Fresh Fracture, Challenging Bone Repair, and Nonunions

J. Tracy Watson, MD, FAAOS

Dr. Watson or an immediate family member has received royalties from Arthrex, Inc., Biomet, NuVasive, and Smith & Nephew; is a member of a speakers’ bureau or has made paid presentations on behalf of NuVasive, Zimmer, and Smith & Nephew; serves as a paid consultant to or is an employee of Bioventus, Radius, and Smith & Nephew; and serves as a board member, owner, officer, or committee member of the American Academy of Orthopaedic Surgeons and the Orthopaedic Trauma Association.

INTRODUCTION

There are no well-defined indications for use of a specific type of bone graft substitute or use of bone growth adjuvants for the treatment of complex fractures or nonunion. With the widespread use of orthobiologics in everyday practice, attention must be directed to substantiate the evidence for their current use for the treatment of fractures or nonunions.

It is important to review the fracture healing cascade and where these materials have their locale of action. The available adjuvants and their indications for use are discussed with this context in mind.

FRACTURE HEALING CASCADE

Following initial trauma, the most common pathway for union is by endochondral bone formation. The inflammatory phase of fracture healing initiates this process via neovascular invasion. This response brings with it migrating pericytes that form undifferentiated mesenchymal stem cells (MSCs). Early callus formation is located adjacent to the fracture site and is initially a cartilage anlage developed from undifferentiated MSCs in the peripheral soft tissues that are recruited, proliferate, and differentiate into cartilage-forming cells.¹ These cells gradually become calcified and are replaced by bone (Figure 1).

Committed osteoprogenitor cells and undifferentiated MSCs derived from the periosteum contribute to the healing process via intramembranous bone formation that occurs on either side of the fracture.² Intramembranous ossification occurs at the periphery of the fracture ends and forms bone without an intervening cartilage phase, also referred to as hard callus (Figure 2).

Direct bone healing requires an anatomic reduction with absolute rigid stability limiting the strain at the fracture site. Direct healing allows the bone to immediately regenerate anatomic lamellar bone and new Haversian canals without any intervening remodeling steps.³ Gaps at the point of contact will still be present and gap healing takes place by recapitulating the fracture healing pathway within these small gaps. Primary cortical healing occurs to reestablish cortical continuity with the aid of so-called cutting cones. Osteoclasts in these cutting cones tunnel directly across points of contact and ultimately provide stability via a resorption process that reestablishes new Haversian systems. Progenitor cells follow and differentiate into osteoblasts and secrete osteoid to bridge the fracture gap.⁴ The callus is replaced by new osteons, requiring minimal participation from the periosteum, external soft tissues, and marrow elements.⁵

The inflammatory phase of fracture healing begins immediately after fracture with clot formation and hematoma development. This is accompanied by the invasion of macrophages, polymorphonuclear leukocytes, and lymphocytic cells.⁶

Platelets found in the hematoma degranulate, releasing various signaling molecules and cytokines involved in the processes of chemotaxis and angiogenesis. They also regulate cell proliferation and differentiation of the cells that have migrated to the site of the fracture.⁷ The inflammatory phase is characterized by neovascularization and ingrowth of proliferative blood vessels. The attachment of undifferentiated MSCs to the extracellular matrix and conductive substrates occurs through the formation of cell adhesion complexes, which consist of integrins and many cytoplasmic proteins, such as alpha-actinin.⁸ Cellular binding to conductive substrates is necessary for circulating inductive factors to stimulate the differentiation of these cells into an osteoblastic lineage⁸ (Figure 1). Thus, any material that induces this process should be considered osteoinductive.

Cellular elements and conductive substrates require each other’s presence to actively function as a viable adjunct for healing of skeletal tissues. Similarly, osteoconductive materials alone work well when filling non-critical-size defects in subchondral locations, but these materials require the migration osteoprogenitor cells into these matrices to incorporate and support the subchondral surfaces. For challenging critical-size defects, all three types of these materials are necessary to achieve efficacy equivalent to autograft.⁹,¹⁰

FIGURE 1 A, Image depicts the inflammatory phase and shows clot formation with platelet degranulation and initiation of neovascularization. B, Histologic slide (50×) from a fracture site demonstrating necrotic bone being resorbed and replaced by fibrous tissue. Empty lacunar spaces without osteocytes denote dead bone. Numerous multinucleate giant cell osteoclasts resorb dead bone spicules (black arrows) and small dark mononuclear inflammatory cells are present. Early vascular channels are developing with the early neovascularization process initiated (white arrows). C, The residual necrotic bone is resorbed and replaced by fibrous tissue and new vessel ingrowth. Fibrous tissue is undergoing early remodeling to early callus by osteolysis and orderly osteoblastic lining of the early collagen (white arrows). Osteoclast multinucleate giant cells can be seen (black arrow) in a receding osseous trabecula. D, Image depicts soft callus phase and shows proliferation of chondroprogenitor mesenchymal stem cells and differentiation into chondrocytes, with the expression of cartilage-specific matrix. Histologic slides show that at the fracture periphery, the cartilage cells have developed prominent nuclear and cytoplasmic vacuoles and appear as hypertrophic chondrocytes. The matrix between these swollen cells becomes calcified as the chondrocytes release calcium into the extracellular matrix. This forms a zone of provisional (preliminary) calcification (clear arrows). E, Soft callous phase (late): islands of young cellular cartilage (black arrows) have become mineralized with interlocking trabeculae of new woven bone rimmed by osteoblasts (black arrows). This intramembranous bone formation has intertrabecular stroma composed of cellular fibrocollagenous tissue (black arrows). Note the vascular channels (clear arrows) in this area of peripheral endochondral ossification.

FACTORS INFLUENCING THE INFLAMMATORY PHASE OF HEALING

Arachidonic Acid Metabolism

Arachidonic acid metabolism exerts complex control over many bodily systems, mainly involved in inflammation and immunity. The enzymes cyclooxygenase (COX)-1 and COX-2 metabolize arachidonic acid to prostaglandin G2 and prostaglandin H₂, which in turn may be converted to various prostaglandins. The classic COX inhibitors are the NSAIDs, which prevent the production of prostaglandin products. They are not selective and inhibit all phases of the inflammatory process, including fracture healing. Because COX-2 is specific to inflamed tissue, there is much less gastric irritation associated with COX-2 inhibitors. This selectivity of COX-2 does not seem to negate other fracture healing side effects of NSAIDs and should be avoided in patients at risk for delayed fracture healing.¹¹

Wnt Pathway

Wnt signaling pathways are a group of signal transduction pathways made of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell. These pathways are activated by the binding of a Wnt protein ligand to a Frizzled cell surface receptor. This leads to regulation of gene transcription and conversion of undifferentiated MSCs into an osteoblastic lineage.¹² Sclerostin
is produced by the osteocyte and binds to a co-receptor group inhibiting the Wnt signaling pathway,¹³ which leads to decreased bone formation. Antibodies against sclerostin have demonstrated promising results in promoting bone formation and increased callus size.¹⁴ An antibody for sclerostin (romosozumab) is available and in clinical use (antisclerostin antibody). Its use has demonstrated increased bone growth and increased bone mineral density of the hip and spine,¹⁵,¹⁶ but its effect on fresh fractures and nonunions is not known.

FIGURE 2 A, Image depicts mature callous phase and shows removal of calcified cartilage with remodeling of early calcified cartilage to secondary woven bone. B, Histologic image (magnification ×50) shows islands of young cellular cartilage (black arrows) have become mineralized with interlocking trabeculae of new woven bone rimmed by osteoblasts (white arrows). This intramembranous bone formation has intertrabecular stroma composed of cellular fibrocollagenous tissue (white arrows) C, Image depicts remodeling phase and shows formation of the woven bone with remodeling to form mature lamellar bone with cortical end plates and cortical structure. D, Histologic image (magnification x50) shows remodeling phase (mature callous). Osseous trabeculae reveal numerous lacunar spaces with prominent osteocyte characteristics of late woven bone in which the lining osteoblast population is prominent (clear arrows). The woven bone is being converted to mature lamellar bone with remodeling into cortical end plates and restoration of the marrow component.

Early Callus Phase

The process of chondrogenesis begins as the early callus phase of healing approximately 7 to 10 days after injury. The migration and proliferation of chondroprogenitor MSCs and differentiation into chondrocytes with the expression of cartilage-specific matrix occur. The formation of a cartilage callus provides immediate mechanical stability to the fracture. Within 2 weeks, protein synthesis is complete and hypertrophic chondrocytes release calcium into the extracellular matrix to precipitate with phosphate ions¹⁷ and calcification begins. Once enough cartilaginous callus is formed, mineralization occurs with the removal of the proteoglycan inhibitors,¹⁸ which prevent mineralization (Figure 1).

Platelet concentrate contains alpha granules that contain more than 30 bioactive proteins, many of which have a fundamental role in hemostasis and/or tissue healing. Of these, platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β) appear to have the most potent effect on the soft callus stage of healing.¹⁹

TGF-β activates fibroblasts to induce collagen formation, endothelial cells for angiogenesis, chondroprogenitor cells for cartilage, and MSCs in an effort to increase the population of factors all crucial to the propagation of the soft callus phase of healing. PDGF also activates macrophages, resulting in débridement of the traumatic site, which then triggers a second source of growth factors released from the host tissues.¹⁹,²⁰

Mature Callus Phase

Calcification of the cartilaginous callus matrix occurs as hypertrophic chondrocytes develop budding of their membrane structures to form vesicularized bodies. These matrix vesicles migrate to the extracellular matrix. The chondrocytes then undergo apoptosis and mineral is laid down on the callus surfaces.¹⁸

Neovascularization brings in perivascular cells and osteoblast progenitors, which infiltrate the calcified matrix surfaces.²¹ Chondroclasts invade along with bone-forming osteoblasts to begin the remodeling of the primary spongiosa (early calcified cartilage) to secondary spongiosa (woven bone), resulting in fracture union by approximately 4 to 5 weeks (Figure 2).

Remodeling Phase

As the formation of woven bone takes place, it remodels to mature lamellar bone and restores the original cortical end plates and cortical structure.²² Osteoblastic cells secrete factors that induce fully differentiated osteoblasts to express ligands that regulate the activity of osteoclasts. Receptor activator of nuclear factor kappa B ligand (RANKL) is essential for the development of osteoclast precursors²² because osteoclasts and osteoblasts play a vital role in normal bone remodeling. RANKL is strongly induced by fracture to increase its activity²² and demonstrates a peak in expression just before calcified cartilage removal begins (remodeling phase) (Figure 2).

Estrogen inhibits the formation and activation of the bone-resorbing osteoclasts via suppression of RANKL signaling within the osteoclast. Selective estrogen receptor modulators are ligands for estrogen receptors that induce these receptors to affect intracellular transcriptional modulators. Raloxifene is such a class of selective estrogen receptor modulator, mimics estrogen’s suppression of RANKL, and thus can also inhibit osteoclast formation. Denosumab is the first RANKL inhibitor to be approved by the FDA. Denosumab inhibits this maturation of osteoclasts by binding to and inhibiting RANKL, thus preventing the binding to RANK.

Parathyroid hormone (PTH) is a polypeptide involved in the regulation of calcium and phosphate metabolism. Bone resorption is caused by osteoclasts, which are indirectly stimulated by PTH. Because osteoclasts do not have a receptor for PTH, PTH binds to the osteoblasts, stimulating them to increase their expression of RANKL. PTH inhibits their expression of osteoprotegerin, which binds to RANKL and blocks it from interacting with RANK. The binding of RANKL to RANK stimulates these osteoclast precursors to fuse, forming new osteoclasts.²³ Although continual exposure to PTH leads to an increase in osteoclast activity and density, intermittent exposure stimulates osteoblasts more than osteoclasts and results in increased bone formation.

Teriparatide is a recombinant form of PTH identical to a portion of human PTH. It is an effective anabolic (ie, bone growing) agent²⁴ used in the treatment of some forms of osteoporosis. Clinically, recombinant PTH has been approved by the FDA for its use in the treatment of osteoporosis and several recent clinical trials have demonstrated that daily systemic treatment with PTH increases bone mineral density and reduces fracture risk in patients with osteoporosis. It is also used off-label to speed fracture healing and treat nonunions and has demonstrated increased fracture callus volume, enhanced mechanical properties, as well as increased bone mineral density, bone mineral content, and total osseous tissue volume.²⁵,²⁶,²⁷,²⁸,²⁹

Diphosphonates are widely used for the treatment of osteoporosis and the prevention of fragility fractures and have two phosphonate [PO(OH)₂] groups. Diphosphonates inhibit the digestion of bone by encouraging osteoclasts to (1) undergo cell death or (2) inhibit the ruffled border with subsequent dysfunction of resorption. Both mechanisms thereby slow bone loss.³⁰,³¹,³²

There are concerns that long-term diphosphonate use can result in oversuppression of bone turnover. It is hypothesized that microfractures in the bone are unable to heal and eventually unite and propagate, resulting in atypical fractures. This phenomenon has been seen most prominently in the proximal femur, often in patients using biphosphates for prolonged periods of time.³³,³⁴

In cases where there is concern of such fractures, teriparatide is potentially an alternative because of reducing damage caused by suppression of bone turnover.³⁵

In cases of atypical femur fracture where teriparatide has been shown to be effective in combination with surgical management, it is proposed that PTH increases bone remodeling resulting in the removal of more densely mineralized bone replaced with new, less-compact normal mineralized bone.³⁶,³⁷

Morphology for Acute Fractures and the Development of Nonunion

Many factors that can affect fracture healing are associated with the characteristics of the injury, including the pattern of bony injury, location of the fracture, status of the soft-tissue envelope, extent of bone loss, and the degree of stability (strain) afforded to the fracture site.³⁸

Motion at the fracture site will allow callus to form and intermittent shear stresses are thought to encourage ossification.³⁹ The larger the stress, the greater the amount of callus formed up to an undefined limit of strain. Interfragmentary micromotion and low amounts of strain can stimulate both intramembranous and endochondral ossification.⁴⁰ If, however, the motion is excessive, a high-strain environment will result in fibrous tissue proliferation and damage the neovascularity, resulting in a deficient inflammatory phase of healing. The ideal degree of mechanical stability has yet to be determined.⁴¹ Studies have shown that excessively rigid
fixation may paradoxically impair fracture healing⁴²,⁴³ via the inhibition of external callus formation, maintenance of a fracture gap aggravated by bone-end resorption, and excessive protection of the healing bone from normal stresses (stress shielding), all leading to adverse remodeling and nonunion. This has been clinically shown with the initial widespread use of locking plates and the overuse of locking screws, minimizing the micromotion and localizing the strain directed at the fracture site. Radiographs documented minimal callus formation and no cortical bridging. All these factors contributed to the high rate of plate failure and nonunion when this fixation montage was used.

Highly comminuted fractures with wide fracture gaps with increased strain fill these gaps with fibrous connective tissue, intervening areas of necrotic bone, and giant cells. This prevents neovascularization response and inhibits the chemotaxis and migration of MSCs. A wide diastasis makes any cellular interaction with circulating inductive factors very difficult (Figure 3).

Atrophic nonunion develops commonly under these circumstances and these types of nonunion have fracture gaps interspersed with dense avascular fibrous tissue, secondary to bone loss or infection. These nonunion types typically occur in concert with mechanical instability and thus both the biologic potential and mechanical stability must be restored. A stimulus to initiate the inflammatory phase of fracture healing and revascularization is necessary along with providing mechanical stability. Biologic adjuvants are then required and are delivered either through the direct application of viable osteoblasts (bone graft) or by providing inductive factors to initiate the chemotaxis and proliferation of MSCs.

FIGURE 3 AP radiograph of the lower leg shows nonmodifiable factors of a fracture that went on to nonunion. Significant open soft-tissue injury, comminution, bone loss, and wide fragment diastasis are seen.

Basic principles of fracture fixation assert that comminuted fractures respond well to bridge plating and relative stability,⁴³ whereas simple fractures should be anatomically reduced, compressed, and rigidly stabilized.⁴¹ Hypertrophic nonunion occurs when the biologic potential is intact and fracture callus is highly proliferative, but because of mechanical instability, large amounts of strain and excessive motion result in fibrous tissue at the fracture site. The treatment of this nonunion requires the augmentation of stability to allow fracture bridging to occur. No additional biologic stimulus is necessary.

CURRENT BIOLOGIC ADJUVANTS

Many of these materials have been shown to be efficacious when used for both the management of acute fracture and the augmentation of nonunion. The best use and indications for these materials will be outlined.

Autogenous Bone Graft

Fresh cancellous autograft provides the quickest and most reliable type of bone graft. These grafts depend on ingrowth of host vessels and perform best in well-vascularized beds. The large surface area of autograft allows for survival of numerous graft cells. Studies document success rates approaching 100% for sub-critical-size defects (1- to 2-cm defects) requiring 20 mL or less of autograft.³⁸,⁴⁴,⁴⁵ However, in many of these studies, multiple graft procedures were required to achieve solid union in defects larger than 3 to 4 cm.⁴⁶,⁴⁷

Iliac crest harvest volumes may limit the utility of this graft because crest volume averages 13 mL anteriorly and 30 mL posteriorly. The reamer-irrigator-aspirator (Synthes) offers a technique to achieve substantial amounts of graft volumes for the treatment of larger segmental defects for management of acute fractures as well as for chronic nonunion defects. The medullary canal of the femur or tibia is reamed with a device designed to collect the reamings and deliver them for grafting procedures.⁴⁸,⁴⁹

Varying amounts of harvested graft have been reported with this technique and range from 30 to 90 mL. Favorable union rates using RIA bone grafting versus autogenous iliac crest bone grafting (AICBG) have demonstrated its equivalency. Further study is required to demonstrate any superiority for larger defects versus the relative efficacy of AICBG.⁴⁸

Investigators have documented elevated amounts of osteoinductive growth factors⁵⁰,⁵¹,⁵²,⁵³,⁵⁴ and osteoprogenitor/endothelial progenitor cell types compared with standard iliac crest grafts. Cell viability and osteogenic potential are similar between bone grafts obtained from both the RIA
system and the iliac crest.⁹ Elevated levels of fibroblast growth factor alpha, PDGF, insulinlike growth factor-I, TGF-β1, and bone morphogenetic protein (BMP)-2 have been measured in the reaming debris as a rich source of growth factors with a content comparable with that from iliac crest.

In summary, both iliac crest bone graft and RIA are excellent sources for autogenous bone graft, the gold standard for augmentation of fracture healing as well as for the treatment of nonunion. RIA is an excellent harvest technique, especially when large volumes are needed, and data support that it supplies active, osteogenic tissue (Tables 1 and 2).

Bone Marrow Aspirate Concentrate

The critical component necessary to all bone formation is the ability to provide viable osteoprogenitor cells. Bone marrow is a plentiful source of musculoskeletal stem cells,⁵⁵ with a high concentration of connective tissue progenitors. One milliliter of iliac aspirate contains approximately 40 million nucleated cells, 1,500 of which are connective tissue progenitors.⁵⁶

Bone marrow aspirate (BMA) has been used as a source of bone marrow-derived MSCs with its relative ease of harvest and low morbidity. The aspirate is typically concentrated by centrifugation to increase the number of MSCs. Concentrated bone marrow aspirate (cBMA) provides both stem cells and growth factors. Injection of cBMA into nonunion sites has had some limited success, but there are no good data showing its effectiveness for its solitary injection into an acute fracture site.

Current use relies on the development of a composite graft. Loading the cBMA onto a highly osteoconductive carrier with a specific three-dimensional architecture facilitates cellular attachment for graft delivery.⁵⁷,⁵⁸ Common materials include cancellous allograft, demineralized bone matrix (DBM), and particulate calcium phosphate ceramics as porous carrier materials. Osseous regeneration is dependent on the number of cells available to participate in bone synthesis. However, the ultimate threshold concentration of cells necessary to promote osteogenesis is not known.⁵⁹,⁶⁰

TABLE 1 Biologic Characteristics of Currently Available Biologic Adjuvants

	Potency of Available Biologic Adjuvants			Osteogenic Living Cells
	Osteoconductive Scaffold	Osteopromotive Facilities Bone Formation	Osteoinductive Growth Factors	Osteogenic Living Cells
Synthetic materials	Ca Ceramics Collagen Degradable polymers	Electromagnetic stimulation Ultrasound stimulation	rBMP-2 rBMP-7 GDF-5	—
Allograft and autograft materials	Allograft struts, dowels, chips Autograft DBM Allograft stem cell prep	Platelet-rich plasma rPDGF	DBM Cancellous autograft RIA cBMA Allograft stem cell prep	Bone marrow elements (cBMA) Cancellous autograft RIA Allograft stem cell prep
Increasing Biologic Activity (ability to form bone)
cBMA = concentrated bone marrow aspirate, DBM = demineralized bone matrix, GDF = growth differentiation factor, rBMP = recombinant bone morphogenetic protein, RIA = reamer-irrigator-aspirator, rPDGF = recombinant platelet-derived growth factor

Many commercially available systems have been developed that have the capability to concentrate progenitor cells three to four times and present them to the surgeon in a usable manner. These devices concentrate the cells via a fully automated closed-loop system for separating nucleated cells from bone marrow. These systems use either a centrifuge or a filtration type of mechanism to accomplish their goals.⁶¹,⁶²,⁶³

Current literature demonstrates perhaps faster healing times with similar union rates when using cBMA combined with cancellous allograft and or DBM compared with conventional autologous cancellous bone graft for treating nonunions with small defects.⁶⁴,⁶⁵,⁶⁶,⁶⁷,⁶⁸,⁶⁹

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