Platelet-rich plasma in orthopedic therapy: a comparative systematic review of clinical and experimental data in equine and human musculoskeletal lesions

Background This systematic review aimed to present and critically appraise the available information on the efficacy of platelet rich plasma (PRP) in equine and human orthopedic therapeutics and to verify the influence of study design and methodology on the assumption of PRP’s efficacy. We searched Medline, PubMed, Embase, Bireme and Google Scholar without restrictions until July 2013. Randomized trials, human cohort clinical studies or case series with a control group on the use of PRP in tendons, ligaments or articular lesions were included. Equine clinical studies on the same topics were included independently of their design. Experimental studies relevant to the clarification of PRP’s effects and mechanisms of action in tissues of interest, conducted in any animal species, were selected. Results This review included 123 studies. PRP’s beneficial effects were observed in 46.7% of the clinical studies, while the absence of positive effects was observed in 43.3%. Among experimental studies, 73% yielded positive results, and 7.9% yielded negative results. The most frequent flaws in the clinical trials’ designs were the lack of a true placebo group, poor product characterization, insufficient blinding, small sampling, short follow-up periods, and adoption of poor outcome measures. The methods employed for PRP preparation and administration and the selected outcome measures varied greatly. Poor study design was a common feature of equine clinical trials. From studies in which PRP had beneficial effects, 67.8% had an overall high risk of bias. From the studies in which PRP failed to exhibit beneficial effects, 67.8% had an overall low risk of bias. Conclusions Most experimental studies revealed positive effects of PRP. Although the majority of equine clinical studies yielded positive results, the human clinical trials’ results failed to corroborate these findings. In both species, beneficial results were more frequently observed in studies with a high risk of bias. The use of PRP in musculoskeletal lesions, although safe and promising, has still not shown strong evidence in clinical scenarios. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0403-z) contains supplementary material, which is available to authorized users.


Background
Musculoskeletal lesions are a common consequence of physical overstrain, which negatively impacts quality of life and athletic performance. Specifically among both, equine and human athletes, treating persistent or slow healing injuries poses a challenge for clinicians. These lesions, which frequently result in inadequate tissue reorganization and thus in a high re-injury rate, are often related to a long period of incapacity or to an unsatisfactory return to performance [1].
Hemoderivatives have been reported to be beneficial in scenarios in which efficient, cost-effective and safe forms of orthopedic interventions are necessary to restore the normal function and structure of musculoskeletal components and in which health care professionals are tempted to explore promising forms of therapy. The use of patients' own biological materials for tissue healing and therapeutic purposes offers a safe and interesting alternative to conventional treatments, and such materials often lack side effects. Presently, several blood-derived products are available for intra-lesional injection, such as platelet-rich plasma (PRP) or plasma rich in growth factors, autologous conditioned serum, autologous blood preparations and autologous protein concentrate [1][2][3][4][5][6].
The rationale behind the injection of autologous blood preparations lies in the exploitation of advantageous mechanisms of the body's natural response to injury, whether the platelets' ability to induce hemostasis and to release growth factors [7,8] or the production of antiinflammatory cytokines by blood components [5,9]. Blood is an important and unique source of cellular and protein products that has been explored more intensively over the last three decades for the production of biomaterials for clinical use [4]. A considerable number of data have suggested beneficial effects associated with their use, but these findings have not been unanimous when experimental and clinical trial' results have been compared.
Platelet-rich products, in particular, have gained popularity for their increased concentrations of growth factors, but their compositions encompass much more than these factors. A myriad of other blood-derived substances such as fibrin and leukocytes are contained within these products, characterizing PRP as a complex and unique mixture, with donor-related properties and a yet unrevealed spectrum of active blood components [10]. Platelet concentrates with distinct compositions, and therefore distinct applications, are referred to as PRP. Some authors have proposed a more accurate terminology, along with a thorough description of the platelet concentrate. Using this terminology four different products can be recognized according to their fibrin architecture and leukocyte content: pure platelet-rich plasma, leukocyte and platelet-rich plasma, pure platelet-rich fibrin and leukocyte-and platelet-rich fibrin [11].
The cellular and molecular content of platelet concentrates also depend on the method or kit used for their preparation. Until now, there has been no consensus on which processing method yields the best platelet-related product for a particular purpose, and patented and nonpatented technologies and procedures have been used in clinical and experimental scenarios [12].
During athletics, both horses and humans are subjected to escalating stresses to their appendicular systems, to compete successfully. These increasing demands are accompanied by a multitude of orthopedic lesions, for which conventional therapies have proven partially helpful.
The objectives of this systematic review were to assess the effectiveness of PRP in the healing of tendon, ligament and articular lesions in equine and human patients, to compare the results from clinical and experimental studies in both species, and to verify the existence of a relationship between the study designs and results of clinical trials. This review also aimed to evaluate critically the existing evidence to provide clinicians with high-quality information and a more thorough appreciation of the effects of the diversity of PRP' processing techniques.

Methods
This systematic review was performed in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement, an evidencebased, established guideline for systematic reviews published by the CONSORT group [13,14].
A comprehensive literature search addressing the use of hemoderivatives in orthopedic lesions was conducted in July 2013 for all relevant articles in English, French, German, Spanish and Portuguese without publication or date restrictions, and all of the authors (PMB, JJM, TSLM, RYAB) were involved. The Embase, Bireme, Medline, PubMed and Google Scholar databases were used, with the search terms "platelet rich plasma", "PRP", "tendon", "joint", "articular", "ligament", "musculoskeletal injuries", "human" and "equine". Additional studies were identified by searching the reference lists of eligible articles. Studies that used PRP in conjunction with stem cells or other biomaterials, as regenerative/ anti-inflammatory therapies for other target tissues (e.g., bone) or in other medical fields (such as ophthalmology, craniomaxillofacial or plastic surgery) were excluded.
Equine clinical studies, because of their scarcity, were included independently of their design or level of evidence if they described the effects of PRP on tendon, ligament or articular injuries. Human clinical studies were included if they reported the use of PRP in tendon, ligament or articular lesions and were either double-blind RCTs or prospective/retrospective cohort studies or if they were case series with a control group.
Experimental studies with PRP conducted in several species, both in vivo and in vitro, were selected if they were controlled and relevant to the clarification of the effects and mechanisms of action of PRP in tissues of interest. Studies that used products not derived from blood processing, despite an analogous function or a similar composition to PRP, were not included [15][16][17].
Preliminarily, the abstracts and titles were reviewed to select manuscripts for full-text review. Relevant data were then extracted from the selected articles based on predefined data fields and were sorted in tables corresponding to clinical and experimental studies, independently, by all of the authors. The tables were intended to (1) facilitate the identification of the lesion/tissue/population studied; (2) characterize the control and intervention groups; (3) state the outcome measures and methods employed for the evaluation of effects; (4) describe in detail the method of preparation, composition and protocol of administration of PRP; and (5) present the results and rate them as positive or beneficial (+), partially positive (±) or negative (impartial) (−). This assessment was not made by the review's authors and is in accordance to the results presented, when confronted with the selected outcome measures. This overall view of the studies' design and contents was followed by annotation of weak points that could have negatively affected the generation of quality evidence or that could have biased the studies' results. Experimental studies with in vivo and in vitro ramifications had their results presented on separate lines to allow for a thorough analysis.
The risk of bias of the selected clinical trials was presented in tables according to PRISMA guidelines, and the studies were assigned as having a high or a low risk of bias if they exhibited, respectively, more or fewer bias criteria. This information, together with the analysis of the clinical trials' results, was used to verify a possible association between these two variables and was also presented in tables.

Results
Our search parameters yielded 7415 results: 3563 from Embase, 2817 from Bireme, 101 from Medline, 122 from PubMed and 812 from Google Scholar. Fifteen studies were identified in reference lists from the selected articles. The titles and abstracts of the retrieved records were screened for eligibility, and 5926 studies were thus excluded. One thousand five hundred four articles were fully assessed. One hundred twenty-three (123) articles were selected after the exclusion criteria (i.e., the exclusion of duplicates, review articles, non-controlled human trials, studies in which other tissues were examined and experimental studies investigating PRP preparation particularities) were applied ( Figure 1).
Among these 123 studies, a relatively homogeneous distribution between clinical trials (60 studies or 48.8%) and experimental studies (63 studies or 51.2%) was observed. These 123 articles yielded 126 results because two experimental studies comprised both in vivo and in vitro experiments, which were considered separately.
Side effects associated with PRP use in the clinical setting were observed in two studies (3.3%); in one study [18] they referred to swelling at the injection site and in another [45] they related to infection after platelet-rich fibrin matrix application.

Discussion
The use of hemoderivatives for tissue healing has gained increasing popularity for the treatment of musculoskeletal lesions. Among these derivatives, PRP has already been established as a part of the repertoire of possibilities for the treatment of orthopedic conditions [4,6,10,[141][142][143][144][145][146][147].
Despite widespread acceptance of its ambulatory use, research continues for the purpose of providing convincing evidence of clinical benefits associated with this hemoderivative's administration. Uncontrolled or biased reports of PRP's efficacy have been excessive and have not strengthened the existing evidence, suggesting (but not definitively demonstrating) the beneficial effects of PRP.
The overall quality of the study design was less than ideal in the majority of the selected studies and the quality was inversely correlated with the performance of PRP in clinical trials. Rigorous study design and a low risk of bias were associated with negative outcomes in PRP clinical trials. Because a high-quality study design was a much more common feature of human studies and a rare feature of equine studies, it was not surprising to find more negatively affected outcomes in human clinical trials and positive outcomes in equine clinical trials. Other authors have emphasized the importance of welldesigned clinical studies for the evaluation of PRP's efficacy and limitations and these authors have warned about their scarcity [141,146,[148][149][150][151][152][153][154][155][156], but the present review presented a quantitative link between study design and outcome.
Accordingly, 76.9% of the studies with negative results were classified as RCTs, while RCTs comprised only 42.8% of the studies with positive results. While the inclusion of studies with different designs can be a subject to debate if, in one hand, the inclusion of RCTs, cohorts and controlled case series caused heterogeneity of our study sample, on the other, the diversity and the large number of studies included allowed for comparisons and for the establishment of a quantitative relationship between study design and outcome after PRP intervention. This relationship was more consistent in the human clinical trials, given the scarcity of RCTs and controlled studies in the equine species. As expected, the majority of less than ideally-designed equine studies yielded positive results, and the only negative result among equine clinical trials originated from the only equine RCT. The same was true for including data of clinical and experimental studies, but, again, the comparison between results of PRP´s efficacy with these different methodologies revealed important results.
Another purpose of the current review was to analyze and summarize the most common flaws in study design and to evaluate their implications in the results of PRP interventions. The most striking feature contributing to the debatable quality of the clinical trials included in this review was the lack of a true placebo control group. This observation referred mainly to the human clinical trials because the majority of the equine clinical trials lacked a control group, thereby preventing comparison.
Because several of the selected studies adopted subjective outcome measures and lacked a true placebo control group, their results could have been impacted. However, a gold standard treatment, if one exists, can be assigned to the control group for comparison with a new proposed treatment. Then, researchers would be comparing two active treatment groups, without a placebo group [157]. This comparison was frequently observed in this review, and PRP's effectiveness was often compared with that of hyaluronic acid, corticosteroids, autologous blood injections, no treatment, dry needling, physiotherapy or combinations of these therapies. Nevertheless, treatment controls, such as sham acupuncture and intra-articular hyaluronic acid, have a greater effect size than the average placebo effect [158]. Although control groups were often assigned to some sort of treatment in the selected articles, we could not identify relationships between positive or negative results and the lack of a true placebo group. In addition, as other authors [159] have mentioned, the use of PRP combined with other biological therapies could pose a challenge to the evaluation of PRP's individual effects and confound interpretation of the results. Therefore, studies that chose to do so were not included in this review.
Poor hemoderivative characterization was the second most common flaw contributing to the assignment of study design as not ideal. Inconsistencies associated with PRP preparation and administration have contributed to the lack of strength and to the disparities of the generated evidence regarding PRP's efficacy, in both clinical and experimental studies [160]. The most important consequence of the confounding diversity of the methods employed for PRP processing was the difference in resulting final products, which precluded a comparison of the treatment's results [143,149,156]. Similar to any autologous blood-derived product, PRP has unique, nonreproducible and donor-related features that can jeopardize a comparison of the results [161,162]. Therefore, the hemoderivate composition should be verified and clearly presented [81,163] to minimize these effects.
This review confirmed the existing diversity of preparation methods, commercial or laboratorial, for obtaining PRP, as already indicated by other researchers [12,81,149,156,[164][165][166]. These methods might include single, double or triple centrifugations, filtration and plateletpheresis, with and without the aid of activating agents. Most of the selected clinical studies employed one centrifugation step, and more positive results were observed with this method. Previous studies have demonstrated that a double centrifugation method resulted in higher platelet concentrations [167,168], but caused more alterations in platelet morphology and was more sensitive to small errors during preparation [168,169] compared with the single centrifugation method. This trend toward more positive outcomes with PRP prepared with one centrifugation step must be confirmed with further research. Overall, PRP characterization was more adequate in the experimental studies than in the clinical trials.
Another controversial topic in PRP preparation has been the need for activationor the lack of it [163,[170][171][172]. Until a consensus is reached, comparing activated to non-activated products is inevitable. Most of the clinical and experimental studies activated PRP before injection, and clinically more positive outcomes were observed without PRP activation. The percentages of clinical and experimental studies that employed activation were similar. Experimentally, there was no relationship between the use of activation before PRP administration and favorable or unfavorable outcomes.
The administration protocols also varied greatly among the studies -clinical and experimental -regarding injection or application techniques, volume of hemoderivative employed and timing and frequency of administration. Again, all of these variables limited our ability to compare the results from different articles [148,166].
Poor blinding was the third most frequent negative feature of the studies designs. Blinding limits bias in outcome evaluation, and whenever feasible, outcome assessors should not be aware of the treatment allocations of the patients in a clinical study [173]. Blinding of outcome assessors is one of the safeguards to assure the internal validity of a trial, and there has been strong evidence that their unblinding exaggerated treatment effects. Particularly when scoring subjective outcomes, for instance pain scores, biased findings can result from inadequate blinding [157,174]. Blinding becomes less important in reducing observer bias, as the outcome measure becomes less subjective [157]. In addition to the blinding of outcome assessors, trial participants and investigators should be unaware of an assigned intervention for similar reasons.
The adoption of a short follow-up period and the enrollment of a small sample were the fourth most commonly encountered weaknesses in study design, particularly in horses. Inadequate duration of follow-up or treatment compromises the external validity of an RCT. Clinicians treating patients with a variety of conditions have called attention to the contrast between the beneficial effects of treatments in short-term RCTs and the less encouraging experiences with long-term treatment in clinical practice [175]. Long-term follow-up evaluations should be a priority [81]. In this review, the classification of short study duration was evenly distributed between studies with positive and negative results and did not particularly affect outcomes, although a few clinical trials showed the shortterm efficacy of PRP in improving knee function and quality of life [55,176].
The detrimental effects of enrolling a small sample on the power of a study have been well documented. When a sample is too small, a study is particularly susceptible to a type II errors; that is, the study could be insufficiently powerful to detect real differences between observed groups. Calculations of explicit sample size or power to anticipate this error have rarely been performed before the start of a research study [177]. However, in the selected articles, smaller samples were not particularly associated with positive or negative outcomes. Nevertheless, we encourage authors to enroll sufficient numbers of subjects in their studies to assure adequate power and significance to their findings.
The adoption of inadequate outcome measures was the fifth most frequently encountered weakness in study construction in this review. As already mentioned subjective outcomes present great opportunities for bias [174] and frequently were the only type of employed outcome measure in the selected studies.
In orthopedic research, health status can be assessed by a number of methods, which are classified either as objective (e.g., radiological changes, range of motion) or as subjective (those relying on responses obtained directly from patients about their perceptions of health and illness) and as either generic or disease-specific [174]. Most authors have accepted that a combination of objective and subjective measures is desirable for conducting a complete assessment. Often, a single given parameter employed for outcome evaluation suits certain circumstances but not others, and there has rarely been a single most appropriate rating system or outcome measure [173,178,179]. Employing different outcome measures allows for the capture of diverse aspects of overall function, and lack of agreement between patient-reported and objective measures reflects this diversity, rather than indicating a weakness in one method or the other [180].
In this review, 48.3% of the selected studies only evaluated PRP treatment's efficacy with a few questionnairereported or subjective measures, as opposed to the ideal multifaceted evaluation; therefore, they were classified as having poor outcome measures. It is important to choose the most adequate measures for a particular task, condition and setting; otherwise, the results of clinical research can be misleading. In particular, the inclusion of mechanical tests resulted in more outcomes that were negative in the experimental studies. This association might help to explain why the consistently positive responses induced by PRP in vitro, particularly in cell cultures, were not correlated with similar improvements in outcomes in vivo. Another aspect that added confusion to the comparison of results of PRP treatments among the studies was the variety of lesions treated. PRP's performance was compared in acute and chronic; experimentally induced and naturally occurring; tendon, ligament and articular disorders without due distinction for the particular tissues' characteristics. Biomechanical particularities, such as those of the flexor and extensor tendons; lesion localization, such as insertional versus body tears in ligaments; and staging of lesions, such as advanced osteoarthritis versus mild cartilage injuries, were not considered when evaluating PRP's treatment efficacy [166,177].
The localization of lesions in the clinical trials was associated with both positive and negative outcomes. PRP intervention yielded more positive results in tendinopathies than in arthropathies and desmopathies in humans. In a large survey of PRP's effects in the equine species in which 191 subjects were treated for desmopathies, tendinopathies and arthropathies, good results after intralesional injection were obtained, but in tendinopathies results were more impressive [181]. The reasons for these findings are unknown, and given the heterogeneity of tissues and stage of diseases, these results should be interpreted with care. Other researchers failed to provide definite recommendations for PRP intervention on a tissue-related basis [164]. Clinical and experimental scenarios, as well as interventions in distinct phases of lesion progression, different species and particularly-affected structures do affect performance of a treatment modality and must be considered.
This review demonstrated that scientific evidence of the beneficial effects of PRP in clinical settings remains lacking. While the vast majority of experimental in vitro studies yielded positive results, the same promising outcomes were not verified in the clinical trials after intervention with PRP, particularly in humans. Corroborating these observations, we found that all of the negative results from experimental works came from in vivo studies. Other authors have made similar remarks when comparing in vivo and in vitro studies of PRP [78,[182][183][184], but the present review furnished numeric data for making comparisons and drawing conclusions. Altogether, these numbers demonstrated that the high expectations created by PRP's outstanding performance when tested in vitro have not been fulfilled when studies have been performed in living subjects. Given the small number of experimental studies with negative results, factors that could have adversely affected the outcomes were searched. Among the experimental in vivo studies that yielded negative results, four included mechanical evaluations in their outcome measures. Two studies exhibited side effects associated with PRP administration, and one study failed to show improvements during the histological evaluation of experimentally induced tendon lesions treated with PRP. The trend toward failure when mechanical tests were applied in PRP-treated tissues was already noted in in vivo studies with mixed results and was more evident when the follow-up period lasted longer after the PRP intervention. Mechanical tests should be considered when testing PRP's effects in experimental models.
Why in vitro positive results outpaced the positive results observed in clinical and in vivo PRP studies by such a large margin remains to be determined. The particular characteristics of these distinct environments may provide clues about this discrepancy. The in vivo environment presents several variables that are absent outside of a living organism and that could interfere with PRP's effectiveness. However, the primary advantage of in vitro research is that it allows for an enormous level of simplification of the system under study and of the effects of a compound on a system. As a drawback, in vitro experiments fail to replicate the precise cellular conditions of a living organism. They are conducted in a closed system that tend to allow for higher and longer exposure of cells or explants to a given molecule than the exposure found in body tissues, which are half open systems. In vivo, substances cannot easily reach target cells and are subject to biokinetics, which can result in underestimation of their effects [185].
Another relevant aspect that could harm the extrapolation of in vitro results to the in vivo scenario was the lack of consideration of interspecies differences. Numerous experimental in vivo studies have enrolled different animal models, and this finding could further affect the interpretation of the results of the efficacy of PRP in horses and humans.
Overall, PRP has proved to be a safe therapeutic tool, with few adverse effects observed in the selected articles. This finding was similar to reports by other authors [55,164,177,181,186]. Although the in vitro antibacterial effect of human PRP against methicillin-resistant Staphylococcus aureus has been demonstrated [187], as well as those of equine platelet concentrates and plateletpoor plasma [188] the potential risk for bacterial contamination during PRP processing must be considered.

Conclusion
PRP has demonstrated the potential to exert beneficial effects in the healing of tendons, ligaments and cartilage, but definitive clinical evidence of its efficacy remains lacking. A great amount of literature has been dedicated to the topic, and well-constructed clinical trials, with sufficient power and duration to detect differences between treatments, as well as standardization of products, procedures and conditions to be treated, are as important as they are scarce.
Our results confirmed that biased, poorly designed studies, that are not properly controlled or blinded and that adopt inadequate outcome measures, favored the observation of positive results. As a consequence of this finding, the majority of equine clinical studies, which lacked randomization; blinding; and adequate power, outcome measures and control groups, yielded positive results. Similarly, human clinical trials with analogous undesirable features tended toward positive outcomes. Although the human clinical trials were better constructed than the equine studies, some aspects of their design must nevertheless be improved to generate strong evidence regarding the use of PRP in clinical scenarios.

Additional file
Additional file 1: Table S1. Characteristics of 60 clinical studies that provided evidence regarding PRP intervention.
Additional file 2: Table S2. Characteristics of 63 experimental studies that provided evidence regarding PRP intervention.