Platelet-rich plasma application in chondrogenesis.

Platelet-rich plasma (PRP), an autologous derivative of whole blood, has been recently used in surgical treatment. PRP contains growth factors including transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) and also bioactive proteins that influence the healing of tendon, ligament, muscle, and bone. This article describes the current clinical applications of PRP in chondrogenesis. This study reviews and evaluates the studies that have been published in the field of chondrogenesis. All aspects of using PRP in chondrogenesis are reviewed.

INTRODUCTION

During the last decade, the term platelet-rich plasma (PRP) has received wide and growing attention in the field of regenerative medicine.[1] PRP is defined as the portion of the plasma fraction of blood having a platelet concentration above the baseline value.[23] The significance behind using PRP is due to the abundance of growth factors and protein in a well-prepared PRP concentrate involved in tissue engineering.[4] There are some advantages of using PRP. First of all, it is easy to obtain PRP from patient's own blood. Secondly, by regulating the processing technique and activation protocol, it is possible to control the dose of growth factors released on activation.[5] PRP has been used in medical fields such as oral and maxillofacial surgery to enhance hard and soft tissue healing and it has gained attention in orthopedic and sports medicine as a treatment for various problems, including bone, cartilage, ligament, and tendon pathologies.[678910] Due to the poor regenerative capability of articular cartilage and currently limited clinical treatments, recently cartilage repair, through tissue engineering, has been considered as an alternative approach.

There are two promising cell sources for cartilage tissue engineering: Mesenchymal stem cells (MSCs) and chondrocytes. Both can be differentiated in 3D culture[1112] in the presence of growth factors such as transforming growth factor-β (TGF-β), insulin-like growth factor-1, and bone morphogenic protein-6 (BMP-6).[13]

The importance behind using PRP in cartilage tissue engineering field is that PRP is rich in growth factors, including those that promote proliferation of chondrogenic cells and secretion of cartilaginous matrix, such as TGF-β, platelet-derived growth factor (PDGF),[14] insulin-like growth factor (IGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF).[151617] There are three main types of vesicles detected in a platelet: 1) a-granules, 2) β lysosomes, and 3) dense-core granules. Many of the key ingredients such as growth factors are stored in a-granules.[18] The PRP growth factors and their role in chondrogenesis are given in Table 1.

Table 1

Growth factors present in platelet-rich plasma and their roles in chondrogenesis

This review will provide an overview of the studies featuring the role of PRP in chondrogenesis of chondrocytes and stem cells.

PREPARATION OF PRP

In all available PRP techniques, blood is collected with an anticoagulant such as ethylenediaminetetraacetic acid a mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD)[26] and then it is immediately processed by centrifugation. The time for platelet concentrate preparation is about an hour. The first centrifugation step is designed to separate the blood into three layers: Red blood cells (RBCs) are located at the bottom, acellular plasma or platelet-poor plasma (PPP) is at the top, and a “buffy coat” layer appears in between, in which platelets are concentrated. The goal of the subsequent steps is to discard both the RBC layer and the PPP to collect only the buffy coat layer. Finally, the obtained platelet concentrate is applied to the desired site.[27]

PRP was applied in three forms, including fresh platelets, activated platelets by freeze –thaw, and through thrombin cycles in research and clinical trial. The concentration of the growth factors released was quantified with enzyme-linked immunosorbent assay (ELISA) kits. The highest concentrations of epidermal growth factor (EGF) and fibroblast growth factor (FGF) were found in frozen platelets while the maximum TGF-β1 was detected in thrombin-activated platelets.[28]

EFFECTS OF PRP ON THE CHONDROCYTES’ PROLIFERATION AND MATRIX SYNTHESIS

To investigate the effect of PRP on chondrogenesis, it is necessary to consider the changes that occur on the chondrogenic markers such as SOX9, aggrecan, and collagen type II. A summary of all the studies is presented in Table 2. It has been shown that using 10% PRP in place of 10% fetal bovine serum in the dulbecco's modified eagle medium (DMEM) for culturing porcine chondrocytes in alginate beads produced more proteoglycans, glycosaminoglycan (GAG), and DNA.[29] The increased production of GAG was seen when 3% PRP was mixed with gelatin hydrogel and then injected intra-articularly to the rabbit model of osteoarthritis (OA).[30]

Table 2

PRP effects on the chondrocytes in vitro and in vivo

Interestingly, PRP in the form of medium supplement in vitro and in the gel form that encapsulated cell brick stimulated the synthesis of collagen type II by chondrocytes[2931] and caused upregulation of the SOX9 gene expression and when was used in monolayer as a medium supplement or was mixed with hydrogel.[3233] it has the same effects. The proliferative effect of PRP on chondrocytes from different sources such as bovine articular and nasal septal chondrocytes,[34] rabbit articular chondrocytes,[35] and sheep articular chondrocytes[36] was reported even when PRP was used in the form of platelet lysate. However, the effect of PRP on matrix accumulation was not confirmed in some studies.[3435] These inconsistent results may be due to the activation form of PRP or the source of chondrocytes. The 10% PRP also stimulates upregulation of TGF-β, VEGF, and chondromodulin-I (ChM-I) by rabbit chondrocytes.[37] This indicates that culturing chondrocytes in the presence of PRP may alter their gene expression profile. PRP can be considered as a successful injectable carrier to study the chondrocytes’ differentiation potential. Results showed that PRP as a gel could provide ideal conditions to preserve the chondrocyte phenotype in vivo and in vitro.[3839]

THE EFFECTS OF PRP ON THE CHONDROGENESIS AND PROLIFERATION OF MSCs

Due to the problems related to the autologous chondrocyte implantation method[40] for cartilage defect treatment, much attention has been paid to find other cell sources for cartilage tissue engineering. MSCs are a promising cell population for regeneration of mesenchymal tissues such as cartilage. The main chondrogenic inducer MSCs belong to TGF-β family.[4142] Due to the complexities involved in the safety and efficacy of either exogenous or genetically induced growth factor delivery, investigators are trying to find[43] substitutions such as PRP. The core ingredient of PRP is TGF-β1.[44] A summary of all the studies is presented in Table 3.

Table 3

PRP effects on the MSCs in vitro and in vivo

Injection of muscle-derived stem cells (MDSCs) with PRP into the knees of rat model of OA produced more collagen type II and decreased the number of apoptotic cells in articular cartilage, promoted proliferation, adhesion, and migration of MDSCs, and finally enhanced the integration of the transplanted cells in the repair process.[45] The effect of human PRP on the chondrogenesis of human subchondral progenitor cells in pellet culture system has been established. At the same time, the upregulation of cartilage hypertrophic marker collagen type X was detectable.[46] It seems that presence of different components of inductive medium such as dexamethasone, insulin–transferrin–selenium (ITS), and ascorbic acid for chondrogenic induction of human umbilical cord derived mesenchymal stem cells (HUCMSCs)[47] and adipose-derived stem cells (ADSCs) (unpublished data) is necessary. The gene expression of aggrecan and SOX9 were enhanced when bone marrow derived stem cells (BMSCs) were in the presence of 10% buffered PRP[16] This shows that probably activation step is not mandatory. Application of 10% PRP as a medium supplement in monolayer culture of BMSCs and ADSCs caused upregulation of collagen type II, aggrecan, and SOX9. When PRP was used as a scaffold for BMSCs and ADSCs, it produced the hyaline cartilage.[48] The mentioned results show the positive effects of PRP in chondrogenesis of MSCs from different tissues.

THE ANTI-INFLAMMATORY EFFECTS OF PRP ON THE CHONDROCYTES

The anti-inflammatory effects of PRP on the chondrocytes seem to correlate with the upregulation of cannabinoid receptor type 1 (CB1). It is reported that CB1 agonists have analgesic and anti-inflammatory effects and reduce joint damage in animal models of arthritis.[334950] The anti-inflammatory effects of PRP are due to the reduction in the transactivation of nuclear factor-kappa B (NF-κB), the critical regulator of the inflammatory process. Activated PRP has an enhanced concentration of hepatocyte growth factor (HGF) and tumor necrosis factor-a (TNF-a). These growth factors, by disrupting the transactivation of NF-κB, are the key ingredients that contribute to PRP anti-inflammatory effects. The second mechanism is decreasing the expression of inflammatory enzymes cycloxygenase 2 and 4 (COX-2 and COX-4).[51] Decreasing the gene expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and prostaglandin-endoperoxide synthase (PTGS) 2 is another mechanism used by PRP to avoid inflammation in chondrocytes.[52] It was shown that PRP could have pro-inflammatory effect on the human chondrocytes after the initial reduction of COX-2;[53] may be, PRP has a dual effect on the chondrocytes’ response to inflammatory conditions.

CONCLUSION

There are several potential advantages of using PRP in tissue engineering, especially in chondrogenesis. PRP can keep the phenotype and differentiation potential of chondrocytes in terms of proliferation, and synthesis of proteoglycan and collagen type II.

It seems that PRP induce chondrogenesis of MSCs through the secretion of various growth factors, especially TGF-β, since its concentration is high.

The limitation of using PRP is due to two reasons. Firstly, there is not a standard preparation protocol and various platelet activation methods sometimes give different results that do not support each other, thereby making it difficult to compare them. Secondly, there is lack of knowledge about the growth factors and their concentration at which they exert PRP chondrogenic effect. To solve these issues, it is necessary to determine the concentration of PRP growth factors in each study. Since MSCs’ chondrogenesis induced by PRP led to production of hypertrophic cartilage, a better understanding of the mechanisms of chondrogenesis is necessary. For OA, however, it needs further investigation.