of the Platelet-Rich Fibrin. These types appeared initially in the Dohan classification and refer to the fibrin clots that are formed either by centrifuging the blood without anticoagulants or by activating the liquid PRP and waiting for the formation of fibrin net, as mentioned above. A product derived from this is the hyperacute serum that is obtained with a procedure similar to that of the PRP but without using anticoagulants. Thus, after centrifugation of the blood, the upper fraction is a fibrin clot (Platelet-Rich Fibrin), which is squeezed to obtain the hyperacute serum [47]. It contains all the plasma and platelet biomolecules without coagulation proteins such as fibrinogen, namely, the product obtained is almost identical to the exudate gradually released from the fibrin net achieved after the activation of PRP. However, many growth factors present in the hyperacute serum will be eliminated quickly after its injection into the affected area due to its short half-life, whereas if they are released in a controlled manner as in the activated PRP, its time

The lack of standardization is one of the main limitations in the application of PRP. Although all these products are called PRP, their composition may differ from many others and as a consequence their biological effects and clinical results. For instance, the presence of leukocytes determines the levels of pro-inflammatory molecules, and the activation or not of platelets affects the biomolecule release kinetics. Therefore, the comparison of PRP studies, assuming that it is the same product, yields contradictory data, so it is necessary to specify the type of PRP used

different biological effects but also the classification systems (**Table 1**).

*DOI: http://dx.doi.org/10.5772/intechopen.90543*

promote tissue repair [40].

**3.3 Types of Platelet-Rich Plasma**

possible the different PRP products (**Table 1**).

of action will be longer [48].

in these works [49].

*Isolation, Activation, and Mechanism of Action of Platelet-Rich Plasma and Its Applications... DOI: http://dx.doi.org/10.5772/intechopen.90543*

exogenous biological elements such as thrombin. It also prevents local hypocalcemia that can be caused by the calcium-chelating anticoagulants previously used in blood collection to prepare the PRP. Thus, PRP can be used as an injectable liquid formulation immediately after activation or as a fibrin membrane-clot minutes after adding the activator. In this case, and due to its consistency, this formulation can be used as a biological and autologous scaffold in surgical interventions that promote tissue repair [40].

#### **3.3 Types of Platelet-Rich Plasma**

*Regenerative Medicine*

**Figure 3.**

When separating the PRP from the rest of the blood fractions, there is the option to include or not the leukocyte layer, thus obtaining different PRP products, which will be detailed below. Although in some musculoskeletal disorders the use of Leukocyte-Rich PRP (LR-PRP) need further research, there is an increasingly broad consensus by which the use of leukocyte-poor PRP (LP-PRP) preparations is recommended for joint degeneration [36]. The inclusion of leukocytes in the PRP generated pro-inflammatory molecules that had negative effects on cell proliferation and chondrogenic differentiation as well as a worse regeneration of articular cartilage [37]. However, the fraction of red blood cells must be discarded in order to avoid the presence of erythrocytes in the PRP. The presence of erythrocytes in the PRP entails their own degradation processes such as hemolysis and eryptosis. As a result, products that promote inflammation and cellular stress are generated, which

*Obtaining Platelet-Rich Plasma. After blood fractionation, the platelet-enriched plasma fraction is obtained. The activation of this fraction causes the release of the platelet content that together with the plasma molecules constitutes the effector biomolecules of the PRP. It also generates the polymerization of the fibrinogen that will create a network of fibrin where these biomolecules will be trapped, and that will be released progressively.*

The last step in the process to obtain PRP is the activation of platelets, through which its platelet content not only is released but also triggers the polymerization of fibrinogen in a fibrin mesh that traps the molecules. Thus, a controlled release system that delivers the molecules as it degrades is obtained. Activation can be exogenous either by physical methods such as freeze–thaw cycles or by the addition of certain substances (calcium chloride, thrombin). Some methods propose endogenous activation in which PRP is administered without prior activation and platelets are physiologically activated inside the body [39]. However, the use of exogenous activation allows a more versatile PRP, and depending on the time that has elapsed since the activation, different formulations are achieved at the point of care. The addition of calcium chloride as an activation method avoids the use of

would hinder the beneficial action of PRP [38].

*3.2.3 Platelet-Rich Plasma activation*

**74**

As mentioned above, many variables may be involved in the obtaining process. It is not the intention of this chapter to delve into the large number of PRP types that exist both in the market and in the scientific literature. However, it is important to mention the variables that condition not only the type of PRP and therefore the different biological effects but also the classification systems (**Table 1**).

The three main variables that condition the obtaining of PRP, namely, number of platelets, presence or not of leukocytes, and activation, generate many different products under the PRP term which are necessary to differentiate. Not only a wide variety of products have emerged but also several classification systems that have attempted to clarify the inconsistency that accompanies the term PRP. Initially, the main difference was the presence or not of leukocytes. In the first classification of Dohan et al., PRPs could be distinguished in leukocyte-poor PRP and Leukocyte-Rich PRP, besides contemplating the fibrin presence [41]. Subsequently, Mishra [42] and DeLong [43] took into consideration the number of platelets and the activation of PRP. In the following classifications, the presence of erythrocytes [44] was also mentioned, and in recent years aspects such as recovery efficiency or centrifugation and application methods were addressed [45, 46], trying to classify as much as possible the different PRP products (**Table 1**).

As if that were not enough, new denominations are being coined in products derived from blood but that share the fundamental principles of PRP. This is the case of the Platelet-Rich Fibrin. These types appeared initially in the Dohan classification and refer to the fibrin clots that are formed either by centrifuging the blood without anticoagulants or by activating the liquid PRP and waiting for the formation of fibrin net, as mentioned above. A product derived from this is the hyperacute serum that is obtained with a procedure similar to that of the PRP but without using anticoagulants. Thus, after centrifugation of the blood, the upper fraction is a fibrin clot (Platelet-Rich Fibrin), which is squeezed to obtain the hyperacute serum [47]. It contains all the plasma and platelet biomolecules without coagulation proteins such as fibrinogen, namely, the product obtained is almost identical to the exudate gradually released from the fibrin net achieved after the activation of PRP. However, many growth factors present in the hyperacute serum will be eliminated quickly after its injection into the affected area due to its short half-life, whereas if they are released in a controlled manner as in the activated PRP, its time of action will be longer [48].

The lack of standardization is one of the main limitations in the application of PRP. Although all these products are called PRP, their composition may differ from many others and as a consequence their biological effects and clinical results. For instance, the presence of leukocytes determines the levels of pro-inflammatory molecules, and the activation or not of platelets affects the biomolecule release kinetics. Therefore, the comparison of PRP studies, assuming that it is the same product, yields contradictory data, so it is necessary to specify the type of PRP used in these works [49].


#### **Table 1.**

**77**

**Figure 4.**

*potential and decrease their aberrant and senescent forms.*

*Isolation, Activation, and Mechanism of Action of Platelet-Rich Plasma and Its Applications...*

As PRP is a product that contains a large number of bioactive molecules, it is wrong and impossible to define the effect of the PRP based on the isolated actions of each molecule. The biological effect of PRP depends not only on its molecules but also on its synergistic effect, which also considers interactions between molecules. Indeed, many PRP molecules are activated in the presence of others, or on the contrary many have antagonistic effects, conditioning the final

*Biological effects of Platelet-Rich Plasma on joint degeneration. The inhibition of the intracellular signaling pathway NF-Kβ, the reduction of reactive oxygen species, and the promotion of M2 macrophages cause the drop of pro-inflammatory molecule levels, achieving an anti-inflammatory effect. In addition, this decrease in pro-inflammatory molecules such as prostaglandin E2 achieves an analgesic effect, which is also favored by the activation of endocannabinoid systems. On the other hand, PRP modulates the cellular response, stimulating the proliferation of chondrocytes and synoviocytes, which increase the production of the substances responsible for lubrication. This modulation also affects mesenchymal stem cells, which increase their chondrogenic* 

*DOI: http://dx.doi.org/10.5772/intechopen.90543*

effect of PRP.

**4. Platelet-Rich Plasma and joint degeneration**

**4.1 Therapeutic potential of PRP in joint degeneration**

*Variables analyzed in the different classification systems.*

*Isolation, Activation, and Mechanism of Action of Platelet-Rich Plasma and Its Applications... DOI: http://dx.doi.org/10.5772/intechopen.90543*
