Orthobiologics and platelet rich plasma.

Orthobiologics have evolved to the extent that they significantly influence modern orthopedic surgical practice. A better understanding of the role of various growth factors and cells in the process of tendon healing, ligament repair, cartilage regeneration and bone formation has stimulated focused research in many chronic musculoskeletal ailments. Investigators have published results of laboratory as well as clinical studies, using orthobiologics like platelet rich plasma, stem cells, autologous conditioned serum etc., with variable results. However, a clear consensus over the best orthobiologic substance and the method of preparation and usage of these substances is lacking. Much of the confusion is due to the fact that studies ranging from RCTs to case reports present variable results, and the interpretations are wide-ranging. We have reviewed the available orthobiologics related data with a focus on platelet rich plasma in orthopedic conditions.

INTRODUCTION

Sports related injuries among professional and recreational athletes are increasingly encountered and diagnosed and demand a quick return to preinjury level of sporting activities.1 “Orthobiologics”, refers to the use of biological substances to help musculoskeletal injuries heal quicker. They are used to improve the healing of fractured bones and injured muscles, tendons and ligaments and are derived from substances that are naturally found in body.2 When they are used in concentrations many times the normal, they can potentially help speed up the healing processes.2

The substances include bone grafts, autologous blood, platelet-rich plasma (PRP), autologous conditioned serum and stem cells.2 Bone grafts act by their osteoinductive, osteoconductive and osteogenic properties to stimulate new bone formation and have no effect on the healing of muscles, tendons and ligaments.3 On the other hand, autologous blood, PRP and autologous conditioned serum deliver growth factors to the diseased areas to stimulate the repair process.456 Stem cells are unique in the sense that they provide a means to replenish the dead or dying cells in areas where the cells have limited regenerative capabilities.7 Each of these biologic substances has some advantages and disadvantages, which would be further elaborated in this article.

RATIONALE FOR ORTHOBIOLOGIC SUBSTANCE USE

Though the bones and joints make up the basic skeletal framework of the body, the musculo-tendinous units are the prime mediators of movement. While the muscle cells have adequate blood supply, they lack the ability to regenerate after injury; on the other hand, tendons are precariously supplied by blood vessels, hence injuries to the musculo-tendinous areas are notorious for inadequate healing and chronicity.89

Injuries to the musculo tendinous structures may be acute or chronic, with the chronic conditions being more problematic. These chronic conditions are believed to be a result of overuse, which occurs as a result of multiple micro-traumatic events that cause disruption of the internal structure of the tendon and degeneration of the cells and matrix. This fails to mature into normal tendon and at times, such injuries result in tendinosis,8 which is often accompanied by what is called as an angiofibroblastic degeneration.9 This type of injury is seen in lateral epicondylar tendinopathy, rotator cuff injuries, patellar tendinopathy, Achilles tendinopathy and plantar fasciitis; consequently most of the research in the field of orthobiologics is being done in these areas.4

Additionally, the orthobiologics are now being explored in early osteoarthritis of knee,10 cruciate ligament injuries and fracture nonunions.36

ORTHOBIOLOGIC OPTIONS

The concept of employing growth factors for healing of musculoskeletal lesions is relatively recent. Researchers have shifted focus from recombinant growth factors (considering the high cost, short life span, inefficient delivery system and the requirement of high doses for achieving therapeutic effects) to autologous blood products.6 Historically, autogenous fibrin gel was initially described in 1990.11 PRP was used for the first time in bone repair by Marx et al.,12 (1998) who published a case series of 88 patients having mandibular continuity defect and were treated with bone grafting. In half of these patients they had added PRP to the graft and these showed an increase in maturity and consolidation of graft in subsequent radiographs.

Autologous blood injection (ABI) is the simplest method of delivering blood derived growth factors. The procedure includes drawing of venous blood from the patient and then injecting it at the desired area. Although this delivers growth factors contained in platelets, red and white blood cells are also delivered, which have no healing properties. Consequently, the results of ABI have been variable. Edwards et al.,13 Connell et al.,14 Ozturan et al.,15 Creaney et al.,16 and Thanasas et al.17 have reported good results with ABI in lateral epicondylitis, while Kazemi et al.18 found no benefit of ABI over corticosteroids. On the other hand, Suresh et al.19 used ABI for refractory medial epicondylitis and reported encouraging results. James et al.20 reported a significant improvement in VISA (Victorian Institute of Sport Assessment) score with ABI in patellar tendinopathy.

The consideration that growth factors are primarily contained in the alpha granules of the platelet has led to researcher focus being directed towards more effective use of these factors and hence the growth factor delivery system graduated from the use of autologous whole blood to the use of concentrated platelets in the form of PRP. A pubmed search conducted on 13th April 2013 with the keywords “Platelet rich plasma” revealed 6400 results while a similar search that had been done on 16th November 2011 had returned 5928 results— a total 637 articles were added in just 18 months.

Autologous conditioned serum (ACS) has also been researched for its application in orthopaedic conditions. It is prepared by incubation of 50 ml venous blood for 24 h in 5% CO2. Incubation of blood is carried out with small glass beads, which need to be specially processed to make their pH similar to distilled water. Following incubation, centrifugation is done to obtain the ACS.521 Preparation process leads to an accumulation of interleukin-1 receptor antagonist and several growth factors, including insulin-like growth factor-1, platelet-derived growth factor and transforming growth factor-β1, in the liquid blood phase. ACS has been used in human trials by Becker et al.,21 for treatment of lumbar radiculopathy while Moser et al.22 have used it for knee osteoarthritis. Human studies are limited and cannot be sufficiently evaluated to provide any recommendation for the use of ACS at present. Additionally, the preparation procedure is cumbersome and the requirements of special glass beads, laminar air flow system and incubation systems make it a relatively unattractive option.

Stem cells are of two basic types – Embryonic stem cells which are found in the early developmental phase of an individual and mesenchymal stem cells which are found in adults. Mesenchymal stem cells (MSCs) are considered to be an orthobiologic substance. A cell can be called a stem cell if it has the capacity to differentiate into multiple types of cells and the cell is able to self-renew.7 The ability of a stem cell to simultaneously maintain the stem cell pool and generate daughter cells which can terminally differentiate into specialized tissues is the unique capability of such cells. Mesenchymal stem cells can differentiate towards cartilage, tendon and bone cells. They can be isolated from a number of mesenchymal tissues like bone marrow, fat, synovial membrane, periosteum and others. A common source of MSCs is the bone marrow, especially from the iliac crest. The iliac crests are subcutaneous and are easily palpable, thus making the stem cells harvesting process easy. Bone marrow is aspirated from the region of posterior superior iliac spine and the MSCs contained are used either by concentrating the aspirate so as to use the natively available stem cells23 or the cells are separated and cultured24 in laboratory media to increase their numbers before being used. Stem cells have been used with encouraging results in treatment of nonunions, osteonecrosis of femoral head, spinal fusion enhancement and for filling of bone defects and voids.7 In tendon injuries the application of stem cell is presently limited to experimental animal models with no clinical studies available. In view of the fact that the preparation techniques are difficult and resource demanding, along with a relative lack of clinical studies supporting the use of stem cells in various musculoskeletal lesions, a recommendation for the routine use of stem cells cannot be made as yet.

PLATELET RICH PLASMA: CLASSIFICATION AND TYPES

PRP is the plasma fraction of autologous blood which has a platelet concentration above baseline. Other names by which PRP has been known include platelet-enriched plasma (PeRP), platelet-rich concentrate (PRC) and autogenous platelet gel.6 The normal platelet count in whole blood in a healthy individual is between 1.5-4.5 × 105/μL. To be labeled as PRP, a platelet count of 4-5 times of the baseline should be present in the platelet concentrate.

Platelets are formed from megakaryocytes in the marrow. Platelets contain granules namely α, δ and λ. α granules play a pivotal role in platelet function. There are approximately 50 to 80 α granules per formed platelet25 and contain more than 30 proteins including platelet-derived growth factor (PDGF), transforming growth factor (TGF-β, β1 and β2 isomers), platelet factor 4 (PF4), interleukin-1 (IL-1), platelet-derived angiogenesis factor (PDAF), VEGF, epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDEGF), epithelial cell growth factor (ECGF), insulin-like growth factor (IGF) etc., The properties of PRP are based on the production and release of these factors when the platelets are activated. Platelets begin secreting these proteins within 10 minutes of clotting. After initial release of growth factors, the platelets synthesize and secrete additional such factors for the remaining days of their life span.6

Preparation of PRP includes drawing of blood into a bag or a tube containing an anticoagulant (CPDA) followed by centrifugation to separate the red cells from the rest of the blood components. Separation of the platelets from the red cells and the platelet poor plasma leads to retrieval of PRP. This PRP can now be directly used as leukocyte-rich PRP or it may be passed through a leukocyte filter to obtain leukocyte poor PRP. PRP application may be done in two ways – nonactivated and activated. While the activation process consists of simultaneous application of calcium chloride to activate the PRP, the nonactivated application depends on the activation of platelets after coming in contact with collagen of the tissues. For surgical applications, PRP is often treated with calcium chloride before application of thrombin, which forms a gel-like substance for direct application.26

Previously reported studies have used the PRP in either of the two forms leukocyte-rich or leukocyte poor PRP, as the exact influence of leukocytes on the local injection site was not clearly known. Moreover, lack of standardization of the type of PRP used tended to make comparison between studies difficult and unreliable. Mishra et al.27 have proposed the Sports Medicine Platelet-Rich Plasma classification system [Table 1]. This system takes into consideration the platelet count, the activation method and the leukocyte count to divide PRP into four types, with each having two further subtypes.

Table 1

Sports medicine platelet rich plasma classification system

Some recent studies in the literature strongly support the notion that leukocyte poor PRP is better than leukocyte-rich PRP.2829 The platelet concentration process also leads to a simultaneous concentration of leukocytes. While previous studies only speculated that increased residual leukocytes could produce localized inflammation and hence prolonged early postinjection pain,17 studies by Dragoo et al.28 and Mc Carrel et al.29 have conclusively demonstrated significant inflammatory capabilities of leukocyte-rich PRP. Thus, the use of leukocyte-poor PRP may be considered better as just by separating the leukocytes, one may be able to provide better pain relief to the patients. On the other hand, almost all studies published till now have reported no adverse effects by use of PRP in any form.

APPLICATIONS OF PLATELET-RICH PLASMA IN MUSCULOSKELETAL PROBLEMS

PRP has been used in diverse musculoskeletal conditions like lateral epicondylar tendinopathy, rotator cuff tears, sub-acromial impingement, shoulder osteoarthritis, patellar tendinosis, osteoarthritis of the knee, tendo-achilles tears, ACL reconstruction, plantar fasciitis, nonunion of bones and topically to control bleeding after knee arthroplasty.30 Considering that there are numerous conditions where PRP is being used, both in clinical or in laboratory settings, it often becomes difficult to get the appropriate evidence for the usage of PRP. In this review, we undertook a focused pubmed based review, with PRP being the common key word and other keywords relevant to the particular orthopedic condition in question [Table 2]. We then segregated the studies into randomized control trials (RCTs), cohort studies and others. Of all the available publications, we attempted to focus on level 1 and level 2 studies only, with significant numbers of patients. For this review, we have focused only on five areas of orthopedic use, i.e. lateral epicondylar tendinopathy, rotator cuff tear, patellar tendinosis, osteoarthritis of the knee and Achilles tendinopathy, as these are the commonest areas for chronicity and confusion regarding orthobiologic interventions.

Table 2

Studies of PRP in orthopedic conditions as searched on pubmed

LATERAL EPICONDYLAR TENDINOPATHY

Though lateral epicondylar tendinopathy can potentially be treated well with conventional established protocols, a significant number of patients do become resistant; these are the patients who could potentially have significant benefit from PRP. Mishra and Pavelko31 were the first to use PRP injection for resistant lateral epicondylitis and reported good results in terms of pain relief and functional improvement in their pilot study. Peerbooms et al.32 conducted a RCT with 100 patients, 51 of whom received PRP injection and 49 received corticosteroid injections and reported better improvement with PRP over a period of 1 year. Gosens et al.33 followed up these patients for the subsequent year and reported a sustained improvement with PRP use in comparison to corticosteroids. In the management of resistant lateral epicondylitis, PRP may be considered better than corticosteroid injections, which were considered for long as the gold standard. In an attempt to increase injection accuracy, Chaudhary et al.34 used ultrasonographic guidance for injection and noted a trend toward an increase in the vascularity at the musculo-tendinous junction of the extensor tendons. Creaney et al.16 reported similar improvements in pain and function scores with PRP and ABI, but noted a lesser rate of conversion to surgery among patients receiving PRP injections. Thanasas et al.17 have also shown encouraging results for PRP use compared to ABI in resistant tennis elbow patients [Table 3].

Table 3

Summary of studies using PRP for resistant lateral epicondylitis

From the available evidences one may offer a PRP injection instead of a corticosteroid injection to patients who have failed to obtain improvement with conservative methods and in whom a surgical treatment option is being considered.

ROTATOR CUFF INJURIES

Rotator cuff tears tend to occur with two age peaks, i.e. in older patients and in younger patients involved in sports. In the older population, some degenerative changes are expected to be present alongside the tear and there are chances that a meticulously performed rotator cuff repair may fail or heal sub-optimally. It is important to note that the distal part of the rotator tendon has inherently poor healing capabilities; PRP with its growth factors may thus be an attractive option for the stimulation of the healing of this tendon. Many shoulder surgeons have started using PRP intraoperatively after performing rotator cuff repairs.353637

Randelli et al.35 were the first to conduct an uncontrolled pilot study of PRP augmentation along with arthroscopic rotator cuff repair. In their 14 patients injected with PRP activated with thrombin at the tendon footprint after repair, they reported statistically significant improvements in VAS, constant and UCLA shoulder scores compared to preoperative values. They subsequently conducted a RCT36 and reported that autologous PRP reduced pain in the first postoperative month and that the longterm results of subgroups of grade 1 and 2 tears suggested that PRP positively affected cuff healing. Rha et al.37 compared ultrasound guided PRP injection with dry needling and concluded that autologous PRP injections lead to a progressive reduction in the pain and disability when compared to dry needling. However, not all studies have shown good results of the use of PRP; Weber et al.38 in their prospective, randomized double blind study reported that platelet-rich fibrin matrix did not show significant improvement in perioperative morbidity, clinical outcomes, or structural integrity. Castricini et al.39 concluded that their study did not support the use of autologous PRP rich fibrin matrix for augmentation of small or medium rotator cuff tear to augment healing [Table 4] summarizes the studies reporting PRP use in rotator cuff tear.

Table 4

Summary of studies using PRP for rotator cuff injuries

The evidences available so far do not provide a clear picture regarding the use of PRP in rotator cuff tears. However, PRP may be used in rotator cuff injuries either as an adjunct to surgery, or as stand alone injections after explaining the prognosis and the limited available evidences to the patient.

OSTEOARTHRITIS OF KNEE

The degenerative process of osteoarthritis of the knee joint results from a gradual and progressive destruction of the articular cartilage and is mediated by metalloproteinases. These enzymes are produced by chondrocytes, which also produce the tissue inhibitors of metalloproteinases (TIMP) 1 and 2, but in amounts insufficient to counteract the proteolytic effect, thus producing overall cartilage destruction. Growth factors contained in alpha granules of platelets have been postulated to be chondro-protective and capable of improving the physiology in osteoarthritic joints. PDGF and TGF-1 are believed to up-regulate the production of endogenous hyaluronic acid levels. PRGF is also believed to be capable of regulating the levels of TIMPs. PRP has been used in patients of early osteoarthritis in an attempt to improve the cartilage structure and to slow down the progression of the disease.

Spakovα et al.40 conducted a RCT on 120 patients with Kellgren and Lawrence Grades 1, 2, or 3 osteoarthritis, comparing PRP injection with hyaluronic acid and concluded that autologous PRP was an effective and safe method in the treatment of the initial stages of knee osteoarthritis. Kon et al.41 concluded that autologous PRP injections showed more and longer efficacy than HA injections in reducing pain and symptoms and recovering articular function. Better results were achieved in younger and more active patients with a low degree of cartilage degeneration. Cerza et al.42 published a RCT with 120 patients reported significantly better clinical outcome with autologous PRP than with HA, with sustained lower WOMAC scores. Patel et al.43 published the results of a RCT conducted on 78 patients (156 knees) with bilateral osteoarthritis and reported improvement VAS scores for pain and in all parameters of WOMAC scores in patients who received PRP injections. The improvement reported was greater among patients with early osteoarthritis (Ahlback I). Importantly, all of these RCTs have reported a good safety profile of the PRP injections and no major complications whatsoever. Published studies of PRP use in knee osteoarthritis have been summarized in Table 5.

Table 5

Summary of studies using PRP for knee osteoarthritis

Osteoarthritis of the knee is one of the commonest degenerative diseases encountered in clinical practice and the prospect of arresting the disease process in its initial stages looks rewarding. The clinical evidences of PRP use in early knee osteoarthritis weighs in favor of PRP; one may offer PRP injections to early osteoarthritis patients but a definite recommendation cannot be made at this stage. Longer follow-ups of current studies would provide a better understanding of the actual benefits of PRP.

ACHILLES TENDINOPATHY

Tendinopathies and tears of Achilles tendon are known to be difficult to heal and PRP has been used in attempts for enhancing healing at this site.

DeJonge et al.44 studied 44 patients with chronic tendinopathy 2-7 cm proximal to the Achilles tendon insertion and injected them with PRP or saline and subjected the patients to an eccentric training program. One year of followup showed no clinical and ultrasonographic superiority of PRP injection over the placebo injection. deVos et al.45 evaluated the tendon structure by ultrasonographic tissue characterization by a double-blind, randomized, placebo-controlled clinical trial in 54 patients; 27 patients received PRP injection and 27 received saline injection. After 24 weeks they concluded that PRP injections did not contribute to an increased tendon structure or altered the degree of neo-vascularization, compared with placebo. Owens et al.46 reported modest improvement in functional outcome in 10 patients who had received PRP injection for mid substance Achilles tendinopathy. Monto et al.47 reported clinical success in 28 out of 30 patients with recalcitrant Achilles tendinosis. The improvement noted was in the AOFAS score and the MR architecture of the tendon.

Enhancement of repair of the Achilles tendon tear with PRP has also been attempted. Though Sanchez et al.26 reported good results in their case control study using platelet-rich matrices for the repair of ruptured Achilles tendon in 12 athletes, Schepull et al.48 concluded that PRP was not useful for the treatment of Achilles tendon ruptures [Table 6].

Table 6

Summary of studies using PRP for Achilles tendinopathy

In summary, the results of use of PRP in Achilles tendon disorders are not as encouraging as in other conditions. While most studies have reported disappointing results, few of the studies do show good results. Differences in PRP preparation and mode of application may be a contributing factor in providing such results. Additionally, each study has used a different outcome evaluation score that may also have some influence on the results. Last but not the least, the properties of the Achilles tendon itself (like the relatively large bulk of the tendon and it being subjected to eccentric loading) may contribute to poor efficacy of this method of treatment.

PATELLAR TENDINOPATHY

Jumper's knee in athletes is a common cause of knee pain. Filardo et al.49 evaluated the efficacy of PRP injections for refractory patellar tendinopathy and concluded that PRP injections had the potential to promote the achievement of a satisfactory clinical outcome, even in chronic refractory tendinopathy. Gosens et al.50 reported on the pain and the activity levels before and after PRP injection in patellar tendinopathy in a prospective cohort study and concluded that patients treated with PRP showed a statistically significant improvement. Vetrano et al.51 reported their results comparing PRP injections with extracorporeal shock wave therapy (ESWT) and reported a significantly better improvement in PRP group over the ESWT group after 12 months of followup [Table 7].

Table 7

Summary of studies using PRP for patellar tendinopathy

In summary, good results of PRP use in patellar tendinopathy have been reported but the studies published so far are poorly conducted. Only one RCT has been conducted till date and hence more studies with encouraging results are required before recommending PRP use in Patellar tendinopathy.

FUTURE OF PLATELET RICH PLASMA AND OTHER ORTHOBIOLOGICS

Orthobiologics, especially PRP, hold a lot of promise as upcoming and novel treatment modalities. Although stem cells and ACS also appear interesting in theory, technical difficulties in preparation and use makes them rather user-unfriendly. If one analyzes the pros and cons of the available orthobiologics, it becomes clear that the lack of adequately powered studies is an important issue preventing their recommendation for routine use. However, even with the limited published scientific data, PRP appears to be the most attractive option available, with minimal side effects; the relative ease of preparation, cost effectiveness and the ability to complete the procedure as a day care procedure goes in favor of PRP. However, caution has to be advocated, as PRP preparation needs to be carried out in a sterile environment and preferably in bio-safety cabinets and the usage standardized. In India, autologous PRP therapy has been used in knee osteoarthritis,43 nonunion,52 lateral epicondylitis, rotator cuff injuries, plantar fasciitis, etc.53 At a few centers it is being utilized for treatment of alopecia.54 The blood banks of these centers are generally preparing the PRP for medical use; some surgeons are using commercially available kits also. However, there is lack of uniformity in the preparation methods amongst various centers and standardization of the final product is not adequately established. Additionally, there are concerns about the need for licensing to prepare such products because all blood banks need to obtain license from the Drug Controller General of India (DCGI) for their functioning, which is generally renewed every 5 years (Drugs and cosmetics act – 1940, MOHF, Government of India). Thus, having a well-equipped and licensed set up for preparation of the PRP is essential. Commercial PRP preparation systems like Biomet GPSIII (Biomet Inc, Warshaw, USA), Arthrex ACP Double syringe system (Arthrex Inc, Naples, USA), RegenPRP (Regen Lab, Switzerland) etc., have yet to become popular in India. This probably is due to the licensing requirements and the cost involved in the use of such systems. The cost of PRP preparation with these systems is somewhere between USD 500 and 1000.55

It is pertinent to mention here that the WADA (World Anti Doping Agency) included PRP in its list of prohibited substances for the year 2010; subsequently in the year 2011 WADA clarified that even though PRP contains growth factors, PRP use in sports injuries is not prohibited. Nevertheless, this episode of banning PRP use in sportsmen and then lifting off the ban, brought significant negative publicity to PRP for routine use.56

What is required at present is an international regulatory authority, which would lay down guidelines for the preparation, handling and use of PRP. The International Cellular Medical Society (ICMS) asserts that a need exists to create standards for PRP preparations, techniques and tracking and has prepared a protocol for the same.57 Quality control regulating the minimum amount of growth factors in the prepared products would also be a welcome addition. Any future trials should follow standardized guidelines and ethics or other relevant committee approval regarding the methodology of PRP preparation and use; the results could be analyzed and made available through the committee also. These few quality control and regulatory steps may lead to more scientific and rational usage of PRP, in well-regulated environments. It may also be noted that it is still illegal for a medical practitioner to charge money for PRP injection in South Korea.58