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

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 effect of PRP.

#### **Figure 4.**

*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 potential and decrease their aberrant and senescent forms.*

Multiple actions are attributed to the PRP in the treatment of pathologies of the musculoskeletal system. However, this chapter will be limited to highlighting the effects that have the greatest impact on improving joint degeneration (**Figure 4**).

#### *4.1.1 Anti-inflammatory effect*

Due to the complex OA pathophysiology, inflammation can be both the cause and consequence of other pathological processes. Because of this, it is important to reverse the pro-inflammatory environment of this pathology and restore homeostasis of the joint to promote tissue repair. Many of the PRP molecules participate in the regulation of these inflammatory processes, which are key in the progression of the pathology. The anti-inflammatory effect of PRP is achieved through the action of its biomolecules at different levels. Molecules such as IGF-1 or HGF restore the original acquiescent state of cell populations from an inflammatory state because of joint degeneration. This effect occurs through the inhibition of the intracellular signaling pathway NF-Kβ by these molecules, and, as a result, the generation of proinflammatory molecules such as IL-β or TNF-α is reduced [50, 51]. The use of PRP rich in leukocytes can be especially important in this mechanism of action since, instead of inhibiting this inflammatory pathway, they activate it due to the presence of certain pro-inflammatory molecules in this type of PRP [37]. This inhibitory effect not only is limited to chondrocytes but also affects other cell populations such as fibroblast, osteoblasts [52], or macrophages [53]. The consequence of silencing this pathway in the different cell types of the joint is the drop in the inflammatory molecular levels of the synovial fluid, relieving the inflammatory environment [54].

Within its anti-inflammatory action, PRP also acts on macrophages by changing its phenotype. This effect may be indirect due to the decrease in pro-inflammatory molecules or direct by a direct action on the PRP components such as the microparticles produced by platelet apoptosis. The result is a phenotype shift of the macrophages from inflammatory (M1) to reparative (M2) phenotype, where the reduction of inflammation is favored and tissue repair is stimulated [55]. This effect is especially important in macrophages present in the synovial membrane. The increase in anti-inflammatory macrophages to the detriment of pro-inflammatories results in a decrease in inflammation of the synovial membrane, which is a hallmark of OA [56]. This polarization towards a reparative state may be due to the action of the interleukin 1 receptor antagonist, present in the PRP, which in addition to avoiding the inflammatory effect of IL-1 promotes the repair phenotype M2 of macrophages [57].

The inflammatory environment in the osteoarthritic joint is also potentiated by the increased presence of reactive oxygen species (ROS), which participate in the OA pathogenesis through synovium inflammation, cartilage degradation, or subchondral bone dysfunction [58]. PRP activates the antioxidant response element through the intracellular signaling pathway NrF2-ARE in osteoblasts [59]. This achieves an antioxidant and protective effect in these cell populations, avoiding damage caused by ROS increment.

The interaction of PRP biomolecules in the mechanisms that trigger inflammation results not only in a decrease in the levels of pro-inflammatory molecules and ROS but also in a promotion of gene expression related to anti-inflammatory action. It has recently been shown that gene expression of enzymes related to aggrecan destruction and metalloproteinase modulation, namely, metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) and tissue inhibitor of metalloproteinases-1 (TIMP-1), are decreased in cartilage and synovium under the presence of PRP. However, gene expression related to the formation of collagen 1 and aggrecan is increased [60].

**79**

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

Pain is one of the most characteristic symptoms of OA and one of the most limiting factors for the patient, affecting its functionality and quality of life. One of the main causes of pain associated with joint degeneration is the inflammation that occurs. Solving the inflammatory problem would partly relieve the pain of the OA patient. This relief is one of the most observed effects in clinical studies since it is the most studied variable. However, it is necessary to deepen the mechanisms of action by which the PRP achieves the analgesic effect. During the inflammatory processes, molecules are generated by resident macrophages outstanding prostaglandin E2 (PGE2), which is one of the main causes of the inflammatory pain [61]. As mentioned earlier, PRP favors the change in macrophages from pro-inflammatory to anti-inflammatory phenotype as a consequence of the production of PGE2 and other pro-inflammatory molecule reductions [56]. In addition, the action of the PRP over the NF-Kβ pathway could also reduce the levels of substances that stimulate the nociceptors of the joint synovitis [62]. Therefore, inhibition of the synthesis of these substances is one of the mechanisms of action by which PRP reduces pain. Although the action on inflammation may be the most predominant mechanism

in pain relief, the implication of other pathways has been studied, namely, the peripheral endocannabinoid-mediated mechanism, which could be a promising therapeutic target in the synovial tissue of OA patients [63]. The influence of PRP on this signaling system is associated with the stimulation that occurs in the cells located in inflammatory environments. In the presence of PRR, these cells would generate analgesic substances such as anandamide and 2-arachidonoylglycerol, which are agonists of cannabinoid receptors 1 and 2. This effect is observed both in vitro and in vivo, with a lower nociceptive response in treated animals [62].

One of the problems associated with osteoarthritis is the lack of lubrication and therefore the increased friction in the joint. In a healthy joint, the synovial fluid has a natural lubricant function due to the presence of hyaluronic acid. The alteration of the components of the synovial fluid worsens the lubrication, deteriorating the cartilage. In addition, this layer of lubricant decreases progressively as the disease worsens, creating a vicious circle [64]. Restoring joint lubrication is one of the priorities to improve the course of the disease, and it is the purpose of intra-articular

The application of PRP also may restore joint lubrication through several mechanisms. First, it has a stimulating effect on the chondrocytes and synoviocytes, due to the fact that it not only enhances its proliferation but also increases the production of hyaluronic acid, improving the lubricating capacity of the synovial fluid [66–68]. Secondly, PRP also influences lubrication through the superficial zone protein (SZP) or lubricin. This protein synthesized by chondrocytes and synoviocytes acts as a chondroprotective barrier against direct contact in joints. PRP improves lubrication both directly, since it contains endogenous SZP, and indirectly

by stimulating the SZP secretion by articular cartilage and synovium [69].

All the effects described above are generated through the interaction between growth factors and cell membrane receptors, triggering intracellular pathways and affecting gene expression that generates the biological effects. In addition to these, which are the most influential in the asymptomatic relief of OA, namely,

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

*4.1.2 Analgesic effect*

*4.1.3 Biolubricanting effect*

hyaluronic acid infiltrations [65].

*4.1.4 Cellular modulating effect*

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

#### *4.1.2 Analgesic effect*

*Regenerative Medicine*

*4.1.1 Anti-inflammatory effect*

Multiple actions are attributed to the PRP in the treatment of pathologies of the musculoskeletal system. However, this chapter will be limited to highlighting the effects that have the greatest impact on improving joint degeneration (**Figure 4**).

Due to the complex OA pathophysiology, inflammation can be both the cause and consequence of other pathological processes. Because of this, it is important to reverse the pro-inflammatory environment of this pathology and restore homeostasis of the joint to promote tissue repair. Many of the PRP molecules participate in the regulation of these inflammatory processes, which are key in the progression of the pathology. The anti-inflammatory effect of PRP is achieved through the action of its biomolecules at different levels. Molecules such as IGF-1 or HGF restore the original acquiescent state of cell populations from an inflammatory state because of joint degeneration. This effect occurs through the inhibition of the intracellular signaling pathway NF-Kβ by these molecules, and, as a result, the generation of proinflammatory molecules such as IL-β or TNF-α is reduced [50, 51]. The use of PRP rich in leukocytes can be especially important in this mechanism of action since, instead of inhibiting this inflammatory pathway, they activate it due to the presence of certain pro-inflammatory molecules in this type of PRP [37]. This inhibitory effect not only is limited to chondrocytes but also affects other cell populations such as fibroblast, osteoblasts [52], or macrophages [53]. The consequence of silencing this pathway in the different cell types of the joint is the drop in the inflammatory molecular levels of the synovial fluid, relieving the inflammatory environment [54]. Within its anti-inflammatory action, PRP also acts on macrophages by changing its phenotype. This effect may be indirect due to the decrease in pro-inflammatory molecules or direct by a direct action on the PRP components such as the microparticles produced by platelet apoptosis. The result is a phenotype shift of the macrophages from inflammatory (M1) to reparative (M2) phenotype, where the reduction of inflammation is favored and tissue repair is stimulated [55]. This effect is especially important in macrophages present in the synovial membrane. The increase in anti-inflammatory macrophages to the detriment of pro-inflammatories results in a decrease in inflammation of the synovial membrane, which is a hallmark of OA [56]. This polarization towards a reparative state may be due to the action of the interleukin 1 receptor antagonist, present in the PRP, which in addition to avoiding the inflammatory effect of IL-1 promotes the repair phenotype M2 of

The inflammatory environment in the osteoarthritic joint is also potentiated by the increased presence of reactive oxygen species (ROS), which participate in the OA pathogenesis through synovium inflammation, cartilage degradation, or subchondral bone dysfunction [58]. PRP activates the antioxidant response element through the intracellular signaling pathway NrF2-ARE in osteoblasts [59]. This achieves an antioxidant and protective effect in these cell populations, avoiding

The interaction of PRP biomolecules in the mechanisms that trigger inflammation results not only in a decrease in the levels of pro-inflammatory molecules and ROS but also in a promotion of gene expression related to anti-inflammatory action. It has recently been shown that gene expression of enzymes related to aggrecan destruction and metalloproteinase modulation, namely, metalloproteinase with thrombospondin motifs-5 (ADAMTS-5) and tissue inhibitor of metalloproteinases-1 (TIMP-1), are decreased in cartilage and synovium under the presence of PRP. However, gene expression related to the formation of collagen 1 and aggrecan

**78**

is increased [60].

macrophages [57].

damage caused by ROS increment.

Pain is one of the most characteristic symptoms of OA and one of the most limiting factors for the patient, affecting its functionality and quality of life. One of the main causes of pain associated with joint degeneration is the inflammation that occurs. Solving the inflammatory problem would partly relieve the pain of the OA patient. This relief is one of the most observed effects in clinical studies since it is the most studied variable. However, it is necessary to deepen the mechanisms of action by which the PRP achieves the analgesic effect. During the inflammatory processes, molecules are generated by resident macrophages outstanding prostaglandin E2 (PGE2), which is one of the main causes of the inflammatory pain [61]. As mentioned earlier, PRP favors the change in macrophages from pro-inflammatory to anti-inflammatory phenotype as a consequence of the production of PGE2 and other pro-inflammatory molecule reductions [56]. In addition, the action of the PRP over the NF-Kβ pathway could also reduce the levels of substances that stimulate the nociceptors of the joint synovitis [62]. Therefore, inhibition of the synthesis of these substances is one of the mechanisms of action by which PRP reduces pain.

Although the action on inflammation may be the most predominant mechanism in pain relief, the implication of other pathways has been studied, namely, the peripheral endocannabinoid-mediated mechanism, which could be a promising therapeutic target in the synovial tissue of OA patients [63]. The influence of PRP on this signaling system is associated with the stimulation that occurs in the cells located in inflammatory environments. In the presence of PRR, these cells would generate analgesic substances such as anandamide and 2-arachidonoylglycerol, which are agonists of cannabinoid receptors 1 and 2. This effect is observed both in vitro and in vivo, with a lower nociceptive response in treated animals [62].

#### *4.1.3 Biolubricanting effect*

One of the problems associated with osteoarthritis is the lack of lubrication and therefore the increased friction in the joint. In a healthy joint, the synovial fluid has a natural lubricant function due to the presence of hyaluronic acid. The alteration of the components of the synovial fluid worsens the lubrication, deteriorating the cartilage. In addition, this layer of lubricant decreases progressively as the disease worsens, creating a vicious circle [64]. Restoring joint lubrication is one of the priorities to improve the course of the disease, and it is the purpose of intra-articular hyaluronic acid infiltrations [65].

The application of PRP also may restore joint lubrication through several mechanisms. First, it has a stimulating effect on the chondrocytes and synoviocytes, due to the fact that it not only enhances its proliferation but also increases the production of hyaluronic acid, improving the lubricating capacity of the synovial fluid [66–68]. Secondly, PRP also influences lubrication through the superficial zone protein (SZP) or lubricin. This protein synthesized by chondrocytes and synoviocytes acts as a chondroprotective barrier against direct contact in joints. PRP improves lubrication both directly, since it contains endogenous SZP, and indirectly by stimulating the SZP secretion by articular cartilage and synovium [69].

#### *4.1.4 Cellular modulating effect*

All the effects described above are generated through the interaction between growth factors and cell membrane receptors, triggering intracellular pathways and affecting gene expression that generates the biological effects. In addition to these, which are the most influential in the asymptomatic relief of OA, namely,

inflammation, pain, and lubrication, there are other trophic and regulating effects that, although they do not have such a drastic clinical outcome, are necessary to promote tissue repair and reverse or slow down the disease.

PRP has demonstrated its biological effect over the chondrocytes of articular cartilage and its consequent impact on cartilage repair. Fice et al. published a systematic review including numerous in vitro and in vivo studies that showed the action of PRP on the cellular response [70]. On the one hand, it acts on cellular behavior, increasing growth, migration, and proliferation rates and reducing negative effects such as apoptosis. On the other hand, PRP enhances the synthesis of glycosaminoglycans (GAGs), proteoglycans, and collagen, improving the production of extracellular matrix.

In addition, stimulation of cartilage repair is also conditioned by the action of MSCs. They are able not only to differentiate into cells with specialized functions such as chondrocytes, osteoblasts, and adipocytes but also to release molecules and cellular signals that regulate the repair processes [71]. The behavior of MSCs in OA is modified, increasing in number in synovial fluid as the severity of the disease increases [72]. These MSCs come from resident joint niches such as the synovial membrane, the surface of the articular cartilage, and the subchondral bone, once again confirming the involvement of all the joint structures in the development of OA [17]. In this pathological environment, these cells have their function altered, losing their restorative activity [73]. Bearing this in mind, mesenchymal stem cells are considered a therapeutic target for the PRP to modulate its behavior and restore its physiological functions. Muiños-López et al. observed a decrease in MSCs in synovial fluid of patients with severe OA after the application of PRP directly into the subchondral bone [74]. The regulatory capacity of PRP on MSCs may be due to the direct action on their cellular response as well as the improvement of the biological environment in which the cells reside. Bone marrow-derived MSCs treated with PRP showed an increase in proliferation and chondrogenic capacity [75]. This increased proliferation was also observed in human adipose-derived stem cells, although their chondrogenic differentiation potential was retained [76]. Restoring tissue homeostasis where MSCs reside, for instance, by decreasing inflammation by inhibiting pro-inflammatory molecules, also improves the action of these cells. The attenuation of a TGF-β-mediated signaling excess in the subchondral bone during OA restores the dysfunction of the MSCs, preventing cartilage degeneration [77]. Liu et al. observed that intraosseous infiltrations of PRP promoted MSC cell proliferation and osteogenesis in an in vivo study, whereas adipogenesis, senescence, and oxidative stress were decreased [78].

#### **4.2 Clinical translation**

The transfer of the PRP from the laboratory to the clinical application has been very fast with extensive worldwide expansion. This has occurred in part because of its ease of obtaining and its high safety as an autologous product, and, as a consequence, more and more clinical studies are being published on the use of PRP in OA. It is not the intention of this chapter to analyze all these studies but to highlight the most relevant aspects of this translation. The latest published meta-analyses concluded that the use of intra-articular PRP infiltrations achieves effects on symptoms such as pain relief or improved function better than the use of hyaluronic acid or placebo especially for the long term [79–81]. Based on these data, it could be accepted that the PRP has evolved from being a promising alternative to a real option for clinicians and patients.

However, it should not be forgotten that it is necessary to continue to carry out high-quality clinical studies to clarify possible doubts and achieve the ideal

**81**

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

protocol for both obtaining and applying PRP products [82]. Some of the clinical studies carried out have attempted to elucidate this type of questions, the presence of leukocytes being one of the most critical issues. Several authors have studied the clinical effect of including leukocytes in the PRP. Mariani et al. studied the pro-inflammatory effect that intra-articular infiltrations of Leukocyte-Rich PRP could have. Surprisingly, and contrary to the in vitro studies, patients who received this treatment did not experience an increase of pro-inflammatory molecules in the synovial fluid or plasma in the short or long term [83]. These data were confirmed in a meta-analysis where there were no differences in adverse reactions between PRP with and without leukocytes, being very rare and local such as pain and inflammation. However, as far as efficacy is concerned, this same work carried out by Riboh et al. showed that PRP poor in leukocytes had significantly better results than those obtained by hyaluronic acid and placebo, whereas this difference did not occur in PRP rich in leukocytes. Therefore, according to the studies carried out in this matter, the inclusion of leukocytes in the PRP does not affect the safety of the product but does diminish its effectiveness in the treatment of knee OA [36]. In spite of these advances, it is necessary to continue studying the rest of the composition variables that may condition the clinical response of the PRP, such as platelet concentration. Recent studies suggest that a concentration below fivefold blood

Not only the variables related to the obtaining or composition of the PRP products condition the clinical effect of this therapy but also the different methods of application. Several clinical studies addressed the effect of a single or repeated administration of intra-articular infiltrations of PRP. A first group of studies focused on analyzing the differences between a single infiltration of PRP and several repeated infiltrations every 1 or 2 weeks. These studies demonstrated that PRP obtained better results than control treatment and, in addition, patients who received repeated intra-articular infiltrations of PRP achieved better clinical response on items such as pain, symptomatology, and function [85–87]. Other studies analyzed the effect of applying several cycles of PRP infiltrations, referring to a cycle as a series of repeated infiltrations in a short period of time. Gobbi et al. compared the efficacy of administration of one PRP cycle against two PRP cycles separated by 1 year, one cycle being three intra-articular infiltrations of PRP in 1 month. In both groups, there was an improvement in patients 1 year after the first cycle, which was accentuated at 18 months after the application of a second cycle [88]. Vaquerizo et al. conducted a similar study comparing patients treated with one PRP cycle with patients treated with two PRP cycles separated by 6 months, one cycle being three weekly intra-articular PRP infiltrations. The results showed that although there were no significant differences in pain improvement, patients who received two cycles had better symptoms and functionality 1 year after treatment [89]. Thus, the different studies that analyze this variable recommend the applica-

Following with the PRP administration modality, it is important to remember that the mechanism of action of PRP biomolecules is cell stimulation and improvement of the biological environment to favor tissue repair. Furthermore, as explained at the beginning of this chapter, OA is an alteration of the whole joint and not just a few elements. Considering these two assumptions, it would be advisable to act on the majority of the tissues involved in the joint and especially on those that perform a more predominant biological function. When PRP is intra-articularly administered, it soaked the articular space, reaching and acting on the cells present both in the synovial membrane and on the articular surface. However, this route of administration does not reach the subchondral bone which communicates with the cartilage, especially in OA case, and it is fundamental both in the maintenance

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

platelet concentration is recommended [84].

tion of repeated PRP injections instead of isolated ones.

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

protocol for both obtaining and applying PRP products [82]. Some of the clinical studies carried out have attempted to elucidate this type of questions, the presence of leukocytes being one of the most critical issues. Several authors have studied the clinical effect of including leukocytes in the PRP. Mariani et al. studied the pro-inflammatory effect that intra-articular infiltrations of Leukocyte-Rich PRP could have. Surprisingly, and contrary to the in vitro studies, patients who received this treatment did not experience an increase of pro-inflammatory molecules in the synovial fluid or plasma in the short or long term [83]. These data were confirmed in a meta-analysis where there were no differences in adverse reactions between PRP with and without leukocytes, being very rare and local such as pain and inflammation. However, as far as efficacy is concerned, this same work carried out by Riboh et al. showed that PRP poor in leukocytes had significantly better results than those obtained by hyaluronic acid and placebo, whereas this difference did not occur in PRP rich in leukocytes. Therefore, according to the studies carried out in this matter, the inclusion of leukocytes in the PRP does not affect the safety of the product but does diminish its effectiveness in the treatment of knee OA [36]. In spite of these advances, it is necessary to continue studying the rest of the composition variables that may condition the clinical response of the PRP, such as platelet concentration. Recent studies suggest that a concentration below fivefold blood platelet concentration is recommended [84].

Not only the variables related to the obtaining or composition of the PRP products condition the clinical effect of this therapy but also the different methods of application. Several clinical studies addressed the effect of a single or repeated administration of intra-articular infiltrations of PRP. A first group of studies focused on analyzing the differences between a single infiltration of PRP and several repeated infiltrations every 1 or 2 weeks. These studies demonstrated that PRP obtained better results than control treatment and, in addition, patients who received repeated intra-articular infiltrations of PRP achieved better clinical response on items such as pain, symptomatology, and function [85–87]. Other studies analyzed the effect of applying several cycles of PRP infiltrations, referring to a cycle as a series of repeated infiltrations in a short period of time. Gobbi et al. compared the efficacy of administration of one PRP cycle against two PRP cycles separated by 1 year, one cycle being three intra-articular infiltrations of PRP in 1 month. In both groups, there was an improvement in patients 1 year after the first cycle, which was accentuated at 18 months after the application of a second cycle [88]. Vaquerizo et al. conducted a similar study comparing patients treated with one PRP cycle with patients treated with two PRP cycles separated by 6 months, one cycle being three weekly intra-articular PRP infiltrations. The results showed that although there were no significant differences in pain improvement, patients who received two cycles had better symptoms and functionality 1 year after treatment [89]. Thus, the different studies that analyze this variable recommend the application of repeated PRP injections instead of isolated ones.

Following with the PRP administration modality, it is important to remember that the mechanism of action of PRP biomolecules is cell stimulation and improvement of the biological environment to favor tissue repair. Furthermore, as explained at the beginning of this chapter, OA is an alteration of the whole joint and not just a few elements. Considering these two assumptions, it would be advisable to act on the majority of the tissues involved in the joint and especially on those that perform a more predominant biological function. When PRP is intra-articularly administered, it soaked the articular space, reaching and acting on the cells present both in the synovial membrane and on the articular surface. However, this route of administration does not reach the subchondral bone which communicates with the cartilage, especially in OA case, and it is fundamental both in the maintenance

*Regenerative Medicine*

tion of extracellular matrix.

oxidative stress were decreased [78].

option for clinicians and patients.

**4.2 Clinical translation**

inflammation, pain, and lubrication, there are other trophic and regulating effects that, although they do not have such a drastic clinical outcome, are necessary to

PRP has demonstrated its biological effect over the chondrocytes of articular cartilage and its consequent impact on cartilage repair. Fice et al. published a systematic review including numerous in vitro and in vivo studies that showed the action of PRP on the cellular response [70]. On the one hand, it acts on cellular behavior, increasing growth, migration, and proliferation rates and reducing negative effects such as apoptosis. On the other hand, PRP enhances the synthesis of glycosaminoglycans (GAGs), proteoglycans, and collagen, improving the produc-

In addition, stimulation of cartilage repair is also conditioned by the action of MSCs. They are able not only to differentiate into cells with specialized functions such as chondrocytes, osteoblasts, and adipocytes but also to release molecules and cellular signals that regulate the repair processes [71]. The behavior of MSCs in OA is modified, increasing in number in synovial fluid as the severity of the disease increases [72]. These MSCs come from resident joint niches such as the synovial membrane, the surface of the articular cartilage, and the subchondral bone, once again confirming the involvement of all the joint structures in the development of OA [17]. In this pathological environment, these cells have their function altered, losing their restorative activity [73]. Bearing this in mind, mesenchymal stem cells are considered a therapeutic target for the PRP to modulate its behavior and restore its physiological functions. Muiños-López et al. observed a decrease in MSCs in synovial fluid of patients with severe OA after the application of PRP directly into the subchondral bone [74]. The regulatory capacity of PRP on MSCs may be due to the direct action on their cellular response as well as the improvement of the biological environment in which the cells reside. Bone marrow-derived MSCs treated with PRP showed an increase in proliferation and chondrogenic capacity [75]. This increased proliferation was also observed in human adipose-derived stem cells, although their chondrogenic differentiation potential was retained [76]. Restoring tissue homeostasis where MSCs reside, for instance, by decreasing inflammation by inhibiting pro-inflammatory molecules, also improves the action of these cells. The attenuation of a TGF-β-mediated signaling excess in the subchondral bone during OA restores the dysfunction of the MSCs, preventing cartilage degeneration [77]. Liu et al. observed that intraosseous infiltrations of PRP promoted MSC cell proliferation and osteogenesis in an in vivo study, whereas adipogenesis, senescence, and

The transfer of the PRP from the laboratory to the clinical application has been very fast with extensive worldwide expansion. This has occurred in part because of its ease of obtaining and its high safety as an autologous product, and, as a consequence, more and more clinical studies are being published on the use of PRP in OA. It is not the intention of this chapter to analyze all these studies but to highlight the most relevant aspects of this translation. The latest published meta-analyses concluded that the use of intra-articular PRP infiltrations achieves effects on

symptoms such as pain relief or improved function better than the use of hyaluronic acid or placebo especially for the long term [79–81]. Based on these data, it could be accepted that the PRP has evolved from being a promising alternative to a real

However, it should not be forgotten that it is necessary to continue to carry out high-quality clinical studies to clarify possible doubts and achieve the ideal

promote tissue repair and reverse or slow down the disease.

**80**

#### **Figure 5.**

*Intraosseous administration of PRP. Intraosseous PRP administrations allow the subchondral bone to be reached and its therapeutic effect to be extended. Intraosseous infiltrations are applied in the femoral condyle (A) and tibial plateau (B) in patients with knee OA and in the acetabulum (C) and femoral head (D) in cases of hip OA.*

of homeostasis and in the pathophysiology of joint degeneration [17]. In order to extend the range of action of the PRP and also act on the subchondral bone, Sánchez et al. described the technique of PRP intraosseous infiltrations (**Figure 5**). This method of application combines conventional intra-articular injection of PRP with intraosseous infiltrations into the subchondral bone of the femoral condyle and tibial plateau in severe cases of knee OA [90]. Afterward, this technique was adapted to treat advanced cases of hip OA, combining intra-articular infiltration with intraosseous infiltrations into the femoral head and acetabulum [91]. In both cases, intraosseous administration must be assisted by imaging, ultrasound, or fluoroscopy, to ensure correct delivery in the required area.

The first published works carried out using this technique provided promising results. In a pilot study performed with patients who presented knee OA of grades 3 and 4 according to the Ahlbäck scale, pain was significantly reduced, and an increase in joint function was observed at 6 months after receiving the combination of intra-articular and intraosseous PRP injections. In addition, the number of MSCs present in the synovial fluid decreased after this treatment [92]. This finding was not observed in patients treated only with intra-articular infiltrations, suggesting the importance of the subchondral bone in the modulation of cellular response in joint degeneration [74]. Following the same trend, an observational study compared the intra-articular administration of PRP versus the combination of intra-articular and intraosseous injections in patients with severe knee OA. The results of this study showed that although there was no difference between both groups at 2 months after treatment, patients who received the PRP intraosseously showed clinically superior results at 6 and 12 months [93]. Su et al. conducted a

**83**

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

clinical trial in which, in addition to comparing intra-articular against intraosseous injections, they used hyaluronic acid as a control treatment. The patients enrolled in this study presented knee OA of grades 2 and 3 according to the Kellgren-Lawrence scale. The results achieved with treatment based on intraosseous infiltrations of PRP were superior to those obtained with both intra-articular PRP and hyaluronic acid [94]. No severe adverse effects were reported in any of these studies, and they were limited to pain after infiltrations. One of the characteristics of the subchondral bone in patients with knee OA is the presence of fibroneurovascular proliferation. Although the PRP contains proangiogenic and profibrotic molecules, no basic or clinical study showed the uncontrolled induction of this effect after the application

Finally, intra-articular injections of MSCs derived from various sources associated with PRP were analyzed in some studies. The vehiculization of MSCs in PRP could entail an improvement in cell viability and may be translated into better clinical results. Although studies performed with both bone marrow [95, 96]- and stroma fraction [97, 98]-derived MSCs showed improvement in these patients after the application of this therapeutic combination, the association of PRP with the MSCs did not lead to a greater clinical improvement in patients. However, the therapeutic potential of the synergy of both therapies justifies further research in

Joint degeneration is a pathology that affects a large part of the population, deteriorating their quality of life being disabling in many cases. It is also related to aging and unhealthy lifestyle habits; thus it is expected that its prevalence will increase in the coming years, assuming a great cost to health systems. Current conventional treatments focus on symptomatic relief without addressing the cause of the disease. Because of this, new treatments based on regenerative medicine are emerging in order to expand the therapeutic arsenal and delay or prevent joint replacement, which is currently the only definitive solution for patients. Moreover, in order to achieve an optimal treatment for joint degeneration, it must be understood that the joint works as a whole organ. All elements of the joint participate in the maintenance of homeostasis, the synovial membrane, cartilage, and subchondral bone

This balance could be maintained or restored by means of several biological therapies such as PRP that is a cocktail of plasma and platelet biomolecules, and it is obtained after fractionating small blood volumes by centrifugation. PRP has a great versatility since it allows its use through different types of formulations, being able to be applied both in outpatient infiltrations and surgical interventions. The therapeutic potential of PRP in joint degeneration lies in its ability to modulate inflammation, lubrication, and pain, acting on different cell populations to create a biological environment conducive to tissue repair. However, the variety in the composition of PRP products leads to different biological effects and consequently contradictory clinical results. It is, therefore, necessary to identify and characterize

The success of the PRP also depends on the method of clinical application. The

administration of PRP has to cover the main joint tissues so that the biological effects of PRP act over the cells of in order to reverse the course of the pathology. Although the safety and ease of obtaining PRP have allowed a quick transfer from the laboratory to the hospital, much is still unknown about this therapy, and further

the PRP used in order to advance both research and clinical practice.

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

of intraosseous PRP [22].

this field.

**5. Conclusions**

being key for biological balance.

basic and clinical research is needed.

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

clinical trial in which, in addition to comparing intra-articular against intraosseous injections, they used hyaluronic acid as a control treatment. The patients enrolled in this study presented knee OA of grades 2 and 3 according to the Kellgren-Lawrence scale. The results achieved with treatment based on intraosseous infiltrations of PRP were superior to those obtained with both intra-articular PRP and hyaluronic acid [94]. No severe adverse effects were reported in any of these studies, and they were limited to pain after infiltrations. One of the characteristics of the subchondral bone in patients with knee OA is the presence of fibroneurovascular proliferation. Although the PRP contains proangiogenic and profibrotic molecules, no basic or clinical study showed the uncontrolled induction of this effect after the application of intraosseous PRP [22].

Finally, intra-articular injections of MSCs derived from various sources associated with PRP were analyzed in some studies. The vehiculization of MSCs in PRP could entail an improvement in cell viability and may be translated into better clinical results. Although studies performed with both bone marrow [95, 96]- and stroma fraction [97, 98]-derived MSCs showed improvement in these patients after the application of this therapeutic combination, the association of PRP with the MSCs did not lead to a greater clinical improvement in patients. However, the therapeutic potential of the synergy of both therapies justifies further research in this field.
