Introduction
Problems associated with non-communicable diseases have exacerbated partly because of a decline in physical activities and energy expenditure. Health-enhancing activities, including sports and exercise, are hindered because current treatments for chronic musculoskeletal conditions often remain unsatisfactory and fail to meet clinical demands. Moreover, demands for effective treatments to accelerate healing in sports injuries have been increasing.
Platelet-rich plasma (PRP) therapies have emerged as a potential approach to enhance tissue repair and regeneration, and their clinical applications have extended across specialties, mostly in oral dentistry, dermatology, ophthalmology, orthopedics, and sports medicine [
1]. PRP application aims to provide supraphysiological concentrations of platelets, including leukocytes in some formulations, to injured/pathological tissues mimicking the initial stages of healing. However, the efficacy of PRP is controversial in some clinical applications because patients’ outcomes exhibit partial improvements [
2–
7]. Despite the lack of safety concerns, routine use of PRP is not recommended in daily practice by public bodies [
8] and is not paid for by insurance companies.
Although relevant treatments have slightly improved, PRP-related approaches should be further developed on the basis of refining formulations and application protocols, such as infiltrating the subchondral bone with leuko-depleted PRP to reduce bone edema in degenerative joint conditions [
9]. New developments can be achieved by expanding customized treatment modalities formulated
à la carte and designed for particular tissues or disease severity stages. A simplified method of customizing PRP for a specific application involves the selection of clinically available formulations, such as leukocyte-rich PRP (L-PRP), pure PRP, and platelet-poor plasma (PPP); platelet and leukocyte dosage, particularly concentration and volume; timing of application, such as disease severity stage; and number of doses. However, these parameters of PRP treatment have yet to be optimized.
PRP therapies can be further enhanced by creating combinatory treatments through identification of specific conditions to associate PRP with selected molecular inhibitors or enhancers for each clinical application. For example, TGF-β neutralization may be desirable in muscle injury because of its involvement in muscle fibrosis [
10,
11]. Nevertheless, the same molecule is beneficial for joint conditions [
12] or vertebral disc regeneration [
13] because TGF-β can stimulate chondrogenic differentiation of precursor cells and synthesis of collagen 2.
In this review, basic knowledge of PRPs were briefly described to enhance our understanding of the potential benefits of creating customized therapies by choosing optimal formulation or by adding drugs or antibodies, cells, and extracellular matrix proteins to PRP. Conservative PRP combinational treatments in the conservative management of soft tissues in orthopedics and sports medicine were also discussed.
Contribution of PRP to healing mechanisms
PRP application can provide benefits because of the simultaneous release of multiple growth factors (GFs), interleukins (IL), and other cytokines involved in basic biological processes essential for tissue healing. PRP is safe and easily obtained. However, researchers experience difficulty in identifying the main factors and pathways that correlate with clinical outcomes in the different stages of tissue healing or in particular chronic diseases because of the high number of signaling pathways that can be activated by PRP secretome (Fig. 1).
Platelets are involved in hemostasis and tissue-healing mechanisms, which are integrated by different processes, including innate immune response, angiogenesis, and tissue remodeling. These processes are based on basic mechanisms, including cell chemotaxis and migration, proliferation, and cell anabolism. In physiology, these mechanisms can regenerate an injured tissue. However, these mechanisms are also involved in diseases. As such, the same molecules can be injurious or have no effects in incorrect contexts.
Inflammation
In some clinical uses, PRP aims to trigger early inflammatory mechanisms mainly directed by platelet secretome. In physiology, platelets initiate an innate immune response by controlling leukocyte traffic through a highly coordinated complex network of molecular signals and cell interactions.
Neutrophils are recruited shortly after platelets are activated and can be attributed to the release of high concentrations of neutrophil-activated peptide-2 (NAP-2) at a micromolar level from α-granules, but proteolytic processing by neutrophils is necessary to produce chemotactically active NAP-2 [
14]. Therefore, neutrophil infiltration is favored by the presence of leukocytes in PRP formulation. Monocytes arrive in a second wave of cell infiltration in response to monocyte chemoattractant protein-1 (MCP-1) and T-cell-specific RANTES protein (CCL5) gradients.
In vivo research has demonstrated that early expression of chemokines, including MCP1 and RANTES, in a healing tendon precedes ingrowth of new nerves, angiogenesis, and emergence of inflammatory cells [
15]. MCP-1 and RANTES contribute to the transendothelial migration of monocytes and T cells through CCR2, CCR1, and CCR5.
Although these processes naturally occur in response to tissue damage, PRP can modify intensity and duration. However, we have yet to verify whether the effects of PRP are transient and whether they may be insufficient to regulate early healing. In this aspect, research data have shown that this network of inflammatory molecules is further amplified by local cells in response to PRP stimulus [
16]. PRP induces the further synthesis of IL-6, IL-8, MCP-1, and RANTES by local cells providing prolonged chemotactic stimulus and additional leukocyte recruitment.
Recruited monocytes/macrophages can polarize into different phenotypes within the M1–M2 spectrum. PRPs can control the switch between pro-inflammatory states toward healing processes through differential molecular secretion. In this regard, PF4, which is the most abundant signaling protein in the whole platelet secretome, skews macrophage polarity toward an intermediary M1–M2 phenotype [
17]. Platelet microparticles generated upon activation with agonists, such as thrombin and Ca
2+, polarize macrophages into a resident M2 phenotype [
18,
19]. Nevertheless, data obtained in tendon research do not provide evidence showing any macrophage activation induced by L-PRP [
20]. In muscle research involving animal models, PRP and controls exhibit similar macrophage infiltration [
11]. Blocking TGF-β1 within PRP enhances macrophage infiltration and increases the number of M2-polarized macrophages.
Some of these apparently contradictory data can be attributed to tissue context and platelet preparation. For example, strong platelet agonists, such as thrombin and calcium, induce rapid α-granule secretion facilitating the formation of microparticles and promote the bioavailability of α-granule secretome, such as NAP-2 or PF4, in leukocytes. By contrast, the lack of activation or slow activation by adenosine di-phosphate or collagen modifies the kinetics of α-granule release and thus alters cytokine bioavailability for macrophage polarization [
21]. Further studies have been conducted on novel systems to control PRP activation and cytokine delivery by using electric pulse fields [
22]. Controlling and understanding the kinetics of cytokine release from PRP may help elucidate adaptations in the biological characteristics of leukocytes for regenerative purposes.
The presence of leukocytes in formulations and particular conditions of host tissues can influence the exacerbating or downregulating effects of PRP on inflammation. Recent data derived from gene expression arrays [
20] performed in the context of tendon research have shown that the main pathway triggered by L-PRP is inflammation, and this process is stimulated by TNF-α and NF-κB activation and oxidative stress enhancement, that is, increased SOD2, NFE2L2, and Prdx1 expression.
However, these results are inconsistent with those of other studies, which have revealed that hepatocyte growth factor (HGF) in the molecular context of PRP reduces canonical NF-κB signaling and enhances IκBα expression in chondrocytes
in vitro [
23]. PRP reduces PGE
2 production in Achilles tendon injuries in a rat model while anti-HGF addition counteracts the anti-inflammatory effects of PRP [
24]. Overall, these data indicate that HGF mediates the anti-inflammatory effects of PRP on injured tendons. HGF also decreases the production of IL-6 and enhances IL-10 in macrophages stimulated by lipopolysaccharide (LPS) [
25].
Current data reveal that L-PRP is more pro-inflammatory than PRP (Table 1), but specific conditions of host tissues can either increase or decrease inflammatory responses and thus compromise data interpretation. PRP formulations may need further refinement based on new clinical and molecular information and our knowledge of chronic diseases because the final effect is a result of the interaction of PRP and host tissues.
Molecular inflammation in osteoarthritis (OA) or tendinopathy is often attributed to the presence of IL-1β in the tissue/organ milieu. Mimicking this situation, PRP downregulated the production of inflammatory proteins, including IL-6, IL-8, and MCP-1, when pure PRP is applied to human tendon cells stimulated by IL-1β [
16]. Similar results have been obtained in other cell phenotypes. For example, microarray analysis has demonstrated an increased expression of matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13), interleukin (IL)-6, IL-8 (CXCL8), CCL5, CCL20, and CXCL10 chemokines when meniscal cells are inflamed with fibronectin fragment (FN-f). Upregulation of these genes is significantly attenuated by PRP administration. Downregulation of inflammation is induced by pure PRP and is associated with Akt and p42/44 activation [
26].
PRP paradoxically exacerbates IL-1α-mediated inflammation and fibrosis in meniscus repair in an animal model [
27]. The IL-1β-mediated production of MMP-1, -3, and-13 is unaffected by PRP treatment in chondrocytes or synoviocytes [
28,
29]. This particular PRP with a high concentration of platelets is prepared by double spinning, but its leukocyte content is unknown.
Inflammation precedes angiogenesis. Thus, the magnitude of PRP-induced inflammation will condition the subsequent angiogenic process. Inflammatory adaptations induced by PRP should be managed with advanced research and updated data.
Angiogenesis
Multiple angiogenic modulators, activators, and inhibitors accomplish coordination of physiological angiogenesis. Platelets mediate angiogenesis through the secretion of several opposite factors, such as pro- and anti-angiogenic molecules. Vascular endothelial growth factor (VEGF) represents the major rate-limiting factor during the angiogenic process. It binds to VEGFR1 (Flt-1), VEGFR2 (FLK-1/KDR), and VEGFR3 (Flt-4) mainly located in endothelial cells. VEGF activities in certain PRP preparations can be suppressed by the presence of the soluble receptor sFlt, which is present in PRPs but not in buffy coat [
30], and this observation suggests that sFlt-1 is derived from plasma.
Among other relevant pro- and anti-angiogenic molecules in PRP are bFGF, HGF, PDGF, angiopoietins and thrombospondin, and PF-4 [
31]. The influx of inflammatory cells further enhances the pool of pro-angiogenic factors by releasing VEGF, angiopoietins, FGF, HGF, and PDGF. However, we have yet to determine whether PRP application triggering angiogenesis can depend on the composition of PRP and the kinetics of cytokine release.
PRP supernatant, which comprises soluble molecules, is more efficient in promoting angiogenesis than “complete” PRP, which is PRP with fibrinogen/fibrin and other insoluble proteins, probably because of potential synergic actions between cytokines and exosomes [
32,
33]. Platelet releasates activate the angiogenic factor receptor AngTie-2 or angiopoietin endothelial cell-specific tyrosine protein kinase receptor in endothelial cells and enhance endothelial cell sprouting and lung epithelial cell budding
in vitro[
34]. Soluble Tie2 receptor, Wnt co-receptor, or low-density lipoprotein receptor-related protein 5 (LRP5) knockdown attenuate the effects of PRP extract after mice are subjected to unilateral pneumonectomy [
35].
Platelet releasates create an angiogenic milieu, which has been used as a priming agent to mobilize peripheral blood stem cells. As a result, cells are polarized toward pro-angiogenic monocytes triggering vessel formation and tissue repair in the ischemic hindlimbs of athymic mice [
36].
Co-localization of proteases with angiogenic factors can regulate their activities. For example, MMP-9 strongly upregulates IL-8 (CXCL8) activity, while MMP-1, -3, and-13 regulate SDF-1α (CXCL12) activity [
37].
Activated leukocytes secrete several kinds of proteases, including MMPs, which expand the concentration of MMPs released from PRPs [
38]. L-PRP and pure PRP contain MMP-2, -3, and-9, whose concentrations are measured in nanograms per milliliter, and MMP activity is dependent on platelet count in a PRP preparation [
39]. MMPs directly participate in angiogenesis. For example, MMP-9 and MMP-2 mainly stimulate endothelial cell survival and proliferation via wnt/β-catenin signaling and migration of vascular cells through basement membrane degradation. Proteases also activate growth and PRP-released chemotactic factors, including CXCL12 (SDF-1α), TGF-β1, and HGF, which favor their organization in tubular structures and sprouting [
40]. The angiogenic effects of PRP may be enhanced by induced ischemia during free-lap harvesting [
41].
In hyaline cartilage, angiogenesis is deleterious. Cartilage degeneration is associated with the presence of VEGF receptors [
42], and the invasion of blood vessels from subchondral bone induces VEGF-mediated cartilage ossification. However, the effects of PRP under hypoxic conditions have yet to be fully investigated. PRP possibly possess anti-angiogenic properties based on the actions of PF-4, TSP-1, or endostatin. TSP1 and endostatin inhibit endothelial cell proliferation by preventing VEGF signaling, while PF-4 inhibits tube formation [
12]. Hence, PRP can induce the downregulation of angiogenesis depending on host context and on prevention of inflammatory cell-induced increase in the pool of pro-angiogenic factors.
Complementary studies on receptor antagonists or specific neutralizing antibodies should be performed to determine individual contributions and define the role of individual PRP cytokines under specific tissue conditions. With this information, new pharmacological approaches may be developed to improve innate immune and angiogenic mechanisms essential for tissue repair.
Tissue anabolism/catabolism
PRPs contain anabolic factors, such as IGF-1 and TGF-b, catabolic molecules, such as MMPs, and molecular regulators of catabolic molecules, such as TIMPs. Binding/latent proteins regulate the biological activities of anabolic growth factors, including IGF-I and TGF-β1. Binding/latent proteins dissociate from GFs under specific conditions determined by the pH of local tissue and the presence of activating proteases. IGF-I is bound by IGFBP3 in PRP, and the presence of MMPs is necessary to degrade IGFBP3 to release bioactive IGF-I. Similarly, the bioavailability of TGF-β1 is regulated by latency-associated protein (LAP) and latent TGF-β1 binding protein (LTBP) [
43]. TGF-β1 becomes activated only when it is removed from LAP [
44]. This regulatory network of proteins can be manipulated if we want to suppress or enhance TGF-β1 or IGF-I actions.
Tailored PRP for the conservative management of musculoskeletal conditions
We address minimally invasive conservative treatments but not tissue-engineering approaches, which are most likely implanted through surgical procedures.
PRP formulations and PRP stability
Defining the optimum formulation for each tissue can optimize PRP therapies. PRP products have been categorized on the basis of different components of PRP formulations: pure PRP, which represents a plasma fraction with enrichment in platelets above peripheral blood, and L-PRP, which includes a relevant concentration of leukocytes [
45]. PRP formulations can be further characterized by their high or moderate concentration of platelets relative to peripheral blood. In clinical applications, PRPs are prepared following commercial protocols and using specific kits, and commercial systems should be carefully selected because the number of platelets and the composition of leukocytes depend on preparation protocols, which are mainly single versus double spinning [
46]. The latter increases the concentration of GFs and other signaling proteins originating from platelets and leukocytes; the kinetics of cytokine and GF release, that is, their bioavailability, depends on specific proteins and activation procedures [
21,
22].
Choosing a suitable formulation for tendon healing
Research on human tendon cells has revealed some differences between the anabolic and inflammatory effects of various PRP formulations
in vitro (Table 1, tendon conditions) [
47–
53]. These data may help determine the PRP formulation that we should use for tendon healing. Overall, current
in vitro studies indicate that leuko-reduced PRP exhibits stronger anabolic properties than L-PRP in terms of collagen synthesis [
47,
48,
51,
53], and L-PRP is more pro-catabolic as assessed by MMP expression [
47,
51,
53].
In terms of platelet concentration, decreasing leukocytes is more important in terms of reducing inflammation and enhancing matrix anabolism than increasing the number of platelets [
47].
In terms of molecular inflammatory status, L-PRP appears to be more pro-inflammatory than PRP [
50,
51]. Compared with those in PPP and PRP, decorin, fibronectin, and aggrecan are downregulated in L-PRP. L-PRP and PRP are also more pro-inflammatory than PPP in terms of IL-6 secretion, but cells in PPP manifest MCP-1
high phenotype. Compared with that in PPP and PRP, CTGF secretion is significantly reduced in L-PRP. The main advantages of L-PRP and PRP are their stronger chemotactic and proliferative properties than those of PPP.
The effects of PRPs differ depending on the severity of tissue damage. Cross
et al. [
48] highlighted the importance of disease stage in determining the effects of PRP by exposing severely and moderately diseased supraspinatus tendon cells to PRP.
A subpopulation of stem cells potentially capable of spontaneous tendon cell differentiation has been identified as a healing source in tendons [
54,
55]. The effects of PRPs in local precursor/stem cells should be determined because these cells can mediate healing. L-PRP and PRP stimulate the proliferation and differentiation of tendon stem/progenitor cells (TSCs) [
53], but the catabolic and inflammatory properties of L-PRP are strengthened by enhancing the production of MMP-1, MMP13, IL-1β, IL-6, TNF-α
, and PGE
2, while pure PRP increases the expression of anabolic proteins and types 1 and 3 collagen. In contrast to pure PRP, L-PRP elicits detrimental effects on TSCs because it is associated with high apoptotic rate, low proliferation rate, and reduced synthesis of type 1 collagen [
52]. Moreover, TSCs grown in L-PRP differentiate into non-tenocytes and produce additional inflammatory factors [
52].
Overall, these data indicate some biological advantages of PRP over L-PRP. However, the translation of these results into clinical applications is not obvious, and recent meta-analysis of clinical outcomes has revealed that L-PRP provides strong therapeutic effects (standardized mean difference of 36.38 and 95% CI= 34.00–34.87 versus 26.77 and 95% CI= 18.31–35.22) [
4]. However, conclusions from this study can be biased because 11 studies are performed with L-PRP versus two studies conducted with pure PRP.
Selecting PRP formulations to treat joint pathology
The choice of PRP formulation appears to be more critical in joint conditions than in other applications because of the complexity of the joint organ, which must consider interactions between the main cell phenotypes, such as synoviocytes, chondrocytes, and meniscus cells (Table 1, joint conditions) [
39,
56–
63]. A subclinical inflammatory state mediated by the innate immune system is a key mediator in the onset and progression of OA, and PRP can alter the intra-articular inflammatory status [
12].
Synovial cells die when leukocytes and red blood cells are included in PRP [
57]. In addition, enhanced production of IL-1β
, IL-6, and TNF-α are measured in L-PRP-treated synovial cells. Nevertheless, the secreted inflammatory molecules in a co-culture model of synoviocytes and chondrocytes do not differ between L-PRP and pure PRP [
62]. Corroborating these results, intraarticular injections of L-PRP did not modify the inflammatory cytokine pattern in OA synovial fluid, as assessed by multiplex cytokine arrays [
64]. Moreover, no significant clinical differences between L-PRP and pure PRP are observed in addition to pain and initial inflammatory reaction in L-PRP-treated patients [
65].
Some cytokines, such as IL-6, a priori considered as inflammatory reaction, possess context-dependent pleiotropic effects. Interactions of platelets with peripheral blood mononuclear cells enhance the anabolic effects of PRP [
66] through IL-6 synthesis and induce the upregulation of anti-inflammatory and anti-fibrotic cytokine IL-10 [
67].
Factors affecting the quality of PRPs
Storage of PRPs
For logistic organization and patient discomfort reduction, PRP can be prepared once and preserved under freezing conditions until the next treatment session when a treatment regimen with PRP requires several doses. Thereafter, activation with thrombin or calcium addition unlikely alters GF release [
68]. Frozen PRP maintains biological properties similar to fresh PRP [
69,
70]. On the other hand, GFs and cytokines are unstable when PRP is stored at 22 °C for 3 or 5 days [
71].
PRP can be lyophilized and remain stable after storage for 4 weeks at room temperature and can be sold as a ready-to-use drug [
72].
Endogenous factors modifying PRP quality
Studies have yet to determine whether the selected PRPs from young healthy donors can provide enhanced healing properties. The main endogenous factors affecting the quality of PRPs are aging, systemic diseases, or medication intake.
Aging increases vulnerability to OA and tendinopathy. Cellular DNA accumulates damage during aging, and cells highly undergo senescence. Current concepts about aging plasticity indicate that tissue regeneration can be improved by exposing old tissues and organs to a young circulatory environment. Studies regarding age as an influencing factor of clinical outcomes after PRP therapies provide further insights into the effects of aging on PRP quality [
73].
Systemic diseases can influence the quality and properties of PRP, but autologous PRP has been effective in healing diabetic foot ulcers [
74]. Conversely, diabetes influences the properties of other regenerative technologies, such as adipose tissue stem cell therapies (ASCs) [
75]. Hyperuricemia can be a risk factor of tendinopathy [
76], but PRP with high concentrations of uric acid (“hyperuricemic PRP”) does not remarkably modify the biological properties of PRP [
77].
Non-steroidal anti-inflammatory drugs and anesthetics are frequently used in musculoskeletal condition, but their negative effects on healing and their PRP-hampering effects are controversial. Experimental data have revealed the cytotoxicity of anesthetics by mediating the production of reactive oxygen species and by activating ERK1/2, c-jun N-terminal kinase, p38, and caspase-3/7 activity levels [
78]. Anesthetics are also toxic for mesenchymal stem cells during chondrogenic differentiation [
79].
Local corticosteroids are used as palliative treatment for joint and tendon conditions. A recent review [
80] of current experimental data, including 18 studies,
in vitro and
in vivo of the effects of local glucocorticosteroids on tendon revealed that cell viability, cell proliferation, collagen disorganization, necrosis, and mechanical property deterioration are reduced. The presence of PRP considerably limits the negative effect on chondrocyte viability at the tested time points for the examined corticosteroids and local anesthetics (
P<0.05) [
81,
82]. Similarly, PRP protects tendon cells against cell death and senescence induced by dexamethasone [
83]. Nevertheless, the addition of anesthetics or corticosteroids to PRP interferes with the positive effects of PRP and thus decreases cell viability [
84].
Systemic anti-inflammatories can interfere with platelet function, as evidenced by a decreased platelet aggregation. Thus, systematic NSAIDs should be avoided before blood is withdrawn to prepare PRP [
85].
Combinational treatments
Combining synergistic therapies can develop efficacious treatments for soft tissue healing.
Tendinopathy
Several combinatorial treatments have been investigated, but few clinical data about their efficacy have been collected. PRP is typically combined with ultrasound-guided tenotomy with positive results in recalcitrant tendinopathies [
86]. The conceptual foundation of this combined treatment stems from inducing micro-injuries and simultaneously delivering healing resources embedded in PRP.
PRP+ cells
Combining PRP with adult cells is straightforward. Implanted cells, that is, skin fibroblasts or mesenchymal stem cells, possibly colonize host tissues, synthesize collagen 1, and differentiate into tenocytes. An alternative mechanism involves paracrine effects of implanted cells interfering with disease progression [
87].
Two clinical studies have explored the combination of PRP with cells for tendon healing. One randomized controlled trial, including 60 patellar tendons treated with PRP or PRP+ laboratory expanded skin fibroblasts, has revealed the decreased hypoechogenicity and tear size in both groups, an increased tendon thickness, and enhanced clinical outcomes after 6 months in the PRP+ cell group [
88].
In another study involving three patients whose stromal vascular fraction (SVF) of adipose tissue is combined with PRP and injected to treat partial thickness interstitial tears, a normal tendon structure is found 12 months post-treatment in all of the three patients [
89], and they recover their full activities after 12 weeks. Improvement has been maintained for 3–4 years.
PRP+ low-level laser therapy
Two different animal models, namely, rat and rabbits, have demonstrated that the combination of PRP with low-level laser therapy is superior to PRP or low-level laser therapy separately in terms of high collagen deposition [
90,
91]. However, this combination has yet to be tested in human clinical trials.
PRP+ HG/IGF-1
IGF-1 injection promotes tendon and ligament healing after collagenase-induced tissue atrophy or ligament disruption in animal studies [
92]. These findings are substantiated by a retrospective study performed in 40 cases of horse superficial digital flexor tendonitis. The intra-lesional administration of 25 or 50
mg of IGF-1 every other day for four or five treatments enhances healing, as shown by the decrease in ultrasonographic lesion severity, but this treatment has not allowed horses to perform sport activities again [
93]. In humans, local injections of recombinant IGF-1 into the patella tendon increase the collagen fractional synthetic rate and procollagen type-I N-terminal propeptide, a marker of type-I collagen synthesis, in the peritendinous fluid of healthy individuals [
94]. Local growth hormone (GH) administration may be useful to improve healing in patients during the rehabilitation of tendon and ligament injuries and post-surgery [
95]. These observations suggest that an enrichment of PRP with HG/IGF-I can be visualized. Elderly people, characterized by an age-dependent decline in the GH/IGF-I activity, may benefit from local GH-IGF-1 administration.
Joint conditions
PRP and cells
The management of joint conditions with cell products, including SVF, bone marrow concentrate (BMC), and purified expanded cells derived from these heterogeneous products, has been reviewed [
87]. SVF is obtained after adipose tissue is fractionated through centrifugation, and protease digestion is used for conservative management. It includes a heterogeneous cell population comprising 2%–10% of adipose-derived stem cells. SVF provides advantages over BMC because harvesting is less invasive and is composed of a high number of mesenchymal stem cells (MSCs).
The effects of BMC injections with follow-up injections of PRP at 8 weeks have been assessed retrospectively in 125 patients with moderate to severe knee, hip, shoulder, or spine OA, and no adverse effects have been observed [
96]. Pain reduction in the hip and ankle is less than that in the knee and shoulder. Patient satisfaction is high, and 91.7% of patients indicated that they would repeat the procedure.
PRP helps not only as a vehicle to confine the cells in the pathological area but also as a source of healing factors; thus PRP contributes to treatment optimization. It is also used for cell expansion in advanced cell therapies [
97]. PRP has also been proposed as a cell-maintained biomaterial to assist in hostile environments during ASC transplantation because the PRP matrix can enhance the stemness properties of ASCs [
74].
In vivo, ASCs pretreated with or without PRP are transplanted into murine models of injured articular cartilage. PRP promotes ASC proliferation and differentiation into chondrogenic cells that strongly express collagen II, Sox9, and aggrecan. Moreover, PRP inhibits the expression of angiogenic factor vascular endothelial growth factor, and PRP-pretreated ASCs consequently improve the healing of injured articular cartilage in murine models compared with that of untreated ASCs [
98].
Intra-articular injections of combination products (SVF+ PRP) have been reported in seven case series [
99–
105]. Adipose tissue has been harvested from an infrapatellar pad (IFP) [
100,
101] or from subcutaneous tissues in the lower abdominal area [
99,
102–
105]. No safety concerns have been observed after the procedure is assessed in 91 patients (hip OA
n = 22; knee OA
n = 74; ankle
n = 2; and low back pathology
n = 2) [
103]; pain and swelling after the procedure was reported in 37% of patients.
A ternary combination product, which includes PRP, hyaluronic acid (HA) and SVF, is injected in three patients with grade 3 knee OA. This injection was followed by three weekly injections of PRP. Pain reduction and improved functionality are reported in three patients at 22 weeks and in MRI images showing cartilage-like tissue regeneration [
104].
PRP and HA
HA is endogenously produced by multiple cell phenotypes. In particular, the production of low-molecular-weight HA is a feature of early healing mechanisms. However, low-molecular-weight HA provides pro-angiogenic properties undesirable in the avascular cartilage, thus only high-molecular-weight HA is appropriate for this application. HA compounds differ in their origin, that is, natural versus synthetic, which is linked to molecular weight and viscoelasticity. High HA concentration is preferred to maintain viscoelastic properties, and PRP combined with HA enhances chondrocyte proliferation and anabolism, as indicated by an increase in glycosiaminoglycans [
106]. The theoretical benefits of the combination of PRP and HA based on synergic and complementary effects have been reviewed [
107].
High adiposity in the IFP induces inflammatory phenotypes in the knee joint and contributes to the development and progression of OA through IFP adipocyte-derived inflammatory cytokines [
108]. HA+ PRP decreases the pro-inflammatory effects of IFP adipocytes through inhibition of cytokines (e.g., IL-1β, COX-2, MMP-1, and MMP-3) and adipokines (e.g., adiponectin and leptin) and recovers IFP-induced dedifferentiation and inflammation in chondrocytes. A procedure to prepare this mixture is clinically available and has been tested in few clinical studies.
Results from clinical trials comparing PRP with PRP+ HA have been reported [
109–
111]. The efficacy of one injection a week for three weeks of PRP+ HA in 40 knees compared with a retrospective cohort treated with PRP is similar after 6 months in terms of pain reduction and improvement of functionality [
109]. A three-arm randomized controlled study involving 111 patients has compared the therapeutic efficacy of PRP, HA, or PRP combined with HA in patients after one injection a week for three weeks in hip OA [
110]. The Western Ontario and McMaster Universities Arthritis Index of the PRP group is significantly better at 2 and 6 months but not at 12 months compared with the two other groups. The addition of HA to PRP did not provide any significant advantage in this study [
110]. A multicenter prospective randomized study has compared the efficacy of three intra-articular injections, namely, PRP, HA, and both, administered bi-weekly to patients with mild to moderate OA. PRP is superior to HA 1 year post-treatment. However, the PRP+ HA combination increases physical function in comparison to PRP at 1 and 3 months but not at 6 and 12 months [
111].
Muscle injuries
The efficacy of PRP in treatments for acute muscle injuries has been evaluated in few controlled clinical trials [
112–
114]. However, current results indicate that PRP in its actual form does not clearly promote the return of injured individuals to active play [
115,
116]. Thus, further research should be performed to determine the positive effect of PRP on some aspects of muscle healing. The timing of PRP application can elicit different effects on various cell phenotypes and determine therapeutic efficacy because of the complex spatiotemporal regulation of wound healing. Denapoli
et al. [
30] revealed that functional results in muscle recovery are enhanced when PRP is injected 7 days after a contusion injury in a rat model. These data contradict current clinical and experimental findings supporting the infiltration of PRP shortly after injury to modulate excessive neutrophil infiltration, which may exacerbate tissue damage [
117].
In vitro and
in vivo experimental studies indicate that PRP may influence myoblast proliferation and differentiation, but the affected tissues exhibit some degree of fibrosis [
118]. Platelets contain three TGF-β isoforms in their α-granules, although the ratio is heavily skewed (4000 TGF-β1: 1 TGF-β2: 10 TGF-β3) [
43]. TGF-β1 and TGF-β2 are pro-fibrotic, but TGF-β3 is not. The overactivation of TGF-β signaling can promote excessive collagen deposition and contribute to fibrosis [
119]. The overproduction of collagen results in a loss of structure and mechanical properties and an increased vulnerability to relapses. Fibrosis is also a problem for muscle fiber re-innervation because it can hamper axonal growth.
TGF-β is secreted in its inactive form in a complex with LAP and LTBP. TGF-β activation occurs once it is dissociated from LAP at low pH or through the action of elastase and thrombospondin or proteases, including MMPs, plasmin, and thrombin [
44].
Inhibiting fibrosis by blocking TGF-β activity has been investigated using different approaches not only in musculoskeletal injuries but also in fibrotic diseases affecting the liver or kidneys [
119]. Inhibition of TGF-β signaling can be performed at a receptor level, and small molecule inhibitors (SMI) of type-II and type-I TGFβ receptor kinases have been wisely explored; the systemic treatment of old mice with a SMI of TβR1 ALK4, 5, and 7 but not a neutralizing antibody or decoy receptor restores the reparative capacity of old muscles [
120]. Integrins are another target of SMI because various integrins (α
vβ
1) mediate the non-proteolytic activation of TGF-β1, which has been shown to protect cells against fibrosis in mouse disease models [
121]. These molecules have yet to be tested in the context of PRPs.
In the context of PRP, direct TGF-β-neutralization with anti-TGF-β1 increases the number of satellite cells and the percentage of regenerating myofibers; as a result, the area of fibrotic tissue is reduced after cardiotoxin is injected into a tibialis anterior muscle and muscle lesions are mimicked in rats [
11]. Moreover, neutralization of TGF-β1-enhanced angiogenesis prolongs satellite cell activation and recruitment of M2 macrophages to the injury site compared with those in control groups [
11].
In addition, indirect TGF-β targeting biologically active molecules, such as curcumin, produced by plants or chemically synthesized molecules, such as decorin, suramin, relaxin, and IFNγ, has been investigated in muscle healing. Observations have linked fibrosis to the local effect of angiotensin II because ACE inhibitors and inhibitors of angiotensin receptors have been demonstrated to decrease fibrosis in the liver, kidneys, and lungs. Angiotensin II promotes the synthesis of large amounts of TGF-β, and TGF-β induces collagen synthesis by acting in an autocrine manner. Lorsatan, a blocker of angiotensin II type-I receptor, inhibits the transcription of TGF-β1 via Smad2/3, and the possibility of combining this drug with PRP is examined in a laboratory study, which has demonstrated a reduction of fibrosis after contusions in the tibialis anterior muscle in rats [
122].
Decorin, a component of the extracellular matrix in musculoskeletal tissues, can balance the profibrotic effects of TGF-β. Kelc
et al. [
10] indicated that the combination of PRP and decorin stimulates muscle commitment and can counteract the pro-fibrotic effects of TGF-β in human CD56
+ myoblast cell line.
Perspectives
Future studies on cytokine-receptor interactions, signal transduction, and cellular responses within acute and chronic tissue injuries will provide new insights into the role of PRPs in traumatic muscle injuries, tendinopathy, and degenerative osteoarticular diseases. The function of individual cytokines within the context of PRP remains unknown and the molecular link between biological mechanisms and clinical outcomes (including pain) has yet to be elucidated.
Further data should also be obtained to elucidate the precise link between the active mechanism of PRP and the biological characteristics of host tissues. Indeed, advancements are impeded by the lack of biomarkers indicating the state of host tissues. Inadequate understanding of the active mechanisms of PRP and the degenerative features of local tissues hinder the identification of optimum combination approaches to enhance healing.
Additional challenges, such as the control of PRP quality and advancements in creating allogenic PRP, remain and must be addressed in future studies.
Currently, PRP therapies have remained unsatisfactory in terms of therapeutic expectations because many individuals do not derive sufficient benefit from PRP. As such, new approaches should be developed. The associations of various treatments with diverse but complementary active mechanisms or with synergistic actions may help establish efficacious treatments for recalcitrant complex musculoskeletal conditions. Research on these combinatory treatments possibly provides insights into the processes involved in physiological healing and pathological failure.
Higher Education Press and Springer-Verlag GmbH Germany
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