Tendinopathies and platelet-rich plasma (PRP): from pre-clinical experiments to therapeutic use.

OBJECTIVES
The restorative properties of platelets, through the local release of growth factors, are used in various medical areas. This article reviews fundamental and clinical research relating to platelet-rich plasma applied to tendinous lesions.


MATERIALS AND METHOD
Articles in French and English, published between 1 January 2012 and 31 December 2014. dealing with PRP and tendons were searched for using the Medline and Scopus data bases.


RESULTS
Forty-seven articles were identified which addressed pre-clinical and clinical studies: 27 relating to in vitro and in vivo animal studies and 20 relating to human studies. Of these, five addressed lateral epicondylitis, two addressed rotator cuff tendinopathies, ten dealt with patellar tendinopathies and three looked at Achilles tendinopathies.


CONCLUSIONS
The majority of pre-clinical studies show that PRP stimulates the tendon's healing process. However, clinical series remain more controversial and level 1, controlled, randomised studies are still needed.


Introduction
The 'restorative' properties of platelets, through the release of growth factors, have been implemented in various medical areas (dentistry, dermatology, orthopaedic and plastic surgery, ophthalmology, etc.), because of their contribution to healing various tissues (bone, skin, muscle, tendons, etc.) [1] . They may be injected, in situ, in the form of platelet concentrates, commonly known as PRP (platelet-rich plasma), obtained from autologous blood [1] .
This innovative treatment has piqued clinicians' interest, particularly specialists in physical medicine and sports traumatology, more so because the production of PRP is relatively easy [2] . Although intratendinous injection can slightly increase the blood concentration of growth factors (particularly in vascular endothelial growth factor (VEGF)) [3] , PRP no longer appears on the list of doping products (www.wadaama.org), in contrast to other labile blood products. PRP can neither improve muscle mass nor alter the transport of oxygen (particularly because the presence of red blood cells alters PRP quality) [4] .
This literature review analyses articles on the use of PRP in tendinous healing (experimental and clinical studies) published between 1 January 2012 and 31 December 2014. Several reviews have already appeared relating to earlier studies [5][6][7][8][9][10][11][12][13][14][15] . The results of both pre-clinical and clinical studies were analyzed separately in this review and finally discussed together in order to trying to improve this therapeutic modality. Articles were searched for using the Medline and Scopus databases, by entering, alone and/or combining, the following In addition to the presence or otherwise of white blood cells in PRP, Galliera et al. observed that platelets actively controlled the recruitment of leukocytes through the intermediary of transforming growth factor β (TGF-β) [18] .
Dragoo et al., compared the inflammatory reaction caused by the infiltration of physiological liquid, blood, PRPs rich in white blood cells and PRPs low in white blood cells in rabbit tendons [19] . Five days after infiltration, the PRPs rich in white blood cells lead to a significant inflammatory cellular reaction, similar to that observed following blood infiltration. However, after 14 [20] . They noted that the PRP with an intermediate concentration of platelets and a high concentration of white blood cells led to higher expression of pro-inflammatory cytokines and catabolic mediators (Interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α)) as well as lower collagen synthesis. Moreover, the PRP with high concentrations of platelets and white blood cells led to a paradoxical effect on collagen synthesis, while also presenting an increase in inflammatory mediators. These observations were confirmed by Boswell et al., who also noted in equine tendon cultures that when platelet concentrations were too high (despite low concentrations of white blood cells) this reduced tendon metabolism and collagen synthesis [21] . Finally, Giusti et al., demonstrated in human tenocyte cultures that concentrations of between 5.10 5 and 1.10 6 platelets/µL stimulated cell proliferation and migration as well as collagen production [22] . On the other hand, higher platelet concentrations have an inhibitive effect both in terms of cell metabolism and collagen synthesis.
In addition to the fact that PRP stimulates cell proliferation and collagen synthesis, Jo et al., demonstrated that platelet activation through thrombin and calcium is more efficient that calcium activation alone [23] .
In vitro, however, PRP does not enable the reversal of degenerative tendinopathies, characterised by the presence of lipid deposits, the accumulation of proteoglycans as well as the presence of calcification [24] . However, still in vitro, PRP does enable the differentiation of tenocyte stem cells and inhibits the differentiation in adipocytes, chondrocytes and osteocytes, which can hamper tendon healing [25] .
Muto et al., demonstrated that PRP protects against the deleterious effects of triamcinolones on cells (reduction in cell viability and stimulation of apoptosis) from human rotator cuffs [26] . Moreover, Carofino et al., studied the effect of two different PRPs obtained from human blood, on isolated tenocytes in the long head of the biceps [27] . These were cultured in PRP and either methylprednisolone, lidocaine or bupivacaine. They observed that corticoids and, particularly, local anaesthetics, alone or combined, reduced the positive effects of PRP on tenocyte proliferation and viability. These observations were corroborated by those of Bausset et al., who showed that local anaesthetics which may be used as painkillers during PRP infiltration in clinical practice, could compromise the potential therapeutic potential of platelets [28] . They reduce platelet aggregation but will not interfere with the release of their growth factors. Finally, to reduce pain during infiltration, they recommend the use of smaller needles (30G), because they do not alter platelet function. During the study of the effects of PRP on biological activity of fibroblasts in human rotator cuffs, Wang et al., observed greater cell proliferation and collagen synthesis when PRP was present in comparison with foetal veal serum [29] . This is supported by other studies which show higher cell proliferation, better alignment of collagen fibres and better tendon biomechanical properties when PRP is concentrated up to five times [30] . Kaux et al., demonstrate, using rats' Achilles tendon lesion models that the early stages of healing (five days) are stimulated by PRP: acute collagen synthesis and better traction resistance [31] . Within this group of PRP, Fernandez-Sarmiento et al., observed early healing progress, better collagen organisation, and a reduction in fibroblast and vascular density [32] . These observations are confirmed by other series using lesion models of rats' rotator cuffs [33] .
In an in vitro model on rabbits' tendon cells and in vivo on rats' Achilles tendons, Zhang et al., proved that the anti-inflammatory effect of PRP is essentially linked to HGF (hepatocyte growth factor) [34] . HGF leads to a reduction in the expression of cyclooxygenases (COX) 1 and 2, as well as prostaglandins (PGE2).
In terms of the growth factors involved in the healing process, Solchaga et al., noted that 10µg platelet-derived growth factor (PDGF-BB) stimulated healing in rats' Achilles tendons, in contrast to 3µg of PDGF-BB, triamcinolone, but also PRP [35] . In contrast to the deleterious effect of VEGF-165, Kaux et al., observed that VEGF-111, another VEGF-A isoform, stimulated the early phases of the healing process in rats' Achilles tendons, by improving bio-mechanical properties [36] .
Finally, in addition to platelet effectiveness on healing in line with a chemical tendinopathy model (platelet and calcaneal tendons in rats), Dallaudière et al., did not show evidence of local toxicity of PRP [37] .
In conclusion, the majority of pre-clinical studies show a beneficial effect of PRP on the proliferation of tenocytes, collagen synthesis and the tendon healing process. In order to optimise efficiency, it would appear that platelet concentration should be lower than 10 6 platelets/µL and that white blood cells should be absent.

B. Clinical studies:
PRPs are used in the context of chronic tendinopathies in injectable form [38] . For some years, it has been used increasingly regularly, more specifically in the area of physical medicine and sports traumatology [11] . The objective is to heal tendinopathies which are particularly resistant to other conservative treatments (eccentric physical therapy, shock waves, etc.) and thus to avoid surgery. In a certain number of cases, the aim is also to reduce the duration of functional impotence and to encourage an early return to physical activity. However, currently the use of these PRPs remains a topic of debate and even controversy [7][8][9][10][11][12][13][14][15] . Table 1 presents the clinical studies discussed in this review.

Epicondylites
In a prospective three-month study, without a control group patients who had suffered from evolving lateral epicondylitis for more than six months and who had received PRP infiltration [39] . They noted that pain, assessed on simple verbal scale, was significantly improved, a well as the Quick disabilities of the arm, shoulder and hand (DASH) score. Ultrasound observations showed the fissure had disappeared in 65% of patients, as well as a reduction in the pathological area and Doppler hyperaemia in 27% of subjects. In their controlled, randomised study (level 1), Omar et al., compared the effect of PRP or corticoid infiltration in 30 patients over a period of six weeks. They observed a positive change on the visual analogue scale (VAS) and DASH score within the two groups, but no significant difference between them [40] .
In their longitudinal six-month study (level 4) of six patients who had received an injection of 3ml of PRP under ultrasound, Chaudhury et al., observed a positive change in the tendon's ultrasound structure, with a trend towards increased vascularisation at the myotendinous junction [41] .
The randomised, controlled, double-blind study (level 1) by Krogh et al., did not show any difference after PRP, corticoid or physiological liquid infiltration in the context of epicondylitis over a period of three months [42] . However, patients who received PRP infiltration started to improve, while those who received corticoid infiltration (resulting in fast improvement in pain in the very short term), indicated the progressive return of pain.
Recently, in their randomised and controlled study (level 1) of 230 patients, Mishra et al., compared a PRP group to an active control group receiving an injection of local anaesthetic [43] . After 12 weeks, there was no difference between the two groups in terms of VAS when extending the wrist against resistance and the patient-rated tennis elbow evaluation (PRTEE) self-assessment score. At the end of the 24 week follow-up period, significantly positive clinical changes were observed in the PRP group of patients.
To summarise, although studies with low levels of proof on the use of PRP for lateral epicondylitis currently appear to be favourable, the small number of series with a high standard of proof are still contradictory and more randomised controlled studies are still needed.

Tendon lesions in rotator cuffs
The randomised, controlled study by Rha et al., (level 1) compared the value of two PRP infiltrations in 39 patients affected by tendinopathy of the rotator cuff, compared to two dry needle insertions guided by ultrasound, over a period of six months [44] . After the first intervention, the two groups evolved identically. However, after the second infiltration of PRP, a clinical improvement (in pain and mobility of the shoulder) was observed in comparison with the control group.
In their randomised, controlled study (level 1), Kesikburun et al., assessed the effect in 40 patients of an injection of either PRP or physiological liquid [45] . After 12 months, no difference could be observed between the two groups in terms of pain, quality of life, handicap and range of motion of the shoulder.
To summarise, the very small number of studies with a high standard of proof on the use of PRP for tendinopathies of the rotator cuff currently remain contradictory. Up to now, it remains hard to draw a conclusion.

Patellar tendinopathies
The prospective, randomised and controlled (level 1) study by Almeida et al., specified that PRP improved healing at the puncture site (patellar tendon) required for ligamentoplasties (Kenneth-Jones type) of the anterior cruciate knee ligament [46] .
The prospective 18-month study (level 3) by Gosens et al., compared 14 patients who had already received treatment (cortocoids or ethoxysclerol and/or surgery) with 22 patients who had never had any infiltrative or surgical treatment [47] . They all took part in eccentric physical therapy before and after the injection. After four weeks, the patients were able to gradually restart their sporting or recreational activities. An improvement in Victorian Institute of Sport Assessment for patellar tendinopathy (VISA-P) scores, VAS and pain during daily activities was seen in both groups, but more significantly so for the patients who had never had 'invasive' treatment before PRP infiltration.
Rowan et al., report on the positive development and return to prior athletic form within six months of a 23-year old elite athlete (level 4) who had received multidisciplinary treatment including PRP infiltration for patellar tendinopathy [48] .
In their longitudinal study (level 3) of 46 patients who had received three PRP infiltrations two weeks apart, with a minimum monitoring period of up to 36 months (average of 49 ± 8 months), Filardo et al., demonstrated a positive evolution two months after infiltration, continuing at six months and until the end of the follow up period [49] . Eighty percent of patients had resumed their sporting activities. Patients with bilateral problems and whose symptoms had lasted the longest, however, evolved less well.
In their randomised and controlled study (level 1), Vetrano et al., assessed the effect of PRP over three treatments with focal shock waves in 46 patients suffering from patellar tendinopathy [50] . After two months, the two groups evolved in a similar way. However, after six and twelve months of follow up, the PRP patients presented better progress than the group treated with shock waves.
As part of their randomised and controlled study (level 1), Dragoo et al., compared the effect of PRP infiltration to the introduction of a dry needle guided by ultrasound in 23 patients [51] . The two groups took part in eccentric physical rehabilitation following infiltration. Although evolution at 12 weeks is better in the PRP group, this beneficial effect dissipates over time and, at 26 weeks, no difference could be seen between the two groups.
Charousset et al., followed 28 high level athletes (level 4) who received three consecutive infiltrations of PRP under ultrasound control [52] . At the end of the two year follow-up period, the subjects reported improvements in their symptoms and function, enabling them to get back to their earlier athletic condition more quickly. Moreover, they observed that tendons regained their normal MRI architecture.
Kaux et al., followed 20 patients suffering from chronic patellar tendinopathy in a longitudinal cohort study (level 3) following 'standardised' PRP infiltration, obtained by an apheresis machine combined with progressively intense submaximal eccentric physical exercise [53] . Evolution at six weeks and three months showed a significant reduction in pain and self-assessed scores, as well as in pain during maximal eccentric effort applied to the quadriceps, but no imaging improvements. The youngest patients appeared to improve more following this PRP treatment.
Two longitudinal studies (level 3 and 4) relating to postinfiltration physical therapy have been published [54,55] . The two programmes were based on eccentric physical therapy following a period of pain-relieving physical therapy and recovery of normal range of movement in the joint. However, the protocol used by Kaux et al., begins eccentric submaximal physical therapy earlier than that of Van Ark et al. These eccentric exercises are gradually combined with isometric work followed by concentric strengthening work on the quadriceps as well as proprioceptive exercises. At the end of the physical therapy treatment, which is supervised by a physiotherapist, the patients can continue self-led treatment in the long term.
Bowman et al., report that three patients (level 4) who had already received PRP infiltration for unresponsive patellar tendinopathy, experienced an increase in painful symptoms leading to an inability to take part in sport, but also in increase in the thickness of the tendon and, in one patient, osteolysis of the patellar pole [56] . Fink et al., also observed an osteolysis of the distal pole of the patella in one patient who had worsening pain and thickening of the patellar tendon after one infiltration of PRP (Level 4). Kaux et al., also published (level 4) the case of a particularly exuberant inflammatory reaction in a Type-1 diabetic patient following a PRP infiltration for patellar tendinopathy. Evolution was favourable at six months under pain-relieving and antiinflammatory treatment [57] .
To summarise, studies on the use of PRP for patellar tendinopathies currently appear to be favourable, but high standard of proof studies remain fairly contradictory. It appears that eccentric physical therapy is necessary following PRP infiltration. More randomised controlled series are needed.

Calcaneal tendinopathies
Over a period of three months, Silvestre et al., followed 32 patients (level 3) with Achilles tendonitis who had received ultrasound-guided PRP infiltration [58] . After one month, 22 patients were completely cured, and after two months, 28 were completely cured, with a favourable evolution in tendinous echostructure and a reduction in Doppler hyperaemia at three months. Only four patients did not see any clinical improvement and were operated upon.
In their four-year longitudinal study (level 3) of 27 patients suffering from chronic Achilles tendinopathy, following three PRP infiltrations two weeks apart, Filardo et al., reported very positive changes in self-assessed status [59] . The patients who had suffered for the longest had more difficulties in returning to sporting activity.
Finally, Murawski et al., conducted a retrospective study (level 4) on 32 patients over six months [60] . At the end of the follow-up period, 25 subjects were asymptomatic and had resumed their sporting activities. The seven others experienced no improvements in their pain and were operated upon. Only four patients presented MRI imaging improvements.
To summarise, studies on the use of PRP for calcaneal tendinopathies currently appear to be positive. However, no high standard of proof studies have taken place since the unfavourable study by de Vos in 2010 which did not report any improvement after an infiltration of leukocyte rich PRP compared to placebo (saline injection) [61] .

Discussion
By releasing different platelet growth factors, PRPs are a new treatment for chronic tendinopathies [15] : the ease of use, the relatively low cost, and the low invasive nature of the treatment are complementary arguments. Up to now, the reported side effects remain benign [56,57] . No relationship has been established between growth factor and possible carcinogenesis. Care should be taken, however, to ensure that any undesirable side effects are actually published. Currently, only a bleeding disorder, a disorder of the skin covering (infection, psoriasis) next to the lesion could be reasonably considered as contra-indication of the use of PRP in the treatment of tendinopathies. This treatment has been used for several years in different surgical specialities with favourable clinical results [62,63] . Despite the effectiveness of PRP on tissue regeneration in vitro and in animals, little clinical proof is currently available in relation to tendinopathies.
The rare controlled, randomised and blind studies which exist appear to contradict one another [40,42,44,50,51,61]. Can the type of tendon change the healing response? Would this be different for certain voluminous tendons (patellar) compared to others (epicondylian, rotator cuff, etc.) [73] ? Studies on the patellar tendon appear to be more positive than other tendons. However, as already observed in previous reviews on PRP [5][6][7][8][9][10][11][12][13][14][15] , the more recent studies cannot conclude that PRP is really efficient in the treatment of tendinopathy, even if most of the low level studies remain very encouraging. Moreover, many of the authors of the articles reporting success of PRP application (essentially with leukocytes rich PRP) have conflicts of interest. PRPs are injected into the tendinous lesion, possibly guided by ultrasound (under aseptic conditions) to improve the accuracy of this treatment by a correct positioning of the needle [74][75][76] . A percutaneous tenotomy using a needle (guided by ultrasound) could also be carried out during the infiltration [77] .
Moreover, studies appear to be difficult to compare because there is no consensus on preparatory methods, qualitative characteristics of PRP (volume, platelet concentration, presence of leukocytes and erythrocytes [64][65][66][67] , the infiltration technique, or the post-injection therapy protocol [11,15,54,55,68] ). Indeed, different techniques collate different platelet volumes and concentrations: lab techniques which have been used in the past from an analytical perspective can be distinguished from recent commercial techniques [64][65][66][67] . Currently, the optimal concentration of platelets used in the treatment of tendinous lesions, has not yet been validated.   However, the literature raises the following elements:  a platelet concentration less than 10 6 platelets/µL (three to four times the blood platelet concentration) would be optimal, while a concentration higher than 12.10 5 platelets would have a paradoxical effect of inhibiting collagen synthesis [20,69]  a total lack of white blood cells, which are likely to slow down healing due to the early occurrence of a higher local inflammatory reaction [19,70] and the presence of pro-inflammatory factors (cytokines and metalloproteinases) likely to damage the extracellular matrix [65,71] . PRPs containing leukocytes reduce collagen synthesis [20].
 the lack of red blood cells because these, once lysed, release various free radicals, similar to those of leukocytes, also likely to damage neighbouring tissues [65,72] .
Anti-inflammatories are forbidden from Day -10 to Day +21, due to their inhibitory effect on PRP action [1,78] . It appears that studies where eccentric physical therapy takes place report better results than those with rest or classic rehabilitation techniques [54,55] ; in fact, the PRPs trigger a healing process, subsequently developed through the vector of eccentric forces [68] .
The IOC coordinated the drafting of three articles on PRP [75,79,80] confirming the medical-sporting value of this treatment. It recommends that the injection is guided by ultrasound in order to ensure the correct position of the needle [75] . A few minutes after infiltration, PRPs diffuse beyond the infiltrated area and the lesion site, singularly questioning the value of ultrasound-guided infiltrations [81] . Moreover, currently there is no consensus on the intratendinous and/or peritendinous site of the infiltration.

Conclusion
Experimentally, PRP, through the release of different growth factors, stimulates tendinous healing. This treatment should optimise healing in pathological human tendons. It should be examined whether PRP improve tendinous healing in the same way in humans as in animals [82] . Up to now, even if PRP seems to be safe and efficient to treat chronic tendinopathies non-responsive to classical conservative treatments, further high standard of proof series should enable this promising technique to be standardised and to optimise the clinical results.