Platelet-Rich Plasma and Cartilage Repair

Abstract

Purpose of Review

To assess the utilization and efficacy of platelet-rich plasma (PRP), for the treatment of articular cartilage injury, most commonly characterized by progressive pain and loss of joint function in the setting of osteoarthritis (OA).

Recent Findings

PRP modulates the inflammatory and catabolic environment through a locally applied concentrate of platelets, leukocytes, and growth factors. Clinically, PRP has been shown to be possibly a viable treatment adjuvant for a variety of inflammatory and degenerative conditions. Recent efforts have focused on optimizing delivery methods that enable platelets to slowly degranulate their biological constituents, which may promote healing and improve OA symptoms for a longer duration.

Summary

There are various factors that affect the progression of OA within joints, including inhibition of inflammatory cytokines and altering the level of enzymatic expression. PRP therapy aims to mediate inflammatory and catabolic factors in a degenerative environment through the secretion of anti-inflammatory factors and chemotaxic effects. There are a growing number of studies that have demonstrated the clinical benefit of PRP for non-operative management of OA. Additional randomized controlled trials with long-term follow-up are needed in order to validate PRP’s therapeutic efficacy in this setting. Additionally, continued basic research along with well-designed pre-clinical studies and reporting standards are necessary in order to clarify the effectiveness of PRP for cartilage repair and regeneration for future clinical applications.

Introduction

Osteoarthritis (OA) is a disease that commonly affects the knee and is described as the progressive loss of joint function and development of pain from gradual deterioration of articular cartilage. The Global Burden of Disease estimated that knee OA affects 3.8% of the global population, and trends favor a greater likelihood of OA development with increased age and more so within the female population (Female 4.8%, Male 2.8%) [1]. Furthermore, the incidence of OA has been shown in the literature to significantly increase following an injury; 5.6 million cases of posttraumatic OA (PTOA) are reported annually with a recent study finding that an estimated 51.6% of patients develop PTOA following ACL injury by 20 years post-surgery [2].

Current approaches for the treatment of OA have recently incorporated the use of biologics that mediate the inflammatory process. The health of articular cartilage depends on a balanced biological milieu of anabolic and catabolic factors. The use of biologics in the treatment of OA is designed to optimize this balance [3–5]. Platelet-rich plasma (PRP) is increasingly used for its ability to affect tissue regulation from the growth factors present in elevated platelet levels, and thereby reduce pain associated with OA [6–9].

To improve the utilization of PRP for articular cartilage treatment, we must understand [1] the catabolic interaction of cytokines inside osteoarthritic environments, [2] the composition of PRP and the mechanism by which it alters the inflammatory processes, and [3] the implications of the current published data on the use of PRP injections in the treatment of articular cartilage disease.

Factors Influencing Osteoarthritis

Inflammatory Cytokines

Inflammatory cytokines influence the production of cytokines and enzymes through intracellular pathways of signal transduction and are believed to be a major factor in OA pathogenesis by altering tissue formation from the promotion of catabolic processes [5]. Two inflammatory cytokines that have been most correlated with the altered environment from OA are interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) [5]. IL-1β and TNF-α are found at elevated levels in fluid and tissue affected by OA [10]. The elevated levels of IL-1β and TNF-α may be contributing factors in cartilage degradation by influencing the expression of chondrocytes to alter structural proteins by inhibiting synthesis of type-II collagen and aggrecan [11, 12], and by promoting the synthesis of matrix metalloproteinases (MMPs), which are harmful to cartilage [13–16]. Additionally, disorders of chondrogenic progenitor cells (CPCs) and induced chondrocyte death have also been reported from elevated levels of IL-1β and TNF-α, resulting in more rapid aging and joint degeneration [17–19]. IL-1β has been found to stimulate production of reactive oxygen species (ROS) which generate free radicals that directly damage articular cartilage [20]. Interleukin-6 (IL-6), usually produced in response to IL-1β and TNF-α by tissue of the affected joint, is a cytokine known for enhancement of the inflammatory response and activation of the immune system [21–23]. IL-6 is believed to play a major role in bone resorption through the formation of osteoclasts of the subchondral bone layer [23–25], while also displaying synergism with IL-1β and TNF-α in influencing decreased type-II collagen synthesis and the increased production of enzymes from MMPs [24, 26].

Anti-inflammatory Cytokines

Interleukin-4 (IL-4) is an anti-inflammatory cytokine that is believed to have a chondroprotective effect by inhibiting both the degradation of proteoglycans in articular cartilage and the secretion of MMPs [27–30], preventing aggrecan breakdown and severe cartilage erosion. This effect is less profound during OA because chondrocytes become less susceptible to IL-4 [31–33]. Interleukin-10 (IL-10) likewise has a chondroprotective effect by stimulating the synthesis of type-II collagen and aggrecan, and significantly reducing the secretion of IL-1β and TNF-α [34].

Interleukin-13 (IL-13) has also demonstrated an ability to inhibit the arthritic pro-inflammatory process by way of inhibiting synthesis of IL-1β and TNF-α [27, 35]. Synovial samples treated with IL-13 have been reported to inhibit the effects of the synthesis of IL-1β, TNF-α, and MMP-3, while also increasing the level of IL-1 receptor antagonist (IL-1Ra) [36], which reduces the binding of fibroblasts between IL-1β and its receptor. Signaling and cell activation by IL-1β occur when an agonist IL-1 family cytokine binds to its respective TIR containing receptor, which subsequently recruits an accessory chain and initiates signaling/cell activation [37]. IL-1Ra blocks the engagement of the accessory chain (IL-1R3) by binding to the ligand receptor (IL-1R1), which prevents the accessory protein from engaging the ligand-receptor complex. This interaction inhibits any subsequent signaling [37].

Enzymes

Low levels of MMP expression contribute to tissue remodeling and turnover of healthy cartilage, but during the development of OA, upregulation of MMP expression occurs from chondrocyte binding to IL-1β and TNF-α through signal pathways involving NF-κΒ [38, 39]. Many catabolic pathways involve the upregulation of MMP-13, a key enzyme in cartilage degeneration through its ability to cleave both type-II collagen [4] and aggrecan [40]. The role of MMP-13 in cartilage degeneration has been shown in multiple mice model studies, where a constitutively active MMP-13 caused OA-like degeneration [41], and knockout MMP-13 resulted in resistance to cartilage erosion [42].

Transcription Factors

Nuclear factor κΒ (NF-κΒ) contributes significantly to the processes involved in cartilage degradation by inducing elevated levels of harmful factors and hindering the cartilage repair process through altered expression of inflammatory cytokines, enzymes, and transcription factors. Once activated by IL-1β and TNF-α, NF-κΒ becomes a positive regulator for the expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 [43], while also regulating promotion of the enzymes MMP-9, MMP-1, and MMP-13 [44, 45]. NF-κΒ suppresses SOX-9, a transcription factor involved in the enhanced expression of type-II collagen and aggrecan [46–48], In addition, NF-κΒ inhibits SOX-9 mRNA in chondrocytes while in the presence of IL-1 and TNF-α, resulting in reduced extracellular matrix protein synthesis (Fig. 1) [55].

Fig. 1.

(1a) Catabolic factors interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) stimulate MMP synthesis. (1b) Matrix metalloproteinases (MMPs) cause degradation of the collagenous framework and induce collagen Type X expression. (1c) Degradation of the cartilage and chondrocytes promotes catabolic activation. (2a) NF-κΒ receptor activator ligand (RANKL) activates in the presence of pro-inflammatory factors and (2b) results in the downregulation of SRY-box 9 (SOX-9) and upregulation of cyclooxygenase (COX-2) and nitric oxide synthesis (NOS). (2c) Inhibition of SOX-9 downregulates collagen Type II. (3a) Pro-inflammatory factors stimulate reactive oxygen species (ROS) and activates catabolic factors. (3b) ROS causes chondrocyte apoptosis and release of catabolic and degenerative factors [3, 48–54]

The inhibition of NF-κΒ reduces the effect of degenerative contributors by suppressing intra-articular signaling of IL-1 and TNF-α. This lessens the upregulation of MMPs and aggrecanases, while reducing the effect upon type-II collagen downregulation in chondrocytes [56–59].

Composition of Platelet-Rich Plasma and Influence on Osteoarthritis

PRP is obtained following the centrifugation of whole blood, yielding a product highly concentrated with platelets. The α-granules within the concentrated platelet solution contain growth factors and proteins vital to the coagulation cascade which, upon activation, may aid in the regeneration of tissues [60]. To combat the catabolic environment of joints affected by OA, PRP is believed to counteract cartilage erosion by inhibiting the catabolic cytokines of IL-1β and TNF-α [61, 62], and by promoting factors associated with cartilage matrix synthesis including fibroblast growth factor, transforming growth factor-β (TGF-β), and others [63–65].

Furthermore, PRP therapy aims to modulate the inflammatory and catabolic environment through its proposed anti-inflammatory effects [66]. PRP activated by thrombin has an enhanced initial content of hepatocyte growth factor (HGF), IL-4, TNF-α, and TGF-β1, along with increases in vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) [67]. In laboratory studies, PRP produces an increase of mRNA and protein levels for type-II collagen and aggrecan [68], in addition to lessening the occurrence of IL-1β-induced inhibition of type-II collagen and aggrecan [66]. Bendinelli et al. [67] proposed that PRP contributes to an anti-inflammatory effect through HGF and TNF-α reduction of NF-κΒ transactivating activity and target gene expression in chondrocytes, while preventing monocyte chemotaxis by expression of TGF-β1 countering the effect on chemokine transactivation by TNF-α. HGF is believed to play a predominant role in the anti-inflammatory effect exerted by PRP, by inhibiting NF-κΒ activity, which, upon the blockade of HGF by the competitive inhibitor NK4, the inhibition on NF-κΒ activity was almost nullified.

In addition, PRP indirectly influences the activity of transcription factor NF-κΒ [69–71], and it may also work to reduce the elevated levels of nitric oxide (NO) [72]. Nitric oxide contributes to cartilage degeneration by inhibiting collagen synthesis [73], inducing chondrocyte apoptosis [74], and increasing production of MMPs [75]. Vuolteenaho et al. [76] reported that TGF-β and a NF-κΒ inhibitor decreased NO production within chondrocytes, but upon testing with PRP releasate, only NF-κΒ activation was counteracted, while NO production was unaltered. [66]

Other way that PRP is believed to influence cartilage degeneration is by alteration of autophagy in chondrocytes. Aging cartilage eventually loses its reversible quiescence, along with its self-renewing capacity [77]. Importantly, studies have shown an increase in chondrocyte quiescence following PRP injection [78], which ultimately may restore this regenerative process through the reestablishment of autophagy and reversal of the senescence process [79].

PRP Applications for Cartilage Defects and Early Onset of OA

There are several operative (microfracture, osteochondral, and tissue engineered grafts) and non-operative (single-molecule agents, hyaluronic acid, corticosteroid injections) treatment strategies for cartilage repair and management of knee OA pain [80]. There is currently a very high demand for immunomodulatory biological approaches to treat cartilage defects and delay progressive OA. Pharmaceutical production of single disease modifying biological agents, such as inflammatory antagonist receptors, has reported some clinical efficacy for the treatment of OA. However, single-molecule agents and targeted biological agents have several limitations in the setting of cartilage defects (CDs) and early onset OA. In contrast, PRP is comprised of several bioactive molecules and proteins that augment the three phases of tissue healing (inflammation, cell proliferation, and remodeling) [80]. Three preparation types have been evaluated in osteochondral lesions and in the early onset of OA: fibrin glue or gel, inactivated leukocyte-poor PRP, and leukocyte-rich PRP [81]. In this section, the pre-clinical and clinical applications of different PRP preparations on cartilage repair and regeneration will be discussed.

Pre-clinical Applications

The activation of PRP results in the secretion of growth factors over the course of up to 7 days and maximizes their effect on both local and migrating cells [81, 82]. A PRP fibrin clot preparation is widely used for the treatment of chondral defects due to its porous fibrin nature and scaffold-like characteristics for the local delivery of growth factors and other molecules [83, 84]. Although liquid PRP has been shown to improve cartilage healing, Milano et al. [83] demonstrated that PRP fibrin glue gel combined with microfracture better restored cartilage tissue compared to liquid PRP and microfracture alone in a sheep model. Few studies have evaluated the efficacy of activated PRP without microfracture, rather they analyzed it in combination with tissue-derived mesenchymal stem cells [68, 85]. Further evaluation of PRP augmented with and without microfracture is necessary to determine the efficacy of these procedures for chondral defects.

Animal models with partial or full-thickness chondral defects have reported improvements when treated with a PRP fibrin clot or in combination with other biological constructs and materials. Cole et al. [86•] combined leukocyte-poor PRP with micronized allograft cartilage as an adjunct to microfracture in an equine model, reportedly resulting in superior cartilage regeneration compared to bone marrow stimulation alone. Elevated leukocytes in PRP formulations have higher pro-inflammatory and degenerative factors that are destructive to the extracellular matrices of cartilaginous tissue [68, 87•]. However, leukocyte-poor PRP also possesses deleterious factors that are destructive for cartilage repair and regeneration. VEGF is secreted by degranulated platelets and responsible for new blood vessel and bone formation [88]. VEGF not only serves as a catabolic molecule in the presence of chondrocytes, but also enhances endochondral ossification through angiogenesis at the site of the chondral defect [88–91]. Soluble fms-like tyrosine kinase 1 (sFlt1), an EGF inhibitor, has been shown to improve articular cartilage regeneration by promoting collagen synthesis in a immunodeficient rat model. [92] These results suggest that suppressing VEGF activity in PRP may help maximize the beneficial effects of PRP for cartilage repair.

Clinical Applications

Over the last 20 years, inactivated and activated PRP products have demonstrated promising results for chondral defects and early onset OA. Variations in PRP preparation have been proven safe, and the evidence of clinical efficacy has also been noted through multiple studies [93–97]. Contemporary literature suggests that PRP is a suitable surgical adjunct to reduce post-operative pain by acting as an analgesic agent for intra-articular applications [98–102].

PRP aims to restore the inflammatory and catabolic environment through potential anti-inflammatory effects. Abrams et al. [103] reviewed the literature and reported that PRP injection therapy for articular cartilage repair demonstrated positive effects in both pre-clinical and human clinical trials. The authors pointed out several limitations in their analysis due to biased patient selection and the lack of standard delivery procedures, which are relevant in that results are often contradictive or difficult to interpret. Nonetheless, Campbell et al. [96] reported on the quality of three meta-analyses that evaluated PRP intra-articular injection therapy for cartilage degenerative conditions and found that the International Knee Documentation Committee (IKDC) scores improved at 6-month follow-up, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) function and visual analogue scores (VAS) for pain improved at 3 and 6-month follow-ups compared to hyaluronic acid (HA). Interestingly, the systematic review was limited to three meta-analyses because of study design variability and reporting inconsistencies [96]. More importantly, variability in patient selection and outcome assessment tools limited the study power and the overall recommendation of PRP therapy for knee OA [96].

Intra-articular PRP injections have demonstrated superior clinical and function outcomes at 6 months of follow-up when compared to HA and saline [104]. All patient-reported outcome and function scores were significantly better than HA at 1-year follow-up; however, there were no significant differences observed between PRP preparation types, such as leukocyte-rich PRP (LR-PRP) and leukocyte-poor PRP (LP-PRP) [104]. PRP preparations that include elevated leukocytes are known to have an anti-microbial effect [105–107] but has also been shown to cause an inflammatory response which can be deleterious at the lesion site [108]. Riboh et al. [109•] evaluated the association between leukocyte concentrations and post-injection reactions and found that there was no significant difference between LP-PRP and LR-PRP preparations in a recent meta-analysis. However, LP-PRP yielded significantly better WOMAC scores compared to LR-PRP, HA, and placebo groups [109•]. Given that there are relatively few robust clinical trials that report the biological differences between LR-PRP and LP-PRP [109•], there is a greater emphasis on reporting the type of PRP that is being studied and also elucidating the role of leukocytes in PRP. Finally, there are few studies that have evaluated long-term follow-up following PRP injection therapy. Filardo et al. [100] conducted one of the only long-term outcome studies and evaluated PRP injection therapy for knee OA. Filardo and colleagues reported that IKDC scores and EQ-VAS pain had significantly worsened by 24 months of follow-up [100]. A temporal effect has been noted in several studies [110, 111], but may be short acting due to a lower metabolic activity and intrinsic interactions over time. Recent efforts have improved the safety and efficacy of PRP by inhibiting deleterious factors and subsequently enhancing factors that initiate musculoskeletal tissue healing [112–114]. There is substantial evidence that neutralizing TGF-β1 with anti-fibrotic agents can enhance VEGF (angiogenic) expression and improve the therapeutic effect of PRP for skeletal muscle healing [112, 114]. In contrast, blocking VEGF in PRP has been shown to improve the therapeutic potential of PRP for cartilage repair [88]. However, few basic science studies within the body of literature have characterized the biological constituents within PRP [115–118]. To improve the therapeutic potential of PRP, robust characterization studies are necessary in order to optimize PRP preparations and delivery methods, such as using microspheres and nanofiber technology [119–123].

Future Directions

PRP-derived growth factors are therapeutically compelling because of their anabolic effects and synergistic roles that can be applied to damaged cartilage tissue. Unlike single targeted biological therapies, PRP is one of few therapies that contains several bioactive factors that are able to reestablish synovial fluid homeostasis [124–126], modulate inflammation [124, 127], and induce cell migration [128–130] and recovery [131, 132•]. Despite the significant clinical and basic science research contributions that have helped define PRP, the lack of robust clinical trials and inconsistencies in reporting standards makes it difficult to translate these results to clinical practice. Therefore, meaningful pre-clinical evaluation and reporting standards are necessary to clarify the effectiveness of PRP for cartilage repair and regeneration.

It is also important to point out that PRP treatment has a higher incidence of adverse events when compared to other conventional and unconventional treatments [93]. The common adverse events include post-injection pain, swelling, and inflammation [96]. The biological mechanisms that cause post-PRP injection adverse events are unclear, but it is possible that the adverse response is a subsequent reaction to the exposure of PRP to the degenerative environment and the infiltration of immunomodulatory factors [133]. Moreover, variations in the delivery methods or the injection volume administered may contribute to post-injection adverse events. Standardization of PRP production methods and administration are necessary in order to accurately measure and assess adverse events as well as outcomes.

While several reports have demonstrated positive short-term outcomes, very few studies have validated the use of PRP for its long-term clinical efficacy [134]. In addition, several biological factors must be considered in order to understand the biological variability of PRP preparations for articular cartilage repair and regeneration. There is evidence that patient demographics, such as age, sex, and body mass index, cause the biological variability of PRP [135–137]. Much less is known about how the variability among individuals affects the biological potential of PRP. In addition, several studies have failed to report on medication use following PRP treatment, and very few studies have evaluated the effect of medications on PRP’s biological potential. [138] Because over the counter non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used after injury to reduce swelling and pain, it is important to evaluate the influence of NSAIDs on the biological potential of PRP. Platelet aggregation impairment and slower activation rates have been demonstrated in users that were administered NSAIDs immediately following orthopedic surgery, for a duration that was less than 1 week [138]. This information suggests that patients should discontinue NSAIDs 1 week prior to their peripheral blood draw and PRP injection.

The methodology used to prepare PRP is yet another area that warrants further research. There are various PRP formulations used for cartilage repair, such as inactivated PRP, activated PRP (PRGF or releasate), and fibrin clots. PRP can be delivered without activating mechanisms to allow for slow release of cytokines, chemokines, and other bioactive factors, while induced PRP activation allows the platelets to rapidly release prior to its delivery. To activate platelets in the presence of an anti-coagulant, exogenous activators (i.e., 10% calcium chloride and recombinant thrombin) cause platelet degranulation before they are delivered to the local defect or intra-articular space. Once the agonists have bound to the platelet, aggregation begins via actin polymerization to form a fibrin clot. The fibrin clot is an adhesive membrane that can be placed directly on the cartilage defect and can serve as a conduit for augmented progenitor cells and other biomaterials [84, 139, 140]. Several studies have measured and compared cytokine and chemokine profiles in different activation protocols and preparation methodologies [115, 117, 118, 141–145]; however, in terms of validated methodologies used to prepare PRP for clinical evaluation, there are very few studies [103, 146, 147]. It is crucial that validated PRP preparation methodologies are considered in future clinical trials that are evaluating the efficacy of PRP treatment for cartilage repair.

Conclusion

The application of PRP in the field of orthopedics and sports medicine is growing and will continue to expand as we improve our understanding of its biological potential. The future direction of PRP and its application for cartilage repair and regeneration will be based on customization and tailoring of biological factors by targeting disease-specific markers.

Conflict of Interest

Dr. Evans reports grants and other from Cooling Systems Inc., outside of the submitted work.

Dr. LaPrade reports royalties from OSSUR, Smith and Nephew, and Arthrex, outside the submitted work.

All other authors declare that they have no conflicts of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.