Platelet-Rich Plasma Therapy in the Treatment of Diseases Associated with Orthopedic Injuries

Abstract

Platelet-rich plasma (PRP) is an autologous platelet concentrate prepared from the whole blood that is activated to release growth factors (GFs) and cytokines and has been shown to have the potential capacity to reduce inflammation and improve tissue anabolism for regeneration. The use of PRP provides a potential for repair due to its abundant GFs and cytokines, which are key in initiating and modulating regenerative microenvironments for soft and hard tissues. Among outpatients, orthopedic injuries are common and include bone defects, ligament injury, enthesopathy, musculoskeletal injury, peripheral nerve injury, chronic nonhealing wounds, articular cartilage lesions, and osteoarthritis, which are caused by trauma, sport-related or other types of trauma, or tumor resection. Surgical intervention is often required to treat these injuries. However, for numerous reasons regarding limited regeneration capacity and insufficient blood supply of the defect region, these treatments commonly result in unsatisfactory outcomes, and follow-up treatment is challenging. The aim of the present review is to explore future research in the field of PRP therapy in the treatment of diseases associated with orthopedic injuries.

Impact statement

In recent years, platelet-rich plasma (PRP) has become widely used in the treatment of diseases associated with orthopedic injuries, and the results of numerous studies are encouraging. Due to diseases associated with orthopedic injuries being common in clinics, as a conservative treatment, more and more doctors and patients are more likely to accept PRP. Importantly, PRP is a biological product of autologous blood that is obtained by a centrifugation procedure to enrich platelets from whole blood, resulting in few complications, such as negligible immunogenicity from an autologous source, and it is also simple to produce through an efficient and cost-effective method in a sterile environment. However, the applicability, advantages, and disadvantages of PRP therapy have not yet been fully elucidated. The aim of the present review is to explore future research in the field of PRP therapy in the treatment of diseases associated with orthopedic injuries, as well as to provide references for clinics.

Introduction

Platelets possess more than 5000 proteins. Upon activation, ∼300 proteins are released, including numerous growth factors (GFs) and cytokines, which induce the proliferation and activation of fibroblasts, mesenchymal stem cells (MSCs), smooth muscle cells, and neutrophils.¹ Platelet-rich plasma (PRP) is prepared by centrifuging autologous whole blood to isolate and concentrate platelets. The platelet count of PRP is consistently threefold to sixfold higher than the baseline of whole blood and ranges from 300,000 to over 1,500,000 platelets/mm³ depending on the force and time of centrifugation, total blood volume, mediators for platelet activation, and donor.² Different donors have been shown to have unique compositions of GFs and cytokines, which are abundant.^3–5 These GFs and inflammatory mediators are released upon activation, and the potential efficacy of PRP is associated with its ease of acquisition, biological safety profile, noninvasiveness, and acceptability, which has led to increased demand for PRP in clinics.⁶

The various GFs in PRP have anti-inflammatory, chemotactic, antiapoptotic, and proliferative effects on fibroblasts and neurons; they stimulate cell migration, differentiation, and proliferation and alter the microenvironment at the impaired site,^5,7 which can relieve pain and reduce recovery time.⁸ PRP is widely used in therapy for hard and soft tissues, such as dentistry,⁹ ophthalmology,¹⁰ osteoarthritis (OA),⁸ sport medicine,^3,11 peripheral nerve injury,¹² and wound healing.¹³ PRP has attracted much attention in the context of tissue repair due to its potential for providing natural physiological deposits of GFs and cytokines involved in tissue regeneration.^3,14 Although PRP is widely used in clinical therapies in many fields and can modify the microenvironments of injury sites¹⁵ and stimulate physiologic healing and tissue regeneration, many issues associated with PRP use have not yet been explored and elucidated.

The motor system is a significant component of the human body, comprising bones, joints, and skeletal muscle that perform various functions, including motion, support, and protection. The motor system is subject to damage over an individual's lifetime, such as orthopedic injuries, which affect not only athletes but also the general public. Orthopedic injuries are caused by acute injury or chronic complication after acute injury and can result in the accumulation of fatigue-associated damage.¹⁶ However, damage to ligaments, tendons, and cartilage heals slowly because of the poor blood supply and limited regeneration capacity of these tissues. Furthermore, cell renewal is slow, and extracellular matrix (ECM) restoration is limited.^12,17,18 The outcomes of treatment of articular cartilage lesions or defects are generally unsatisfactory and often result in the complication of OA, which is associated with pain and activity limitations.^19,20 Muscle injuries are common in sports and lead to disability over time.^21–23 Bone segment defects, which can be caused by high-energy trauma, infection, necrosis, complicated open fractures, or tumors, present healing problems, as the intrinsic regeneration potential associated with these defects is limited. Such defects typically develop into OA, osteomyelitis, and chronic nonhealing wounds,^24,25 making the treatment of these injuries challenging. Peripheral nerve injuries are often attributed to high-energy trauma and complicated open fractures and can lead to disability and/or sensory function. Although autologous bone and nerve grafts are the gold standard treatment for bone and peripheral nerve defects,^26–29 they have significant disadvantages in clinical application, such as donor site morbidity, the limitation of donor sites, and a lack of sufficient volume and quantity of bone and nerve.^{24,25,30–32} Therefore, a replacement treatment is needed. This review summarizes the advances in PRP treatment for diseases associated with orthopedic injuries and related issues.

General Knowledge of PRP

Definition, classification, and preparation of PRP

PRP is a biological product from autologous blood that is obtained through a centrifugation procedure that yields plasma enriched in platelets from whole blood. The term “PRP” was coined by Marx in a maxillofacial surgery reconstruction study in which platelet-rich products were applied to bone grafts.³³ The inchoate definition of PRP was a platelet solution obtained from autologous plasma with the platelet concentration above baseline.³⁴ However, researchers subsequently proposed other definitions.³⁵ It was suggested that the components and production process of PRP be used to classify PRP, because different concentrating processes can yield different PRP products.³⁶ Magalon et al.³⁷ demonstrated that the duration and speed of centrifugation had no effect on the status of platelet activation. According to the procedures and characteristics of preparations, other researchers have suggested classifying PRP by types, such as leukocyte-rich PRP, pure PRP, leukocyte-poor PRP, leukocyte- and platelet-rich fibrin, and pure platelet-rich fibrin.³⁴ Other researchers have advocated for the classification or characterization of PRP based on platelet and white cell counts.³ In multiple studies, the use of different PRP preparations resulted in significant differences in the clinical results of PRP treatment,³⁸ demonstrating the existence of a high level of PRP variability, which supports the establishment of the characterization of PRP before injection.³⁷ PRP preparations vary in the biochemical composition of PRP, with differences observed between preparations from men and those from women, and exhibit proteomic variability.³⁹ Therefore, the potential impact of diverse proteomic profiles of PRP in tissue and clinical responses should be taken into account in clinical applications.

The centrifugation procedure used to obtain PRP includes two centrifugation steps. First, the whole blood is centrifuged for separation into three layers (the platelet-poor plasma layer [top layer], the PRP layer [middle layer], and the bottom red blood cell layer). Subsequently, the red blood cells are removed, and a second centrifugation step is performed, after which the middle layer is harvested as the PRP (Fig. 1),^11–14 which contains greater than 1000 × 10³ platelets/μL.¹⁵ Although PRP has been used in many fields, a standardized procedure is lacking in clinical trials and animal studies (Table 1).

FIG. 1.

The procedure stages of PRP, two centrifugation stages from the whole venous blood. PRP, platelet-rich plasma. Color images are available online.

Table 1.

The Method of Centrifugation of Platelet-Rich Plasma in Clinical Trials and Animal Studies

First author and references	The revolution and time of the first centrifugation stage	The revolution and time of the second centrifugation stage	The count of platelets	Peripheral venous blood	PRP	Specimen source	Activation	Study type
Zahn1	2000 g/20 min	NA	2000 × 103/mm3	NA	NA	Human	NA	Vitro experiment
Veronesi3	400 g/8 min	1100 g/15 min	NA	NA	NA	Rat	Cacl2	Animal study
Okamoto4	250 g/10 min	1000 g/10 min	NA	5 mL	200 μL	Rat	NA	Animal study
Zhang6	1500 rpm/10 min	3000 rpm/10 min	NA	6–8 mL	500 μL	Rat	Thrombin+Collagen	Animal study
Zheng7	400 g/10 min	800 g/10 min	NA	8–10 mL	0.8–1.0 mL	Rat	Thrombin/Cacl2	Animal study
Krüger14	10,000 rpm/10 min	1600 g/10 min	0.6–1.3 × 107/mm3	NA	NA	Human	NA	Vitro experiment
Dwivedi15	160 g/10 min	400 g/10 min	NA	9 mL	1.5 mL	Rabbit	Cacl2	Animal study
Zhang17	500 g/10 min	3000 g/5 min	N/A	NA	NA	Rabbit	Cacl2	Animal study
Symth18	1200 g/17 min	NA	N/A	27 mL	3 mL	Rabbit	NA	Animal study
Di Martino19	1480 rpm/6 min	3400 rpm/15 min	150,000/mm3	150 mL	20 mL	Human	Cacl2	Clinical trial
Liu20	800 rpm/15 min	2000 rpm/15 min	NA	4 mL	0.8 mL	Rabbit	NA	Animal study
Qiu26	360 g/20 min	500 g/10 min	1.7 × 106/mm3	20 mL	1 mL	Minipig	Thrombin/Cacl2	Animal study
He27	2500 rpm/10 min	4500 rpm/3 min	NA	9 mL	NA	Rabbit	Thrombin/Cacl2	Animal study
Wei28	215 g/10 min	863 g/10 min	NA	NA	NA	Rat	Thrombin	Animal study
Sabongi32	1500 rpm/15 min	NA	NA	5 mL	NA	Rat	Cacl2	Animal study
Denapoli34	300 g/15 min	1000 g/10 min	NA	NA	NA	Mouse	NA	Animal study
Xiong39	180 g/10 min	NA	336.3–525.7 × 103/mm3	25 mL	NA	Human	NA	Vitro experiment
Cavallo40	460 g/8 min	730 g/15 min	5–1000 × 103/mm3	9 mL	1 mL	Human	Cacl2	Vitro experiment
Wen41	515 g/5 min	3247 g/6.5 min	150,000/mm3	153–170 mL	30 mL	Human	NA	Vitro experiment
Yang42	2400 rpm/10 min	3500 rpm/15 min	NA	NA	NA	Human	NA	Animal study
Rodrigues45	1258 g/15 min	NA	1200 × 106/mm3	8.5 mL	1 mL	Human	Thrombin	Clinical trial
da Silva46	300 g/5 min	700 g/17 min	1.2 × 106/mm3	NA	NA	Human	NA	Vitro experiment
Zhou47	250 g/10 min	1000 g/10 min	543 ± 166 × 104/mm3	5 mL	NA	Rat	Cacl2	Animal study
Matsui49	450 g/7 min	1600 g/5 min	NA	10 mL	1 mL	mouse	Cacl2	Animal study
Yin51	160 g/10 min	250/15 min	NA	40 mL	4 mL	Human	Cacl2	Clinical trial
Hudgens54	500 g/5 min	700 g/17min	1.4 × 106/mm3	NA	NA	Rat	NA	Vitro experiment
Liu55	200 g/10 min	1500 g/5 min	NA	NA	0.35 mL	Rat	Thrombin	Animal study
Moussa56	1500 rpm/15 min	2800 rpm/8 min	NA	NA	NA	Human	NA	Vitro experiment
Everts58	1660 g/15 min	305 g/3 min	122 ± 326 × 103/mm3	125 mL	4 mL	Human	Cacl2	Vitro experiment
Zhang60	2400 rpm/10 min	3600 rpm/15 min	NA	60 mL	NA	Rabbit	Cacl2	Animal study
Piacentini61	3400 rpm/8 min	NA	NA	14 mL	NA	Human	Cacl2	Clinical trial
Malhotra62	270 g/1 0min	2000 g/10 min	1039 ± 127 × 103/mm3	106 mL	11 mL	Sheep	Cacl2	Animal study
Liu65	200 g/10 min	200 g/10 min	NA	NA	0.35 mL	Rat	Thrombin	Animal study
Kasten66	209 g/16 min	1500 g/12 min	10.05 × 105/mm3	17 mL	NA	Rabbit	NA	Animal study
Berner67	2400 rpm/20 min	3600 rpm/10 min	NA	10 mL	0.3 mL	Rat	Thrombin	Animal study
Kobayashi70	1000 rpm/7 min	3000 rpm/10 min	NA	10 mL	NA	Human	NA	Vitro experiment
Abazari73	250 g/10 min	250 g/15 min	NA	10 mL	3 mL	Human	Freezing+Thawing	Vitro experiment
Kazem-Arki74	2000 rpm/15 min	4000 rpm/18 min	NA	10 mL	NA	Human	Cacl2	Vitro experiment
Liebergall75	1000 rpm/10 min	4000 rpm/10 min	900/mm3	100 mL	5 mL	Human	NA	Clinical trial
Zhang78	500 g/10 min	2000 g/10 min	NA	NA	NA	Human	Thrombin	Vitro experiment
Beck81	1500 rpm/10 min	3000 rpm/10 min	NA	7–8 mL	NA	Rat	Thrombin/Cacl2	Animal study
LaPrade82	400 g/5 min	1000 g/5 min	0.6–1.2 × 106/mm3	NA	NA	Rabbit	Thrombin/Cacl2	Animal study
Yoshida83	2000 g/7 min	NA	8.2 × 105/mm3	10 mL	1.5 mL	Human	NA	Clinical trial
Dallari84	1480 rpm/6 min	3400 rpm/15 min	150,000/mm3	150 mL	20 mL	Human	Cacl2	Clinical trial
Childers85	600 g/30 min	1400 g/15 min	NA	NA	NA	Rat	NA	Animal study
Carr86	610 g/4 min	1240 g/6 min	NA	50 mL	5 mL	Human	NA	Clinical trial
Malavolta87	5800 rpm/15 min	NA	1,185,166 ± 404,472 × 103/mm3	400 mL	40 mL	Human	Thrombin+Cacl2	Clinical trial
Li88	160 g/20 min	400 g/15 min	6.44 ± 0.64 × 106/mm3	NA	NA	Rat	NA	Animal study
Ikumi92	490 g/8 min	NA	NA	20 mL	NA	Rabbit	Cacl2	Animal study
Torul93	580 g/10 min	NA	NA	2 mL	NA	Rat	Cacl2	Animal study
Teymur94	400 g/10 min	800 g/10 min	6.03 × 105/mm3	40 mL	1.5 mL	Human	Cacl2	Vitro experiment
Zheng96	400 g/10 min	800 g/10 min	NA	4 mL	400 μL	Rat	Cacl2	Animal study
Bayram97	2700 rpm/12 min	NA	NA	8 mL	NA	Rabbit	NA	Animal study
Baklaushev99	280 g/15 min	280 g/15 min	5 × 105/mm3	42 mL	3–5 mL	Human	Cacl2	Animal study
Şenses102	3000 rpm/10 min	NA	NA	2 mL	NA	Rat	NA	Animal study
Sánchez103	626 g/8 min	NA	NA	40 mL	1 mL	Sheep	Cacl2	Animal study
Long104	2000 g/15 min	NA	NA	NA	NA	Human	Thrombin/Cacl2	Animal study
Suthar106	NA	NA	NA	40–60 mL	7 mL	Human	Thrombin+Cacl2	Clinical trial
Sriram107	160 g/10 min	400 g/10 min	NA	20 mL	NA	Human	Cacl2	Clinical trial
Velier108	900 g/12 min	NA	NA	10 mL	1 mL	Human	Thrombin+Cacl2	Clinical trial
Thomsen109	3000 g/8 min	3000 g/2 min	NA	18 mL	NA	Human	NA	Clinical trial
Guo110	160 g/10 min	250 g/15 min	NA	40 mL	4 mL	Human	NA	Clinical trial
Burgos-Alonso112	580 g/8 min	NA	NA	9–30 mL	NA	Human	Cacl2	Clinical trial
Yuan113	200 g/10 min	200 g/10 min	NA	30 mL	4 mL	Human	Thrombin+Cacl2	Clinical trial
De Angelis114	3000 rpm/10 min	3000 rpm/15 min	NA	10 mL	NA	Human	Cacl2	Clinical trial
Mei-Dan121	640 g/8 min	NA	NA	18 mL	2 mL	Human	Cacl2	Clinical trial
Cavallo122	3500 rpm/3 min	3000 rpm/22 min	5–1000 × 103/mm3	150 mL	NA	Human	Thrombin/Cacl2	Vitro experiment
Filardo126	1480 rpm/6 min	3400 rpm/15 min	NA	150 mL	20 mL	Human	Cacl2	Clinical trial
Liu127	200 g/10 min	200 g/10 min	NA	9 mL	NA	Human	Cacl2	Animal study
Louis129	130 g/15 min	250 g/15 min	NA	52.5/37.5 mL	4 mL	Human	Cacl2	Clinical trial
Wang132	250 g/10 min	1000 g/10 min	1000–2000 × 103/mm3	18 mL	0.5 mL	Human	Cacl2	Clinical trial
Yanasse133#	200 g/10 min	400 g/10 min	1,000,000/mm3	18 mL	3.2 mL	Rabbit	NA	Animal study
Chang134	300 g/10 min	800 g/10 min	1190 ± 461.9 × 103/mm3	9 mL	1 mL	Rabbit	Cacl2	Animal study
Wang137	2000 rpm/10 min	2000 rpm/10 min	NA	40 mL	4 mL	Human	NA	Clinical trial
Tao138	160 g/10 min	250 g/15 min	NA	40 mL	4 mL	Human	Centrifuged	Clinical trial

N/A, not available; PRP, platelet-rich plasma.

Mechanism and activation methods

PRP activation is required to stimulate tissue regeneration and repair. There are four major approaches to platelet activation, which involve the use of autologous thrombin, CaCl₂, 10% collagen type I, and a mixture of CaCl₂ and thrombin;^14,40 freeze-thaw is another method.⁴¹ Standardized platelet activation is a crucial step for optimizing the release of various GFs and cytokines during application.²⁸ Common activation methods reported in the literature involving animal experiments and clinical trials are the application of CaCl₂ and the application of CaCl₂ in association with thrombin (Table 1). Inflammatory mediators are released simultaneously and are crucial and obligatory factors for tissue repair.⁴² Harrison et al.⁴³ demonstrated that different platelet activators can significantly influence the cytokine release kinetics in PRP and that collagen has a more sustained release pattern, while thrombin can generate instant release. Another study demonstrated that transforming growth factor (TGF)β, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) in PRP were burst released within 2 days, after which the GF release was significantly slowed.⁶ Thus, PRP activation is an issue that is worth considering before application.

Components of PRP

Primary GFs in PRP

Platelets contain three primary types of secretory granules: α-granules, which include many GFs and cytokines; dense γ-granules, which release serotonin, polyphosphates, calcium, adenosine triphosphate, and adenosine diphosphate; and lysosomes, which include various hydrolytic enzymes.⁴⁴ The results of many in vitro and in vivo trial studies have demonstrated that various GFs and cytokines are released from α-granules of concentrated platelets that constitute PRP. The primary GFs in PRP include FGF-2, PDGF, TGF-β, vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF).^{10,27,41,45–47}

VEGF is a highly specific vascular endothelial cell GF that can stimulate vascular permeability, ECM degeneration, and the mitosis, migration, proliferation, and angiogenesis of vascular endothelial cells. VEGF can promote the growth of vascular endothelial cells in vitro and induce vascular hyperplasia in vivo, especially in low-oxygen environments. PDGF is a strong mitogen that can stimulate the differentiation and proliferation of specific cell groups; furthermore, it promotes the growth of smooth and glia-associated muscle cells, osteoblasts, and neutrophils; increases fibroblast formation, collagenase secretion, and collagen synthesis; and stimulates macrophage and neutrophil chemotaxis. TGF-β plays an important regulatory role as a mitogen for fibroblasts and osteoblasts and functions as a regulator of endothelial cells, collagen synthesis, and the secretion of collagenase; a stimulator of MSC proliferation, angiogenesis, and endothelial chemotaxis; and an inhibitor of macrophage and lymphocyte proliferation. EGF stimulates the mitosis of mesenchymal cells, regulates collagenase secretion, and induces endothelial cell chemotaxis and angiogenesis. IGF stimulates mesenchymal and lining cell differentiation and mitogenesis and stimulates osteoblasts to produce type I collagen, alkaline phosphatase, and osteocalcin. FGF is a mitogen of mesenchymal cells, osteoblasts, and chondrocytes that stimulates the growth and differentiation of blood vessels, chondrocytes, fibroblasts, endothelial cells, and osteoblasts and promotes cell migration. VEGF is a target of endothelial cells that causes cell growth, migration, and new blood vessel growth and has antiapoptotic effects.^28,45–48

Numerous studies have shown that VEGF, PDGF, TGF-β1, and EGF can stimulate the maturation of newly formed vessels. Many of these GFs are produced by platelets in PRP.⁴⁹ Following PRP treatment and understimulated angiogenesis and an adequate supply of nutrients, metabolism at the injection site is enhanced, promoting tissue regeneration and reconstruction.

Cytokines in PRP

As participants in the tissue healing process, GFs and cytokines have important functions as cell migration chemoattractants, cell proliferation stimulants, and mitogens to promote tissue healing.⁴³ The cytokines present in PRP include interleukin-1 (IL-1β), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-9 (MMP-9).^27,50,51 IL-1β and TNF-α can activate the nuclear factor-κB (NF-κB) signaling pathway, which has been demonstrated to induce unfavorable effects on tissue regeneration and lead to the production of destructive proteases while enhancing the degradation of ECM and inhibiting ECM formation.^52,53 IL-1β plays important roles in inflammation and matrix degradation and is the primary target for reducing inflammation by manipulating IL-1ra.⁵⁴ MMP-9 is known to degrade ECM molecules and collagen.⁵⁰ Moreover, MMP-9 is a predictor of poor healing, and several studies have demonstrated that MMP concentrations indicate poorly healing or nonhealing wounds.⁵⁵ Previous studies have indicated that PRP may partially inhibit the IL-1β-mediated upregulation of MMPs in vitro.⁴ Moreover, PRP may inhibit the degradative effects of catabolic cytokines, inducing the IL-1β-mediated decrease in aggrecan gene expression and type-II collagen.⁴

Autophagy is an important mechanism of cell survival, and PRP can inhibit this mechanism by altering autophagy and reversing senescence.⁵⁶ In addition, PRP has been demonstrated to decrease the expression of MMP-3, COX-2, and IL-6 while increasing the levels of collagen, aggrecan, and TGF-β and the intracellular anti-inflammatory cytokines IL-13, IL-10, and IL-4.⁵⁶ The TNF-α and NF-κB signaling pathways are mediators of inflammation. In addition, the NF-κB protein is present in nearly all animals and plays a crucial role in inflammatory and immune responses, being involved in the regulation of gene expression. The NF-κB pathway is activated by PRP, which increases the expression of genes associated with cellular proliferation. An intermittent bout of inflammation can trigger the tissue regeneration response.⁵⁴ Interestingly, an injection of PRP into a wound or joint can relieve the inflammatory response and shorten healing time. The inflammatory environment can be controlled through direct injection, as demonstrated in many studies. The molecular mechanism associated with this phenomenon is the PRP-driven inhibition of both the NF-κB cascade (through the prevention of IκBα activation) and NF-κB target gene activation.

Additional components in PRP

PRP is obtained from whole blood, and its biological properties are similar to those of blood, which is a complex of biomolecules. However, these products are more difficult to dispose of and evaluate than synthetic materials, which can be produced on demand.⁵⁷ In addition, because of the intrinsic and versatile characteristics of blood and the various factors in PRP that are not well investigated, PRP is more complex than classical pharmaceutical preparations.⁵⁸ A great deal of attention has been paid to GFs, whereas other factors present in blood-derived products, such as fibrin and leukocytes, which play crucial roles in tissue regeneration and the inflammatory response, have been neglected.⁵⁷ Moreover, PRP contains the full complement of clotting factors and is enriched with a range of chemokines and other plasma proteins.⁵⁹

Clinical Application of PRP

In bone fracture healing

Bone is a unique and dynamic scaffold organ and key component of the motor system of humans. Although bone possesses a high capacity for self-repair, segmental bone defects are commonly due to open fractures, tumor resection, and debridement in osteomyelitis.^12,19,59 In addition, the damage associated with blood circulation around the fracture can result in repair failure and critical size long-bone defects. These defects commonly lead to bone nonunion or delayed union in clinics, representing the transformation of an acute injury to a chronic osteopathy. Repairing bone defects is a challenge in clinics that is important to overcome. Although numerous studies have demonstrated that autografts are the gold standard of treatment, the shortage of donors and associated inevitable complications due to iatrogenic injury limit their application.^12,60–62 Surgical therapeutic intervention is typically required to repair bone defects. Numerous bone defect treatments have been utilized in clinical practice.⁶³ However, the therapeutic procedures involved can cause pin-tract infection, require multiple operations, and entail long recovery time.⁶⁴ In addition, a great deal of pain and economic hardship accompanies available treatment procedures. Furthermore, the disadvantages of donor site damage and other limitations are significant,⁶⁵ causing many researchers to investigate new treatments.

There are three stages of the bone healing progress: The first stage is the early inflammatory stage, spanning the first weeks of healing, which is accompanied by vascular tissue ingrowth. In the second stage, that is, the repair stage, fibroblasts begin to migrate to the fracture site and support vascular ingrowth, collagen matrix is deposited, and osteoid is secreted and mineralized, resulting in the formation of a soft callus around the fracture site. In the third stage, that is, the late remodeling stage, the fracture site recovers its original structure, shape, and mechanical strength.⁶⁶ During all stages, the vascularity of the fracture site is extremely important in determining the success of healing.⁶³ The healing efficacy of PRP was first demonstrated for bone defect treatment by Marx in 1998, and various abundant GFs and cytokines have since been identified and evaluated.²⁹

The significant healing efficacy of PRP is due to the GFs and cytokines it contains, which play crucial roles in inflammation and stimulate the differentiation of marrow-derived MSCs in the rebuilding microenvironment of the defect site. This differentiation may stimulate new bone formation and early revascularization.^14,32 These features of PRP are particularly beneficial for preventing bone nonunion and delayed union. The capacity of PRP to induce the osteogenic differentiation of MSCs is attributable to bone morphogenetic protein-2 (BMP2), which is a component of PRP.⁶⁵ Thus, GFs, such as members of the BMP family, TGF-β, and VEGF are the most commonly used factors to overcome the lack of osteoinductivity.⁶⁷

In clinical studies, the use of PRP with autogenous bone grafts has obtained good outcomes,^62,68 although with the drawbacks mentioned above.⁶⁹ The development of appropriate strategies and advanced biomaterials possesses promise for bone tissue engineering.⁷⁰ Components necessary for bone regeneration include osteoconductive scaffolds, osteoinductive GFs, and cells that possess the capacity for osteogenicity for graft vascularization.⁶⁷ However, because most biomaterials have limited bioactivity, osteoinductive components should be used in combination with PRP. Various studies have demonstrated that combining PRP with biomaterial scaffolds is a favorable method, as it is an efficient, simple, and cost-effective approach that allows for the immobilization of abundant and highly concentrated bioactive factors to produce an optimized microenvironment for tissue regeneration.⁷¹ Stem cells, biodegradable scaffolds, and bioactive factors are well known as the three basic components necessary for tissue engineering.⁷² Mechanical properties that are similar to those of the target tissue with respect to biodegradability and biodegradability are essential traits of polymeric scaffolds, regardless of their origin (natural or synthetic). In addition, scaffold efficacy in tissue engineering can be increased using natural bioactive materials present in the body.⁷³ Recently, PRP-based tissue engineering has shown promise as a treatment for bone and articular cartilage defects, offering significant benefits with respect to biocompatibility, biodegradability, the restorative capability of endogenous GFs, and cost-effectiveness.^60,61,74

Stem cells have the ability of self-renewal and multipotency. Because MSCs can be harvested from multiple sources, such as adipose tissue, peripheral blood, bone marrow, pericytes, synovial membranes, umbilical cords, and placenta, they are considered ideal cells.⁷⁴ The capacity to induce the osteogenic differentiation of MSCs is important for bone healing.⁶⁵

In a randomized and prospective clinical preliminary study, Liebergall et al.⁷⁵ showed that the fracture union time of a PRP and MSC group was shorter compared with a control group and deemed their treatment as a safe and efficient procedure. In an in vitro study, Kashef-Saberi et al.⁷⁶ discovered that PRP-incorporated scaffolds had the greatest effectiveness among several investigated scaffolds in enhancing osteogenic differentiation. Furthermore, tensile mechanical testing revealed improvements in the mechanical properties of the PRP group relative to those of a control group. In addition, Zhang et al.⁶⁰ discovered that the use of allogeneic PRP for bone defect treatment resulted in negligible immunogenicity, significant healing efficacy, potentially more reliable homogeneity, and no necessary health burden to patients.

In ligament, muscle, and tendon injury

Tendons are an indispensable component of the motor system. Acute and chronic tendon injuries are frequently treated in sport medicine and orthopedic-associated practice and are often a result of excessive and repetitive loading.⁷⁷ Due to poor blood supply and limited self-renewal, injured tendons and ligaments heal slowly and incompletely.⁷⁸ The multistage healing procedure of cell proliferation and ECM production is slow and leads to the formation of collagen-rich scar tissue with undesirable mechanical properties that make healed tendons susceptible to reinjury.⁷⁸ In addition, chronic pain, weakness, activity limitations, and loss of stamina⁷⁹ decrease the quality of life.

Tendon stem cells (TSCs) respond to numerous biomechanical and biochemical stimuli and can differentiate into tenocytes and proliferate, allowing them to play an important role in tendon regeneration. Previous studies have demonstrated that activated PRP can stimulate the proliferation of TSCs and the production of tenocytes and abundant collagen.⁷⁸ Kim et al.⁸⁰ investigated the therapeutic effect of bone marrow aspirate concentrates (BMACs) combined with PRP and tendon-derived stem cells (TDSCs) for rotator cuff tendon tears. The results indicated that the proliferation and migration of TDSCs were enhanced by the combination treatment and that the aberrant osteogenic and chondrogenic differentiation of TDSCs was prevented. These findings suggest a mechanistic explanation for the therapeutic benefits of BMAC combined with PRP for the treatment of rotator cuff tendon tears.⁸⁰ Zhang et al.¹⁷ explored the effect of leukocytes in PRP on TSCs after observing the reduced production of GFs in pure PRP. They found that TSC proliferation was significantly reduced following leukocyte-rich PRP application relative to that under pure PRP, with more collagen produced and more tendon-like tissue formed with leukocyte-rich PRP. Furthermore, compared with pure PRP, leukocyte-rich PRP led to the production of more inflammatory factors, greater differentiation of cells into nontenocytes, and increased apoptosis.² Zhang et al. showed that different types of activation of PRP result in different effects on TSCs and tendon healing, suggesting that activated PRP should be applied in clinics to treat various types of tendon injury.⁷⁸

To date, a single application of PRP in both animal studies and clinical trials for ligament, muscle, and tendon injuries has resulted in a variety of outcomes. In a controlled study with a rat model, Beck et al.⁸¹ found that PRP altered the tissue properties of tendons and decreased tendon tissue stiffness without affecting the construct's failure load. Another study with a rabbit model showed that medial collateral ligament healing in all rabbits was evident at 6 weeks. The width of the medial collateral ligament was not significantly different between the PRP treatment group and the control group, whereas the histological and biomechanical properties were improved in the PRP group, indicating that higher concentrations of platelets in the PRP group accelerated the healing of damaged ligament.⁸² Yoshida and Marumo⁸³ reported the results of PRP treatment of chronic medial knee pain with low-grade medial collateral ligament injury. In MRI images at 3 months post-treatment, they observed recovery of damaged medial collateral ligament in both the superficial and deep layers. A study of 34 athletes who underwent treatment for injury to the anterior bundle of the ulnar collateral ligament revealed that 88% of the athletes returned to their sport without any complaints at an average of 12 weeks.⁷⁹ A randomized controlled clinical trial involving PRP injection therapy for Achilles tendinopathy with ultrasound guidance demonstrated that PRP could increase tendon thickness relative to that observed in the placebo group; however, the Victorian Institute of Sports Assessment-Achilles (VISA-A) score did not improve in the PRP group over a 3-month period.⁸⁴ Rotator cuff repairs are one of the most challenging types of repair in orthopedic surgery.⁸⁵ In a randomized controlled trial, an arthroscopic acromioplasty group that had received PRP injection exhibited significantly altered tissue characteristics in the rotator cuff, with reduced cellularity and vascularity and a simultaneous increase in apoptosis.⁸⁶ In a prospective randomized study, Malavolta et al. found that clinical or structural characteristics were not significantly enhanced in a PRP group relative to a control group at 60-month follow-up.⁸⁷

Muscle injuries are frequent, and the recovery of function is incomplete because of fibrosis.⁸⁸ Li et al.⁸⁸ demonstrated that TGF-β1 within PRP not only stimulates muscle regeneration but also significantly reduces fibrosis in rat models with acute muscle injury. However, Reurink et al.⁵ reported no benefit of PRP at 6-month follow-up in a double-blind placebo-controlled trial of 80 competitive and recreational athletes with acute hamstring muscle injuries who received 3-mL PRP intramuscular injections. In a review of literature reporting randomized and quasi-randomized controlled trials, Moraes et al.⁸⁹ concluded that there was insufficient evidence to support the use of PRP for treating musculoskeletal injuries.

In peripheral nerve injury

Peripheral nerve injury is common, and its treatment remains challenging.⁹⁰ The response to damage to a peripheral nerve involves a variety of cell types and molecules with pleiotropic effects and is typically associated with hypoxia, the release of soluble factors, axonal injury, and increased amount of myelin breakdown products.⁹¹ Even if injured axons can regenerate, its regenerative capacity is limited, leading to incomplete functional recovery and long-term disability.^92,93 Nerve grafts are the “gold standard” of treatment for peripheral nerve injury in the clinic⁹⁴; however, significant shortcomings, such as donor site limitations, concomitant morbidity, and length/diameter mismatch⁹⁵ limit their use. Acellular nerve allografts exhibit similar characteristics to autografts in sustaining axonal regeneration in the short peripheral nerve, but their use in repairing larger defects generally fails.⁹⁶ With the development of tissue engineering and material engineering, many nerve conduits for bridging defects have been developed that can act as guides and provide a favorable regenerative microenvironment for cellular proliferation,⁹⁷ allowing axon regeneration. Some authors reported using 2.5 cm of synthetic nerve conduits to bridge nerve gaps; however, complications, such as tube extrusions and fistulization, were noted.⁹⁸ PRP contains abundant GFs and cytokines, and the biomolecules in PRP are generally associated with the regulation of early inflammation, Schwann cell activation, angiogenesis, fibrogenesis, and macrophage polarization, sequentially acting as the keys to nerve function recovery.^16,25

PRP is also of interest as a scaffold component in neuroregeneration.⁹⁹ To be effective, tissue-engineered nerve constructs and scaffolds should support axonal growth, vascularization, and cell migration; allow delivery of GFs; contain guiding cues such as collagens, laminins, and fibronectin; be porous to allow oxygen diffusion; have low antigenicity; and be biodegradable.¹⁶ PRP facilitates neurogenesis and axonal growth and accelerates the migration and proliferation of Schwann cells; these effects are attributed to various GFs (TGF-β1, IGF-1, PDGF-AB, VEGF, GDNF, and NGF) and platelet-derived exosomes, including microRNA and other signaling molecules.^17,100 These characteristics of PRP provide an appropriate biomimetic microenvironment for neuroregeneration.⁹⁹ In a placebo-controlled clinical study, a single PRP ultrasound-guided injection achieved positive effects and relieved symptom in patients who underwent treatment for carpal tunnel syndrome.¹⁰¹

However, different PRP preparations contain different products,³⁸ such as platelet-rich fibrin, and decreased functional recovery in the animal model of sciatic nerve injury has been reported.¹⁰² Thus, the effects of platelet concentrates remain debated with respect to peripheral nerve regeneration.⁹⁷ More studies are needed to standardize PRP dosages and to elucidate additional mechanisms.¹⁰³

In nonhealing wounds

Normal healing of cutaneous skin wounds requires the coordination of many complex biological and molecular events, including cell proliferation, migration, vascularization, remodeling, and ECM synthesis and deposition,¹⁰⁴ which occurs in three dynamic phases: the inflammation, tissue formation, and tissue remodeling phases.¹⁰⁵ However, in some cases, the natural healing process is interrupted,^106,107 which can result in the formation of chronic nonhealing wounds. Some common causes of chronic nonhealing wounds include trauma, venous ulcers, pressure ulcers, diabetic foot ulcers, and arterial ulcers.¹⁰⁸ Chronic ulcer wounds result in decreased quality of life for patients and great social burdens.¹⁰⁹ Although dressing changes and debridement are the typical interventions used for chronic wounds, the outcomes are often unsatisfactory, and new treatments are needed. An ideal regenerative treatment would involve a delivery system to prolong the biological activity half-lives of the naturally derived exogenous proteins to decrease the need for expensive recombinant proteins.¹⁰⁴

Guo et al.¹¹⁰ conducted the first investigation of the activation of the Erk and Akt signaling pathways in PRP, which is the mechanism by which exosomes are created in PRP, and assessed the nature of the signals involved in these processes. They found that exosomes promote proliferation, migration, and angiogenesis. In a randomized clinical trial, recombinant human PDGF, recombinant epidermal EGF, and PRP were found to greatly increase the healing rate.¹¹¹ The feasibility and safety of PRP were confirmed in a randomized controlled pilot study and suggested that PRP represents a safe and effective treatment for venous leg ulcer.¹¹² Yuan et al. demonstrated that PRP could be used to treat large chronic wounds with satisfactory clinical results.¹¹³

The association of hyaluronic acid (HA) with PRP in a biofunctionalized scaffold has the potential to provide faster wound healing than traditional dressings or the single use of PRP or HA and thereby significantly decrease the costs of hospitalization and the pain experienced during the immediate postoperative period. Moreover, the use of PRP and HA together as a biofunctionalized scaffold could contribute to the treatment of chronic ulcers in vivo in case where general grafting tissue is not available in adequate quantity.¹¹⁴

Because stem cells have the abilities of self-renewal and multipotency and because MSCs can be harvested from multiple sources, such as adipose tissue, peripheral blood, bone marrow, pericytes, synovial membranes, umbilical cords, and placenta, they are considered ideal cells for tissue regeneration and repair⁷⁴ with the properties of adhesion, differentiation, proliferation, and chemoattraction. Thus, the combination of stem cells with PRP in scaffolds represents an efficient, simple, and cost-effective approach, allowing for the immobilization of abundant and highly concentrated bioactive factors that produce an optimized microenvironment for tissue regeneration.⁷¹

Yang et al.⁴² reported that full-thickness skin wounds in mice treated with a mixture of heparin-conjugated fibrin (HCF) and PRP exhibited much faster wound closure and dermal and epidermal regeneration than did corresponding skin wounds in control mice. They proposed that this result was potential as HCF-PRP promotes angiogenesis in the wound bed.

In articular cartilage lesions and OA

Articular cartilage lesions are common in clinical practice and are considered among the most challenging issues in orthopedic departments.¹¹⁵ Because articular hyaline cartilage consists of 95% ECM and 5% chondrocytes¹¹⁶ and is not innervated and vascularized, its intrinsic healing capacity is poor.¹¹⁷ Changes in the synovium and the composition of the synovial fluid cause inadequate healing associated with inflammation and vascular pathology, cell apoptosis, meniscus changes, bone reconstitution, and subchondral sclerosis, leading to cycles of progressive joint degeneration that ultimately results in OA.^5,118,119 The specific traits associated with this condition include joint pain, stiffness, and limitations in joint motion.^83,120 Although many treatments exist that include nonsurgical and surgical methods, such as the intraarticular injection of HA, which reduces pain and inflammation and simultaneously increases the levels of endogenous joint fluid,¹²¹ and the microfracture technique, which is the first-line treatment for articular cartilage defects,³¹ optimal efficacy has not been achieved. Developing a less invasive method to improve the status of the chondral surface and allow the swift recovery to full activity is considered highly desirable.¹²²

The disease pathogenesis of OA involves inflammatory mediators, which are produced by the synovium and chondrocytes and differ between the anterior and posterior cruciate ligament.¹²³ Studies have shown that a direct injection of PRP into a joint can control the inflammatory environment.^109,120 Khatab et al.¹²⁴ discovered that intra-articular injections of PRP can simultaneously reduce synovial inflammation, protect cartilage, and reduce pain in mouse models of OA. Similarly, Cole et al.¹²⁵ demonstrated that PRP had the trend toward a decrease in IL-1β and TNF-α and may contribute to improvement of symptoms due to the anti-inflammatory properties. Filardo et al.¹²⁶ reported that an intra-articular injection of PRP significantly improved all clinical scores and the Tegner score of sport activity level. Furthermore, they found no correlation between the demographic factors or articular degeneration level and no difference in clinical outcome between PRP and HA treatment, suggesting that PRP injection is not beneficial for patients who suffer from OA. Liu et al.¹²⁷ showed that pure PRP was more effective than HA and could promote cartilage restoration and alleviate arthritis in a rabbit knee model.

The meta-analysis by Shen et al.,¹²⁸ which contains 14 randomized controlled trials, revealed that at the 3-, 6-, and 12-month follow-up assessments of pain alleviation and self-reported functional improvement, intra-articular PRP injections were more efficacious than injections of HA, saline placebo, corticosteroids, and ozone in the treatment of knee OA. Louis et al.¹²⁹ reported that pure PRP injection significantly improved clinical results in treatment of knee OA.

The 5-year follow-up results of a double-blind, randomized controlled trial demonstrated that all clinical scores investigated were significantly improved in a PRP-treated group relative to an HA-treated group.¹²⁵ However, PRP did not provide superior clinical outcomes, regardless of follow-up points or efficacy duration, consistent with previous results.⁵ Fortier demonstrated that in an equine model, PRP associated with biocartilage (such as the particles of allograft articular cartilage) improved cartilage repair to a greater extent than microfracture alone .¹³⁰

However, the rapid clearance of PRP at the articular cartilage defect sites represents significant drawbacks and inevitably occurs due to a lack of fixation and tissue adhesive ability.⁶ A study by Liu et al.²⁰ demonstrated that a new photocross-linkable PRP-complexed hydrogel glue (HNPRP) that they developed could overcome the drawback, providing convenient formation in situ, achieving controlled release of the GFs, and improving cartilage adhesiveness and PRP-loaded hydrogel integration. In an in vivo rabbit model experiment, an HNPRP hydrogel significantly improved cartilage defect repair. To overcome the disadvantages of PRP treatment, many researchers have investigated scaffold materials into which PRP is loaded, and various studies have attempted to develop potential treatments. The development of tissue engineering is expected to lead to better treatments for articular cartilage regeneration and repair. The crucial elements of tissue engineering are seed cells, GFs, and scaffolds.¹³¹ PRP-based tissue engineering has produced a promising treatment for articular cartilage defects that possess the desirable traits of biocompatibility, biodegradability, restorative capability of endogenous GFs, and cost-effectiveness.¹³² Yanasse et al.¹³³ demonstrated that the use of dental pulp stem cells from humans in PRP scaffolds may be a potential therapy for cartilage repair. They successfully treated full-thickness articular cartilage defects through the use of PRP scaffolds and stem cells in a rabbit model.

Because cell migration and tissue regeneration are supported by biomaterial scaffolding matrices, such matrices have been considered for the repair of osteochondral defects in knee joints. Biomaterials with excellent biocompatibility, biodegradable properties, and biomechanical properties would enhance treatment and limit complications. Chang et al.¹³⁴ reported that a combination treatment involving a polylactic-coglycolic acid scaffold, PRP, and early continuous passive motion exercise to modulate the microenvironment for articular cartilage achieved efficacy for repairing hyaline cartilage and subchondral regeneration, and the regional inflammatory response was modest. With the development of three-dimensional (3D) printing technology, numerous studies have presented methods for cartilage tissue engineering in 3D-prepared scaffolds with subsequent seeding of cells.^131,135 The significant advantage of bioprinting is the distribution of two or more different cells in one construct in the native location with no effect on their activities. Furthermore, bioprinting can mix two or more materials in one construct, adding different cells and factors. This method can produce scaffolds with complex biochemical microenvironments and stabile mechanical properties. However, how to sustain higher cell survival and biomolecular activity while ensuring appropriate biomechanical performance remains unclear, as does how to accurately distribute bioactive molecules, cells, and scaffold materials; thus, additional research is needed.¹³¹

Conclusions

In the treatment of acute trauma and chronic nonhealing after acute trauma, such as bone injury, wounds, skeletal muscle, ligament, tendon, articular cartilage lesions, OA, and nerve injury, PRP therapy plays a prominent role and leads to good outcomes.^{62,63,76,86,87,102,118,128} Trauma commonly causes open cutaneous wounds and acute ligament, tendon, and/or muscle injuries. Because platelets play indispensable roles in the inflammatory phase, coagulation process, and regeneration, PRP can influence the quality and rate of wound repair.¹³⁶ PRP is effective for not only single tissue defect repair but also composite tissue defects. Wang et al. reported a patient who suffered chronic calcaneal osteomyelitis combined with soft-tissue defects and was successfully treated with PRP.¹³⁷ The significant advantages of PRP, such as a rich source, biocompatibility, safety, low cost, simple preparation, and great potential for clinical effectiveness, have been widely shown in experiments and clinical trials.¹¹³

Although the ability of PRP to improve tissue regeneration and healing has been demonstrated by mounting evidence, the optimal processing time and isolation methods for platelets and leukocytes and the optimal concentration of these components for maximal beneficial effects remain unknown (Fig. 2a, 2b). Many differences in the application and effectiveness of PRP are evident in the literature, and there is no consensus on these issues. Moreover, the differences between high and low platelet concentrations and the relative merits remain unclear, and a standardized method for the preparation and administration of PRP remains lacking. The types of PRP used for different injuries need to be considered and investigated in the clinic.

FIG. 2.

The graph of the time and centrifugal revolutions for producing PRP. (a) The time and centrifugal revolutions in the first centrifugation. (b) The time and centrifugal revolutions in the second centrifugation.

The potential of the GFs and cytokines in PRP have attracted a great deal of attention, however, the other components of PRP and their effects have been neglected and need elucidation. Intra-articular injections of PRP produce specific outcomes; however, the optimal frequency and volume of PRP injections remain unknown, the mechanisms of pain relief require further study, and a lack of fixation and tissue adhesive ability of PRP are significant drawbacks that limit its clinical application. One study suggested that lyophilized PRP may be an alternative to the standardization of quality and function in clinical practice.⁴⁶ Although the cell interactions that occur following PRP treatment are unclear, the development of tissue engineering and the application of PRP in combination with stem cells, biomaterial scaffolds, and bioactive factors have yielded desirable results.⁷⁰ Therefore, further research is needed to discover the optimal material scaffold to overcome the drawbacks and promote the clinical use of PRP to help patients, particularly those who suffer from bone defects, cartilage defects, complex tissue defects, or nonhealing.

In addition, high-quality clinical evidence of PRP efficacy in musculoskeletal soft tissue injuries is currently lacking. Most importantly, the molecular mechanisms underlying PRP effects in tissue regeneration are not fully understood. Recently, Tao et al. discovered that exosomes derived from human PRP could prevent osteonecrosis of the femoral head (ONFH) caused by glucocorticoid-associated endoplasmic reticulum stress in rat through the Akt/Bad/Bcl-2 signaling pathway.¹³⁸ In addition, they proposed a potential method for the treatment of ONFH. Therefore, there may be variety of unknown properties and mechanisms of PRP awaiting discovery.

In brief, while advantages of PRP with respect to treatments in sports medicine have been demonstrated, it also has disadvantages, and multicenter studies of the effects of PRP over multiple follow-ups and the interactions of GF are needed.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the National Key R&D Program of China (2017YFA0104702), the National Natural Science Foundation of China (31771052), the Beijing Municipal Natural Science Foundation (7172202), and the PLA Youth Training Project for Medical Science (16QNP144). This study was approved by the committee of medical ethics of the participating hospitals.