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Biology and Enhancement of Skeletal Repair
Introduction
The rapid development of contemporary fracture fixation devices has occurred as the concepts of minimally invasive surgical techniques have advanced. Percutaneous instrumentation, the refinement of insertion portals, and the development of blocking screw techniques have expanded the range of intramedullary (IM) nailing from midshaft injuries to the articular margins. Locking plates have extended the role for periarticular fracture fixation using minimalistic exposures. Fully implantable limb lengthening nails and IM devices with bone transport capabilities are coming on line, and these devices will further define the ability to perform complex deformity correction, limb lengthening procedures, and bone transport, all without the need for cumbersome and patient-demanding external fixation devices.
Similarly, the understanding and use of growth factors and other biologic adjuvants for the treatment of long bone fracture healing, subchondral defects, and nonunion repair continue to evolve. It is increasingly important that the orthopaedic surgeon be familiar with the biologic characteristics and clinical application of these therapies. Multiple materials have become available to enhance fracture healing through a myriad of biologic pathways. The purpose of this chapter is to review the basic fund of knowledge on the topic of orthobiologic surgical adjuvants and the newer arena of orthobiologic manipulation by pharmacologic agents, and to provide a comprehensive review of the use of these currently available adjuvants for approved clinical use. The current levels of evidence supporting the use of these various materials will be reviewed if available.
Biology of Graft Substitutes
The biology of bone grafts and their substitutes is appreciated from an understanding of the bone formation processes of osteogenesis, osteoinduction, and osteoconduction.
Graft Osteogenesis
Graft osteogenesis is the ability of cellular elements within a donor graft, which survive transplantation, to synthesize new bone at the recipient site. Osteogenesis can occur in two ways. Surface osteoblasts can survive the transplantation by receiving nutrition through diffusion at the recipient site, and then proliferate to form more living bone tissue. There is more surface area in cancellous graft, and thus it has more potential for surviving cells than a cortical graft. Likewise the transplantation of marrow elements alone has demonstrated this ability to survive and form bone.
The ability to provide a threshold concentration of viable bone-forming elements presents a viable mode of intervention for the treatment of fractures or nonunions provided the recipient site provides the appropriate biologic environment to allow these cells to produce their osteogenic stimulus.
These transplanted cells or circulating pluripotential cells must have the appropriate substrate to attach to, or become attached to, once the cells have localized to the site of defect or injury. This substrate site for cellular attachment has to have the appropriate three-dimensional architecture to allow for these cells to proliferate. Subsequently, these cells then use this substrate as a scaffolding through which to build bone. This three-dimensional process involves vascular proliferation and ingrowth of capillaries along the open spaces in the substrate and thus the porosity of these materials is critical. Attachment is then followed by the differentiation of cells into bone-forming cell types and the production and remodeling of bone then proceeds.
Providing a competent three-dimensional architecture for cellular attachment is the next area of intervention in a process termed graft osteoconduction.
Graft Osteoconduction
A major category of available graft substitutes for defect management intervention is to provide a lattice-like substrate material that will facilitate the attachment and proliferation of osteoblastic cellular elements. This is followed closely by vascular proliferation and ingrowth of capillaries along the open spaces in the substrate. The porosity of these materials is critical to facilitate the revascularization of these grafts. Attachment is then followed by the differentiation of cells into bone-forming cell types and the production and remodeling of bone then proceeds. The conductive substrate then undergoes incorporation into the developing bone or degradates by a chemical dissolution process. The incorporation kinetics and timing are material dependent as these materials have a host of properties that are unique to each individual substrate.
Graft Osteoinduction
As all skeletal tissues evolve from mesenchyme, undifferentiated mesenchymal cells make a genetic commitment to a particular cellular lineage early in the developmental or repair process. In the case of repair, some stimulus must signal the undifferentiated mesenchymal cells to differentiate along a chondro-osteogenic pathway. Before this differentiation into an osteogenic lineage, these cells are affected by multiple growth factors that provide chemotactic and mitogenic stimuli to these cells. They influence these cells to migrate, attach, and multiply at the locale, which provides a competent osteoconductive substrate as a site of cellular attachment. This phenomenon, known as osteoinduction, is defined as “a process that supports the mitogenesis of undifferentiated mesenchymal cells leading to the formation of osteoprogenitor cells with the capacity to form new bone.” Thus any material that induces this process could be considered to be an osteoinductive growth factor.
Growth Factors
Proteins secreted by cells that bind to receptors on target cells to carry out a specific action are known as growth factors. This action may be autocrine, paracrine, or endocrine. Autocrine signaling occurs when a cell secretes a growth factor that binds to that same cell, resulting in self-activation. Paracrine signaling refers to the secretion of growth factors to neighboring cells, where the signal only travels a short distance to act in a local fashion on other cells. Endocrine signaling occurs when a growth factor is released into the circulation and acts on a cell at a distant site. Once a growth factor binds to a receptor, the receptor is activated and undergoes a change in its conformation. This change in conformation activates an intracellular protein known as a transcription factor, which travels to the nucleus by various messenger factors and induces the expression of a new gene or set of genes.
This cascade of activity is jump-started by graft-derived growth factors such as transforming growth factor–β (TGF-β), insulin-like growth factors (IGFs) 1 and 2, platelet-derived growth factor (PDGF), and others. They influence these cells to migrate, attach, and multiply at the locale that provides a competent osteoconductive substrate as a site of cellular attachment. This phenomenon, known as osteoinduction, is defined as “a process that supports the mitogenesis of undifferentiated mesenchymal cells leading to the formation of osteoprogenitor cells with the capacity to form new bone.” Thus any material that induces this process could be considered osteoinductive.
Several aspects of this cascade are important to understand when considering the therapeutic potential of each individual growth factor. First, two different growth factors may have the same effect on a cell. Thus it is simplistic to assume that each growth factor works alone. These proteins work in concert with one another, and the homeostatic and regulatory mechanisms that serve to modulate the activity of these factors is just now beginning to be understood. Although each growth factor family has its own corresponding family of receptors, many of these receptors activate the same set of genes. Therefore different growth factors may lead to the same cellular response. Many growth factors display pleiotropic activity in that a single growth factor may elicit a variety of different effects in the same cell, in the same cell at different stages of development, or in different cells. This is due to the fact that the signals transmitted to the nucleus frequently activate several genes at once. This has important clinical implications, as growth factors may also activate deleterious cellular effects in addition to their advantageous ones.
The temporal relationship of growth factor action is also dependent on the timing of application of the particular adjuvant, as well as the dose response that is variable depending on the timing of the factor application. Many of these factors act within a specific stage of fracture healing. The concept of osteoinductive new bone formation is realized through the active recruitment of host mesenchymal stem cells (MSCs) from the surrounding tissue, which differentiate into bone-forming osteoblasts. This process is facilitated by the presence of growth factors within the graft, principally bone morphogenetic proteins (BMPs). However, many factors take part in the fracture healing cascade before the potent action that the BMPs impart on the pluripotential cells.
It has been shown that the growth factors expressed during fracture healing include BMPs, TGF–β, fibroblast growth factor (FGF), and PDGF. On the basis of these findings, these growth factors have been most extensively studied in the context of fracture healing. The use of each of these growth factors in the treatment of fracture healing is discussed indivually.
Growth Factor Effects on the Phases of Fracture Healing
Fracture healing is reviewed in Chapter 4 ; the particular growth factors that affect each stage of fracture healing are reviewed here. Many of these factors are now available for clinical application, and it is important to understand their locale of intervention and overall mode of action.
Platelets found in the hematoma degranulate, releasing various signaling molecules that are contained within each platelet. These include factors such as TGF-β and PDGF. These cytokines and signaling molecules are involved in the processes of chemotaxis and angiogenesis and also regulate cell proliferation and differentiation of the cells that have migrated to the site of the fracture. The TGF-β family of growth factors, which includes all of the TGF-βs, BMPs, and growth and differentiation factors (GDFs), controls a number of processes during this initial phase of skeletal repair. BMP-2 most likely induces both chondrogenesis and periosteal osteogenesis, leading to the initiation of the endochondral healing response and intramembranous ossification.
The inflammatory phase of fracture healing is characterized by neovascularization and ingrowth of proliferative blood vessels. In addition to this oxygen-rich environment, it is vitally important that the mesenchymal cells that have recently migrated to the site of injury have a suitable osteoconductive surface for their implantation.
Cellular attachment to the extracellular matrix (ECM) and conductive substrate is a basic requirement for fracture healing. This is accomplished with integrins, which are transmembrane receptors that are the bridges for cell-cell and cell-ECM interactions. Essentially, they act as “glue” to allow the attachment of cells to the conductive matrix.
Plasma fibronectin has been found in the fracture hematoma within the first 3 days after fracture. Its production by cells associated with the callus appears to be greatest in the earliest stages of fracture healing. This is consistent with its function to establish provisional fibers and attachments in the developing cartilaginous matrices.
When activated, integrins in turn trigger chemical pathways to the interior (signal transduction), such as the chemical composition and mechanical status of the ECM, which results in a response (activation of transcription) such as regulation of the cell cycle, cell shape, and/or motility; or new receptors being added to the cell membrane. Integrins give the cell critical signals about the nature of its surroundings. Together with signals arising from receptors for soluble growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), PDGF, and many others, they enforce a cellular decision on what biologic action to take, be it attachment, movement, death, or differentiation. Thus integrins lie at the heart of the early inflammatory process. The actual attachment of the cells takes place through formation of cell adhesion complexes, which consist of integrins and many cytoplasmic proteins, such as α-actinin. This cellular binding to a competent conductive substrate is necessary for the circulating inductive factors (BMPs) to then differentiate these cells into an osteoblastic lineage.
Factors Influencing the Inflammatory Phase
Arachidonic Acid Metabolism and the Action of Nonsteroidal Antiinflammatory Drugs
Arachidonic acid metabolism exerts complex control over many bodily systems; it is mainly involved in inflammation and immunity. Two families of enzymes catalyze fatty acid oxygenation of arachidonic acid and produce either prostaglandin or leukotriene factors, both of which can affect the inflammatory phase of healing with their actions. These actions include a direct role in the stimulation of platelet aggregation and vasoconstriction, both important for the activation and degranulation of platelets. As leukocyte chemotaxis is activated these factors may directly activate gene transcription of the attached MSCs.
The enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) metabolize arachidonic acid to prostaglandin G2 and prostaglandin H2, which in turn may be converted to various prostaglandins. The classical cyclooxygenase (COX) inhibitors prevent the production of prostaglandin products. They are not selective and inhibit all phases of the inflammatory process. The main COX inhibitors are the nonsteroidal antiinflammatory drugs (NSAIDs). However, the selectivity for COX-2 inhibition was the main feature of this newest member of this drug class. Because COX-2 is usually specific to inflamed tissue, there is much less gastric irritation associated with COX-2 inhibitors, with a decreased risk of peptic ulceration. The selectivity of COX-2 does not seem to negate other fracture healing side effects of NSAIDs. Specifically, COX-2 inhibitors and other NSAIDs in general should be considered a potential risk factor for fracture healing and therefore be avoided in patients at risk for delayed fracture healing. It appears as though the temporal time for their action is during the inflammatory phase of fracture healing and thus this has been firmly established within the first 3 to 4 weeks of injury. Thus if possible these medications should be avoided during this time frame.
Wnt Pathway
Wnt signaling pathways are a group of signal transduction pathways made of proteins that pass signals from outside a cell through cell surface receptors to the inside of the cell. Wnt proteins form a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during development and adult tissue homeostasis. Three Wnt signaling pathways have been characterized. All three Wnt signaling pathways are activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, which passes the biologic signal to the protein Disheveled inside the cell. The canonical Wnt pathway leads to regulation of gene transcription and thus is important for the conversion of undifferentiated MSCs into an osteoblastic lineage that takes place under the influence of inductive factors during the inflammatory phase of fracture healing. Induction of the Wnt signaling pathway promotes bone formation, whereas inactivation of the pathway leads to osteopenic states. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway.
Sclerostin is produced by the osteocyte and has antianabolic effects on bone formation. It binds to the LRP5/LRP6/Frizzled coreceptor group inhibiting the Wnt signaling pathway. The inhibition of the Wnt pathway leads to decreased bone formation. Although the underlying mechanisms are unclear, it is believed that the antagonism of BMP-induced bone formation by sclerostin is mediated by Wnt signaling, but not BMP signaling pathways. Antibodies against endogenous antagonists, such as antisclerostin, have demonstrated promising results in promoting bone formation and increased callus size. Improved fracture healing rates are equivocal and more studies are needed in this regard. Sclerostin production by osteocytes is inhibited by parathyroid hormone (PTH) and cytokines, including prostaglandin E2. Sclerostin production is increased by calcitonin ( Fig. 5.1 ).
An antibody for sclerostin is available in clinical trials (antisclerostin ab). Its use has increased bone growth in preclinical trials in osteoporotic rats and monkeys. In a phase 1 study, a single dose of antisclerostin antibody (romosozumab) increased bone density in the hip and spine in healthy men and postmenopausal women and the drug was well tolerated. In a phase 2 trial, 1 year of the antibody treatment in osteoporotic women increased bone density more than bisphosphonate and teriparatide. Monthly treatments of the antibody for 1 year increased the bone mineral density of the spine and hip by 18% and 6%, respectively, compared with the placebo group. A recent study reported that postmenopausal women with osteoporosis who received 12 months of treatment with romosozumab followed by alendronate had a significantly lower risk of fracture than did those who received alendronate alone. However, the study also demonstrated an increased risk of cardiovascular adverse events and has not obtained full US Food and Drug Administration (FDA) approval. These results demonstrate the interplay that these signaling pathways have on bone formation and fracture healing, and in the future will likely be available for fracture augmentation.
Factors Affecting the Early Callus Phase
Platelet concentrate contains alpha granules, which contain more than 30 bioactive proteins, many of which have a fundamental role in hemostasis and/or tissue healing. These proteins include PDGF (including aa, bb, ab isomers), TGF-β (including β1 and β2 isomers), platelet factor 4, interleukin-1, platelet-derived angiogenesis factor, VEGF, epidermal growth factor, platelet-derived endothelial growth factor, epithelial growth factor, IGF, osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin, and thrombospondin-1. Of these, PDGF and TGF-β appear to have the most potent effect on the soft callus stage of healing (see Fig. 5.1 ).
TGF-β activates fibroblasts to induce collagen formation, endothelial cells for angiogenesis, chondroprogenitor cells for cartilage, and mesenchymal cells in an effort to increase the population of factors crucial to the propagation of the soft callus phase of healing.
The main function of PDGF is stimulating cellular replication (mitogenesis). This growth factor increases populations of healing cells, including MSCs and osteoprogenitor cells. PDGF also activates macrophages, resulting in débridement of the surgical or traumatic site. The macrophage activation then triggers a second source of growth factors released from the host tissues under the influence of the macrophage action. Recombinant PDGF has been approved by the FDA for use as an alternative to autograft for ankle and/or hindfoot fusion indications (Augment). This recombinant growth factor is combined with an osteoconductive beta–tricalcium phosphate (β-TCP)–collagen matrix, which is also injectable. Studies have shown that this material was an effective alternative to autograft for ankle and hindfoot fusions. These results from multiple studies demonstrated comparable fusion rates in these patients when using this material compared with autograft. Additionally, these patients had a marked decrease in pain and fewer side effects compared with the treatment groups where autograft was used for the fusion procedure.
Remodeling Phase
As osteoblastic formation of the woven bone takes place, subsequently the formation of more organized secondary bone also begins. The newly formed woven bone remodels to recapitulate the mature lamellar bone and restore the original cortical end plates and cortical structure. Although the major cell type responsible for fracture callus remodeling is the osteoclast, it is the interaction between osteoblastic and osteoclastic function that leads to successful remodeling.
Recent evidence suggests that cells in the osteoblastic lineage, including mesenchymal stromal cells, secrete factors that induce fully differentiated osteoblasts to express ligands that regulate the activity of osteoclasts. One of these, the receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL), has been shown to be essential in the development of osteoclast precursors and thus modulates this remodeling activity. This surface-bound molecule RANKL found on osteoblasts serves to activate osteoclasts, which are critically involved in bone resorption. Osteoclastic activity is triggered via the osteoblasts’ surface-bound RANKL activating the osteoclasts’ surface-bound receptor activator of NF-κB (RANK). A molecule that can block the binding of RANKL to RANK would successfully block the formation of osteoclasts.
When the hemopoietic mononuclear osteoclast precursor binds the osteoblast through the interaction of RANKL and RANK, these mononuclear cells are induced to fuse and form multinucleated osteoclasts with bone-resorbing capacity. Through the binding of RANKL, osteoclasts and osteoblasts play a vital role in normal bone remodeling. The osteocyte produces RANKL and sclerostin to control bone turnover and production in response to the mechanosensors in the osteocytes.
Studies show that RANKL is expressed at very low levels in unfractured intact bones but is strongly induced by a fracture to increase its activity. The coregulation of this process takes place through the action of macrophage colony-stimulating factor, which shows a peak in expression just before calcified cartilage removal begins (remodeling phase). Overproduction of RANKL is implicated in a variety of degenerative resorptive bone diseases, such as rheumatoid arthritis and psoriatic arthritis.
Factors Affecting the Remodeling Phase: New Venues for Effective Pharmacologic Intervention
Activation of RANK by RANKL promotes the maturation of preosteoclasts into osteoclasts. Denosumab is the first monoclonal antibody that binds to be approved by the FDA. Denosumab inhibits maturation of osteoclasts by binding to and inhibiting RANKL. This mimics the natural action of osteoprotegerin, an endogenous RANKL inhibitor, that presents with decreasing concentrations (and perhaps decreased avidity) in patients who are suffering from osteoporosis. This protects bone from degradation and helps counter disease progression. This has the effect of inhibiting the differentiation of the osteoclast precursor into a mature osteoclast. The first human monoclonal antibody for the treatment of osteoporosis, denosumab is approved for use by the FDA for postmenopausal women with risk of osteoporosis and fractures. Emerging data indicate that denosumab may also reduce the risk of osteoporotic fracture in patients with steroid-induced osteoporosis.
PTH is an 84-amino-acid polypeptide involved in the regulation of calcium and phosphate metabolism. Bone resorption is the normal destruction of bone by osteoclasts, which are indirectly stimulated by PTH. Stimulation is indirect because osteoclasts do not have a receptor for PTH; rather, PTH binds to the osteoblasts and indirectly stimulates these precursors to fuse, forming new osteoclasts, which ultimately enhances bone resorption. Although continual exposure to PTH leads to an increase in osteoclast activity and density, intermittent exposure stimulates osteoblasts more than osteoclasts and results in an increase in bone formation. PTH given in intermittent doses inhibits osteoblast apoptosis, thus prolonging the synthetic life span of this bone-forming cell (see Fig. 5.1 ).
Teriparatide is a recombinant form of PTH. Teriparatide is identical to a portion of human PTH, and intermittent use activates osteoblasts more than osteoclasts, which leads to an overall increase in bone. It is an effective anabolic (i.e., bone-growing) agent used in the treatment of some forms of osteoporosis.
Clinically, PTH has been approved by the FDA for its use in the treatment of osteoporosis in postmenopausal women and men. Several recent clinical trials have demonstrated that daily systemic treatment with PTH increases bone mineral density and reduces fracture risk in osteoporotic patients. It is also occasionally used off-label to speed fracture healing and treat nonunions. Numerous animal studies using PTH to augment fracture healing have consistently demonstrated increased fracture callus volume and enhanced mechanical properties as well as increased bone mineral density, bone mineral content, and total osseous tissue volume. These findings have supported the initiation of clinical trials to study the role of systemic administration of PTH in fracture patients.
Recent data using this as an adjuvant for fracture healing in patients with atypical femoral shaft fractures when given immediately after surgical repair suggest a trend for superior healing of these fractures and improved bone mineral density compared with those who received teriparatide in a delayed fashion. The number of patients was too small to achieve statistical significance. In the context of a patient who has experienced an atypical femoral fracture after receiving bisphosphonate treatment, therapy with teriparatide for 24 months increased bone mineral density and reduced the risk of additional fractures resulting from osteoporosis. Again, patient cohort size was too small to achieve significance, but these results do lend support for the adjuvant use of teriparatide after surgical repair of atypical femoral shaft fractures.
Atypical femoral shaft fractures have been shown to have a high association with chronic bisphosphonate therapy for osteoporosis. Nitrogenous bisphosphonates act on bone metabolism by binding and blocking multiple enzymatic pathways that are essential for connecting some small proteins to the cell membrane. Inhibition of these specific proteins can affect osteoclastogenesis, cell survival, and cytoskeletal dynamics. In particular, the cytoskeleton is vital for maintaining the “ruffled border” that is required for contact between a resorbing osteoclast and a bone surface. Thus inhibition of the ruffled border with subsequent dysfunction of resorption is the main osteoclast effect seen with this class of bisphosphonates. Unfortunately the biphosphonate inhibition of the ruffled border has long-lasting effects and thus simple discontinuation of this drug does not immediately reverse the osteoclast inhibition effects. Several animal models have shown increased callus volume, trabecular bone volume, and bone mineral content with bisphosphonate treatment but have also shown delayed maturation and remodeling of the callus. There are concerns that long-term bisphosphonate use can result in oversuppression of bone turnover. It is hypothesized that microcracks in the bone are unable to heal and eventually unite and propagate, resulting in atypical fractures.
Such fractures tend to heal poorly and often require some form of bone stimulation, for example, bone grafting as a secondary procedure. This complication is not common, and the benefit of overall fracture reduction still holds. However, with these uncertain effects on bone repair, their role in fracture healing is unclear. In cases where there is concern of such fractures occurring, teriparatide therapy is potentially a good adjuvant to assist in healing of these fractures due to its ability to reduce damage caused by suppression of bone turnover.
Graft Incorporation and Healing of Fractures
Fracture healing is a complex process in which a series of molecular and cellular events leads to the formation of new bone. This process requires osteogenic cells, osteogenic factors, and an osteoconductive scaffold under the proper mechanical stimuli and with adequate vascularization. The majority of fractures heal uneventfully, but it has been reported that 5% to 10% of fractures either fail to unite or demonstrate a delay in healing. Therefore alternative strategies designed to enhance fracture healing and to improve the treatment of delayed unions and nonunions are required.
Therapies aimed at enhancing fracture repair act through one of the following mechanisms: (1) directly increase the conductive substrate and available sites of competent cellular attachment, (2) increase osteoblastic activity, (3) indirectly recruit progenitor cells that will mature to osteoblasts, (4) inhibit osteoclastic activity, or (5) stimulate tissue revascularization. Growth factors, by virtue of their ability to regulate cell behavior, may influence a number of these processes.
All bone graft and bone-graft-substitute materials’ mechanism of action can be described through these processes. Although fresh autologous graft has the capability of supporting new bone growth by many of these mechanisms, it may not be necessary for a bone graft replacement material to have all the properties inherent in that material to be clinically efficacious ( Table 5.1 ). When inductive molecules are locally delivered on a scaffold, ultimately MSCs are attracted to that site and are capable of reproducibly inducing new bone formation provided minimal concentration and dose thresholds are met. In some clinical studies, osteoinductive agents have been shown to potentially perform superiorly compared with conductive materials alone.
Table 5.1
Available Fracture Healing Adjuvants With Their Inherent Properties
| Osteoconductive Scaffold | Osteoinductive Growth Factors | Osteogenic Living Cells | |
|---|---|---|---|
| Synthetic Ca + ceramics | • | ||
| Marrow concentrates | • | ||
| Bone morphogenetic proteins (BMPs) | • | ||
| Autograft | • | • | • |
| Platelet-rich concentrates | Osteopromotive indirect cellular effect No intracellular transcription |
||
| Banked demineralized bone matrix (DBM) | • | • |
When bone marrow aspirate (viable stem cells) is applied to an osteoconductive scaffold, these cells are still reliant on the local mechanical and biologic inductive signals to ultimately form bone. Similarly, osteoconductive materials work well when filling noncritical-sized defects that would normally heal easily without any additional adjuvants added. However, in more challenging critical-sized defects, all three types of these materials are necessary to achieve efficacy equivalent to autograft.
Stages of Bone Graft Incorporation
Bone grafts and bone graft substitutes incorporate via a well-defined pathway that can be divided into five distinct stages of host response, with the duration of each phase depending on the type of graft or adjuvant material used.
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Stage 1: Initial injury and hemorrhage. Initiates the pathway with the degranulation of platelets at the site of injury. (Platelet-derived factors.)
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Stage 2: Inflammation. Under the influence of many active cytokines that are produced at the site of injury. These cause migration of cells to the site of injury. (Growth factor intervention.)
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Stage 3: Vascular proliferation and ingrowth. Under the influence of many cytokines, invading capillaries bring perivascular tissue with mesenchymal cells that can differentiate into osteoprogenitor cell lines. (Autograft, marrow cellular therapies.) A competent conductive substrate is required for this process to occur. (Allograft and alloplastic conductive substrates.) This vascular invasion can be significantly inhibited by nonsteroidal antiinflammatory medications, which will inhibit this process and thus alter the fracture healing pathway. (NSAIDs.)
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Stage 4: The fourth stage consists of osteoclastic resorption of the avascular (dead) bone graft lamellae and simultaneous production of new bone matrix by osteoblasts. (Pharmacologic manipulation.)
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Stage 5: In the final stage, the newly formed bone is remodeled and reoriented based on the mechanical environment of the host site. (Pharmacologic manipulation.)
Orthobiologic interventions have been specifically designed to target these stages to achieve a specific effect. Each broad category of intervention will be discussed in terms of the desired effect these materials have on the specific stage of graft incorporation. The gold standard and prime material that all others are compared with is autogenous iliac crest bone graft (AICBG).
Urist and colleagues identified a protein that they termed bone morphogenetic protein (BMP). Other molecules were soon identified and helped to characterize an entire family of osteoinductive molecules. This family of specific factors now contains at least 15 BMPs and is part of the larger TGF-β superfamily of molecules.
Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract (BMP) that direct cartilage and bone formation, as well as the constitutive elements supplied by the animal during this process, have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expressions of the recombinant human proteins have been obtained.
Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-β supergene family, and the third, BMP-1, appears to be a novel regulatory molecule. BMP factors can be synthesized by recombinant gene technology or derived from autologous bone, allogeneic bone, or demineralized bone matrix (DBM).
DBM is allogeneic bone that has undergone the acid extraction of the mineralized ECM of the allograft bone. In theory, the noncollagenous proteins, including osteoinductive proteins such as the BMPs, remain viable while the structural portion of the allograft has been removed.
All bone graft and bone-graft-substitute materials can be described through these three processes. Although fresh autologous graft has the capability of supporting new bone growth by all three mechanisms, it may not be necessary for a bone graft replacement material to have all three properties inherent in that material to be clinically efficacious (see Table 5.1 ). When inductive molecules are locally delivered on a scaffold, ultimately MSCs are attracted to that site and are capable of reproducibly inducing new bone formation provided minimal concentration and dose thresholds are met. In some clinical studies, osteoinductive agents have been shown to potentially perform superiorly compared with conductive materials alone.
Autogenous Iliac Crest Bone Graft
Fresh cancellous autograft provides the quickest and most reliable type of bone graft. Its open structure allows rapid revascularization; a 5-mm graft may be totally revascularized in 20 to 25 days. These grafts depend on ingrowth of host vessels and perform best in well-vascularized beds. The large surface area of harvested autograft allows for survival of numerous graft cells. It is estimated that approximately 25 to 30 mL of graft can reliably be harvested from an anterior iliac crest. Cadaver studies also indicate that on average 35 mL is achieved from a posterior crest harvest and 18 mL on average is harvested from a proximal tibial site. However, many other sites of harvest have been described, differing only in the amount of graft obtained from each harvest site (see Table 5.1 ). Using volumetric computed tomography (CT) scans on healthy volunteers the amount of potential graft harvested from the pelvis has been estimated. It has been shown that more cancellous and total corticocancellous bone can be harvested from the posterior than the anterior iliac crest, together with similar or smaller volumes of cortical bone. Sex of the donor (not age) was an important factor in terms of the amount of bone than can be harvested, with a wide range of volumes individually from both iliac crests. Literature has demonstrated histologic differences between iliac crest and tibial bone graft, suggesting superiority of iliac crest in terms of osteogenic and hematopoietic progenitor cell content. Studies document success rates approaching 100% for subcritical-sized defects (1- to 2-cm defects) requiring 20 mL or less of autograft.
Many issues exist regarding AICBG due to the limited quantity available and the reported rates of postoperative pain from the graft harvest site. Substantial complication rates related to the harvest site have been reported. It has been thought that this technique is restricted to short defects in the range of 4 to 6 cm. Numerous studies report favorable union results for critical-sized defects up to 4 cm. However, in many of these studies multiple graft procedures were required to achieve solid union.
The ability to obtain substantial amounts of autogenous graft material would appear to be an advantage for the treatment of critical-sized defects. The reamer irrigator aspirator (RIA, Synthes; Paoli, PA) offers a technique to achieve substantial amounts of graft volumes for the treatment of larger segmental defects. The medullary canal of the femur or tibia is reamed with a device designed to collect the reamings and deliver them for potential grafting procedures. Variable amounts of harvested graft with this technique have been reported in the literature and range from 30 to 90 mL. A comparison between a historical control group using anterior iliac harvesting (40 patients) versus a study group using femoral shaft RIA harvesting (41 patients) documented on average 25 to 75 mL of harvested RIA graft (average, 40.3 mL). The authors reported a favorable union rate with RIA bone grafting (37 of 41 patients) versus AICBG (32 of 40 patients), although not statistically significant. There were significantly lower postoperative harvest site pain scores from the RIA group versus the AICBG group at 48 hours, 48 hours to 3 months, and greater than 3 months ( P = 0.001, 0.001, and 0.004, respectively). There were a total of two complications related to the graft harvest site in the RIA group (one perforation of the distal anterior femoral cortex treated conservatively and one excessive reaming of the femoral neck treated with prophylactic cannulated screws) versus 12 harvest site complications in the AICBG group (3 infections, 1 hematoma, and 8 patients with numbness).
This study has several limitations, including the concurrent use of bone morphogenetic protein-2 (BMP-2) in most cases. This somewhat limits the ability to draw strong conclusions regarding the relative efficacy of RIA bone graft versus AICBG from this study.
A study reported on the treatment of 20 bone defects ranging from 2 to 14.5 cm (average = 6.6 cm) using RIA bone graft. Eighteen of the 20 patients were initially treated with an antibiotic cement spacer using the Masquelet technique (see Fig. 5.1 ). The average graft volume obtained using the RIA was 64 mL. Seventeen of 20 bone defects ultimately healed, although 7 of these required repeat surgery. The authors reported no significant complications related to the bone graft harvest site ( Fig. 5.2 ).
Numerous basic science studies have demonstrated the biologic potential of RIA bone graft. Investigators have documented elevated amounts of osteoinductive growth factors and osteoprogenitor/endothelial progenitor cell types compared with AICBG. The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion. Medullary autograft cells harvested using RIA are viable and osteogenic. Cell viability and osteogenic potential were similar between bone grafts obtained from both the RIA system and the iliac crest.
Elevated levels of FGFa, PDGF, IGF-I, TGF-β1, and BMP-2 were measured in the reaming debris compared with iliac crest curettings. However, VEGF and FGFb were significantly lower in the reaming debris than from iliac crest samples. In comparing platelet-rich plasma (PRP) and platelet-poor plasma (PPP), all detectable growth factors, except IGF-I, were enhanced in the PRP. In the reaming irrigation FGFa (no measurable value in the PRP) and FGFb were higher, but VEGF, PDGF, IGF-I, TGF-β1, and BMP-2 were lower compared with PRP. BMP-4 was not measurable in any sample. The bony reaming debris is a rich source of growth factors with a content comparable to that from iliac crest.
The irrigation fluid from the reaming also contains growth factors. Proteins present in the RIA effluent water indicate the potential for clinical use of this filtrate as an adjunct for enhancing bone production, healing, and remodeling. This finding has encouraged the development of processing protocols for viable use of the effluent RIA waste water. Similarly cells cultured from RIA compared favorably to those from iliac crest bone grafts (ICBGs) with respect to their potential bone formation.
The use of the device has been associated with pathologic fractures, due to the eccentric reaming caused by malposition of the reaming guide wire. An eccentric canal can significantly compromise a long bone's torsional strength, more than if reamed concentric to a larger diameter having the same minimum wall thickness. This puts the shaft at risk for pathologic fracture with trivial forces. In addition significant perioperative blood loss remains a prevalent complication during RIA reaming, as well as persistent postoperative pain in the reamed extremity. A recent series found that 44% of patients undergoing RIA graft harvest required transfusion with a mean hematocrit drop of 113.7 across all subjects, which is significantly greater than that associated with ICBG.
Although the early evidence regarding RIA bone grafting is encouraging, there is currently a lack of high-level comparative evidence; however, a randomized controlled trial (RCT) comparing RIA grafting to ICBG revealed similar rates of union of the grafted bone, with significantly less donor site pain in the RIA patients (Level I). RIA also demonstrated a greater volume of graft compared with anterior ICBG and a shorter harvest time compared with posterior ICBG. For larger volume grafts, cost analysis favored using RIA, primarily related to the more invasive crest harvest with attendant longer operating room (OR) times.
A study published in 2010 compared the osteoprogenitor and endothelial progenitor cells obtained from RIA versus hip aspirate. Patients in both groups did not significantly differ regarding their age, gender, or preexisting health conditions. In comparison to aspirates obtained from iliac crest, the RIA aspirates from the femur contained a significantly higher percentage of CD34+ progenitor cells, a significantly higher concentration of MSCs, and a significantly higher concentration of early endothelial progenitor cells (EPCs). The results demonstrated that RIA aspirate is a rich source for different types of autologous progenitor cells, which can be used to accelerate healing of bone and other musculoskeletal tissues. The major flaw of this study was that for the clinical use of marrow aspsirates to be effective, a threshold concentration needs to be obtained. This study only evaluated the cellular types and found comparative numbers of active cells.
Osteoconductive Graft Substitutes
There is considerable interest in creating osteoconductive matrices using nonbiologic porous structures implanted into or adjacent to bone. The host substrate must mimic the cancellous bony architecture and have very specific surface kinetics to facilitate the migration, attachment, and proliferation of MSCs, which then differentiate into osteoprogenitor cells (augmentation of stages 2 and 3 of graft incorporation). Broad categories of these materials are available and in general are classified as calcium ceramics. These include the specific materials of calcium sulfate (CS), calcium phosphate, synthetic tricalcium phosphate and beta tricalcium phosphate, and coralline hydroxyapatite. All of these materials have been used as carriers for bone marrow aspirate concentrate (BMAC), all with comparable results.
The history of bioceramics dates back to 1892 with the use of CS for space-occupying lesions. CS has the distinction of being the alternative that is both one of the simplest and the one that has the longest clinical history as a synthetic bone graft material—spanning more than 100 years.
The original material was plaster of Paris, which is noninflammatory and nonreactive and encouraged bone healing in a contained lesion. Peltier took commercial-grade plaster of Paris, which was then mixed with water, poured into molds made of wax paper or aluminum foil, and then allowed to set forming small pellets or columns. He performed a series of bone defect studies in dogs to determine the role these materials had in the ability to heal these defects. From his experiments he determined that the plaster of Paris itself does not stimulate osteogenesis. Its chief effect was found to be a mechanical one of preventing the collapse of the periosteal tube and favoring regeneration. In this way the material provided a supportive scaffolding. There is no doubt, however, that subperiosteal resections in which plaster of Paris columns were inserted regenerated, in whole or in part, more frequently than was the case in subperiosteal resections alone.
Subsequently, coralline hydroxyapatite was reported for similar lesions followed by other ceramics. All of these bone graft substitutes have the advantage of being nonimmunogenic, noninflammatory, in an unlimited supply, and packaged sterile.
Calcium Sulfate Substitutes
CS was one of the first orthobiologic materials to be used commercially as a bone graft substitute. CS has a crystalline-independent rate of incorporation and is very consistent in terms of dissolution/incorporation. The crystalline structure is very consistent throughout a whole range of materials, with a very constant rate of osteointegration. In contrast to calcium phosphate, CS behaves as a true salt; that is, if it egresses into the joint it quickly dissolves into sulfate and calcium ions and is then absorbed into the synovium. This resorption mechanism involves a fluid exchange mechanism and may promote excessive drainage if used in wounds with questionable soft tissue coverage or integrity. A low but consistent complication rate, specifically serous drainage from the wound as the calcium sulfate absorbs, has been reported. This complication is higher when the material is used in higher volumes (greater than 20 mL) or in subcutaneous bones (tibia, ulna).
This may cause a potential increase in osmotic load at the site of implantation. Therefore to avoid subsequent drainage at the graft site, the CS product should be reserved for situations with adequate blood supply and competent soft tissue coverage. Additionally, this material should be used in contained defects only. Studies document that implanted CS pellets in contact with joint synovial fluid are at risk for resorption without any significant bony healing response. If CS pellets are to be implanted in periarticular locations, complete bony containment is necessary.
CS hemihydrate has been used for many years as a self-setting biomaterial due to its good setting properties. The fairly rapid degradation rate of these materials that occurs in 3 to 4 months was once viewed as an advantage. However, as these materials began to be used to support articular subchondral surfaces in cases of periarticular plateau and pilon fractures, this rapid degradation became a distinct disadvantage. Transition to full weight bearing occurs normally at 3 to 4 months postsurgery, and many cases of late articular collapse have been subsequently reported due to this rapid incorporation with simultaneous loss of articular support. Additionally, this material demonstrates rapid loss in its mechanical compressive strength after implantation, compared with the phosphate ceramics.
This combination of rapid degradation rate, speedy loss of compressive strength, and lack of bioactivity has limited its application for bone defect management. Three case series examining the use of calcium sulfate for the treatment of bone nonunion revealed a significant failure rate, suggesting that this material, used in isolation, is not optimal to promote union in that setting.
The current best use of this material appears to be that of a carrier for adjuvant antibiotics as a treatment for osteomyelitis. The characteristics regarding rapid resorption and degradation are now advantageous for delivering high dose antibiotics. McKee et al. demonstrated results for the treatment of chronic osteomyelitis and infected nonunions, using an antibiotic-impregnated CS pellet. Recent studies have evaluated the strategy of débridement, with removal of any implants, and excision back to bleeding bone. A composite of CaSO plus tobramycin preformed pellets (OSTEOSET T) was packed into a contained defect or the intramedullary canal with further bony stabilization and soft tissue reconstruction ( n = 7) undertaken as required. The average follow-up was 15 months. Union rate after tibial reconstruction was 100%. Wound complications were encountered in 52%: a wound discharge in the early postoperative period was noted in seven patients (33%) independent of site of pellet placement. This is thought to be due to an osmotic load as CS dissolves by chemical dissolution and acts like a salt in a fluid dynamic, as previously discussed.
In cases where the soft tissues may be incompetent, this material then leaks onto the surface and can be mistaken for infection. This is a sterile exudate from local wound fluid overload. Surgeons must be familiar with this common phenomenon if they use this material. The authors concluded that this material was an effective adjunct in the treatment of chronic tibial osteomyelitis after trauma. This was based on the low incidence of residual infection; however, wound drainage is an issue that must be recognized (Level III).
There is Level I and II evidence (one randomized trial, one case-control study, one prospective cohort study) that antibiotic-impregnated bioabsorbable calcium sulfate has the potential to reduce the number of procedures and surgical morbidity associated with the surgical treatment of chronic osteomyelitis and infected nonunion while maintaining a high rate of infection eradication. Calcium sulfate remains an inexpensive, safe, reliable bone void filler that can also serve as an absorbable delivery vehicle for antibiotics or other compounds.
In addition to antibiotic delivery, these materials have also been used recently for the management of dead space. This is a fundamental aspect of surgery. Residual dead space after surgery can fill with hematoma and provide an environment for bacterial growth, increasing the incidence of postoperative infection. Materials for managing dead space have historically included polymethylmethacrylate (PMMA), which is nonresorbing and usually requires removal in a second surgical procedure. The use of CS offers the advantage of being fully absorbed and does not require subsequent surgical removal. As calcium phosphate has historically been used as a bone void filler, there are some concerns for the risk of heterotopic ossification (HO) when implanted adjacent to soft tissue, and thus it may not be an ideal material for dead space management.
A recent study assessed the osteoinductive potential of CS and identified and characterized residual material present in muscle tissue using histology, energy-dispersive radiograph spectroscopy analysis, and scanning electron microscopy. CS beads with and without antibiotic were implanted in intramuscular sites in both athymic rats and New Zealand white rabbits. At 28 days after implantation no signs of osteoinduction or HO were observed. At 3 weeks after implantation near-complete bead absorption was confirmed and thus this material appears to be an excellent alternative to bone cement and may help avoid additional surgery for removal ( Fig. 5.3 ).
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