Platelet-Rich Fibrin Scaffolds for Cartilage and Tendon Regenerative Medicine: From Bench to Bedside

Nowadays, research in Tissue Engineering and Regenerative Medicine is focusing on the identification of instructive scaffolds to address the requirements of both clinicians and patients to achieve prompt and adequate healing in case of injury. Among biomaterials, hemocomponents, and in particular Platelet-rich Fibrin matrices, have aroused widespread interest, acting as delivery platforms for growth factors, cytokines and immune/stem-like cells for immunomodulation; their autologous origin and ready availability are also noteworthy aspects, as safety- and cost-related factors and practical aspects make it possible to shorten surgical interventions. In fact, several authors have focused on the use of Platelet-rich Fibrin in cartilage and tendon tissue engineering, reporting an increasing number of in vitro, pre-clinical and clinical studies. This narrative review attempts to compare the relevant advances in the field, with particular reference being made to the regenerative role of platelet-derived growth factors, as well as the main pre-clinical and clinical research on Platelet-rich Fibrin in chondrogenesis and tenogenesis, thereby providing a basis for critical revision of the topic.


Introduction
The development of cost-effective biomaterial scaffolds to regulate inflammation and enhance wound healing processes is one of the most intriguing challenges in modern regenerative medicine and tissue engineering (TE). Materials of both biological and synthetic origin have been extensively investigated for regenerative purposes. In particular, naturally derived biomaterials offer the advantages of receptor-binding ligand presentation and susceptibility to cell-triggered proteolytic degradation and remodeling [1].
Among natural biomaterials, Platelet-rich fibrin (PRF) has recently aroused widespread interest as a biophysical and biochemical milieu that delivers growth factors (GFs), cytokines and immune/stem-like Among natural biomaterials, Platelet-rich fibrin (PRF) has recently aroused widespread interest as a biophysical and biochemical milieu that delivers growth factors (GFs), cytokines and immune/stem-like cells for immunomodulation and tissue healing purposes [2]. PRF was first introduced by Choukroun and Collaborators [3] as a leukocyte-and platelet-rich biomaterial for oral and maxillofacial surgery applications. The preparation protocol consists of blood collection by venipuncture and subsequent centrifugation to form a strongly polymerized fibrin clot. The technique requires neither an anticoagulant nor thrombin/calcium gluconate [4] (Figure 1). Choukroun's PRF has several advantages over Platelet-rich plasma (PRP), including the denser fibrin network, which allows for easier handling and suturing, as well as slower degradation rates after application, and consequently, delayed GFs and cells release profiles [5,6]. Moreover, this blood-derived membrane is enriched with leukocytes, which play a key role not only in immune and antibacterial responses, but also in the wound healing process [7,8]. Since Choukroun's PRF was first described, many variations of the original protocol have appeared, resulting in the production of PRF-like materials with different architectures and cell contents [9][10][11][12][13][14][15][16][17]. The fundamental challenge to be overcome remains the concentration of platelets, which should be increased to a minimum of 5 times above baseline values for the hemocomponent to be considered "platelet rich" [18]. Platelet concentrates preparation protocols. Schematic drawing of the classical preparation protocols of PRP (Platelet Rich Plasma) and PRF (Platelet Rich Fibrin) hemocomponents. According to PRP protocol (a), blood is collected by venipuncture in the presence of anticoagulants. Thereafter, a two-step centrifugation procedure occurs. The first centrifugation yields three layers: RBCs (red blood cells), "buffy coat" and PPP (platelet poor plasma); hence, PPP and "buffy coat" are transferred into another tube and centrifuged again. After discarding the PPP fraction, the resulting PRP is suspended and activated by fibrin. As regards PRF protocol (b), venous blood is withdrawn without anticoagulants and centrifuged, causing the coagulation and stratification of blood components. In the middle of the tube, between the PPP and the RBC layers, a PRF clot develops, which naturally entraps platelets, leucocytes and molecules like growth factors and fibronectin. To be considered "platelet rich", hemocomponents should be 5 times concentrated in platelets. Thus, the drawing is intended to be representative of the PRP and PRF manufacture steps, as many variations of the protocols are reported in the literature. There is general consensus in referring to Choukroun's protocol [3] as the first method for PRF development. Platelet concentrates preparation protocols. Schematic drawing of the classical preparation protocols of PRP (Platelet Rich Plasma) and PRF (Platelet Rich Fibrin) hemocomponents. According to PRP protocol (a), blood is collected by venipuncture in the presence of anticoagulants. Thereafter, a two-step centrifugation procedure occurs. The first centrifugation yields three layers: RBCs (red blood cells), "buffy coat" and PPP (platelet poor plasma); hence, PPP and "buffy coat" are transferred into another tube and centrifuged again. After discarding the PPP fraction, the resulting PRP is suspended and activated by fibrin. As regards PRF protocol (b), venous blood is withdrawn without anticoagulants and centrifuged, causing the coagulation and stratification of blood components. In the middle of the tube, between the PPP and the RBC layers, a PRF clot develops, which naturally entraps platelets, leucocytes and molecules like growth factors and fibronectin. To be considered "platelet rich", hemocomponents should be 5 times concentrated in platelets. Thus, the drawing is intended to be representative of the PRP and PRF manufacture steps, as many variations of the protocols are reported in the literature. There is general consensus in referring to Choukroun's protocol [3] as the first method for PRF development.
In cartilage regeneration studies, PRF is currently emerging as a biological tool to deliver supraphysiological levels of GFs and cytokines to the site of injury. Besides their physiological role, the in situ administration of platelet-derived GFs was demonstrated to stimulate in vitro proliferation and differentiation of chondrocytes [27] and to promote in vivo healing of cartilage [28]. Therefore, platelet GFs might offer promising treatments for the enhanced regeneration of focal articular cartilage defects.
The effect of platelet-derived GFs in chondrogenesis has been investigated both in vitro and in vivo by using platelet concentrates in the form of medium supplements or gels that encapsulate cells (i.e., chondrocytes, mesenchymal stem cells) [29,30]. Interestingly, Gaissmaier and Collaborators [31] demonstrated an increase of human chondrocyte proliferation rates following the addition of 1% and 10% human platelet supernatant to the culture medium. Similarly, the in vitro study performed by Akeda and Colleagues [32] highlighted the stimulating effect of 10% PRP-enriched medium on porcine chondrocyte proliferation, as well as collagen and proteoglycan biosynthesis. The positive effects of PRP on articular cartilage regeneration have been pointed out by several in vivo studies based on the administration of the hemocomponent in animal models of osteochondral defects, revealing an efficient capacity to promote cartilage healing [33,34]. Like PRP, PRF also contains a plethora of GFs and cytokines released from the platelets. Thus, it could positively influence articular cartilage regeneration through the same mechanisms described for PRP, ensuring superior mechanical performance which better meets the demands of the target tissue.
Regarding the role of platelet-derived factors in chondrogenesis, PDGF is first GF present in a wound, where it serves to promote tissue healing by collagen and protein synthesis. It exhibits mitogenic activity to fibroblasts, vascular muscle cells, glial cells and chondrocytes [35]. In particular, PDGF has been reported to stimulate chondrocyte proliferation through the extracellular signal-regulated kinase 1/2 pathway [36], as well as inhibiting the IL-1β-mediated activation of NF-kB and cell apoptosis [37].
IGF-1 has been shown to regulate articular cartilage metabolism in both healthy and disease conditions. In vitro studies on normal chondrocyte cultures have demonstrated that this GF stimulates ECM synthesis and decreases matrix catabolism [28]. Moreover, IGF-1 is able to induce chondrogenic differentiation of Mesenchymal Stem Cells (MSCs), exerting even higher stimulation when used together with TGF-β1 [38]. In animal models of Osteoarthritis (OA), IGF-1 has demonstrated its potential to enhance cartilage repair and protect the synovial membranes from chronic inflammation [39]. However, the capacity of chondrocytes to respond to IGF-1 seems to decrease with age [40] and in OA [41].
TGF-β1 stimulates ECM synthesis by chondrocytes and inhibits the catabolic activity of IL-1 [42]. Both in vitro studies on MSC cultures and in vivo investigations on embryonic cartilage development have demonstrated the role of TGF-β1 in mediating the shift from proliferation to differentiation of chondrocytes [43]. Moreover, TGF-β1 has been shown to induce in vitro chondrogenesis of synovial lining and bone marrow-derived MSCs [44]. In vivo studies on rabbits provided evidence of TGF-β1-mediated repair of cartilage defects [45]. Recently, some authors have also described scaffold functionalization with TGF-β1 in order to deliver this bioactive factor to the site of cartilage injury [46,47]. bFGF can be found in relative abundance in the pericellular matrix of cartilage, and it is believed to play a key role in preserving the chondrocyte phenotype during expansion. On joint loading, its binding to cell surface receptors activates anabolic signaling pathways which lead to a decrease of aggrecanase activity, not affecting proteoglycan content [35]. In human OA, bFGF catabolic effect on dedifferentiation depends on the upregulation of the matrix metalloproteinases MMP1 and MMP13, and the downregulation of aggrecan and collagen II [48].
EGF signaling strongly influences chondrogenesis by upregulating the expression of SOX9 [49]. Due to this, EGF has been recognized as a potent mitogen of cultured chondrocytes [50]. In addition, an in vitro study on human articular chondrocytes have demonstrated that EGF treatment protracted extracellular signal-regulated kinases (ERK1/2) and Akt phosphorylation to regulate the chondrocyte ECM deposition [51].
Generally known as a potent stimulator of vasculogenesis and angiogenesis, VEGF has been found to be secreted by hypertrophic chondrocytes. Its angiogenic activity can attenuate hypoxic states, which is necessary for chondrocyte phenotype preservation [35]. The role of VEGF in chondrogenesis has been mostly investigated by knockout studies, demonstrating that it inhibits migration and accelerates proliferation of chondroprogenitor cells in vitro, as well as playing a crucial role in chondrocyte metabolism [52].
Clearly, the synergistic action of different GFs is needed to enhance and regulate chondrogenesis; based on that, the therapeutic use of PRF as a reservoir of several bioactive agents could constitute a valid strategy to accelerate the cartilage healing process.

PRF in Cartilage Tissue Engineering
Cartilage tissue engineering is mainly focused on the treatment of articular joint defects. Being an avascular and hypocellular tissue, articular cartilage (AC) shows limited intrinsic ability for self-repair, which implies that cartilage injuries cause progressive and degenerative joint diseases, such as OA [53].
Over the last two decades, several therapeutic strategies have been tested for AC regeneration, including scaffold-free approaches, cell-free methods, and advanced therapies associating cells with different biomaterials [54]. The first strategy relies on the only use of cells at high densities to promote cell-to-cell interactions and cell-based extracellular matrix neo-deposition. Autologous chondrocyte implantation represents the clinical option of choice [55,56], but it has recently been shown to be ineffective as a long-term treatment due to the dedifferentiation of chondrocytes during in vitro expansion [57]. Among alternative cell types that are under investigation for cartilage repair, MSCs from different sources (i.e., bone marrow, adipose tissue, umbilical cord) have gained considerable interest by virtue of their ease of isolation, high expansion capacity and chondrogenic differentiation potential [54,56,57]. Regarding cell-free strategies, the implantation of unseeded scaffolds made of both natural (collagen, hyaluronan, acellular matrices) and synthetic [poly(lactic-co-glycolic acid)] materials is combined with microfracture procedures [58] for the recruitment of bone marrow-derived MSCs to repair cartilage lesions [54]. Finally, the production of advanced tissue substitutes by associating cells and scaffolds seems to promise breakthroughs in the clinical practice of osteochondral regeneration [54]. In this regard, innovative strategies such as microfluidic biofabrication and cartilage bioprinting are offering unique possibilities to combine cells and biomaterials in an ordered and predetermined way for the manufacture of organized three-dimensional tissue constructs [54,59,60].
Despite progress in the field, the biological and functional outcome of most current treatments still remains controversial, mainly because the repaired tissue often shows fibrocartilaginous characteristics, not resembling the mechanical properties or ECM zonal organization of the native articular cartilage [61]. Thus, an intriguing challenge for orthopedic surgeons is represented by the development of an ideal treatment option which promotes hyaline cartilage neo-formation and subchondral bone preservation. From this perspective, the use of blood-derived products such as PRF has recently been recognized as a promising strategy to improve functional cartilage regeneration, by virtue of the presence of bioactive factors which stimulate the proliferation of chondrogenic cells and deposition of cartilaginous ECM [62].

In Vitro Studies
In vitro studies of the chondrogenic potential of PRF are quite scant (Table 1). This can probably be ascribed to the fact that the beneficial effects of platelet-derived GFs on chondrocyte and MSC proliferation, as well as on cell deposition of cartilaginous matrix, have been extensively demonstrated by in vitro investigation on PRP [63,64]. In the attempt to define the effect of PRF-derived GFs on chondrocyte proliferation and cartilage-specific ECM synthesis, Chien and Colleagues [65] investigated the incorporation of human PRF exudates into biodegradable fibrin (FB) scaffolds made from bovine fibrinogen and thrombin. In parallel, FB scaffolds alone and agarose (AG) scaffolds were used as controls. Before cell culture experiments, the concentrations of PDGF-BB, TGF-β1, IGF-1 and bone morphogenetic protein (BMP-2) into PRF exudates were quantified in comparison with FB scaffolds and three blood derivatives (i.e., serum, plasma and fibrin). Enzyme-linked immunosorbent assay (ELISA) demonstrated that the amounts of these GFs and cytokines were higher in PRF exudates rather than in the blood-derived products, except for TGF-β1. In this case, PRF exudates resulted to be richer in TGF-β1 when compared to serum and fibrin, but did not reach the concentration level of plasma samples. Subsequently, FB scaffolds +/− PRF exudates and AG scaffolds were seeded with primary chondrocytes from the knee cartilage of osteoarthritic patients and a human chondrosarcoma cell line, SW-1353. Both 2D-and 3D cultures of each cell type were tested. Overall, experimental data highlighted that cell growth rate, type-II collagen and Glycosaminoglycan (GAG) mRNA expression, as well as GAG and proteoglycan protein accumulations, were significantly increased when chondrocytes were cultured on FB scaffolds added with PRF exudates [65]. More recently, in vitro investigations based on rabbit chondrocyte cultures were carried out to evaluate the effect of an injectable PRF (i-PRF) on osteochondral regeneration [61]. Different from standard PRF, which consists of a 3D fibrin matrix, the development of the low speed centrifugation concept made it possible to obtain a new formulation of PRF for injectable purposes (i-PRF), to be used in clinical applications where this administration strategy is preferable. Interestingly, i-PRF can be injected similarly to PRP, with the advantage of forming a fibrin clot shortly after administration. Thus, this new formulation of PRF was compared to PRP by treating rabbit chondrocytes with culture media conditioned with 20% of each platelet concentrate releasates, in normal conditions or in the presence of interleukin (IL)-1β to mimic an osteoarthritic microenvironment. Gene expression studies on chondrocytes in normal conditions have demonstrated that i-PRF up-regulated chondrogenesis-related genes (SOX9, COL2A1 and ACAN) better than PRP. In the osteoarthritis-like environment induced by IL-1β, the inhibition of pro-regenerative genes (i.e., SOX9, COL2A1 and ACAN) was observed, coinciding with the up-regulation of genes related to arthritic disease progression (i.e., ADAMTS4, PTGS2 and MMP13). Remarkably, i-PRF addition to IL-1β-conditioned cultures has been shown to be superior to PRP treatment in modulating the inflammatory environment, by up-regulating the expression of pro-regenerative genes and down-regulating osteoarthritis-related markers [61].
A detailed characterization of PRF-induced chondrocytes harvested from rabbit cartilage was performed by Wong and Co-workers [66]. First of all, the PRF prepared from rabbit blood samples was characterized for (a) PDGF, IGF-1 and TGF-β1 release, (b) mechanical behavior, and (c) ultrastructural morphology by Scanning Electron Microscopy. After that, the chemotactic stimulus induced by PRF on chondrocytes was evaluated using both isolated cell populations and cartilage fragments co-cultured with the hemocomponent. In particular, cell exclusion zone and transwell migration assays revealed the ability of PRF scaffolds to attract and nourish chondrocytes during both in vitro and ex vivo tests. Moreover, chondrocytes were treated with PRF-conditioned media in different concentrations (25%, 50%, 100%), confirming that the hemocomponent increased cell proliferation rate in a dose-dependent manner. Finally, PRF was proven to up-regulate cell chondrogenic markers, such as type-I and type-II collagen and Aggrecan, as well as to stimulate the deposition of cartilaginous matrix (i.e., GAG accumulation) [66].
The chondrogenic potential of PRF-derived factors were assessed not only on chondrocyte cultures, but also on different cell types, such as rabbit meniscocytes [67] and human adipose-derived stem cells (ASCs) [68]. The work by Wong and Colleagues [67] on meniscocytes is very similar to the study on chondrocytes previously reported by the same research group [66]. Also, in this case, rabbit PRF demonstrated the ability to stimulate cultured meniscocytes in terms of cell migration, proliferation and deposition of cartilaginous matrix.
To the best of our knowledge, the only in vitro study on stem populations was reported by Souza and Colleagues [68] and considered the stimulation of human ASCs with eluates recovered from human Fibrin rich plasma (FRP) membranes. This treatment was successful in increasing cell proliferation in 2D cultures, as well as specific differentiation towards the chondrogenic lineage of micromass 3D cultures, as demonstrated by the induction of mucopolysaccharides and aggrecan synthesis.
Interestingly, the liquid formulation of PRF, called i-PRF, was tested not only in vitro to improve chondrocyte proliferation and differentiation (see Section 3.1), but also in vivo, through the injection into rabbit models of osteochondral lesions [61]. Finally, rather than using PRF as a 3D matrix delivering regenerative cells and GFs, Wu and Colleagues [53] tested the addition of PRF releasates (PRFr) to MSC implantation for osteochondral defect repair.
Remarkably, some pre-clinical investigations also reported comparisons between PRF and PRP therapeutic effects on cartilage regeneration, mainly with the aim of verifying whether the fibrin network offered better structural support to cartilage and subchondral bone repair [61,62,71,73].
Overall, pre-clinical investigations revealed a significant improvement of cartilage regeneration after PRF/i-PRF/PRFr treatment, with general evidence of enhanced healing after combining the hemocomponent with autologous cartilage [66,69,75] or MSC administration [53,72,74,76,77]. In particular, ICRS macroscopic evaluation made it possible to attribute significantly higher scores to PRF-treated defects regarding degree of damage repair, integration with the border zone, regularity of repair tissue surface and overall repair grading [61,62,[70][71][72][73][74]. In parallel, higher scores were registered also by a histological grading system, PRF-treated groups presenting normal cell distribution and cartilage mineralization, a higher number of chondrocyte-like cells, hyaline cartilage-like formation and no subchondral abnormalities, as well as good integration with the surrounding cartilaginous tissue [53,61,62,66,67,[69][70][71][72][73][74][75][76]. According to these criteria, PRF proved to be superior to PRP in promoting cartilage healing in most cases [61,62,73]. Regarding chondrogenic marker expression, Aggrecan, SOX9 and type-II collagen resulted in up-regulation in the repair of cartilaginous tissue after PRF treatment [62,77]. Remarkably, staining for type-I and type-II collagen highlighted the fact that PRF better promoted the formation of hyaline-like cartilage rather than fibrocartilage in the defect site [73].
In addition to the orthotopic implant, the viability of cartilage grafts embedded in PRF has been evaluated through subcutaneous implant, from the perspective of applications in rhinoplastic surgery [78,79] (Table 2). Autologous cartilage grafting is a standard procedure in rhinoplasty, diced cartilage representing the most popular option [79]. In the effort to maintain its structure, function, and viability upon implantation, as well as to avoid tissue scattering and subcutaneous irregularities formation, diced cartilage graft is commonly wrapped into acellular cadaveric dermis (e.g., AlloDerm), oxidized regenerated cellulose (e.g., Surgicel) or autologous fascia. Although they are widely used in clinical practice, these materials still present some important drawbacks, such as high resorption rates, high costs and the need for a second incision [78,79]. In this context, PRF has been investigated as a bioactive material which could extend cartilage graft viability during rhinoplastic procedures of nasal dorsum augmentation/correction. To this end, diced rabbit cartilage was wrapped with autologous PRF and implanted into dorsal subcutaneous pockets of rabbit models. In parallel, diced cartilage wrapped with acellular cadaveric dermis, oxidized regenerated cellulose or autologous fascia were implanted for a direct comparison. The in vivo studies highlighted that PRF can ensure better cell viability of the cartilage graft with greater biocompatibility and less inflammation and fibrosis. Thus, PRF may represent an ideal autologous biomaterial for the transfer of diced cartilage grafts during rhinoplastic surgery [78,79].
Despite the satisfying outcome of in vivo animal studies, an important limitation in the current pre-clinical research is represented by the fact that PRF regenerative effect was mainly validated in rabbit models of osteochondral defects. As known, rabbits show high potential for spontaneous cartilage healing that is not seen in larger animal models and humans, losing favor with researchers for in vivo investigations of osteochondral repair [80,81]. Thus, pre-clinical studies on larger and more relevant animals (i.e., sheeps, pigs, dogs, horses) should be deepened to best highlight PRF efficacy and safety in experimental conditions which are as similar as possible to intended human use.

Clinical Trials
Although pre-clinical studies in pathological animal models is important to test the safety and biocompatibility of a scaffold, the final proof of regenerative potential is given by implanting in far more complex human patients. Regarding PRF, its clinical use could be prompted by the fact that it is mainly an autologous product which needs no/minimal manipulation before being implanted into the patient.
To our knowledge, only four studies can be found in the literature about the clinical application of PRF for cartilage repair (Table 3). In 2015, Buda and Collaborators [82] reported a retrospective study about the treatment of hemophilic ankle arthropathy by the use of a collagen matrix seeded with bone marrow-derived cells (BMDCs) and added to autologous PRF. In hemophilic patients suffering from joint cartilage degeneration due to hemarthrosis, regenerative approaches, such as bone marrow-derived cell transplantation (BMDCT) and Matrix Autologous Chondrocyte Implantation (MACI) have poorly been considered [82,83]. The aforementioned study described the arthroscopic implantation in the ankle joint of a collagen membrane loaded with BMDCs and PRF. After a mean follow-up of 2 years, patients were evaluated by means of American Orthopaedic Foot and Ankle Society (AOFAS) scores, radiographs, magnetic resonance imaging (MRI) and Mocart scores, detecting signs of osteochondral regeneration and no progression of joint degeneration. Three of the five treated patients were also able to return back to sporting activities, leading to the conclusion that BMDCT coupled to PRF could represent a valid strategy for promoting cartilage restoration in mild ankle hemophilic arthropathy [82]. Two subsequent clinical trials aimed at testing PRF capacity to regenerate knee chondral defects. In both cases, a novel approach to cartilage repair was investigated by performing microfractures in combination with platelet concentrate administration. In particular, Papalia and Colleagues [84] described a retrospective clinical study on 48 patients treated by three methods: (i) microfractures and intra-operative administration of PRF; (ii) microfractures and post-operative injections of PRP and (iii) microfractures alone. After 2 and 5 year-follow ups, clinical scores [International Knee Documentation Committee (IKDC) functional scores, Visual Analogue Scale (VAS) pain], as well as MRI and Mocart scores revealed that the addition of platelet concentrates significantly improved microfractures outcome, with PRF ensuring better and earlier results than PRP [84]. Similarly, the work by D'Antimo and Collaborators [85] retrospectively investigated the regenerative effect of microfracture technique and the concomitant application of an autologous leucocyte-and platelet-rich fibrin membrane (CLP-MB). This hemocomponent is produced through blood collection by multiple apheresis cycles to obtain a leucocyte-platelet concentrate which is added to a plasma cryoprecipitate [9,10]. The final membrane is highly enriched with platelets, leukocytes, monocytes/macrophages, fibrinogen, and CD34 + cells. Our research group performed a detailed in vitro characterization of the CLP-MB membrane, highlighting its regenerative properties in terms of GF release, mechanical behavior and biodegradation profile [12]. The CLP-MB exhibits good elasticity and high deformation capacity, demonstrating itself to be suitable for implantation in the Knee joint for articular cartilage repair. Moreover, the membrane has been shown to sustain over time the release of GFs and cytokines (i.e., PDGF-BB, VEGF and IL-10) responsible for cell proliferation/differentiation, angiogenesis stimulation, as well as regulation of inflammatory condition. In vitro biodegradation studies showed good preservation of the fibrin network up to 21 days, with a progressive loss in cellular elements (Figure 2).   The subcutaneous implantation of CLP-MB into athymic rats demonstrated that it progressively biodegraded and was replaced by connective tissue, suggesting that it could work as a biomimetic scaffold which degrades with time to be substituted by neo-regenerated tissue (Figure 3) [12]. The subcutaneous implantation of CLP-MB into athymic rats demonstrated that it progressively biodegraded and was replaced by connective tissue, suggesting that it could work as a biomimetic scaffold which degrades with time to be substituted by neo-regenerated tissue (Figure 3) [12]. Regarding the clinical trial, 25 patients with focal chondral lesions underwent a single-step Autologous Matrix-Induced Chondrogenesis (AMIC) treatment. Briefly, after performing Steadman microfracture procedure [58], the CLP-MB membrane was placed upon the defect site and then covered by a homogenous layer of collagen-based injectable gel (Cartifill) [85]. During arthroscopic surgery, the CLP-MB membrane appeared to be easy to handle, adapting well to the cartilage lesion. After 1-, 6-and 12-month follow-ups, IKDC and VAS scores were considered to evaluate the short-term safety and efficacy of the therapy. Overall, clinical data showed that one-stage AMIC based on the implantation of CLP-MB with Cartifill significantly reduces pain perception and provides functional improvement. Of utmost importance, the preparation of CLP-MB for implantation was subject to quality control tests during all the production steps, assuring that a standardized, traceable and safe product was obtained [85].
PRF efficacy was clinically assessed not only in arthroscopic knee surgery, but also in rhinoplastic procedures for dorsal nasal augmentation purposes. Notably, Kovacevic and co-workers [86] experimented with, for the first time, a novel technique for cartilage graft stabilization by the use of autologous platelet concentrates. This retrospective clinical study presents the data collected from 48 patients subjected to rhinoplasty and implanted with heterogeneous grafts made of (a) cartilage scales, (b) a cartilage pâté, and (c) an autologous platelet concentrate to stabilize the construct. Interestingly, different platelet-rich products were used: liquid Plasma-Rich Growth Factors (PRGF) and PRF prepared according to two distinct protocols to obtain a liquid Regarding the clinical trial, 25 patients with focal chondral lesions underwent a single-step Autologous Matrix-Induced Chondrogenesis (AMIC) treatment. Briefly, after performing Steadman microfracture procedure [58], the CLP-MB membrane was placed upon the defect site and then covered by a homogenous layer of collagen-based injectable gel (Cartifill) [85]. During arthroscopic surgery, the CLP-MB membrane appeared to be easy to handle, adapting well to the cartilage lesion. After 1-, 6and 12-month follow-ups, IKDC and VAS scores were considered to evaluate the short-term safety and efficacy of the therapy. Overall, clinical data showed that one-stage AMIC based on the implantation of CLP-MB with Cartifill significantly reduces pain perception and provides functional improvement. Of utmost importance, the preparation of CLP-MB for implantation was subject to quality control tests during all the production steps, assuring that a standardized, traceable and safe product was obtained [85].
PRF efficacy was clinically assessed not only in arthroscopic knee surgery, but also in rhinoplastic procedures for dorsal nasal augmentation purposes. Notably, Kovacevic and co-workers [86] experimented with, for the first time, a novel technique for cartilage graft stabilization by the use of autologous platelet concentrates. This retrospective clinical study presents the data collected from 48 patients subjected to rhinoplasty and implanted with heterogeneous grafts made of (a) cartilage scales, (b) a cartilage pâté, and (c) an autologous platelet concentrate to stabilize the construct. Interestingly, different platelet-rich products were used: liquid Plasma-Rich Growth Factors (PRGF) and PRF prepared according to two distinct protocols to obtain a liquid injectable PRF (i-PRF), or a so called advanced-PRF (a-PRF), prepared as a tear-resistant and flexible membrane. For PRGF, a few drops of the hemocomponent were released on the top of the cartilage scales-cartilage pâté compound graft, which was then incubated at 37 • C to improve fibrin polymerization. Regarding i-PRF, the platelet concentrate was sprayed on the top of the compound graft and completed coagulation in a few minutes, obtaining cartilage scales embedded in the fibrin matrix. The application of a-PRF was chosen when the graft was based only on cartilage pâté, which was placed on a layer of a-PRF and stabilized with i-PRF. This kind of graft was especially used for corrections of minimal dorsal irregularities or during tip surgery. Clinical data demonstrated satisfactory dorsal nasal augmentation in 47 cases, with only one patient showing graft displacement 3 months after surgery; however, no further displacement was registered. The implantation of a cartilage graft combined with platelet concentrates did not lead to dorsal irregularities, nor signs of resorption, erythema or inflammation, demonstrating itself to be a promising alternative to current surgical strategies for dorsal nasal augmentation [86].

PRF-GFs in Tenogenesis
To date, several strategies have been developed to ameliorate clinical outcomes in cases of acute or chronic injuries of the tendons; however, as yet, none is used routinely to treat patients, proving that tendon injuries are a significant clinical problem and that they remain a challenge in clinical practice [87]. In fact, tendons have the ability to heal naturally after injury, but with significant fibrosis that compromises mechanical outline and alters their in vivo performance, also making them more susceptible to further damage [88].
Even if tendons consist primarily of water and type-I collagen, with smaller amounts of other collagens and matrix materials and various types of cells, mainly fibroblasts [26], effective treatments are lacking, possibly due to a low level of comprehension of their biology with respect to that of other components of the musculoskeletal system. Hence, to understand the molecular basis of tendon regeneration, considering growth factors involved in tendon wound healing and adhesion may serve as a guide for customizing effective treatments based on the latest Regenerative Medicine methods [89].
As for other tissues, tendon injury is characterized by the onset of inflammation, cell proliferation, reparation by collagen deposition and ECM production up to remodeling [26]; these events are stimulated by the release of GFs and cytokines including interleukins, tumor necrosis factor (TNF), connective tissue growth factor (CTGF), VEGF, PDGF, bFGF, TGF-β1, EGF and IGF-1 [26,87,90]. Different roles have been recognized for GFs; they affect their own activity as well as the expression of other molecules. Considering their function, GFs support/control different stages of healing. IGF-1, PDGF and bFGF are essential during the early and intermediate stages of tissue regeneration as they aid fibroblasts in migration, proliferation and in synthetizing ECM. Regarding TGF-β1 and VEGF, they have some role in these processes too, but they are mainly involved in angiogenesis of the injured area where they regulate tissue remodeling [26]. However, embryological experiments and genetic analyses proved that TGF-β1 and bFGF are the main GFs with a role in tendon development [91,92] GFs powerfully regulate cell biological responses; hence, their exogenous addition can further stimulate tissue recovery and the differentiation of stem cells into the tenogenic lineage [93]. Ectopic administration of a single GF has been considered for tendon healing but administering a pool of bioactive molecules may boost regeneration. According to this assumption, hemocomponents have been investigated.
The eminent role of IGF-1 in tenogenesis has been supported by much evidence. It increases collagen expression in adult human tenocytes in vitro and also collagen content and fibril diameter in tendon constructs [94]. However, its temporal expression in cases of injury has been debated. In a study by Dahlgren et al. [95], IGF-1 levels decreased of about 40% in the first 2 weeks in a horse model of lesioned flexor tendon compared to normal tendon; and they increased to exceed those of normal tendon at week 4. Conversely, other authors claimed to have identified IGF-1 during all phases of healing, even if it was mostly present in the early period of tendon development, highlighting its importance in the preliminary stages of regeneration. Yet, the idea that the exogenous administration of IGF-1 after injury may exert therapeutic advantages, i.e., by enhancing the metabolic response of tendon fibroblasts, is commonly accepted. It appears to aid the migration and proliferation of fibroblasts, also stimulating gene expression of collagen, and protein and extracellular matrix synthesis both in vivo [94] and in vitro [96]. This evidence confirms previous data by Murphy and Nixon [97], also observing in an in vitro study on equine tendon that IGF-1 acts in a dose-dependent manner.
At the time of tendon injury, PDGF peaks rapidly, being released from degranulating platelets; Therefore, it is thought to play a significant role in the early stages of healing [98]. It has many functions, including fibroblast mitogenesis, angiogenesis and activation of macrophages. Interestingly, it also promotes the production of other GFs including TGF-β1, IGF-1 and VEGF, confirming its leading role in healing processes [96].
According to many in vitro and in vivo studies, bFGF is involved in regulating cell proliferation and migration in addition to stimulating angiogenesis and collagen synthesis [26,96]. It is produced by normal tendons fibroblasts; moreover, its expression increases at the injured sites in various animal models of tendons injury [91].
TGF-β1 is produced by most cells involved in the healing process [26], and it is similarly active throughout the injury recovery period; in particular, TGF-β1 increases fibroblast stimulation, cell proliferation and collagen production, but it also recruits macrophages [96].
VEGF is only relevant toward the end of the inflammatory process [96], as confirmed by Petersen et al. [99], showing that its levels are neglectable in normal human Achilles tendons and increase in cases of rupturing. It is released by a variety of cells including platelets and tenocytes, thereby exerting a prominent role in wound healing as it initiates angiogenesis [90]. Conversely, with experimental tendon healing, it seems to have a rather deleterious effect, perhaps because of its angiogenic activity and by stimulation of matrix metalloproteinases [100].

In Vitro Studies
Surprisingly, in vitro studies describing the use of PRF are rare in comparison to pre-clinical or clinical trials (Table 4). However, solid in vitro evidence could be useful to understand the mechanisms underlying successful or unsuccessful therapies with PRF which, to date, have not found consensus among clinicians.   To succeed in the reconstruction of injured ligament, controlled administration is required of chemotactic, mitogenic, and angiogenic factors that will promote cell metabolism, the formation of new blood vessels, and tissue remodeling [95]. To our knowledge, only Anitua and colleagues [101] have combined a preliminary in vitro to an in vivo study evaluating the effectiveness of autologous fibrin matrices. In particular, the authors compared the behavior of freshly isolated human tendon cells on homologous fibrin, platelet-rich (PR-) and platelet-poor (PP-) matrices. The platelets entrapped in the fibrin matrices boosted tendon cells proliferation, also increasing the synthesis of structural ECM-protein collagen-I, with no difference between PR-and PP-matrices. This result was unexpected, considering that TGF-β1 levels were higher in PR-matrix samples (i.e., with or without tenocytes) than PP-matrices. Probably, other platelet-released molecules, including hepatocyte growth factor (HGF) (i.e., a mitogen for endothelial cells [120]), may hide the effect of TGF-β1. Additionally, cultured tenocytes also synthetized VEGF and HGF; VEGF, but not HGF, was significantly higher in the presence of platelets. The eminent and debated role of TGF-β1, which stimulates VEGF secretion by tendon cells but also drives fibrogenesis up to scar tissue deposition, was later considered also by Visser et al. [102]. Three different hemocomponents, namely PRF matrix, PRF membrane and whole blood clot, were analyzed. Both PRF-derived constructs, characterized by dense fibrin scaffold, allowed for increased concentrations of eluted TGF-β1 and tendon cells proliferation. Evidence about the deep relationship between the physical form of the hemocomponent and GFs release was also highlighted by other Authors. In fact, Zumstein et al. [111] proved that matrices based on leukocyte-and platelet-rich fibrin (L-PRF) can guarantee higher levels of eluted GFs than normal blood and thus compared their release from a standard/gelatinous matrix versus a dry/compressed matrix. L-PRF clots showed a continuous slow release with an increase in the absolute release of growth factors TGF-β1, VEGF and myeloperoxidase-content (MPO) in the first 7 days in vitro; for IGF-1, PDGF-AB and platelet activity factor (CXCL4) it boosted in the first 8 h reaching zero at 28 days. Moreover, CXCL4, IGF-1, PDGF-AB, and VEGF were released in high levels from the standard/gelatinous matrix compared to the dry/compressed matrix; this represents behavior not encountered for MPO and the TGF-β1.
In addition to its role as a GFs delivery system, PRF may be also considered as a scaffold for cells adhesion and proliferation. In particular, considering the potential of MSCs in tissue regeneration, Beitzel et al. [103] evaluated their response to five different scaffolds: fresh-frozen human rotator cuff tendon (i.e., allograft); human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA); porcine non-cross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland); human platelet-rich fibrin matrix and fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Even if MSC differentiation successfully occurred, adhesion was significantly greater to both the non-cross-linked porcine collagen scaffold and platelet-rich fibrin matrix. Considering proliferation, the non-cross-linked porcine collagen scaffold was much more effective compared with PRF-M and fibrin matrix based on platelet-rich plasma. Thus, stiffness of the matrix significantly affects cell behavior, suggesting the importance of preliminary studies dedicated to the development of the manufacture protocol. The role of the matrix is not only that of a mere surface; its intrinsic characteristics modulate cell behavior, and its mechanical properties have an additional, instructive role that, in the case of PRF, can also descend by its ability to entrap/release platelets and/or growth factors.

Pre-Clinical Studies
The literature reports on pre-clinical studies aiming to develop new approaches PRF-based towards, Achilles tendon rupture [101,114], regeneration of the tendon-bone insertion [108], healing of flexor tendon [115,116] and patellar tendon [117], also including medial collateral ligament reconstruction [118].
Different animal models of injuries were considered; the first experimental works were carried out on species with a complex neurological development, including sheep [101,114] and dogs [117]. Later, ethic issues, a growing awareness of the value of responsible animal research, together with mature surgical experience with the use of hemocomponents for regenerative purposes, have prompted researchers to favor species with simpler neurological developments, like rabbits [115,116,118] and rats [108].
The authors described the manufacture of different PRF-derived products according to the content in platelets and/or structure, but a shared preparation-protocol, as well as a strong characterization study to allow for comparisons, is lacking. Visser et al. [117], Hasan et al. [108], Liao et al. [116] studied PRF; Anitua et al. [101] considered both platelet-rich and platelet-poor matrices; others included plasma in the fibrin matrix (i.e., platelet-rich plasma fibrin matrix-PRPFM) [114,115] and then compared its healing effect versus PRP [115], while Matsunaga et al. [118] described a novel protocol to prepare a Compact Platelet-Rich Fibrin Scaffold (CPFS) resembling a sheet. Concerning surgical practice, hemocomponents were used either alone as a booster of tissue healing after surgery [101,108,[115][116][117][118], or combined to an acellular porcine dermal (APD) patch [114].
Interestingly, regarding the origin of hemocomponents, both autologous [101,114,116,117] and allogenic preparations [108,115,118] were investigated. Assessing the effectiveness of allogenic products verifies the efficacy of these therapeutic approaches, even in patients for whom there are no clinical indications for autologous preparations (e.g., thrombocytopenic patients). Thus, an optimal characterization study may identify the quality parameters needed to assure the clinical efficacy of such products. As reported in Table 5, many different aspects were considered to evaluate the role of hemocomponents in tissue regeneration. In particular, newly-formed tissue was assessed for cell density and tissue morphology, vascularization, absence of inflammation, matrix deposition, adequate integration of the scaffold without adhesions, thickness of the repaired tissue, and the mechanical parameters.
Both Anitua et al. [101] and Sato et al. [115] proved the effectiveness of fibrin matrices to enhance healing and the remodeling of tendons without side effects (i.e., edema or adhesions), to reduce risk of repeated rupture after surgery and to help in early rehabilitation. A positive outcome in using fibrin products was later suggested also by Matsunaga et al., [118] showing that CPFS accelerates healing of tendons and ligaments, acting as a provisional support for graft augmentation. Conversely, to gain regeneration in an injured Achilles tendon model, no decisive role of fibrin matrix was highlighted by Sarrafian et al. [114]. In fact, the authors, who mainly focused on a porcine dermal patch, observed that the addition of PRPFM to APD did not exert a substantial role in regeneration. A negative outcome after using fibrin products was also found. Visser et al. [117] claimed that PRF membrane did not enhance the rate or quality of tendon healing in cases of defects, also increasing the amount of repair tissue within and surrounding the defect. Similarly, the study results of Hasan et al. [108] showed that PRFM did not make it possible to recapitulate the native enthesis, but rather, induced a disorganized healing response characterized by fibrovascular scar tissue. Also, Liao et al., [116] reported undesirable effects on the biomechanical properties of repaired tendon, suggesting that sometimes PRF may hinder, rather than enhance, healing.
Besides the different pre-clinical study outcomes, which are likely due to different preparation protocols, the lack of data on functional recovery of the operated limb is also evident. The authors mainly focused on the quality of the regenerated tissue from both a histological and biomechanical perspective; however, assessments on mobility could provide interesting and useful data for a complete evaluation of the efficacy of fibrin-based products too.

Clinical Studies
The scientific rationale behind the use of PRF-based products is related to the intrinsic nature of the entrapped platelets, acting as a reservoir of many GFs; they accelerate the healing process, controlling pain and inflammation [21]. In addition, PRFs are easy to obtain, and so are promptly available for clinical use. Despite this, controversies in the literature regarding the benefits and the clinical outcomes cannot be neglected (Table 6).
Considering tendons, PRF has been mainly adopted to treat rotator cuff injuries [104][105][106][107]109,110,112]. Recently, in 2017, Alviti et al. [113] considered their use for acute ruptures of the Achilles tendon, while, Saltzman et al. [119] addressed the repair of the gluteus medius tendon. In all these clinical trials, PRF was used as an additional treatment to standard suture bridging (i.e., single or double row), and clinical outcomes were compared to those of routine-surgery approaches or, in a study by Alviti et al. [113], to the characteristics (i.e., gait analysis) of healthy subjects, suggesting the need for a strategy assuring a functional recovery which superimposable to that of normal tendon. In fact, demographic data about patients enrolled for surgery revealed the involvement of a working-age population, with an average age of about 53 and a lower range of 30.
As for both in vitro and pre-clinical studies, the results obtained from tendon surgery with PRF-based products were different. Most authors asserted that the use of PRF-based products does not play a key role in tissue healing, pain or re-tear rate, and also, that functional outcome scores were not improved [105,107,110,112,119]. However, as suggested by Castricini et al. [107], this may depend on tear size and the heterogeneity of available PRF preparation products. Only two studies expressed a negative opinion on PRF products according to regression analysis and re-tear rate data [106,109]. Conversely, Alviti et al. [113], through a gait analysis study, demonstrated that the approach by suture and PRF augmentation in acute ruptures of the Achilles tendon is responsible for improvements without differences with respect to the healthy-control group.
Operative time was considered only by two groups [106,110], and discrepancies were pointed out; however, this parameter is deeply related to both the surgeon's ability and experience, as well as to the specific clinical-case, so it may only act as a general indicator. Finally, infections were a sporadic event; only Bergeson et al., [106] described their occurrence in 2 of 16 patients implanted with a platelet-rich fibrin matrix.
Considering the overall results, the major limitation of these studies is attributable to the low number of individuals included therein; thus, a factual and critical analysis of PRF effectiveness requires not only more careful attention to the manufacture protocol, but also the possibility to involve more candidates for surgery to assure the feasibility of an accurate statistical analysis.

Conclusions
To date, the literature demonstrates the interest of scientific community for the safe therapeutic approach represented by platelet-rich hemocomponents, which can be prepared as inexpensive autologous products with high regenerative potential. In particular, PRF is considered as a second-generation platelet concentrate which may offer many advantages over first-generation PRP, by virtue of its three-dimensional fibrin matrix, ensuring favorable mechanical properties and the prolonged release of GFs, cytokines and regenerative cells. This defines PRF as a true biomaterial delivering the key players of the healing process, with improved potential for application in a wider range of clinical fields. Nevertheless, one of the main issues to deal with in the clinical translation of PRF is the great variability of preparation protocols and equipment [121,122], which impedes the obtainment of a unique and standardized product. In addition, PRF biomaterials have an intrinsic variability depending on a multitude of factors. Autologous hemocomponents prepared with the same system but at different times may yield different results in bioactive factors content [122]. The peculiar characteristics of the patient/donor (i.e., life style, contingent or chronic disease including drugs treatments) affect the final product properties and responses to the treatment. For example, data about PRF from patients suffering from coagulation disorders treated with proper medicines (i.e., heparin, warfarin or platelet inhibitors) are scant [123]. Moreover, as recently assessed by Xiong et al. [124], age and sex may also be considered as variables [125,126]. With this in mind, better standardization of manufacturing procedures allowing for consistent outcomes to be predicted is urgently needed.
Regarding the therapeutic effects of PRF, the synergistic action of platelet GFs is recognized to be the key element for enhancing and regulating tissue regeneration. Thus, a detailed characterization of PRF releasates and their mechanism of action (i.e., cell signaling pathways) seems to be recommended to better understand their potential for clinical application. As an important gap in knowledge, the literature is lacking specific biomolecular characterization of PRF products, with a few studies trying to describe their content, but which are only focused on platelets counts and main GFs quantifications [12]. PRF appears to be very difficult to characterize, as it is a complex blood-derived preparation that concentrates hundreds of regenerative elements hard to be singularly investigated [127]. Also considering the heterogeneity of PRF products, developing a method to specifically identify their composition in terms of cell content, type and concentration of the released GFs/cytokines, as well as fibrin matrix fibers, would help to predict the therapeutic outcomes of hemocomponent clinical implantation.
Despite the aforementioned concerns which need to be addressed, the therapeutic efficacy of PRF has recently gained attention, also in the field of cartilage and tendon TE, which is still seeking ideal bioactive materials promoting adequate functional tissue recovery. Although the stages of healing are similar for various tissues, for cartilage and tendons, the regeneration process naturally occurs at a slower rate than that of other connective tissues, probably because of their dense and hypocellular composition. As it gathers all molecules that are physiologically involved in the healing response, autologous PRF is potentially useful to recover all tissue integrity after injury, and it would be of prime importance for tissues like cartilage and tendons, showing an intrinsic slow or often inadequate regeneration ability resulting in fibrosis.
The reviewed studies reporting PRF-mediated chondrogenesis or cartilage healing demonstrate a large consensus on the beneficial effects exerted by the hemocomponent on cartilaginous tissue regeneration. We found general agreement on the preparation protocol, referring to Choukroun and Collaborators [3] in most of the reported studies. The characterization of PRF composition and biomechanics, as well as in vitro assessment of its chondrogenic potential, appeared to be neglected; nevertheless, the therapeutic outcome of hemocomponent implantation for the treatment of full-thickness osteochondral defects has been proven to be generally successful by pre-clinical research and clinical trials.
Conversely, studies regarding the use of PRF for tendon repair are often conflicting, showing different results in animal models and clinical trials. As previously stressed, a possible explanation for this variability could be the lack of a standardized preparation protocol.
However, apart from result variability, which could be district-dependent, the use of PRF in cartilage and tendon regenerative medicine is paving the way for new therapeutic strategies which may overcome the limits of the current surgical approaches. Although further in vitro and in vivo investigations are required to fully estimate the potential benefit of this new regenerative technology, the autologous origin and graft versatility of PRF, as well as its high content in regenerative elements, seem to be strongly encouraging and worthy of further, in-depth exploration.
As for future perspectives, a noteworthy application of PRF and its released GFs could be represented by the functionalization of biosynthetic scaffolds to improve their capacity to support cell adhesion and proliferation, as well as to promote functional cartilage and tendon regeneration. Also, the manufacture of composite scaffolds mixing or combining polymers and the PRF as different layers may be an intriguing and vanguard manner of working with this hemocomponent.