Evaluation of the Effects of Platelet-Rich Plasma (PRP) Therapy Involved in the Healing of Sports-Related Soft Tissue Injuries

Abstract

Introduction

According to the World Health Organization (WHO), musculoskeletal injuries are the most common cause of severe long-term pain and physical disability, and affect hundreds of millions of people around the world⁽¹⁾. The impact such injuries have on everyday life is substantial. Thus, the primary aim of orthopaedic surgeons is a safe and full recovery for their patients with a return to pre-injury level of activity as quickly as possible. The traditional management of orthopaedic and sports related injuries includes everything from conservative “RICE” treatment and physical therapy to corticosteroid injections and surgical intervention. Recently, advances in biomedicine and biotechnology have enthused the use of cell therapy, tissue engineering, and autologous blood concentrates to enhance healing and stimulate growth in bone and soft tissue injuries.

One of the most popular methods used to biologically enhance healing in the fields of orthopaedic surgery and sports medicine includes the use of autologous blood products, particularly, platelet rich plasma (PRP). PRP is an autologous concentration of human platelets to supra-physiologic levels. It is produced from a patient's peripheral vein and centrifuged to achieve a high concentration of platelets within a small volume of plasma. It is then re-injected at a site of injury or inserted as a gel or other biomaterials during surgery.

Historically, platelets have been used to treat patients with thrombocytopenia or hemorrhage^{(2, 3)}. Other blood products such as fibrin (in the form of surgical glue) have been utilized for wound healing^(4-6). In fact, fibrin (in the form of a clot) has been used in a few studies to augment healing in meniscal repairs^{(7, 8)} and also to augment graft healing in ACL reconstruction⁽⁹⁾. Because of its limited use only for surgical procedures and the inability to modify the platelet concentration, fibrin in its various forms is not as extensively used as PRP for sports related injuries.

PRP therapy has grown in popularity over the past few years. Not only has there been an increasing number of basic science and clinical studies, there has also been a rising level of public awareness secondary to the use of PRP to treat high-profile athletes. In fact, one of the most cited articles to date on PRP therapy was published in the New York Times showcasing its use in two professional athletes⁽¹⁰⁾.

Many clinicians feel that PRP therapy is safe given its autologous nature and long-term usage without any reported major complications. For this reason in addition to its easy availability, it is readily used in clinical and surgical settings. Despite its widespread unregulated use, the efficacy of PRP therapy has yet to be established^(11-13). There are many unanswered questions concerning the composition of PRP, individual blood product characteristics, different protocols of production, different methods of administration, and mechanisms of action exerted by PRP and its individual components on a cellular level.

The actual mechanisms of action of PRP are extensive because of the release of a myriad of bioactive factors.

There is a general consensus in PRP research that the injection of concentrated platelets, once activated, results in an exponential increase in numerous growth factors (Table 1) at the sight of injection. However, the function of many growth factors, chemokines, cytokines, and inflammatory mediators has not been elucidated, nor have the interactions between factors and their influence on neighboring cells. As such, the primary purpose of this article is provide an overview of PRP-mediated effects by discussing current research on growth factors and proposed mechanisms by which PRP exerts its downstream effects on healing muscles, tendons, and ligaments. This review will first address the natural healing process for soft tissue injures; followed by proposed mechanisms of action of PRP and associated growth in tendon and ligament injuries, and muscle contusion. Finally, current and future areas of research will be discussed to address common unanswered questions concerning the applicaiton of PRP for sports-related injuries.

The Natural Healing Process in Soft Tissue Injuries

Acute sports-related soft tissue injuries result from a single, traumatic event (macro-trauma) such as muscle contusion or ligament sprain/tear. Chronic soft tissue injuries often result from repetitive mechanical stress (micro-trauma) or overuse followed by inflammation and take time to develop such as in the case of tendinopathies (e.g. rotator cuff tendinopathy and Achilles tendinopathy). Regardless of the type of injury, the wound healing process is shared among all soft tissues, with differences in timing, duration of phases, and interactions between key mediators^(14-16).

The general healing cascade involves four overlapping phases: (1) hemostasis; (2) inflammation; (3) cellular and matrix proliferation, which begins within days of an injury and comprises the most important phase of healing; and (4) wound remodelling, the longest phase, which may involve scar tissue formation^{(17, 18)}.

Immediately following injury, capillary leak allows for the recruitment of hemostatic factors and inflammatory mediators. The coagulation cascade is activated leading to platelet aggregation, clot formation, and development of a provisional extracellular matrix construct⁽¹⁹⁾. Platelets adhere to exposed collagen and circulating extracellular matrix proteins, which triggers the release of bioactive factors from alpha granules⁽²⁰⁾. These bioactive actors include growth factors, chemokines, and cytokines, in addition to pro-inflammatory mediators such as serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine⁽²¹⁾.

The inflammatory phase follows in a highly orchestrated fashion. Chemoattractant agents begin to summon neutrophils to the injured site within 1-2 hours in the early inflammatory phase. Later (around 48 – 72 hours post-injury), macrophages appear in the wound and play the leading role in wound debridement and regulation of inflammation. They are also involved in recruiting fibroblasts and endothelial cells. The last cells to enter the wound during the late inflammatory phase are lymphocytes.

The cellular and matrix proliferation phase is arguably the most important phase of wound healing; in part, because the cells involved serve as a metabolic engine driving tissue repair⁽²²⁾. These cells originate from pluripotent progenitor cells in adjacent tissues including infrapatellar fat pad for anterior cruciate ligament, muscle derived stem cells⁽²³⁾ and satellite cells⁽²²⁾ for muscle. After 2-3 days of wound healing, macrophages and chemotactic, mitogenic, and angiogenic growth factors recruit fibroblasts and epithelial cells to infiltrate the site of injury⁽²²⁾. Once in the wound, fibroblasts synthesize collagen and change to their myofibroblast phenotype to facilitate wound contraction. Angiogenesis and the formation of granulation tissue are also important aspects during the proliferative phase of healing.

The final phase of the healing process involves wound maturation and remodelling. During this phase, growth factors such as PDGF and TGF-β, and fibronectin stimulate fibroblasts proliferation, migration, and synthesis of the components of extracellular matrix^{(24, 25)}. The remodelling phase is tightly regulated to maintain the balance between degradation and synthesis. Type I collagen replaces Type III collagen, proteoglycan, and fibronectin through a process referred to as “creeping substitution” to form a more robust matrix with increased tensile strength⁽²⁶⁾. The maturation phase varies in duration depending on the extent of the wound pathology, individual characteristics, as well as specific tissue healing capabilities of the tissue involved^{(27, 28)}. Additionally, pathophysiological and metabolic factors can affect wound healing. They include local causes such as ischemia, tissue hypoxia, infection, and growth factor imbalance, as well as systemic causes such as metabolic disease and nutritional status. In such unfavorable environments, PRP has been shown to be a viable therapeutic adjunct for soft tissue injuries⁽²⁹⁾.

PRP: Definition, Properties, Preparation and Composition

Platelet-rich plasma (PRP) is an autologous concentration of human platelet to supra-physiologic levels. Platelets are irregularly shaped, non-nucleated cytoplasmic bodies derived from fragmentation of megakaryocyte precursors. They circulate in the blood of mammals expressing glycoproteins on their cell membranes and play a pivotal role in hemostatis and wound healing via the formation of fibrin clots^{(30, 31)}. At baseline levels, platelets function as a natural reservoir for growth factors including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF-I), to name a few^(32-36). Such growth factors are released from the alpha granules of activated platelets and are involved in important cellular processes including mitogenesis, chemotaxis, differentiation, and metabolism⁽³⁷⁾. Hence, the rationale for increasing platelet concentration in compromised (or injured) tissue lies in the belief that additional platelets will result in the exponential release of multiple bioactive factors, and subsequently, enhance the natural healing process.

Many clinical studies have demonstrated the beneficial effects of PRP therapy in oral-maxillofacial, plastics, and orthopaedic surgery. In fact, autologous blood products including platelets were first popularized in the 1990s in oral and plastic surgery^(38-42). Despite the increasing evidence of PRP as an augmenter of natural healing, its clinical efficacy in the management of sports-related injuries is still a matter of debate^(11-13).

Platelet-rich plasma is produced from blood obtained by phlebotomy, which is centrifuged to achieve a high concentration of platelets within a small volume of plasma. The platelet-rich product is then re-injected at a site of injury or prepared as a gel or other biomaterial and inserted during surgery. There are numerous protocols and commercial systems for producing PRP. Traditionally, two centrifugation steps are used to isolate the erythrocyte fraction from the buffy coat (plasma containing platelets, leukocytes, and clotting factors). The second step separates the platelet-poor plasma (PPP) from the platelet-rich fraction. Single-step systems are also available. Centrifugation itself is likely the most critical step during the PRP preparation process, as centrifugal forces in spins greater than 800x gravity (g) may result in significant loss of granular loads of platelets, which would subsequently dilute growth factor concentrations⁽⁴³⁾.

A major limitation in evaluating the clinical effects of PRP is variation in established preparation protocols. Many different commercial systems are available that utilize different centrifugation machines and protocols (Table 2). Platelet and growth factor concentrations can vary depending on the system used⁽⁴⁴⁾. Furthermore, the presence or absence of leukocytes, which produce VEGF⁽⁴⁵⁾ and possess antimicrobial properties⁽⁴⁶⁾, and activating factors such as calcium chloride or thrombin further influence the quality of PRP and resulting effects.

Additional variation in PRP products results from patient differences in age, medical comorbidities (particularly hematologic disorders), and healing capabilities. In essence, the effects of PRP are influenced by substantial differences in the content of platelet concentrates as well as individual characteristics, which likely contributes to its variable findings in the literature.

Orthopaedic Applications of PRP

PRP is commonly used in orthopaedic practice for sports-related injuries of skeletal muscle, tendons, and ligaments. It is thought to exert its effects through many downstream events secondary to release of growth factors and other bioactive factors from its alpha granules. These effects may vary depending on the location of injury and the concentration of important growth factors involved in various soft tissue healing responses.

Future Directions of Research

Major strides have been made in PRP research with findings influencing the management of orthpaedic injuries. Despite the multitude of clinical and basic science studies published within the last year, many important questions remain unanswered including those concerning the dosing, timing, and frequency of PRP injections; different techniques for delivery and location of delivery (over the injured tissue, within the injured tissue, or intra-articularly); optimal physiologic conditions for injections; and the concomitant use of recombinant proteins, cytokines, additional growth factors, biological scaffolds, and stems cells (Table 3).

In a recent systematic review evaluating clinical outcomes of PRP use in orthopaedic injuries, the authors concluded that one of the most challenging barriers to critical evaluation is lack of standardization in preparation and dosage of autologous blood concentrates⁽¹²⁵⁾. Specific alterations in growth factor concentration, supplementation of platelet activators, leukocytes, and or other cellular components decrease the generalizability of clinical and basic science findings, rendering any results difficult to interpret.

Individual patient characteristics also contribute to the observed variation in PRP content and quality as well as the cellular responses to autologous blood products. Age or skeletal maturity has been shown to influences ACL cell metabolic activity, apoptotic rate, collagen production, and response to PRP in a porcine model⁽¹²⁶⁾. The diversity of the human population can affect physiologic responses to PRP. Thus, future research studies should account for individual differences in age, gender, healing capabilities and whole blood characteristics.

Another important topic for consideration in PRP research is defining the optimal dose or concentration of platelets and growth factors in PRP. Although a correlation was found between increased platelet count and increased ACL graft strength⁽¹²²⁾ – “more” platelets and growth factors may not necessarily be more effective. In a study using a porcine model to assess optimal platelet concentration in PRP, increased concentration did not seem to have an effect on functional outcomes of primary ACL repair⁽¹²⁷⁾. PRP containing 5x the baseline systemic platelet count and PRP containing 3x the baseline systemic platelet count did not compromise ligament stiffness and resulted in similar biomechanical outcomes despite histological differences⁽¹²⁷⁾. The 5x group ACLs had increased cellularity, more organized collagen bundles, and more elongated fibroblasts. The finding of similar biomechanical outcomes despite differences in histology raises another important question in PRP research: is there a relationship between cellular findings following PRP treatment and actual clinical implications? Previous animal studies have demonstrated that increased cellularity during early wound healing may have a positive impact on ligament ⁽¹²⁸⁾ and tendon ⁽¹²⁹⁾; however, others have found no such relationship^{(130, 131)}. From a basic science standpoint, elucidation of specific cellular responses to PRP involves the evaluation of the inflammatory cells involved, their interactions, and behavior in an altered cellular environment. This response will likely vary depending on the injury site and thus, provides another avenue for continued PRP research.

To our knowledge, there are no studies reporting the negative effects of PRP. The assumption that autologous products are intrinsically safe should be critically evaluated. When cells are in their natural environment, they behave “naturally” meaning many of their activities, interactions, and released bioactive factors can be predicted. However, when cells and cell fragments are exposed to unnatural environments, for instance, a high-speed centrifugation process, the resulting products and exerted effects may be less predictable. Furthermore, there is no guarantee that platelets (in the form of PRP) will remain localized at the site where they are injected. Dissemination may result in unexpected results in surrounding tissues or even systemically. As such, the safety of autologous blood products – especially in the presence of cell, growth factor, and platelet activator adjuncts – proves yet another aspect of PRP therapy warranting research.

Conclusion

There is not one overarching mechanism of action of PRP given the differences in platelet content, resulting growth factor concentrations, and varying downstream cellular responses. What is known is that PRP contains a high concentration of platelets and that these platelets, once activated, release numerous growth factors into the surrounding environment. The resulting cellular effects are both pro-inflammatory and anti-inflammatory in nature and appear to be dependent on many different factors including the stage of the natural healing process, the site of the injury, and cellular environment. The interplay between individual growth factors and their resulting effects are currently being evaluated through well-designed basic science studies. Despite the volumes of basic science research supporting the use of PRP for sports related injuries, clinical evidence for PRP therapy has not been well established due to many of the unanswered questions addressed above. Continued basic science research elucidating the downstream effects of PRP can help drive clinical research. Strong scientific and clinical findings can then be used in conjunction to help develop clinical recommendations for the use of PRP in the management of sports related injuries.

References