Platelet-rich plasma (PRP): methodological aspects and clinical applications.

http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–13 ! 2014 Informa UK Ltd. DOI: 10.3109/09537104.2014.881991

REVIEW ARTICLE

Platelet-rich plasma (PRP): Methodological aspects and clinical applications Laı´s Fernanda Marques1, Talita Stessuk2, Isabel Cristina Cherici Camargo1, Nemi Sabeh Junior3, Lucineía Dos Santos1, & Joaõ Tadeu Ribeiro-Paes1 Department of Biological Sciences, University of the State of Saõ Paulo, UNESP, Assis, Saõ Paulo, Brazil, 2Post Graduation in Biotechnology ICB, IPT, I. Butantan, University of Saõ Paulo, Saõ Paulo, Brazil, and 3On Reabilita Clinic, Assis, Saõ Paulo, Brazil

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Abstract

Keywords

The clinical use of platelet-rich plasma (PRP) is based on the increase in the concentration of growth factors and in the secretion of proteins which are able to maximize the healing process at the cellular level. Since PRP is an autologous biologic material, it involves a minimum risk of immune reactions and transmission of infectious and contagious diseases, and it has been widely used for the recovery of musculoskeletal lesions. Despite the great potential for applicability, the implementation of the therapeutic employment of PRP as a clinical alternative has become difficult, due to the lack of studies related to the standardization of the techniques and/or insufficient description of the adopted procedures. Therefore, it is required establish standard criteria to be followed for obtaining a PRP of high quality, as well as a larger number of studies which should establish the proper concentration of platelets for the different clinical conditions. In this context, the purpose of this review is to discuss some methodological aspects used for achieving the PRP, as well as to discuss the bioactive properties of PRP, and to point out its therapeutic use in different fields of regenerative medicine.

Bone, cartilage, muscle, platelet-rich plasma, tendon

Introduction The cicatrization process comprehends four main stages, namely: clotting, inflammation, cell proliferation and repair of the matrix, epithelialization and remodeling of the cicatricial tissue [1]. Following the injury, the platelets are stimulated to aggregate themselves and to secrete growth factors, cytokines and other homeostatic factors required for the clotting cascade which characterize, therefore, the first stage [1, 2]. Clotting and platelet degranulation lead to the inflammatory stage by means of the release of serotonin and histamine, bioactive factors which increase the capillary permeability which will allow the arrival of larger inflammatory cells to the place of would, such as leukocytes, macrophages and neutrophils, which act on the phagocytosis of the material resulting from the cellular lysis [1, 3]. Once this stage is over, the number of anti-inflammatory cells is reduced and fibroblasts synthetize collagen, elastin and other components of the matrix. Residual epithelial cells or those which have migrated to the injured area multiply themselves and form scars which are initially reddish and elevated. The scar is then remodeled from a balance between the degradation by proteases and the production of extracellular matrix, besides the reduction of fibroblasts and capillaries by means of the apoptosis process [1]. Growth factors have been known since the 1950s, when Cohen and Levi-Montalcini [4], after extracting venon from snakes, isolated factors which could stimulate the growth of nerve fiber. The study along with the description of the epidermic

Correspondence: Joaõ Tadeu Ribeiro-Paes, MD, PhD, Department of Biological Sciences, University of the State of Saõ Paulo, UNESP, Av. Dom Antonio, 2100, 19.806-900, Assis, Saõ Paulo, Brazil. Tel: +55-183322-5856. E-mail: [email protected]

History Received 29 November 2013 Revised 27 December 2013 Accepted 7 January 2014 Published online 10 February 2014

growth factors by Cohen [5] in 1962, are considered pioneer events for the development of treatments. However, only in 1982 growth factors deriving from platelets themselves were described in detailed form [6, 7]. Platelets derive from megakaryocytes, and have no nucleus; therefore, are unable to divide themselves. From the morphological aspect, is worth observing the occurrence of a dense cytoplasmic granulation. The alpha-granules store growth factors that are responsible for the chemotaxis, proliferation, cell differentiation and angiogenesis [8]. The dense granules carry the required bioactive factors for tissue recovery process such as serotonin, dopamine, histamine, adenosine and calcium. The substances produced by the platelet granules are secreted during the process of platelet activation, and they regulate the fundamental aspects of the tissue repair [2]. The rational for the clinical use of platelet-rich plasma (PRP) is based on its ability to stimulate the production and, accordingly, the increase in the concentration of growth factors and in the secretion of proteins which are able to maximize the healing process at the cellular level [2]. The use of PRP speeds the vascularization of grafts, reduces post-surgery morbidity, improves the regeneration of different tissues, as well as reduces the formation of a scar, since it accelerates the maturity and regeneration of the epithelium of wound [9]. Furthermore, is estimated that the PRP acts increasing the recruitment, proliferation and differentiation of the cells involved in the tissue regeneration [2]. The modulation of the inflammatory process is an important aspect in terms of the mechanism of action and repair of lesions mediated by PRP treatment. In the context of inflammatory lesions, PRP acts via secretion of growth factors, inflammatory mediators such as cytokines and chemokines, as well as


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expression of chemokine receptors. The enriched concentration of activated platelets presents a balance of pro- and anti-inflammatory factors well-orchestrated in order to resolve inflammation. In this sense, the PRP is capable of modulating secretion and recruitment of key inflammatory cells, such as monocytes and leukocytes, in the injury site. Therefore, the therapeutic action and tissue regeneration PRP-mediated process seems to result from the control of the local inflammatory response [10, 11]. The first clinical use described in the literature dates back to year 1987, when Ferrari et al. [12] described the use of autologous PRP as an additional element for transfusion in cardiac surgeries, for the purpose of avoiding the use of homologous products. However, only in the 90’s decade PRP started to be widely used as an adjuvant in different surgical procedures, mainly in the areas of dentistry and orthopedics. Thus, this article presents a brief review of the literature, emphasizing methodological aspects of PRP isolation, as well as the main clinical applications in bone, cartilage, tendon and muscle lesions, besides aesthetic plastic and reconstructive surgery.

Platelet-rich plasma PRP consists of a volume of plasma with platelet concentrations higher than the basal levels, which are achieved by means of centrifugation. Generally two centrifugations are employed with set a time and speed previously defined in different experimental protocols. After the first centrifugation, the red blood cells are separated from the plasma in function of the different densities, and sediment in the bottom portion. Just above, between the erythrocytes and the plasma, the buffy coat is formed, followed by

Platelets, Early Online: 1–13

the PRP, the platelet-middle plasma (PMP) and the platelet-poor plasma (PPP) [13]. The plasma and the buffy coat are collected and undergo a second centrifugation for an increase in platelet concentration. The two superior thirds of the achieved plasma (corresponding to the middle- and poor-platelet plasma) are discarded and the remaining portion is mixed with the erythrocyte button deposited in the bottom, which originates the PRP. In order to set a standard PRP volume to be achieved, some authors have established the production of 1 ml of PRP for each 10 ml of collected blood. Therefore, after the second centrifugation, the superior portion corresponding to the plasma is collected so that the remaining plasma volume plus to the erythrocyte button provides 1 ml of PRP [14, 15]. Before being administrated to the patient, PRP must be activated in order that the platelets release their factors (Figure 1, Table I) and other products related to the regeneration process. It is usually employed calcium chloride, bovine thrombin or collagen to platelet activation [2]. The use of autologous PRP has the advantage of eliminating the risk of crossed contamination, as well as the transmission of microbial diseases or immune reactions [25]. In this regard, several authors are against the inclusion of bovine thrombin in some protocols, for the activation of the platelets due to the risk of coagulopathy through the production of antibodies against clotting factors. Therefore, the use of autologous thrombin must be reconsidered. Alternatively, the use of the collagen to the detriment of thrombin providing lower clot retraction and without the reduction of growth factors [26]. The use of PRP in liquid or gel form has shown an improvement in the cicatrization process. However, the gel preparation requires a longer time for the achievement of the autologous thrombin, thus leading to a more complex and longer

Figure 1. Process for obtaining the PRP. 1 and 2: After the collection, the blood undergoes the first centrifugation process. 3 and 4: The superior phase corresponding to the PPP, PMP, PRP and buffy coat is removed to the second centrifugation. 5 and 6: Afterwards, the two superior thirds corresponding to the PPP and PMP are discarded and the portion corresponding to the PRP is then mixed to the erythrocyte button and the buffy coat deposited in the tube bottom.

PRP: Methodologies and clinical applications

DOI: 10.3109/09537104.2014.881991

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Table I. Growth factors with the sources of obtaining and respective functions.


Growth factor

Source

TGF-b

Platelets, neutrophils, macrophages, monocytes, natural cell killers, Th1 cells, bone extracellular matrix and cartilaginous matrix

FGF

Platelets, macrophages, chondrocytes, osteoblasts and mesenchymal cells

PDGF a-b

Platelets, macrophages/monocytes, endothelial cells, osteoblasts and smooth muscle cells

Epidermic growth factor

Platelets, macrophages/monocytes

VEGF

Platelets, endothelial cells

IGF

Platelets, macrophages, osteoblasts, bone matrix and mesenchymal cells

Function

References

Regulates the mitogenic effect of other growth factors, stimulates the proliferation of undifferentiated mesenchymal cells, fibroblast and osteoblast mitogen, endothelial regulator and regulator of the collagen synthesis and secretion of collagenase, stimulates angiogenesis and endothelial chemotaxy, inhibits the proliferation of macrophages and lymphocytes Mitogen for mesenchymal cells, chondrocytes and osteoblasts, stimulates the growth and differentiation of chondrocytes and osteoblasts Stimulates the chemotaxy and mitosis of fibroblasts, smooth and glia muscle cells, regulates the secretion of collagenase and collagen synthesis, mitogen for mesenchymal cells and osteoblasts, stimulates the chemotaxy of macrophages and neutrophils Stimulates mitosis of mesenchymal cells, regulates the secretion of collagenase, stimulates chemotaxy and angiogenesis of endothelial cells Stimulates mitosis of endothelial cells, increases angiogenesis and permeability of the vessel Stimulates the differentiation and mitogenesis of mesenchymal cells and of lining cells, stimulates osteoblasts and the production of type I collagen, osteocalcin and alkaline phosphatase

[6, 15, 16]

preparation [26]. Another difficulty is the loss of the material which has a tendency to flow off until the gel formation is completed [26, 27]. The direct injection of PRP at the site of the lesion, without the need for activation, is an attractive and palpable alternative because activation can be supposedly attributed to the trauma caused by the needle and/or the residual collagen, implying in the reduction of costs and the preparation time [27]. Generally, the therapeutic use of PRP in the past 20 years has demonstrated to be a safe and effective treatment; however, there is some conditions that the indication must be availed with caution, as in cases of thrombocytopenia, platelet dysfunction syndrome, septicemia, hypofibrinogenemia, recent fever condition, anemia, cancer, skin lesions in the area of the injection, use of corticosteroids (in up to 2 weeks before the procedure), or non-steroid anti-inflammatories (in up to 48 hours before procedure) and active infections with Pseudomonas, Klebsiella or Enterococcus [28–31]. Nevertheless, the literature has reported conflicting results on the benefits of PRP. The number of participants in different studies, is usually small and the techniques used are not standardized [2, 28, 32]. Different platelet concentrations are achieved by means of different methodologies with results which are sometimes not well defined as to the improvement of cicatrization [33]. The increase in the rotation force is known to provide a higher platelet concentration; however, too high forces may lead to the loss of growth factors in the supernatant plasma due to an early activation of the platelets and to the rupture of the tubes which mean losses to therapeutic efficiency of PRP [14, 34]. In general, the unsatisfactory results reported may be associated with the quality of the material obtained, since the platelet concentrations obtained are highly divergent among the studies. Other important variables are related to the maintenance of platelet integrity and effective activation of the material, which often are not elucidated in the studies.

Clinical applications Bone lesions Bone defects may arise out of mechanical traumas, pathologies and physiologic stress. Due to the high prevalence of injuries,

[6, 17, 18] [6, 15, 19]

[6, 20, 21] [6, 21, 22] [6, 23, 24]

bone is the second most transplanted human tissue, overcome only by hematopoietic tissue. It is estimated that annually 2.2 million people are subjected to grafting procedures to repair bone defects in orthopedics, odontology and neurosurgery [35]. The use of autologous grafts is considered a gold standard among the biomaterials employed in filling of bone cavities. However, the need for two surgical procedures, the limitation of tissue available, risks of infection, necrosis and re-absorption in up to 30% of patients, motivated the proposition on synthetic biomaterials, which by turn are not biologically functional and adapted to remodeling bone tissue [36, 37]. The use of biological factors, such as PRP and bone morphogenetic proteins (BMP), associated or not with graft, has shown promising results in regard to bone reconstructions, since they are directly associated with the normal physiology of this tissue (Table II). Platelet growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-b (TGF-b), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF)-A and insulin-like growth factor (IGF-1) regulate bone regeneration through migration, proliferation and differentiation of osteoblasts, justifying the therapeutic use. Since the 1990s, the use of the PRP in the treatment of bone lesions has shown significant results. It is used as an alternative to fibrin glue or platelet gel frequently employed in maxillofacial reconstructions. The therapeutic benefits that extend beyond the reparative power, consisting of one action faster than conventional treatments maximized by autologous growth factors and therefore free from complications of immune origin [25, 38, 39]. Batista et al. (2011) [40] compared the PRP action with the concentration of bone marrow, had better consolidation and greater bone quantity by area in the PRP group. The superior result obtained can be explained by the immediate recruitment of all proteins necessary to start the healing cascade, while the concentrated of bone marrow demanded longer time to recruit these elements. Thus, it can be assumed that the monitoring for a period of time up to 4 weeks, this group might have had similar results of consolidation. However, there were no new studies that could confirm this hypothesis. Several studies reporting the association of PRP and artificial bone grafts showed improvement in the quality of healing. However, when it was employed only PRP the results obtained

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Table II. Use of PRP in animal models with bone lesions.


Lesion site

PRP production

Therapeutic (treated group)

Therapeutic (control group)

Outcomes

References

Defects at the distal region of the femur Mice

Ten milliliter of peripheral blood of isogenic mouse were collected and centrifuged to enact blood phase separation

PRP one application

Femurs were alternated between the right and left sides

There was no increase in the inflammatory process. Showed a tendency to increase bone matrix formation.

[54]

Bone defect (3.3 mm in diameter) in the proximal metaphysis of rabbit’s right tibia

The blood was centrifuged for ten minutes at a speed of 1200 rpm at room temperature. PRP received 0.2 ml of 10% calcium gluconate.

PRP with b-tricalcium phosphate one application

bone marrow concentrate with b-tricalcium phosphate

PRP promoted better consolidation than the centrifugation of the bone marrow group

[40]

Critical-sized defects measuring 2 cm rabbit Tibias

Blood with EDTA was centrifuged two times: first at 150 g for 20 minutes and second at 450 g for 10 minutes

Three groups: PRP, coragraft, PRP and coragraft

No PRP or coragraft

PRP with bone grafts improved the quality of healing bone

[46]

Mandibular intraosseous defects Dogs

The centrifugations: at 5600 rpm for 10 minutes At 2400 rpm for 10 minutes. The PRP was actived with thrombin and CaCl2.

Bony cavity on the right side filled with autogenous PRP

The left side was empty and used as a control

Increase bone cell proliferation

[47]

were inferior, probably due to the lack of connective tissue which provides adequate reparation [41, 42]. In cell culture, the use of the PRP has evidenced the potential of growth factors by means of the modulation of cellular processes. The use of homologous PRP, in osteoblasts derived from patients with periodontitis, showed an increase in the proliferative response [43]. This result was corroborated by another study on osteogenic potential of PRP, showing increase in cell proliferation and osteogenic differentiation in vitro, as well as the increase in the bone formation around the acellular graft and a high arterial perfusion around the bone defect [44]. Although different studies indicate satisfactory results, some researchers have reported conflicting results [25, 45]. The application of PRP in combination with bone marrow concentrate on defects of tibia and in combination with bovine cancellous bone critical defects in the skull increased bone healing in rabbits [46]. When applied on mandibular bone lesions in dogs, there was bone regeneration of the PRP group in a shorter period of time compared to the control group [47]. On the other hand, the use of the PRP combined with bone graft has not shown better results compared to graft alone in dogs and rats [48–50]. The conflicting results may be related to the type of methodology employed to produce the PRP, which can result in inadequate concentration of growth factors required for the repair of bone lesions. Although the use of calcium and thrombin for activation of PRP is associated with increased proliferation and migration of bone marrow stem cells, it is known that high concentrations may interfere with angiogenesis due to the impairment of migration and proliferation of vascular endothelial cells and reducing the release of VEGF [51, 52]. Another important aspect is related to possibility of activated platelets compromise the osteogenic differentiation [53]. The search for alternative forms of platelet activation may be the key to obtain better therapeutic results of PRP employment. The use of tiny concentrations of thrombin

supplemented with collagen and mechanical stimulation led to increased levels of VEGF compared to using only thrombin and calcium chloride [44]. Considering the homeostatic function performed by platelets, it is natural to conclude that the concentrate contributes to repair injuries. However, the presence of different factors, sometimes antagonists, tends to create an imbalance unable to promote tissue recovery. Thus, it is important to emphasize the need for more detailed studies on the concentration of each growth factor obtained, since it can vary between individuals, and especially among the different animal species (Table II) that are widely studied in order to validate the therapeutic properties of PRP in bone lesions. Cartilage lesions The practitioners of physical activities have a high incidence of articular cartilage lesions, while treatment remains a challenge to sports medicine due to the limited regenerative capacity of cartilaginous tissue [55]. Therefore, PRP has been offered as an alternative treatment to articular cartilage lesions of knee, hip and ankle [8]. Different studies have shown the action of growth factors in stimulating the proliferation of chondrocytes, the chondrogenic differentiation of mesenchymal stem cells derived from bone marrow and control of the synthesis of cartilaginous matrix [56–58]. Studies of migration and chondrogenic differentiation of human subchondral progenitors, cultured in the presence of PRP, confirms the chemotaxy potential in the chondrogenic migration and differentiation. PRP-stimulated cells achieve a significant increase in the formation of a cartilaginous matrix, and the analysis of gene expression shows the presence of chondrocyte, adipocyte and osteocyte-marker genes, although an evident differentiation of these last two cell types did not exist [59].

[68]

[78]

[75]

Hyaluronic acid Intra-articular application of autologous PRP

Untreated lesions or PLGA administered alone PRP–PLGA Scaffold

Grade 1, 2 or 3 osteoarthritis

Two centrifugations: at 4 C for 15 minutes at 800 rpm and at 4 C for 15 minutes at 2000 rpm Three centrifugations: 15 minutes at 3200 rpm at 22 C, 10 m at 1500 rpm to separate the leukocytes and centrifugation step at 3200 rpm for 10 minutes Articular cartilage defects of rabbits

Two centrifufations

Chondral loose body Human Rabbit model OA

Two centrifugations: at 1.800 rpm for 15 minutes and a second at 3.500 rpm for 10 minutes produced a unit of 20 ml of PRP Blood was centrifuged at 1800 rpm for 8 minutes with trisodium citrate Degenerative changes in the joint Human

3% PPP

Improved articular cartilage Healing functional and accelerated Return to activity Increased cartilage matrix metabolism; investigators postulated that may have preventative implications in OA management PRP with scaffold stimulated osteochondral formation with cartilaginous matrix and type II collagen Statistically significantly better results –

Knee arthroscopy reattached the fragment, supplemented with PRP 3% PRP or PRP in biodegradable gelatina hydrogel microspheres

[61]

Results positive at 6 months follow-up, with mild degradation of the scores at 1-year follow-up – PRP intra-articular knee injections administered every 21 days

References Outcomes Therapeutic (control group) Therapeutic (treated group) PRP production Lesion site

Table III. Clinical studies and animal models with the use of PRP in cartilage lesions.


In another study of cell culture involving chondrocytes derived from humans with osteoarthritis, at different concentrations of platelet lyse, showed a reduction of the inflammatory effects, including the inhibition of the nuclear factor-kB (NFkB), the chief factor involved in osteoarthritis pathogenesis [60]. Sanchez et al. [61] were the pioneers in using PRP in the treatment of avulsion of knee articular cartilage in football players. The avulsed fragment was fixed with absorbable pins and the existent gaps between the fragment and the receiving area were filled with PRP. After using a platelet concentrate obtained by a single centrifugation, named by the authors as preparation rich in growth factors (PRGF), the clinical results obtained were very satisfactory, with the acceleration of the cure and functional recovery of the lesion. In total knee arthroscopy (TKA), several studies obtained the reduction in the need of blood transfusions, less infectious processes, less post-surgery complication, as well as a shorter time in the hospital [62–64]. The direct application of PRP in athletes with chronic patellar tendinitis, in patients with knees joint osteoarthritis (OA) and osteochondral lesions of the talus, demonstrated functional improvement and pain management [65–67]. Spakova´ et al. [68], compared the efficiency of PRP with hyaluronic acid in patients with different degrees of OA and obtained more satisfactory results in the group treated with PRP, although the two treatments have been statistically significant. Regarding the treatment of anterior cruciate ligament injuries, application of PRP has shown significant effects from a clinical standpoint, resulting in an improved biomechanical and a faster recovery [69, 70]. However, some authors have pointed to the absence of significant results [71–73]. The potential of growth factors in cartilage repair is well established regarding the regulation of the physiology of articular cartilage. Growth factors such as TGF-b and FGF have been shown to stimulate the chondrogenic proliferation and biosynthesis of the extracellular matrix of articular chondrocytes in vitro, allowing to presume the obtaining of clinically satisfactory results [59, 74–76]. However, the discrepancies between the results obtained can be related to different methods of obtaining the PRP, forms of activation and isolated application or aggregated with other elements (Table III). The use of PRP in soluble form cannot support the biological activity by being rapidly degraded, and employment in the gel form does not contain high levels of growth factors, besides the application with needles to be hampered by high viscosity. Depending on the location and extent of the lesion it is possible to choose the way that best fits the needs of the patient, but it is important to note that the newly obtained PRP may have an acidified pH (6.9–7.0) and can interfere in the functions naturally performed by cytokines [76]. Studies have reported that the use of alkaline PRP (7.4) has allowed a stimulating effect on cell proliferation due to the release of additional components [78, 79]. The general proposition that PRP stimulates chondral anabolism and reduces catabolic processes may explain the improvement of clinical symptoms. Filardo et al. (2011) [80] indicate that PRP should act in articular homeostasis, resulting in the reduction of hyperplasia of the synovial membrane and modulating cytokine levels, although these mechanisms of action have not been clearly determined. Thereby, more detailed studies related to PRP obtainment, concentration of growth factors, gender, age and degree of degeneration should be considered when analyzing the results, since they may interfere with treatment. Another aspect to be considered is the follow-up of patients to determine whether the original structure of the joint was resumed or if the recovery was only temporary, aiming to standardize the number and time between applications in order to enable the routine use of this therapy in clinical practice and avoid invasive surgical procedures.

[66]


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[88]

[84]

Minor recovery period, stimulation of myogenesis Minor recovery period Actovegin/Traumeel

Arthroscopy without PRP

PPP or no treatment

Four applications of 100 ml of PRP or PPP Autologous conditioned serum (ACS)

[91]

[94] –

Injection ultrasound-guided injection Application of PRP guided by arthroscopy

Tibialis anterior muscles of Sprague Dawley men Muscle trains

One centrifugation activation with autologous thrombin Apheresis with Leukoreduction using citrate dextrose gel with 10% sodium gluconate 3 ml PRP not activated PRP obtained by Symphony II platelet concentration system One centrifugation: 3.500 rpm at 10 minutes Grade II lesions

Rotator cuff (430 mm, anteroposterior)

Outcomes Therapeutic (control group) Therapeutic (treated group) PRP production

Muscle lesions are usually caused by direct trauma or by decompensation of the eccentric load during muscle contraction, and they are very common, especially in elite athletes [2]. In the majority of the cases, the recommended treatments are conservative and the best therapies have not been clearly defined. Thus, muscular injuries represent a challenging problem for traumatology and sport medicine [81]. Therefore, the more clinically serious muscle injuries are among the most indicated for PRP treatment [82]. The background of previous lesions represents a great risk factor of muscle injuries, probably due to the formation of scar tissue on the trauma area. Since muscle lesions imply a high morbidity rate within amateur and professional sports, the use of PRP means a promising alternative since it accelerates muscle regeneration and minimizes the occurrence of new lesions [83, 84]. The muscle regeneration process is regulated by the presence of growth factors and cell interactions, and it features a high concentration of cytokines found in muscles. Growth factors, especially the IGF, improving muscle regeneration and increased strength [85, 86]. It is also known that PRP induces the proliferation of muscle cells, as well as the differentiation of satellite cells and are active in the angiogenesis process [87]. The first clinical study to employ growth factors in the recovery of muscle lesions used conditioned autologous serum. Eight professional athletes were enrolled in the study and compared with 11 athletes with similar previously treated lesions (control group). The treatment started 3 days after the lesions, all of them ranked as moderate degree lesions, and it led to the reduction of the edema and bleeding of the treated group, with full functional recovery within 2 weeks, which represented half the expected time for regeneration of these kinds of lesions [85]. Other studies with elite athletes showed that percutaneous PRP injections in the muscle injured area improved the functional recovery, and they enable the continuation of sports activities [88]. Cugat et al. [89] used PRP in muscle lesions caused by mechanical traumas. Aided by ultrasound equipment, the author injected PRP directly into the injured area, and this way recovery time was reduced in half. Studies conducted by three independent teams, which were seeking to cure the limited regenerative capacity of the rotator cuff led to the evaluation of its repair potential in the presence of PRP. Patients were randomized for the treatment by arthroscopy with and without PRP. None of the assessed scores presented statistically significant difference, and one of the studies also hints at negative effects. However, one of the teams reported the achievement of satisfactory results in small and medium-size lesions. In this way, authors suggest the use in bigger-sized lesions, since the different methods commercially available may lead to more efficient preparations [90–92]. Despite the discrepancies between the results obtained by different research groups, the rationale for the use of PRP in muscle lesions is substantiated by the release of growth factors linked to acceleration of tissue recovery, once it acts in proliferation of muscle precursors, vascularization improvement, reduction of infectious processes and symptoms of pain, leading to functional improvement and rapid return to sports activities. It is known that the PRP appears to act better on the regeneration phase, through the action of IGF which stimulates muscle cells to proliferate and form muscle tissue free from fibrous scar tissue associated with relapses. Another important aspect is based on the hypothesis that PRP can have no therapeutic effect when applied in the first 24 hours after injury [93]. The extensive description of conflicting results in the literature (Table IV) makes clear the need for additional studies that should be strictly standardized in relation to the size and location of the

Table IV. Use of PRP in animal models with muscle lesions.


Muscle lesions

Pain relief, functional improvement; to sports activities Absence of significant differences between groups

References


Lesion site

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lesion, age of the patients studied, methodology used to obtain the PRP, leukocyte and platelet count in order to obtain a product having adequate physiological concentrations of growth factors and other bioactive factors related to muscle recovery, searching for routine clinical application of a therapy highly effective and, at the same time, minimally invasive.


Tendinous lesions Tendon is a durable and flexible tissue, anchored to the bone by a mineral resistant connection. However, due to the fact that this tissue is subject to much mechanical loading and stress, there is great risk of tendinopathies, chronic tendons injuries, since the continuous effort may cause successive lesions of the collagen fibers [55]. The tissue of tendons is not very well vascularized, the metabolic rate and oxigen consumption are low, so it regenerates slowly, in the variable manner and with risk of reinjury. Therefore, scars may impair tendons function and increase the risk of repeated injuries, as well as chronic inflammation and pain [8]. Tendinous lesions outstand among musculoskeletal lesions, and impact both practitioners of intense physical activities, as well as inactive people [95, 96]. It is estimated that these lesions represent from 30 to 50% over all lesions related to sports [97]. In this context, and by taking into account the pieces of evidence that tendinitis is an intrinsic degenerative disorder, PRP has been widely used in the treatment of Achilles tendon, elbow, rotator cuff and patellar lesions [98]. The use of PRP as therapeutic strategy in tendon injuries has been especially considered in patients with chronic inflammation, over 12 months and refractory to previous treatments [99]. Concomitant with the applied research, PRP has generated interesting results also in vitro models for the purpose of tendon repair. At the cellular level, tendons are formed mostly by tenocytes, cells responsible for the maintenance and tissue repair. In this regard, in vitro studies related to the action of PRP on tenocytes have shown results of great importance and clinical interest. There is evidence in the literature suggesting an increased in tenocytes proliferation inder action of the PRP, and this effect is dose dependent. Still, although controversial, there are results that indicate the ability of PRP to act on the expression of collagen (COL1 and COL3) by tenocytes in order to contribute to the quality of the repair [100]. One important exception, however, must be emphasized, as in vitro models are not capable of mimicking the microenvironment of chronic inflammatory tendinopathy and thus it is not possible to draw conclusions categorical, since the laboratory response may be different from the clinical reality. Animal models, similar to the in vitro tests are not able to provide the identical condition to human tendinopathy. However, the results indicate interesting therapeutic properties of PRP on tendon lesions surgically induced when there is comparison with control groups [100]. When PRP is used in acute tendon injuries, the repair process happens faster and the organization of fibroblasts and collagen bundles is optimized in relation to controls without administration of PRP in equine model [101], murine [102] and cunicular [103]. However, in most of the studies reported, there are few data and cytological investigations of the PRP employed, and thus impediment of therapeutic elucidation. Further, the treatment is carried out in animal models in the acute phase in contrast, therefore, to most clinical cases. Mishra and Pavelko [65] published the first study in human patients using PRP in the treatment of tendinosis, or chronic tendinitis of the elbow. From the 140 evaluated patients, only 20 with a background of failure with non-surgical treatments underwent the experiment, and five were used as a control group (treated with bupivacaine). Within 8 weeks after one application of PRP, there was an improvement in 60% of the treated patients,


7

and within 6 months, 81% of patients felt better, and this improvement reached 93% two years afterwards, without complication. Among patients treated with PRP was possible to observe a return of daily activities (99%) and of sports activities (94%). However, the authors did not consider the variation in the duration of pain and neither the age of the patients to assess outcomes between treated and control groups. Still, the work presents a reduced number of control patients (n ¼ 5), and 60% of these (n ¼ 3) were excluded from the study, becoming impossible a statistical analysis [76]. Another relevant site of tendinopathy, lesions in the rotator cuff are among the most common disorder of shoulders and usually need surgical intervention, due to the inexistence of therapeutic agents which enable the cure of the tendon. The treatment of 14 patients with partial thickiness rotator cuff and a background of failure in the answer to conventional treatments involving steroids, physiotherapy and non-steroid anti-inflammatories, were assessed as to the possible answer to PRP. Within 8 weeks, 12 patients involved in the study achieved significant improvement of pain, strength and muscle resistance [104]. A study proposed by Sańchez et al. [105], pointed out the potential of PRP in the treatment of tendinous lesions. In said study, six athletes having Achilles tendon lesions underwent the conventional surgery combined with PRP (called by the author as a platelet-rich fibrin matrix), and an improvement in the cure and functional recovery were achieved, in comparison with the control group. In another study which entailed the use of PRP in chronic tendinopathy of no-insertion of the tendon calcaneus, with a background of previous and unsuccessful treatments, achieved a significant clinical improvement, which was further confirmed by ultrasonography studies. After 18 months, all patients showed an improvement in acute symptoms, increase in the vascularization and in the diameter of areas previously noted as having fibrillary interruption. The authors reported the increase of the PRP vascular activity to its involvement with surrounding tissues related to the tendinous cicatrization. The significant functional improvement, the lack of complications followed by functional improvement and satisfaction of patients are pointed out by the author as encouraging initiatives for the use of said therapy [106]. The use of PRP in patellar tendon during the reconstruction of the anterior cruciate ligament, also points out the benefits of therapy, and it has shown significant reduction of the size of the lesions and pain during the post-surgery period [107]. Different studies do not present uniformity of results and procedures, thus it is necessary to make further randomized trials with larger numbers of patients, in order to enable a consensus or methodological reference, allowing conclusions more consistent about the use of PRP on tendon injuries. Table V presents some works using of PRP in different types of tendon injuries. As seen in Table V, there are a different number or even conflicting parameters in regard to the preparation, quality, dosage and interval of injection of PRP in various clinical studies in tendon injuries. Thus, the analysis of therapeutic results becomes complex, since there may also the involvement of several cell types such as lymphocytes and neutrophils, in accordance with the methodology used to obtain the PRP. Furthermore, inflammatory cytokines with short lifetime, released after platelet activation, may start an acute inflammatory response in compromised tendinous fibers, causing a proliferative phase synthesis of collagen, essential for tendon repair [108]. Another flagrant methodological distinction between the different clinical protocols refers to the previous activation of PRP, while in other studies the PRP is injected without previous activation. Another interesting fact refers to the discrepancy of clinical outcomes. Clinical responses range from significantly positive in

Whole blood (55 ml) and sodium citrate processed in accordance with the kit adopted for obtaining the non-activated PRP Whole blood (27 ml) and sodium citrate (3 ml) processed according to the kit adopted for obtaining non-activated PRP Whole blood (510 ml) and sodium citrate centrifuged for 15 minutes at 2000g. Non-activated PRP (1.5 ml) obtained from the buffy coat.

Whole blood (9 ml) and trisodium citrate centrifuged for 6 minutes at 1100 rpm. The plasma was centrifuged for 25 minutes at 4500g. PRP activated with calcium chloride. Whole blood (9 ml) centrifuged in two steps. PRP activated with calcium chloride Whole blood (54 ml) and citrate (6 ml) centrifuged for 15 minutes at 3200 rpm, 6 ml of non-activated PRP

[113]

[114]

Reduction of pain and significant functional improvement in the treated group compared to the control Similar functional improvement in treated and control groups

Twenty patients, intratendineous injection of saline (5 ml) Five patients, intratendineous injection of bupivacaine (2–3 ml) Forty nine patients, intratendineous injection of 1 ml of corticosteroids and bupivacaine Seventy patients, two intratendineous injections of blood (1.5 ml) with an interval of 1 month

Fifteen patients, intratendineous injection of PRP (2–3 ml)

Fifty patients, intratendineous injection of 1 ml of corticosteroids and bupivacaine Eighty patients, two intratendineous injections of PRP (1.5 ml) with an interval of 1 month

[112] There was no significant difference in pain reduction, functional improvement and quality of life between groups Constant and significant improvement in pain in the treated group compared with the control group

Thirty nine patients, arthroscopy (suture surgery)

Forty patients, application of PRP clot associated with suture during arthroscopy Twenty patients, intratendineous injection of PRP (5 ml)

[76]

[92]

There was no significant difference in improvement between the groups

Forty five patients, arthroscopy

[90]

[111]

[106]

[110]

[109]

[104]

References

Forty three patients, application of PRP clot during arthroscopy

Twenty seven patients submitted to arthroscopy

The treated group showed significant improvement, absence of complications and faster return to physical activities when compared to the control group There was no significant difference between the groups with respect to pain relief and return to physical activities Minimal functional improvement without changing the magnetic resonance imaging Functional improvement, pain reduction, positive change to examination by Doppler ultrasonography Pain reduction and significant functional improvement in short-term treated group. In long term (46 months) there was no difference between groups. There was no difference in magnetic resonance imaging. There was no significant difference between the groups in relation to Constant Shoulder Score or changes in magnetic resonance imaging.

Outcome


Elbow, epicondylitis



Rotator cuff

Rotator cuff

Rotator cuff

Rotator cuff

–

Whole blood processed in accordance with the kit adopted for obtaining the non-activated PRP Whole blood processed in accordance with the kit adopted for obtaining the non-activated PRP Whole blood (54 ml) and citrate dextrose solution (6 ml) centrifuged for 15 minutes at 3200 rpm. PPP centrifuged for 2 minutes at 2000 rpm. PRP activated with autologous thrombin.

Achilles tendon, chronic tendinopathy


Twenty seven patients randomized, intratendineous injection of saline (4 ml) –

Twenty seven patients randomized, intratendineous injection of non-activated PRP (4 ml)

Whole blood (54 ml) and citrate centrifuged for 15 minutes, non-activated PRP


Ten patients, intratendineous injection of non-activated PRP (6 ml) Fourteen patients, intratendineous injection of non-activated PRP (3 ml) Twenty six patients submitted to arthroscopy, intratendineous injection of activated PRP (6 ml)

Six athletes, open suture surgery

Six athletes, open suture surgery followed by intratendineous injection of activated PRP (4 ml)

Whole blood and trisodium citrate centrifuged at 460 g for 8 minutes. One milliliter above the erythrocyte fraction activated with calcium chloride.

Achilles tendon, no more than 2 weeks after rupture

Therapeutic (control group)

Therapeutic (treated group)

PRP production

Lesion site

Table V. Clinical studies with the use of PRP in patients with tendinopathies.


8 Platelets, Early Online: 1–13

Whole blood (27 ml) and citrate dextrose solution (3 ml) processed in accordance with the kit adopted for obtaining the non-activated PRP Whole blood (150 ml) and sodium citrate (21 ml), centrifuged for 15 minutes at 1800 rpm. Plasma centrifuged for 10 minutes at 3500 rpm. Non-activated PRP (20 ml). Whole blood (150 ml) and sodium citrate (21 ml), centrifuged for 15 minutes at 1800 rpm. Plasma centrifuged for 10 minutes at 3500 rpm. Non-activated PRP (20 ml).

Knee, patellar tendinopathy


Knee, patellar tendinosis


Whole blood (450 ml) fractionated in portion erythrocyte and PPP, PRP activated with autologous thrombin and calcium chloride. Whole blood (150 ml) centrifuged for 6 minutes at 1480 rpm. Plasma centrifuged for 15 minutes at 3400 rpm, generating 20 ml of PRP, PRP activation of calcium chloride.

Whole blood (30 ml) and ACD-A anticoagulant centrifuged for 15 minutes at 3200 rpm, non-activated PRP


Knee, patellar tendinosis

Whole blood and anticoagulant centrifuged for 15 minutes at 3200 rpm, nonactivated PRP


Functional improvement, significant reduction of pain compared to the control group in the short term. Increased improvement in 6 months when physiotherapy was inserted in the PRP group. Functional improvement and reduction of pain higher in the group treated (short term). No significant difference in the long term (6 months) between groups Stability of functional improvement, pain reduction, return to activities, patient satisfaction

For 114 patients, intratendineous injections of bupivacaine (2–3 ml) –

–

Sixteen patients, physiotherapy treatment

Fifteen patients, reconstruction of the anterior cruciate ligament

For 116 patients, intratendineous injections of PRP (2–3 ml)

Eight patients (6 athletes), intratendineous injection of PRP (3 ml) Twenty athletes, 3 intratendineous injections of PRP (5 ml) every 15 days

Fifteen athletes, 3 intratendineous injections of PRP (5 ml) every 15 days

Twelve patients, reconstruction of the anterior cruciate ligament with implant of PRP gel (20– 40 ml) Forty three patients, 3 intratendineous injections of PRP (5 ml) every 15 days –

Significant pain reduction in the shortterm treated group. In long term (43 months), there was no difference between groups Functional improvement and reduction of pain superior in the treated group. Significant differences in favor of the treated group in alternating periods. Functional improvement and positive change in the magnetic resonance imaging. Total return to sports 12 weeks after injection. Significant reduction of pain and stiffness, functional improvement and return to activities at 6 months after treatment

Fourteen patients, intratendineous injections of blood (3 ml)

Fourteen patients, intratendineous injections of PRP (3 ml)


[119]

[107]

[118]

[65]

[117]

[116]

[115]

DOI: 10.3109/09537104.2014.881991

PRP: Methodologies and clinical applications 9

10


the short and/or long-term, positive but not significant in the short and/or long term, or the results are similar when compared to control groups. Chronic tendinopathy, who comprise the majority of clinical studies have pathologies cell subtypes that range over time and thus the PRP could be beneficial and contribute to the morphological and functional improvement in some of these stages, but not in others. Finally, the tendency observed in the literature, as well as clinical experience on the treatment of tendinopathy with PRP, support the importance of the combined treatment. In this sense, physical therapy and a program of activities after injection of PRP, adopted in most studies, demonstrate better results in tendon lesions and should be considered for this type of clinical application.


Conclusion In the past twenty years, PRP application has proved to be a safe and effective in experimental animals models and in the clinical treatment of human patients. Its therapeutic use in regenerative medicine has reported favorable effects in connection with safety, and a simple methodology of preparation. Another important advantage relates to the low cost of obtaining the preparations of PRP. Nonetheless, the implementation of its therapeutic use as a clinical alternative has been impaired, or even made unfeasible, for a series of aspects related with the insufficient description of the adopted procedures, the large variability of the studies in regard to platelet quality and concentration, small sample and lack of control groups. Therefore, new research are required, with greater accuracy and experimental refinement, employing a larger numbers of patients, randomization of samples, standardization of methodological procedures, as well as the definition of a consensus to establish more precisely the clinical conditions for which the PRP should be properly and precisely employed.

Declaration of interest The authors report no conflicts of interest. The authors thank Fundaçaõ para o Desenvolvimento da Unesp (FUNDUNESP), Prefeitura Municipal de Assis and CIVAP/Sau´de (Assis, SP) for the financial support. T.S. is financially supported by Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES).

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