Growth Factors

Introduction

There have been significant advancements over the last several decades in the field of orthopedic surgery with regard to our understanding of biomechanics, tissue healing, and the pathogenesis of musculoskeletal diseases. Biologics and regenerative medicine have received significant attention over the last decade with an aim to accelerate the healing process and potentially reverse degenerative processes. As research continues into current technologies such as bone marrow aspirate, platelet-rich plasma, and adult stem cells, newer biologic treatment options are on the horizon. This chapter will review the biological basis for healing as well as current biological treatment options and indications for orthopedic injuries.

The Healing Cascade

Acute orthopedic injuries result from a single, traumatic event such as a fracture, muscle contusion, or ligament sprain/tear. Chronic bony and soft tissue injuries often result from repetitive mechanical stress, followed by a prolonged inflammatory state such as in the case of stress fractures or tendinopathies (e.g., rotator cuff tendinopathy and Achilles tendinopathy). Regardless of the type of injury, the general wound-healing process is shared, with differences in timing, duration of phases, and interactions between key mediators.

The general healing cascade involves four overlapping phases: (1) hemostasis; (2) inflammation; (3) cellular and matrix proliferation, which begins within days of an injury and comprises the most important phase of healing; and (4) wound remodeling, the longest phase, which may involve scar tissue formation. Immediately after injury, capillary leak allows for the recruitment of hemostatic factors and inflammatory mediators. The coagulation cascade is activated leading to platelet aggregation, clot formation, and development of a provisional extracellular matrix construct. Platelets adhere to exposed collagen and circulating extracellular matrix proteins, which triggers the release of bioactive factors from platelet alpha granules. These bioactive actors include growth factors, chemokines, and cytokines, in addition to proinflammatory mediators. The inflammatory phase follows in a highly orchestrated fashion. Chemoattractant agents begin to recruit neutrophils to the injured site within 1–2 hours in the early inflammatory phase. Later, macrophages appear in the wound and play the leading role in wound debridement and regulation of inflammation. They are also involved in recruiting fibroblasts and endothelial cells. The cellular and matrix proliferation phase is arguably the most important phase of wound healing because the cells involved serve as a metabolic engine driving tissue repair. After 2–3 days of wound healing, macrophages and chemotactic, mitogenic, and angiogenic growth factors recruit fibroblasts and epithelial cells to infiltrate the site of injury. Once in the wound, fibroblasts synthesize collagen and facilitate wound contraction. Angiogenesis and the formation of granulation tissue are also important aspects during the proliferative phase of healing. The final phase of the healing process involves wound maturation and remodeling. During this phase, growth factors such as platelet-derived growth factors (PDGFs) and transforming growth factor-beta (TGF-β), and fibronectin stimulate fibroblasts proliferation, migration, and synthesis of the components of extracellular matrix. The remodeling phase is tightly regulated to maintain the balance between degradation and synthesis. Type I collagen replaces type III collagen, proteoglycan, and fibronectin to form a more robust matrix with increased tensile strength. The maturation phase varies in duration depending on the extent of the wound pathology, individual characteristics, as well as specific tissue-healing capabilities of the tissue involved. In addition, pathophysiological and metabolic factors can affect wound healing. They include local causes such as ischemia, tissue hypoxia, infection, and growth factor imbalance, as well as systemic causes such as metabolic disease and nutritional status. In such unfavorable environments, PRP and other mediums rich in growth factors have been shown to be a viable therapeutic adjunct for acute and chronic orthopedic injuries.

Types of Growth Factors

PRP and other autologous blood products primarily function through the release of growth factors, such as PDGFs, epidermal growth factors, TGF-β1, vascular endothelial growth factor (VEGF), basic fibroblast growth factor, hepatocyte growth factor, and insulin-like growth factor 1 (IGF-1). These growth factors are released from the alpha granules of activated platelets and are involved in important cellular processes including mitogenesis, chemotaxis, differentiation, and metabolism.

VEGF has received significant attention given its critical role in the formation and maturation of new blood vessels at the site of injury during the healing process. For this, VEGF acts by binding to its receptors VEGFR-1, VEGFR-2, and VEGFR-3, activating signaling cascades that promote the migration, proliferation, and survival of endothelial cells.

PDGF has also been extensively studied due to the role it plays in regulating osteoblast replication and bone collagen degradation, controlling the proliferation of repair cells, and inducing cartilage and bone formation. As a multiple mitogen, PDGF is released by platelets and facilitates blood clotting via the adhesion between platelets and blood vessels. Previous studies demonstrated that PDGF was a stimulator for bone fracture healing and was responsible for bone metabolism processes, including cell proliferation, migration, and apoptosis. Nash et al. reported in a rabbit model that bone marrow cavity volume and bone mineral density were markedly increased after injecting an isoform of recombinant human PDGF into rabbits with tibial fractures, indicating that PDGF participates in the stimulation of fracture healing. In addition, human cartilage cells cultured with PDGF and TGF-β1 exhibited a significantly higher proliferation rate in an in vitro study by Brandl et al., suggesting that PDGF participates in the proliferation of chondrocytes and plays a role in the repair of cartilage tissue.

TGF-β1 has been found to play a significant role in the osseous and soft tissue–healing processes but may have detrimental effects on tissue healing and regeneration. TGF is a superfamily of proteins that primarily functions to enhance the proliferative activity of fibroblasts, stimulates biosynthesis of type 1 collagen and fibronectin, induces deposition of bone matrix, and inhibits osteoclast function/bone resorption. This family of growth factors also includes bone morphogenetic proteins, which function to maintain tissue homeostasis, stimulate bone and cartilage formation, and promote vascular remodeling. Although these attributes of TGF-β growth factors can enhance tissue repair, they can also lead to extensive tissue fibrosis. TGF-β has been implicated in the development of fibrosis in skeletal muscle, as well as other tissues, and may contribute to the association between PRP and muscle fibrosis through collagen deposition and conversion of skin fibroblasts to myofibroblast-like cells. Furthermore, TGF-β has been found to inhibit myogenic differentiation, myoblast fusion, and expression of various muscle-specific proteins. Owing to these characteristics of TGF-β, some have advocated the concomitant use of antifibrotic agents such as losartan or TGF-β neutralization antibodies with PRP injection ( Table 3.1 ).

Platelet Rich Plasma

Research into PRP has increased exponentially over the past decade with over 80 randomized controlled trials (RCTs) performed. This exponential increase in research coincides with a global market for PRP projected to exceed $451 million in the next decade.

PRP is classically described as a volume of plasma that has a platelet count more than that of whole blood. In clinical use, platelet concentration is increased 4- to 10-fold the baseline levels. Platelets are irregularly shaped, non-nucleated cytoplasmic bodies derived from fragmentation of megakaryocyte precursors. As discussed previously, platelets serve as a natural reservoir of growth factors and are released from the alpha granules of activated platelets. Therefore the rationale for creating and using PRP is to increase platelet concentration in injured tissue, resulting in the exponential release of multiple bioactive factors, and to subsequently enhance/stimulate the natural healing process.

To create PRP, autologous whole blood is collected in the presence of an anticoagulant that binds calcium and prevents the initiation of the clotting cascade by inhibiting the conversion of prothrombin to thrombin. PRP can be produced in the absence of an anticoagulant as well; however, the time required between blood draw and PRP injection must be significantly shortened. Although several anticoagulants are available, acid citrate dextrose-A, citrate phosphate dextrose, and sodium citrate are commonly used because of their ability to maintain the structural and functional integrity of platelets. Once the anticoagulated whole blood is collected, it is separated into its individual components using plasmapheresis, a 1- to 2-phase centrifugation process that separates blood components based on their size and density. Owing to their inherent morphologic differences, centrifugation separates the whole blood into a clear plasma layer on top, a buffy coat layer consisting of the white blood cells (WBCs) and platelets in middle, and red blood cells (RBCs) at the bottom. If a two-phase centrifugation process is used, the second phase is performed to separate the platelet-poor plasma from the platelet-rich fraction ( Table 3.2 ).

Although the term PRP suggests a mixture of only platelets and plasma, it encompasses a broader group of products that may also include RBC and/or WBCs. In addition, there can be significant variations in PRP preparations with regard to the volume of whole blood, concentration of platelets in plasma, amount of growth factors, volume of PRP, the presence and/or concentration of RBCs and/or WBCs, the presence of platelet activators, and pH of the solution. More than 40 commercial PRP systems are available, and each product may contain differing concentrations of platelets, leukocytes, and growth factors. Owing to this variation, the success of specific PRP products cannot be generalized to all preparations, limiting the ability to evaluate the clinical efficacy of PRP for various indications.

PRP can be activated via platelet activators such as thrombin or calcium chloride which results in a rapid release of the growth factors. Ninety percent of the growth factors contained within the platelets will be released within the first 10 minutes after activation. Many growth factors have very short half-lives, suggesting platelet activation should be performed at the time of injection or just before injection to be most efficacious. Owing to this, most commercial PRP kits for use in soft tissue do not activate PRP. Using unactivated PRP results in a more physiologic activation by the tissue into which it is injected or applied.