Advances with Platelet-Rich Plasma for Bone Healing

Blake M Bacevich; Richard David James Smith; Alec M Reihl; Augustus D Mazzocca; Ian D Hutchinson

doi:10.2147/BTT.S290341

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Review

Advances with Platelet-Rich Plasma for Bone Healing

Authors Bacevich BM, Smith RDJ, Reihl AM, Mazzocca AD, Hutchinson ID

Received 5 October 2023

Accepted for publication 17 January 2024

Published 25 January 2024 Volume 2024:18 Pages 29—59

DOI https://doi.org/10.2147/BTT.S290341

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Shein-Chung Chow

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Blake M Bacevich,¹ Richard David James Smith,¹ Alec M Reihl,¹ Augustus D Mazzocca,^1,² Ian D Hutchinson¹

¹Division of Sports Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Massachusetts General Brigham, Boston, MA, USA; ²Medical Director, Division of Sports Medicine, Department of Orthopaedic Surgery, Massachusetts General Brigham, Boston, MA, USA

Correspondence: Ian D Hutchinson, Division of Sports Medicine, Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, Tel +1 781-487-6166, Fax +1 781-487-6826, Email [email protected]

Abstract: Despite significant advances in the understanding and delivery of osteosynthesis, fracture non-union remains a challenging clinical problem in orthopaedic surgery. To bridge the gap, basic science characterization of fracture healing provides a platform to identify and target biological strategies to enhance fracture healing. Of immense interest, Platelet-rich plasma (PRP) is a point of care orthobiologic that has been extensively studied in bone and soft tissue healing given its relative ease of translation from the benchtop to the clinic. The aim of this narrative review is to describe and relate pre-clinical in-vitro and in-vivo findings to clinical observations investigating the efficacy of PRP to enhance bone healing for primary fracture management and non-union treatment. A particular emphasis is placed on the heterogeneity of PRP preparation techniques, composition, activation strategies, and delivery. In the context of existing data, the routine use of PRP to enhance primary fracture healing and non-union management cannot be supported. However, it is acknowledged that extensive heterogeneity of PRP treatments in clinical studies adds obscurity; ultimately, refinement (and consensus) of PRP treatments for specific clinical indications, including repetition studies are warranted.

Keywords: platelet rich plasma, bone regeneration, fracture healing, fractures, ununited

Introduction

Bone healing stands as a complex and pivotal process within the realm of orthopedics, carrying with it substantial clinical and financial burdens. In 2019, the global incidence of bony fractures stood at a staggering 178 million cases, underscoring the pervasive nature of this often-debilitating health concern.¹ While the majority of fractures heal successfully, approximately 5–10% of patients encounter a formidable obstacle: non-union.² The Food and Drug Administration (FDA)³ has defined a non-union as a fracture that persists at 9 months post-injury, exhibiting insufficient signs of healing over three consecutive months. This condition manifests in two distinct forms—hypertrophic non-union, due to inadequate stability at the fracture site, and atrophic non-union, attributed to a deficiency of fracture biology and bone healing.⁴ The management of fracture non-union is considered on an individualized basis and involves surgical strategies to optimize stability (and strain) at the fracture site while identifying and addressing any deficits in fracture biology manifested in the bone healing response [183]. The costs associated with non-union treatment can be staggering, with tibial non-unions, for example, incurring an approximate cost of $25,556 USD compared to $11,686 USD for tibial fractures without non-union complications.⁵

The physiological process of bone healing is intrinsically sophisticated, typically progressing through three stages: the inflammatory phase, where hematoma forms and inflammatory cells infiltrate the site; the reparative phase, which involves the formation of a soft callus that gradually mineralizes; and the remodeling phase, where the callus is replaced by mature bone tissue.⁶ Each of these stages is marked by a distinct cascade of cellular and molecular events, underscoring the potential for therapeutic interventions that can modulate these processes.

Historically, the treatment of bone injuries has evolved from rudimentary splinting techniques to advanced surgical interventions, reflecting a deepening understanding of bone biology and healing processes. In the quest to enhance bone healing, regenerative medicine has introduced several innovative therapies, of which platelet-rich plasma (PRP) therapy has emerged as a significant contender. PRP therapy, in comparison to other regenerative approaches like stem cell therapy or bone morphogenetic proteins (BMPs), offers a unique blend of autologous growth factors and cytokines, potentially reducing the risk of immune rejection and other complications associated with allogeneic or synthetic materials.⁷ Initially recognized for its role in tissue sealing as fibrin tissue adhesives, PRP subsequently garnered attention for its potential to emulate the initiation of the natural healing cascade.⁸ The rationale behind PRP therapy lies in its ability to release biologically active factors and adhesion proteins, offering the potential to stimulate the resolution of chronic pathological processes.⁹ Specifically, PRP is replete with growth factors such as Platelet-Derived Growth Factor (PDGF), Transforming Growth Factor-beta (TGF-β), and Insulin-Like Growth Factor-1 (IGF-1), which are critical mediators in the bone healing process.¹⁰ These growth factors and cytokines play key roles in regulating inflammation, angiogenesis, and osteoblastic activity, making them vital to the various phases of bone repair.^11–13

Despite promising pre-clinical data supporting the potential of PRP, clinical trials have yet to unequivocally demonstrate its benefits in bone healing. Moreover, the absence of a standardized PRP injection protocol(s) hinders efforts to generalize findings or collate the data of individual studies. Dosage and timing intervals remain uncertain, and the composition of PRP varies widely in terms of leukocyte and platelet count, growth factor concentration, and red blood cell contamination due to patient characteristics and the preparation kit used.^8,14 This heterogeneity in PRP formulations further complicates its clinical application to date, given the current absence of a universally accepted PRP injection protocol. Therefore, the aim of this narrative review is to provide a comprehensive platform to evaluate the evidence regarding the use of PRP for bone healing.

Biological Activity of PRP: Influence on the Bone Regeneration Process

The pursuit of optimal strategies for bone healing has driven the exploration of PRP therapy due to its ability to serve as a concentrated source of autologous growth factors and cytokines. Our current understanding of PRP’s biological activity in bone healing has been predominantly centered on three key aspects: inflammatory cytokines, growth factors, and angiogenic factors (Table 1). These factors orchestrate the intricate process of cell signaling, tissue regeneration, and angiogenesis during the bone healing process.

Table 1 Function of the Growth Factors and Cytokines Found in PRP on Bone Regeneration

Inflammatory Cytokines

The initial phase of bone healing is characterized by inflammation, a crucial process that dictates the subsequent stages of repair, and platelets in PRP have been shown to successfully modulate this inflammatory response.^10,15 Once activated, the platelets in PRP release a spectrum of inflammatory cytokines from their alpha-granules such as Interleukin-1 (IL-1), Interleukin-6 (IL-6), and Tumor Necrosis Factor-alpha (TNF- α).^13,16 These cytokines play pivotal roles in the initiation of fracture repair by recruiting immune cells and initiating a cascade of biochemical and cellular alterations that set the stage for subsequent stages of bone repair. IL-1 stands out as a main regulator of the initial inflammatory responses in bone healing. Its release at the fracture site follows a biphasic pattern, characterized by an initial peak during the onset of the fracture healing process, succeeded by a subsequent peak during the shift from chondrogenesis to osteogenesis in the phase of endochondral maturation.^17,18 This cytokine plays a multifaceted role, influencing the recruitment of immune cells to the injury site and initiating a multitude of cellular responses necessary to the bone healing process.^19–21 Additionally, TNF-alpha also follows a biphasic pattern in its expression during the healing process and plays a pivotal role in recruiting osteoblasts to the injury site.^17,18 These bone-forming cells are crucial for the synthesis of new bone tissue and studies^20–22 have indicated that both TNF-α and IL-1β recruit osteoblasts, highlighting their collaborative role in bone regeneration. Furthermore, IL-6 is a multifunctional cytokine involved in bone repair. Studies using IL-6 knockout mice²³ have shown that this cytokine plays a role in callus remodeling and mineralization, indicating its significance in the later stages of bone healing. Additionally, IL-6 has been implicated in recruiting osteoblasts, further contributing to bone formation.^24,25 Thus, the orchestrated release of inflammatory cytokines in the early phases of bone healing is crucial for initiating the repair process and may be able to be amplified through the application and activation of PRP.

Growth Factors

PRP’s effectiveness in bone healing can be attributed significantly to the rich assortment of growth factors contained in the alpha-granules of platelets. Of the numerous growth factors that have been defined in the literature,^26–29 the three that appear to play the most prominent role in bone healing include PDGF, TGF-β, and IGF-1.¹⁰ PDGF is a critical growth factor in PRP that plays a pivotal role in the early phases of bone healing by initiating several essential processes upon release from activated platelets. It stimulates revascularization, an essential step in bone repair, by promoting the growth of new blood vessels.^11,30 This improved blood supply may facilitate the delivery of oxygen and nutrients to the injury site, accelerating the healing process. PDGF also has a profound impact on collagen synthesis, a key component of bone tissue. It encourages the production of collagen, enhancing the formation of a robust extracellular matrix (ECM) essential for bone regeneration.^11,31,32 Moreover, PDGF can directly influence mesenchymal stem cells (MSCs), inducing their migration and osteogenic differentiation.^33,34 These MSCs are crucial for generating new bone tissue, making PDGF a potent stimulator of bone formation.

TGF-β is also abundantly present in PRP and holds a multifaceted role in bone healing. It functions by exerting both paracrine and autocrine effects, influencing various cell types involved in long-term healing, bone regeneration, and bone modeling.²⁹ One of TGF-β’s most crucial functions is its ability to initiate the signaling pathway of osteoprogenitor cells, which synthesize BMPs.¹² These BMPs have demonstrated the potential to play a pivotal role in regulating the expression of growth factors in bone and cartilage tissue, further promoting bone healing and regeneration.^35,36 TGF-β’s influence also extends to fibroblasts and pre-osteoblasts, stimulating the biosynthesis of type I collagen and fibronectin, supporting the formation of a robust ECM.^37–39 Additionally, TGF-β promotes the deposition of bone matrix, contributing to the early stages of bone repair.⁴⁰ Furthermore, it inhibits osteoclast formation and bone resorption, tilting the balance toward bone formation over resorption.⁴¹

IGF-1 is another significant component of PRP that plays a vital role in bone regeneration. This growth factor is deposited in bone matrix, endothelial cells, and chondrocytes and is released during the bone regeneration process.⁴² IGF-1 is responsible for orchestrating the complex interaction between bone formation and bone resorption. IGF-1’s presence in platelets influences osteoblasts and pre-osteoblasts, initiating osteogenesis and inhibiting the apoptosis of bone cells.⁴³ Additionally, IGF-1 affects the expression of mesenchymal collagen enzymes, decreasing their degradation and enhancing collagen synthesis within the ECM. This leads to improved structural integrity and strength in the newly formed bone tissue.⁴³ The growth factors found in PRP, including PDGF, TGF-β, and IGF-1, work synergistically to enhance bone healing. They promote angiogenesis, collagen synthesis, ECM formation, and osteogenesis, contributing to the regeneration and repair of bone tissue. These growth factors play distinct but interconnected roles, collectively facilitating the intricate process of bone healing and regeneration.

Angiogenic Factors

Angiogenesis plays a sustained role in delivering oxygen, nutrients, and precursor cells to the site of injury.^44–46 PRP has demonstrated the ability to serve as a potent facilitator of angiogenesis, promoting the formation of new blood vessels that are crucial for supporting the regenerative processes in bone repair. Among the angiogenic factors found within PRP, Vascular Endothelial Growth Factor (VEGF) stands out as a principal driver of neovascularization. VEGF is a signal protein and its primary function is to stimulate angiogenesis.⁴⁷ Upon the application of PRP to the bone defect site, the release of VEGF from platelets sets in motion a cascade of events. VEGF initiates a signaling cascade, acting as a potent mitogen and chemoattractant for endothelial cells, promoting their proliferation and migration to the area surrounding the bone defect.^48–50 Once recruited, endothelial cells start to organize into primitive vascular structures, sprouting and elongating to form capillaries that infiltrate the damaged tissue.^48,51 This neovascularization process serves two essential purposes in bone healing. Firstly, it ensures a continuous supply of oxygen and nutrients to the healing site, facilitating the metabolic demands of reparative cells. Secondly, it provides a conduit for the migration of osteoprogenitor cells and mesenchymal stem cells, which are crucial for the formation of new bone tissue.^10,52,53

While VEGF primarily influences the growth of new vessels, angiogenin, another angiogenic factor found in PRP, contributes to the development of collateral circulation, which can be particularly relevant in cases where the primary blood supply to a bone defect may be compromised.¹⁰ Enhanced blood supply increases the resilience of the healing process, ensuring that adequate resources are available to support the regenerative demands of the damaged bone tissue. In the intricate orchestration of bone healing, angiogenesis is a fundamental process that ensures the delivery of essential resources to the site of injury. PRP therapy, enriched with angiogenic factors such as VEGF and angiogenin, plays a central role in promoting neovascularization and collateral circulation. By stimulating the formation of new blood vessels and alternate circulation pathways, PRP creates an environment conducive to optimal bone regeneration.

Other Bioactive Factors

In addition to growth factors, PRP contains a diverse array of bioactive factors stored within the dense granules of platelets, including serotonin, histamine, dopamine, calcium, and adenosine.^54,55 These factors exert fundamental effects on the biologic aspects of wound healing, influencing inflammation modulation and cell function. In the context of PRP therapy, serotonin, histamine, and dopamine contribute to wound healing by increasing capillary permeability. This effect facilitates the influx of inflammatory cells to the site of injury, promoting an initial immune response and the activation of macrophages.^56,57 Furthermore, calcium is essential for blood clotting, and its release from platelet granules upon activation is crucial for the formation of a stable fibrin clot at the site of injury.⁵⁸ The clot not only prevents excessive bleeding but also provides a scaffold for cells involved in tissue repair to attach and proliferate. Additionally, adenosine receptor activation has been shown to modulate inflammation during wound healing, promoting an anti-inflammatory environment.⁵⁹ Attenuation of local inflammation may be beneficial in the early stages of bone repair, as it may help mitigate excessive inflammation and tissue damage.^10,59

PRP Separation: Optimizing Platelet Concentration

Over the past decade, substantial efforts have been devoted to refining PRP preparation techniques, with the aim of optimizing platelet concentration—a critical factor influencing its therapeutic effectiveness in bone healing. While numerous studies have demonstrated PRP’s positive effects on the differentiation and proliferation of human osteoblasts, at present, there exists no unanimous agreement on the ideal PRP dosage. Marx et al initially defined PRP as containing a minimum platelet concentration of 1,000,000 platelets/µL, however, the US Food and Drug Administration (FDA) mandates that PRP products must possess a minimum platelet concentration of 250 × 103/mL.⁶⁰ Several additional investigators^61–63 have also reported that a platelet concentration approximately two times greater than that found in peripheral blood positively affects osteoblast proliferation in vitro and significantly reduces bone healing time. However, Jovani-Sancho et al⁶⁴ reported that an optimal platelet concentration of four times that of peripheral blood was necessary for optimal results. Other studies^65–67 have indicated that concentrations below approximately 0.85 × 10⁹/mL had no significant effect on osteogenesis. In contrast, however, Choi et al⁶⁸ found that lower PRP concentrations, ranging from 1% to 5% of peripheral blood levels, stimulated the viability and proliferation of osteoblasts. Furthermore, it is crucial to exercise caution when considering platelet concentration, as adverse events have been observed at higher dosages.^69–71 Fernandez-Medina et al⁷² indicated that cell viability and migration assays demonstrated detrimental effects on human osteoblasts when the PRP concentration exceeded 60%. Similarly, Al-Hamed et al⁶¹ reported that platelet concentrations greater than 8.21 ± 0.4 × 10⁹/mL inhibited osteogenic proliferation and Graziani et al⁶⁵ observed that a platelet concentration approximately 3.5 times greater than that of native blood led to a reduction in cell proliferation. These findings underscore the complexity of determining the precise platelet concentration required for optimal bone healing, as different concentrations of PRP may produce varying effects.

Centrifugation separates individual cells within blood based on their individual density gradients, thus the overlaps and proximity of the density of platelets and leukocytes present the possibility of contamination (Figure 1). Similar to other indications, the optimal concentration of leukocytes within PRP for bone healing remains not fully understood. Proponents of incorporating leukocytes argue that the antimicrobial properties of WBCs could mitigate the risk of infection, particularly when PRP is utilized intraoperatively.^73–76 Moreover, studies by Zimmermann et al⁷⁷ have revealed that leukocytes in leukocyte-rich PRP (LR-PRP) contribute significantly to the increased variability of growth factors, such as PDGF-αβ, PDGF-β1, and VEGF, in comparison to leukocyte-poor PRP (LP-PRP). This suggests that the concentration of white blood cells can be manipulated to optimize growth factor levels, potentially influencing the healing process positively. However, critics of leukocyte incorporation argue that the existence of WBCs can result in immediate pain and discomfort post-injection, while their catabolic and proinflammatory attributes may adversely impact the process of articular cartilage recovery as a result of the increased release of proinflammatory cytokines.^78–80 Clinical investigations have further validated concerns regarding increased acute swelling and pain after intra-articular LR-PRP injection.^81,82 Nevertheless, it is noteworthy that both LR-PRP and LP-PRP have demonstrated statistically significant improvements in clinical outcomes. Recent research, however, has added to this debate by highlighting the importance of matching the type of PRP with the specific clinical context. The prevailing evidence suggests that the choice of leukocyte concentration should be guided by the injection site.^73,83 For intra-articular applications, LP-PRP appears to be more beneficial, as indicated in the treatment of knee osteoarthritis. LR-PRP has shown adverse effects on synovial cells, resulting in cell death and proinflammatory mediator production.^81,84 In contrast, for the treatment of chronic tendinopathy, leukocyte-rich PRP has demonstrated superiority over leukocyte-poor PRP.⁸⁵

Figure 1 Density Gradients of Cells Contained within Blood Aspirate.

Notes: (A) Platelets, (B) Monocytes, (C) Lymphocytes, (D) Basophils, (E) Neutrophils, (F) Erythrocytes, (G) Eosinophils.

Unfortunately, a lack of universal preparation standardization and compositional reporting hampers our ability to collate data from individual studies and to gain consensus on findings. In most cases, blood is drawn from a patient, treated with an anticoagulant, and then centrifuged within an hour of collection. The methods then employed for isolating platelets and growth factors from whole blood can be broadly categorized based on two different distinctions: plasma vs buffy coat-based systems and single-spin vs double-spin processes. Plasma-based systems utilize a slower, shorter spin to isolate plasma and remove WBCs, resulting in platelet 2–3x’s baseline levels (Figure 2A). ⁶⁹ Contrary, buffy coat-based systems utilize a longer, double spin to isolate a platelet-poor layer (Figure 2B). This allows for an obtained platelet concentration of 3–8x that of baseline levels, however, because of the density it also keeps a concentration of WBCs.⁶⁹ Furthermore, single-spin processes, represented by many clinically used commercial devices, encompass variations such as low-platelet PRP (PRPLP) and high-platelet PRP (PRPHP). These one-step methods offer a more straightforward and less resource-intensive approach to PRP preparation. Conversely, double-spin processes (PRPDS) have historically been favored in basic science investigations due to their ability to produce PRP with higher platelet concentrations.⁸⁶ These methods often involve two sequential centrifugation steps, allowing for the separation of platelets from other blood components more effectively. However, recent studies have provided conflicting insights into the strengths and limitations of single-spin and double-spin processes. Notably, Mazzocca et al⁸⁷ demonstrated that PRPHP produced significantly higher platelet and white blood cell concentrations compared to both the single-step PRPLP and two-step PRPDS procedures. However, no significant differences were observed between PRPLP and PRPDS. Conversely, Saqlian et al⁸⁸ and Nagata et al⁸⁹ reported a greater platelet and WBC quantity following PRPDS compared to single-spin techniques. Additionally, when considering specific growth factors, Han Oh et al⁸⁸ demonstrated that PRPDS resulted in a significantly greater concentration of PDGF and VEGF whereas single-spin methods produced a significantly greater concentration of TGF and FGF. While findings by Mazzocca et al underscore the potential efficacy of one-step procedures and suggest that the increased time required for two-step procedures may not necessarily be advantageous for producing therapeutic PRP preparations, other studies still provide support to the historical superiority of PRPDS.

Figure 2 (A) Plasma-Based PRP Preparation. (B) Buffy Coat-Based PRP Preparation.

With the clinical advent of PRP use for bone and soft tissue indications, numerous commercial PRP preparation kits have entered the market. These devices offer the convenience of pre-packaged, standardized protocols, which can be especially beneficial for clinical applications. However, while designed to serve a common purpose, these kits exhibit noteworthy differences in multiple aspects of PRP preparation which manifests as variations in platelet, WBC, and RBC concentrations in the final PRP product. Numerous comprehensive reviews of currently available devices reveal substantial variability in their methodologies and the resultant PRP compositions. Dejnek et al⁸⁹ extensively evaluated four commonly used commercial PRP systems: Arthrex Autologous Conditioned Plasma (ACP), Mini GPS III, Xerthra, and Dr. PRP. Among the systems evaluated, Mini GPS III notably stood out, yielding significantly higher concentrations of platelets, WBCs, and RBCs compared to the other three systems. Additionally, in a systematic review of the 10 most referenced commercially available PRP systems, Oudelaar et al⁸³ found significant variations in platelet and leukocyte concentrations. The highest concentration of platelets was produced by the Cascade system, while the lowest concentration of platelets was generated by the ACP system. Notably, the GPS III system exhibited a significantly higher concentration of leukocytes compared to other systems. Furthermore, the study reported that the GPS III and SmartPrep systems had the highest platelet enrichment factors, while the ACP, RegenPRP, and Cascade systems showed lower platelet enrichment factors. Furthermore, when analyzing 33 different commercial systems, Fadadu et al⁷³ found a significantly positive correlation between maximum centrifuge spin force, platelet concentration, and PDGF concentration, however, spin time demonstrated no significant relationships. Additionally, 3 of the 33 systems resulted in a platelet count less than that of whole blood. A review by Magalon et al⁹⁰ also demonstrated that of the 36 PRP preparation systems analyzed, 11 resulted in a final product made up of more RBCs than platelets. These findings emphasize immense variability in commercially available PRP preparation systems. Thus, the choice of a specific commercial device plays a substantial role in determining PRP composition and underscores the importance of selecting the most suitable system based on the intended clinical application. Despite the profound importance of optimizing PRP’s platelet concentration, the challenge of defining a singular optimal value is exacerbated by the variability in research methods employed by past studies.^87,91 Consequently, tailoring PRP to individual clinical contexts remains a dynamic process that considers the nature of the injury, the patient’s unique characteristics, and the desired treatment outcomes.

Activation of PRP: Unleashing the Healing Potential

The activation of PRP is an important phase in its therapeutic application, as it serves to transform concentrated platelets into a biologically active state, primed to effectively stimulate the regenerative process. This activation process encompasses two key elements. Firstly, it involves the degranulation of platelets, liberating GFs from α-granules. Secondly, it triggers the cleavage of fibrinogen, initiating matrix formation—a clotting process that facilitates the development of a platelet gel, effectively constraining the secretion of molecules to the designated site.^92,93 Consequently, the choice of activator during PRP preparation becomes a critical determinant of its efficacy, influencing both the quantity and release kinetics of GFs from platelets within PRP. Research on the activation of PRP has unveiled a complex interplay of factors that significantly influence its clinical efficacy and therapeutic potential and activation methods have undergone significant development, with multiple techniques devised to unlock the potential of growth factors and other bioactive molecules.

One traditional method of activation involves the addition of bovine thrombin to PRP. Thrombin serves as a rapid activator of platelets, promoting degranulation and facilitating the conversion of fibrinogen into fibrin, resulting in the formation of a stable clot that effectively traps platelets at the target location.⁹⁴ This entrapment promotes immediate degranulation and the release of growth factors and cytokines. Prior studies have demonstrated that the rapid action of thrombin resulted in an immediate release pattern of approximately 70% of stored growth factors within 10 minutes and nearly 100% released within 1 hour.^29,95 While this method offers swift and substantial growth factor activation, it comes with a challenge—released growth factors are swiftly cleared, precluding their long-term stimulatory effects on cells. This concept has been supported by studies that have found that the rapid activation triggered by bovine thrombin results in a reduction in the overall quantity of growth factors accessible at the tissue location over time.^69,93 If not promptly utilized upon release, GFs risk degradation before additional tissue receptors become available.^95,96 Additionally, bovine thrombin has been shown to carry the potential for complications arising from the generation of antibodies that may result in immune-mediated coagulopathy.⁸⁶

An alternative approach utilizes calcium chloride to convert autologous prothrombin to thrombin, resulting in platelets being trapped in a fibrin matrix. Numerous studies^97,98 have demonstrated that using calcium chloride as an activator can lead to higher concentrations of specific GFs, such as TGF-β(1) and PDGF-AB. Additionally, calcium-based activators have been shown to induce a gradual and extended process of platelet activation, leading to the progressive release of platelet content.^93,95 This sustained activation results in the gradual accumulation of endogenous thrombin, facilitating a slower and more extended release of growth factors spanning several days.⁹⁹ Consequently, this extended-release pattern addresses the need for sustained growth factor delivery necessary for the prolonged nature of bone regeneration.^100–102 Additionally, calcium chloride activation was observed to have advantages beyond its release kinetics. Zhuang et al⁹⁷ demonstrated that calcium chloride can avoid the complications related to antibody formation and immune-mediated coagulopathy associated with bovine thrombin due to the autologous formation of thrombin from prothrombin. However, calcium chloride activation is not without potential shortfalls as well. An excess of calcium has been shown to trigger the swift activation of the clotting cascade, inducing rapid degranulation.¹⁰² Additionally, elevated calcium levels may enhance the activity of protein C, protein S, and antithrombin III, potentially destabilizing the fibrin clot and consequently shortening the therapeutic window for platelets.^102,103

Fufa et al¹⁰⁴ initially proposed the concept of Type-1 collagen as a safe and effective alternative to bovine thrombin for clot activation in PRP. Their initial findings supported this notion by demonstrating a reduction in clot retraction and comparable levels of PDGF-AB and VEGF release. However, recent research has cast some uncertainty on its efficacy. While numerous studies^8,95 have observed a more sustained cytokine release pattern with Type-1 collagen compared to bovine thrombin, a contrasting perspective emerged from Cavallo et al,⁹³ indicating that collagen’s platelet-activating capabilities were relatively weak, leading to a lack of clot formation and notably lower GF release compared to bovine thrombin and calcium chloride. While this approach holds promise, further investigations are imperative to elucidate the genuine activation potential of Type-1 collagen in PRP applications.

In some cases, PRP may be applied without exogenous activators. During local infusion, the presence of the natural clotting factor, thrombin, often suffices to activate platelets effectively.^105,106 This simplified activation process, however, may lead to variations in growth factor release contingent upon the specific clinical context. Additionally, in a recent meta-analysis⁹⁹ comparing activated and non-activated PRP, it was observed that non-activated PRP did not yield any substantial clinical improvements in terms of pain relief or functional scores when compared to a placebo. The choice of activator, whether it be calcium chloride, thrombin, collagen, or others, has a profound impact on clot formation, release kinetics, and the therapeutic potential of PRP. Understanding these factors is essential for tailoring PRP preparations to specific clinical needs and optimizing their effectiveness in various medical applications. In the context of bone healing, achieving sustained and controlled release of growth factors is often desirable as this aligns with the gradual and intricate nature of bone regeneration. Ongoing research continues to shed light on this dynamic field, enhancing our ability to harness the therapeutic potential of PRP for improved patient outcomes.

Delivery: Tailoring Application Methods

The manner in which PRP is delivered to the target site also plays a role in optimizing PRP’s therapeutic potential for bone healing. Clinically, PRP is often given through direct injection, topical application, or in combination with a surgical procedure and remains a widely employed and versatile clinical delivery method for bone healing. By injecting PRP directly into the affected site, clinicians can promote a concentrated release of growth factors precisely where they are needed most. Thus, this method expedites the regenerative process by providing a high concentration of growth factors directly to the injury site. Precision of delivery to the targeted tissue using ultrasound may also enhance clinically efficacy.^107–109 Additionally, topical application of PRP has gained recognition as an effective clinical approach for surgical or wound site(s), promoting tissue repair, reducing inflammation, and accelerating the healing process.^110–112 Furthermore, in addition to standalone PRP delivery, clinicians frequently combine PRP with surgical procedures involving bone grafts. This approach aims to optimize the integration of graft materials and enhance the overall success of the surgical intervention. For instance, PRP may be mixed with bone graft materials such as autografts, allografts, or synthetic grafts before implantation. However, this combination has shown contradicting effects on the enhancement of the graft’s osteogenic potential and ability to accelerate bone healing and reduce the risk of graft rejection.^113–116

To address the need for sustained growth factor release, researchers have employed scaffolds as delivery vehicles for PRP. In the context of bone healing, scaffolds can play a critical role in maintaining the integrity of the injury site, preventing migration of PRP, and enabling controlled and sustained release of growth factors.¹¹⁷ Scaffolds such as hydrogels, sponges, and nanofiber-based structures offer the ability to tailor the release kinetics of PRP-derived growth factors.¹¹⁸ Thus, the choice of scaffold material can influence factors like degradation rate, which, in turn, affects the release profile of PRP components. Hydrogels and sponges, composed of materials such as alginate and gelatin, have demonstrated their efficacy as delivery systems for PRP in bone regeneration. These systems offer the advantage of tailorable scaffold degradation, which affects the release of incorporated factors, making them ideal for sustained delivery and enhanced bioavailability of growth factors at the injury site.¹¹⁹ In support of this, Lin et al¹²⁰ incorporated PRP into an alginate hydrogel, demonstrating that the growth factors released from the hydrogel stimulated the osteogenic differentiation of human MSCs in vitro. Lu et al¹²¹ further investigated the growth factor release kinetics of PRP-incorporated alginate hydrogels, showcasing the varying release profiles based on carrier type and the potential of these factors to promote osteoblast-like cell proliferation and activity. In addition to alginate, gelatin, a denatured collagen derivative, has gained attention as a base material for scaffolds in bone healing. Gelatin shares functional groups with collagen, the primary organic component of bone, yet is easier to obtain and less expensive making it an attractive option.^119,122 Animal studies by Hokugo et al^123,124 have demonstrated that PRP growth factors can be immobilized within gelatin hydrogels, leading to growth factor release correlating with hydrogel degradation. Such studies highlight the potential of hydrogels and sponges to offer controlled and sustained delivery of PRP-derived growth factors, contributing to enhanced bone healing both in vitro and in vivo. Incorporation of bioactive inorganic calcium phosphates, such as carbonated hydroxyapatite (CHA), into PRP-based scaffolds holds significant promise for bone healing and regeneration. Kaur et al¹²⁵ conducted a study in which they explored the combination of PRP and CHA, finding that this hybrid scaffold yielded significantly enhanced histological bone formation. This suggests that the integration of CHA into PRP delivery systems can enhance the osteogenic potential, potentially accelerating bone healing. Additionally, a study by Liu et al,¹²⁶ focusing on the inclusion of platelets in calcium phosphate cement, indicated promising outcomes for angiogenesis and osteogenesis. Furthermore, an animal study by Qiu et al¹²⁷ adds to the growing body of evidence supporting the positive impact of PRP in combination with calcium phosphate cement on bone regeneration by demonstrating favorable results in minipigs. These studies underline the versatile applications of PRP, especially when combined with calcium-based materials, in promoting both vascularization and bone tissue formation. PRP has also been covalently or ionically bonded onto plasma polymers, showcasing enhanced scaffold properties.¹²⁸ Specifically, it has been reported that the application of poly-ε-caprolactone (PCL) nanofibers coated with PRP substantially enhances the survival and growth of human MSCs.¹²⁸ These findings emphasize the diverse strategies available for optimizing PRP delivery systems and their potential to enhance bone healing through various approaches, including surface modifications and the development of novel biomaterials.

A Review of Pre-Clinical and Clinical Studies

Vitro Pre-Clinical Studies

This comprehensive review identified 24 in vitro studies that investigated the effects of PRP or a related derivative, on various cell types.^{31,32,61,67,72,129–147} The diverse spectrum of cell types included osteoblasts, fibroblasts, osteocytes, myocytes, tenocytes, human umbilical vein endothelial cells, bone marrow mesenchymal stem cells, marrow stromal cells, and human osteosarcoma cell lines. Among these studies, 14 (58%)^{32,61,67,129,136–140,142–144,146,147} of the 24 studies reported PRP increased cell proliferation, 7 studies (29%)^{67,132,134,135,139,141} reported PRP increased expression of bone-related genes and growth factors, and 5 studies (21%)^{31,132,143,146,147} reported PRP increased cell migration. Notably, several of these investigations highlighted the effects of PRP were dose-dependent with differing cell responses at different concentrations.^{61,67,72,142,144,146} In addition, 2 studies (8%)^140,141 provided evidence suggesting that PRP has the potential to facilitate osteogenic differentiation of pluripotent stem cells. Furthermore, 1 study (4%)¹³⁷ reported PRP’s ability to induce tubular formation in human umbilical vein endothelial cells (HUVECs). Another study (4%)¹⁴⁵ indicated an increase in osteoblast viability and adhesion following PRP exposure. Conversely, 2 studies (8%)^130,131 did not discern any notable impact of PRP on cell behavior. Lastly, 1 study (4%)⁷² found that concentrations of PRP exceeding 60% decreased cell viability and migration. A summary of the main details of all in-vitro pre-clinical studies can be found in Table 2.

Table 2 Effects of PRP on Cell Behavior in vitro

Vivo Pre-Clinical Studies

A total of sixty pre-clinical in vivo animal studies were identified which investigated the impact of PRP on bone healing.^{63,67,123,139,141,148–202} The animal models employed in these studies exhibited a notable variation in usage, with rabbits being the most commonly utilized model in 25 studies (42%), followed by rats in 12 studies (20%) and sheep in 8 studies (13%). Conversely, the less frequently employed animal models included goats in 2 studies (3%), pigs in 2 studies (3%), and mice in 1 study (2%). Regarding the bones studied, the tibia was the most frequently examined bone in 23 studies (38%), followed by the femur in 18 studies (30%) and the radius in 11 studies (18%). In contrast, the skull/forehead was among the least studied bone in 3 studies (5%), along with the fibula and metatarsal, each studied in 2 studies (3%).

Out of the 43 in vivo animal studies that incorporated scaffolds-based delivery methods, a variety of scaffold types were employed. Examples included calcium phosphate, bone autograft, bone allograft, gelatin hydrogels, titanium mesh, collagen, ceramic-coated hydroxyapatite, and coral. In several instances, studies compared the effectiveness of PRP delivery with and without a scaffold, with the most favorable outcomes generally observed when PRP was administered alongside a scaffold.^{123,150,154,161,168,175,176,178} Notably, the dose-dependent response of PRP observed in in vitro studies was also echoed in some of the in vivo experiments.^67,190

Of the 45 pre-clinical animal studies that evaluated radiographic bone healing, 36 studies (80%) reported improvements when PRP was employed, whereas 7 studies (16%) did not reveal any radiographic improvement, and 2 studies (4%) even indicated reduced radiographic bone healing. Similarly, out of the 58 pre-clinical animal studies assessing histopathologic bone healing, 43 studies (74%) reported positive outcomes when PRP was applied. Conversely, 13 studies (22%) did not detect any histopathologic improvement, and 2 studies (3%) reported reduced histopathologic bone healing in association with PRP. Biomechanical properties also displayed favorable trends, with 11 out of 13 pre-clinical animal studies (85%) reporting improvements in bone healing when PRP was employed. Only 2 studies (15%) did not observe any biomechanical improvement, and none indicated a reduction in biomechanical properties when PRP was used.

In summary, pre-clinical in vivo animal studies generally demonstrate overall positive effects of PRP on bone healing. However, the substantial variability in study designs and protocols makes direct comparisons challenging. Moreover, several studies combined PRP with other factors like stem cells or scaffolds, complicating the isolation of PRP’s specific effects. Additionally, many studies compared interventions to untreated negative controls, which may not be ideal, and a few studies lacked control groups entirely. Furthermore, subjective evaluations and a lack of statistical comparisons were observed in several studies.^{152,154,155,158,167,171,198} Lastly, the use of blinded analysis of specimens was inconsistent, with only 20 studies (33%) reporting its implementation. Table 3 presents a concise overview of the key information pertaining to all in-vivo pre-clinical studies.

Table 3 Effects of PRP on Bone Healing in Pre-Clinical in vivo Animal Models

Vivo Clinical Studies

There were 24 clinical studies that evaluated PRP to treat fractures in human patients (Table 4). ^203–226 Among these studies, 11 were prospective randomized control trials, 8 were prospective case series, and 5 were retrospective case series. The bones predominantly examined were the tibia in 9 studies (38%) and the femur in 5 studies (21%). In terms of PRP delivery methods, 11 studies (46%) utilized PRP injection alone at the injury site, while 9 studies (38%) incorporated PRP with a scaffold, such as bone graft. Four studies (17%) involved the injection of PRP in combination with other substances like bone marrow aspirate or stem cells. The average number of patients per study was 52±52, with a range of 10 to 200 patients, and an average follow-up period of approximately 16±15 months (range 2–72 months). Of the clinical studies, 19 (79%) reported favorable clinical outcomes associated with the use of PRP to improve bone healing. Three studies (13%) demonstrated equivocal outcomes, while two studies (8%) indicated negative effects of PRP on bone healing. As with the pre-clinical studies, there is considerable variability among clinical studies, making it challenging to draw direct comparisons between outcomes. Notably, 9 studies (38%) lacked a control group, rendering it impossible to draw definitive conclusions due to the absence of a comparative baseline. Additionally, 14 studies (58%) did not employ any form of randomization in patient assignment to different treatment types within the study. It is worth noting that, to the best of our knowledge, there are no published double-blinded randomized control trials of PRP in the context of bone healing. Considering the existing body of research, which encompasses a reasonable number of patients and follow-up periods, future clinical investigations should prioritize the use of double-blinded randomized control trials to ascertain the true efficacy of PRP in promoting bone healing.

Table 4 Effects of PRP on Bone Healing in Clinical Studies

Conclusion

Recent evidence gathered in this extensive review of in vitro pre-clinical, in vivo pre-clinical, and clinical studies underscores the growing significance of PRP as a valuable adjunct in the domain of bone healing. In vitro investigations have demonstrated PRP’s potential to stimulate various cell types, promoting proliferation, gene expression, and migration, thereby substantiating its regenerative potential at the cellular level. Pre-clinical animal investigations, despite the inherent diversity in experimental models and methodologies, affirm the positive impact of PRP on radiographic, histopathologic, and biomechanical aspects of bone regeneration. However, the landscape of pre-clinical research reveals the complexity of direct comparisons due to the variable inclusion of adjunctive factors. In the clinical arena, a majority of studies extend support for the beneficial role of PRP in bone healing yet emphasize the demand for more rigorous methodologies to delineate its precise therapeutic potential. Furthermore, investigations delving into dose-dependent PRP effects and the differentiation between PRP formulations concerning platelet concentration and leukocyte content also represent areas meriting further exploration.

Overall, PRP has emerged as a promising adjunctive tool in the context of bone healing, offering multifaceted advantages that encompass augmented cellular responses, accelerated tissue restoration, and potential expedited rehabilitation. However, advancing its integration into evidence-based medical practice necessitates meticulous and standardized clinical investigations, encompassing larger and more diverse patient cohorts, and employing well-defined outcome measures. These endeavors are poised to deepen our comprehension of PRP’s therapeutic implications, particularly in the dynamic field of regenerative medicine, offering renewed optimism for individuals seeking enhanced musculoskeletal recovery.

Abbreviations

PRP, Platelet-Rich Plasma; LR-PRP, Leukocyte-Rich PRP; LP-PRP, Leukocyte-Poor PRP; PRPLP, Low-Platelet PRP; PRPHP, High-Platelet PRP; PRPDS, Double-Spin PRP; FDA, Food and Drug Administration; GF, Growth Factor; IL-1, Interleukin-1; IL-6, Interleukin-6; TNF-α, Tumor Necrosis Factor-alpha; PDGF, Platelet-Derived Growth Factor; TGF-β, Transforming Growth Factor-beta; IGF-1, Insulin-Like Growth Factor-1; ECM, Extracellular Matrix; MSCs, Mesenchymal Stem Cells; BMPs, Bone Morphogenetic Proteins; VEGF, Vascular Endothelial Growth Factor; WBC, White Blood Cell; ACP, Autologous Conditioned Plasma; CHA, Carbonated Hydroxyapatite; PCL, Poly-ε-Caprolactone; HUVEC, Human Umbilical Vein Endothelial Cell.

Disclosure

ADM disclosures include research support and consulting with Arthrex Inc., Naples, FL, as well as consulting and stock interests in Restor3d. All other authors report no conflicts of interest in this work.

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