Chapter 5: Biologic and Biophysical Technologies for the Enhancement of Fracture Repair

Eric Wagner, Thomas A. Einhorn, Sanjeev Kakar

Chapter Outline

Introduction

Fracture repair is a well-orchestrated biologic process that includes multiple signaling pathways and is regulated by both local and systemic factors. However, any abnormality within this well-orchestrated cascade has the potential to impair healing, leading to between 5% and 10% of fractures failing to achieve complete union.96 In many instances, the cause of the impairment is unknown and may be related to inadequate reduction, instability,73 the systemic state of the patient,97,215 or the nature and extent of energy associated with the traumatic insult itself.245,326 In addition, the local environment or blood supply can predispose fractures to an abnormal or impaired healing process. For example, open fractures of the tibia have a wide range of delayed union rates of between 16% and 100% depending on the grade of injury.135 In the scaphoid and femoral neck, fracture healing is dependent on an intact blood supply from a single vessel, and disruption of that vessel leads to high rates of nonunion.83,137,255 Finally, the subtrochanteric region of the femur is at an increased risk since the mechanical loads occurring there are among the highest in the skeleton.162 
While fracture healing typically occurs without incident, complications related to delayed union or nonunion can be severe with regard to patient morbidity and medical treatment costs. For example, Busse et al.58 found that the direct costs associated with the treatment of a tibial nonunion are approximately $7,500, while this estimate can be over $17,000 when indirect costs, such as loss of work productivity, are taken into account. To improve and expedite repair, surgeons may consider the use of bone grafts, biologic agents, or physical stimulation. This chapter will review the current use and development of these approaches in the restoration of skeletal function. 

Bone Grafts and Bone-Graft Substitutes

It is estimated that more than 2.2 million bone-graft procedures are performed worldwide each year, with over 200,000 performed within the United States.147,201 Indications for their use include malunions, nonunions, arthrodesis, and reconstructive procedures.239 Their successful incorporation depends on osteoinductive growth factors, an osteoconductive extracellular matrix, and osteogenic pluripotent stem cells residing in the bone marrow. Osteoinduction refers to the recruitment and differentiation of pluripotent mesenchymal stem cells (MSCs) into bone-forming osteoprogenitor cells, mediated by graft-derived growth factors such as bone morphogenetic protein (BMP).308,329 Osteoconduction involves the creation of the bone scaffold that supports the ingrowth of blood vessels and perivascular tissue, as well as the attachment of osteoprogenitor cells. This occurs in an ordered sequence which is dependent on the three-dimensional structure of the graft, the local blood supply, and the biomechanical forces exerted on the graft and surrounding tissues.308 Osteogenesis refers to the process of bone formation after the terminal differentiation of osteogenic progenitor cells into mature osteoblasts. These three processes create the signals, scaffolds and cells necessary for the initial phases of fracture healing (Table 5-1).239,308 
 
Table 5-1
Properties of Types of Autologous Bone Grafts
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Table 5-1
Properties of Types of Autologous Bone Grafts
Property Cancellous Nonvascularized Cortical Vascularized Cortical
Osteoconduction +++ + +
Osteoinduction ++a +/– +/–
Osteoprogenitor cells +++ ++
Immediate strength +++ +++
Strength at months ++ ++, +++ +++
Strength at a year +++ +++ +++
 

Reprinted with permission from: Finkemeier CG. Bone-grafting and bone graft substitutes. J Bone J Surg Am. 2002;84:454–464.

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Bone grafting stimulates a sequence of events similar to most tissue regeneration. After initial hematoma formation, there is a release of cytokines, including platelet-derived growth factors (PDGFs), transforming growth factor-betas (TGF-betas), and fibroblast growth factors (FGFs) that lead to the recruitment of circulating progenitor cells and the production of regenerative, angiogenic, and inflammatory factors.42 The recruited cells then begin the process of graft incorporation, as osteoclasts resorb necrotic graft material. Pluripotential mesenchymal cells respond to local growth factors and differentiate into osteoblasts that produce osteoid. While osteoblasts and endosteal cells on the surface of the graft may survive the transplantation and contribute to the healing, the main contribution of the graft is to act as an osteoinductive and osteoconductive substrate. These properties provide the necessary mechanical and chemical requirements to support the attachment, proliferation, migration, and differentiation of bone forming cells. The final stages in the process involve mineralization of the osteoid, remodeling of the callus, and incorporation of the remaining graft. The process of remodeling of the callus (composed of woven bone) involves the coordinated activities of osteoblastic bone formation and osteoclastic bone resorption, with woven bone ultimately being replaced by lamellar bone. 
Autogenous bone grafting is considered to be the “gold standard” as it utilizes osteoinductive growth factors, an osteoconductive matrix, and osteogenic stem cells to provide consistent results with regard to healing and integration.38,198,285,305 However, the morbidity associated with graft harvesting, such as donor-site pain, nerve or arterial injury, and infection rates of between 8% and 10%,23,111,134,317,349 as well as a limited number of donor sites and the increased operative time required for harvest, have prompted extensive research into alternatives. One alternative that avoids many of these limitations and complications is allograft bone.53,88,138,153,291 However, allogeneic graft is limited by its lack of osteoinductive capabilities32 and increased costs.254 Furthermore, although perhaps unfairly as the current donor selection process has greatly reduced any risk, many patients and surgeons remain concerned regarding the risk of disease transmission.24,156 As a result, there have been investigations into bone-graft substitutes and other tissue engineering strategies for fracture management. 

Autologous Bone

Autologous bone grafting remains the gold standard to which all materials and technologies to enhance bone healing are compared. Possessing excellent osteoinductive, osteoconductive, and osteogenic potential, it is the ideal bone graft. Furthermore, graft versus host disease and any disease transmission risk are eliminated since it is the patient’s own bone. 
Either cancellous or cortical bone can be harvested depending on the procedure. In some cases, it is necessary to augment healing via a vascularized graft, usually from the fibula, rib, or distal femur. Careful planning is needed to ensure that the proposed harvest site will contain both the correct type and amount of graft. For example, a large segmental defect in the radius would need a large structural cortical graft,105,231 whereas a tibia plateau fracture with a depressed fragment may require only a small amount of cancellous graft. The most common and best-described sources of autologous bone include the pelvis, the distal radius,315 the fibula,198 the proximal tibia,248 the ribs,208 the greater trochanter,239 and the olecranon.239 

Autologous Cancellous Bone Graft

Cancellous bone is the most commonly used bone-graft source, serving as an effective graft material for fractures that do not require immediate structural support from the graft. Instead, it serves as a scaffold for the attachment of host cells and it provides the osteoconductive and osteoinductive functions required for the laying down of new bone. However, it lacks immediate structural stability and strength and is therefore unable to support force transmission alone. 
Although cancellous bone graft lacks mechanical strength, its main advantage lies in its tremendous biologic activity. Lining the trabeculae of the graft material are pluripotent progenitor cells capable of differentiating into multiple different lineages, including osteoid-producing cells.294 Furthermore, the large surface area leads to immediate graft incorporation. In fracture healing, the initial phase involves progenitor cell recruitment and proliferation under the control of local cytokine release. As resorption ensues, more cytokines are released, leading to the formation of granulation tissue and neoangiogenesis, or the formation or new blood vessels. These cytokines also direct the differentiation of osteoprogenitor cells into mature bone forming cells. Within the first couple days, the graft appears to be completely vascularized, while bone formation occurs within weeks.28,91 
The cancellous graft also serves as a scaffold to be resorbed as the mature osteogenic cells lay down a new osteoid matrix.308 This begins the graft incorporation and can take many months to complete. When seen on x-ray, this phase represents the gradual loss of a clear delineation between the native bone and fracture lines or graft incorporation.184 The mechanical properties begin to be restored after the initial weeks of graft incorporation. The process by which the graft is replaced by new bone is known as “creeping substitution”306 and is usually complete within 1 year (Fig. 5-1, Table 5-1). 
Figure 5-1
Low-power photomicrograph showing creeping substitution.
 
Newly formed woven bone, containing osteoblasts with basophilic-staining nuclei, is laid down upon dead lamellar bone identified by the presence of empty osteocytic lacunae (hematoxylin and eosin stain, original magnification 10×).
Newly formed woven bone, containing osteoblasts with basophilic-staining nuclei, is laid down upon dead lamellar bone identified by the presence of empty osteocytic lacunae (hematoxylin and eosin stain, original magnification 10×).
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Figure 5-1
Low-power photomicrograph showing creeping substitution.
Newly formed woven bone, containing osteoblasts with basophilic-staining nuclei, is laid down upon dead lamellar bone identified by the presence of empty osteocytic lacunae (hematoxylin and eosin stain, original magnification 10×).
Newly formed woven bone, containing osteoblasts with basophilic-staining nuclei, is laid down upon dead lamellar bone identified by the presence of empty osteocytic lacunae (hematoxylin and eosin stain, original magnification 10×).
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While cancellous graft does not provide structural support by itself, through impaction and aided by internal fixation, it is used for areas of bone loss. Examples of such use are in the treatment of bone loss associated with depressed tibial plateau fractures or revision hip and knee arthroplasty.322,323 Excellent results have also been demonstrated with nonunions and arthrodeses, due to its rapid incorporation and osteogenic regeneration potential (Fig. 5-2).47,90,250 
Figure 5-2
A 52-year-old laborer developed a scapholunate advanced collapse (SLAC) and scaphoid nonunion, which are shown in plain radiographs (A), as well as on CT and MRI (B).
 
He underwent a total wrist arthrodesis augmented with iliac crest bone grafting and demonstrated good healing of the fusion at 6 months (C) and (D).
He underwent a total wrist arthrodesis augmented with iliac crest bone grafting and demonstrated good healing of the fusion at 6 months (C) and (D).
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Figure 5-2
A 52-year-old laborer developed a scapholunate advanced collapse (SLAC) and scaphoid nonunion, which are shown in plain radiographs (A), as well as on CT and MRI (B).
He underwent a total wrist arthrodesis augmented with iliac crest bone grafting and demonstrated good healing of the fusion at 6 months (C) and (D).
He underwent a total wrist arthrodesis augmented with iliac crest bone grafting and demonstrated good healing of the fusion at 6 months (C) and (D).
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Autologous Cortical Bone Graft

Cortical bone can provide good structural support, but it has much weaker osteoconductive and osteoinductive properties. Its use is indicated when immediate structural support is necessary, but it has slightly limited long-term healing potential.239 This is partly due to the thickness of the cortical matrix, limiting the diffusion of nutrients, and subsequent neovascularization and osteogenesis (Table 5-1).81 This density also limits the remodeling process, with the bone incorporation relying on osteoclasts instead of osteoblasts. This resorption phase during the first 6 months leads to progressive mechanical weakness, which is eventually restored around a year after the procedure.108,132 Remodeling proceeds and creeping substitution can require up to 2 years for completion.56,104,295 Cortical bone grafts are usually harvested from the ribs, fibula, or crest of the ilium (as a so-called tricortical graft) and can be transplanted with or without a vascular pedicle (Table 5-1). 
Vascularized bone-graft biology is different from its nonvascularized counterpart, not only in terms of the rate of repair but also in the way in which remodeling occurs.81 Most of these grafts are harvested from the iliac crest with the deep circumflex artery, the fibula with peroneal artery branches, the medial femoral condyle with descending genicular artery branches, the distal radius with supraretinacular artery branches, or the ribs with the posterior intercostal artery.51 Once implanted with its viable vascular pedicle, the independent blood supply leads to significant biologic activity and regeneration potential with retention of up to 90% of the graft’s osteocytes.108,239,247 Dell et al.81 examined vascularized and nonvascularized grafts histologically and graded the amount of necrosis based on the presence or absence of osteocytes. At 2 weeks, the vascularized graft remained mostly viable, with the only area of necrosis noted at the periphery, while the nonvascularized grafts showed diffuse necrosis of the medullary cavity, taking up to 24 weeks to resemble the vascularized grafts. The increase in osteocyte survival and the early vascularity leads to a rapid incorporation of a vascularized bone graft.130,295 The initial differences in strengths are due to the remodeling processes. While nonvascularized grafts are incorporated through creeping substitution, vascularized grafts do not induce a robust inflammatory and angiogenic response compromising the early mechanical strength. 
In the treatment of critical-sized defects, or bone defects that will not heal without grafting, both vascularized and nonvascularized grafts are indicated. For defects up to 6 cm in length where immediate structural support is desired, nonvascularized cortical autografts can be used.108 Controversy exists regarding the best alternative for defects between 6 and 12 cm, while defects greater than 12 cm are good candidates for vascularized grafting procedures.81,105 Vascularized grafts are also indicated for reconstruction of defects where the microenvironment of the host is inadequate to initiate an effective biologic response. Examples include acute traumatic injuries with extensive soft tissue damage and impairment of blood supply, atrophic nonunions, and irradiated or severely scarred tissue.81,105 For example, free vascularized medial femoral condyle flaps have been successfully used to treat many upper-extremity nonunions, including those of the distal radius, scaphoid, and humeral shaft (Fig. 5-3, Table 5-1).65,172,173 
Figure 5-3
A 22-year-old man with a scaphoid nonunion that has led to a humpback deformity, seen in preoperative radiographs (A) and CT (B).
 
He was treated with internal fixation augmented with a vascularized medial femoral condyle graft. C: Radiographs immediately postoperatively and then every 3 months demonstrate gradual healing of the nonunion with graft incorporation. D: A CT scan at 1 year postoperatively demonstrates good healing of the nonunion and graft incorporation.
He was treated with internal fixation augmented with a vascularized medial femoral condyle graft. C: Radiographs immediately postoperatively and then every 3 months demonstrate gradual healing of the nonunion with graft incorporation. D: A CT scan at 1 year postoperatively demonstrates good healing of the nonunion and graft incorporation.
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Figure 5-3
A 22-year-old man with a scaphoid nonunion that has led to a humpback deformity, seen in preoperative radiographs (A) and CT (B).
He was treated with internal fixation augmented with a vascularized medial femoral condyle graft. C: Radiographs immediately postoperatively and then every 3 months demonstrate gradual healing of the nonunion with graft incorporation. D: A CT scan at 1 year postoperatively demonstrates good healing of the nonunion and graft incorporation.
He was treated with internal fixation augmented with a vascularized medial femoral condyle graft. C: Radiographs immediately postoperatively and then every 3 months demonstrate gradual healing of the nonunion with graft incorporation. D: A CT scan at 1 year postoperatively demonstrates good healing of the nonunion and graft incorporation.
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Complications of Autologous Grafting

In addition to longer operative times associated with harvesting autologous bone grafts, the morbidity associated with the donor sites is a significant concern. The most common donor site for harvesting autograft is the iliac crest.239 Arrington et al.15 reviewed 414 cases of iliac crest bone-graft harvesting and found that patients experienced multiple superficial hematomas and infections as well as a variety of more significant complications including 4 deep hematomas, 2 incisional hernias, 6 neurologic injuries, 3 vascular injuries, 2 iliac wing fractures, and 7 deep infections. Another similar review of iliac crest grafting by Younger et al.349 of 243 iliac crest harvests demonstrated 57 (24%) superficial complications, such as infection or hematoma, and 15 (6%) deep complications, such as deep hematomas and infections. Other reviews have demonstrated similar rates of adverse events, with deep major complications also including sacroiliac injury, ureteral injury, and vascular injury, in addition to deep hematomas, infections, and iliac crest fractures.91,134,239 According to a review of the literature by Myeroff and Archdeacon,239 superficial or minor complications occur in 7.1% to 39% of patients, while major or deep complications occur in 1.8% to 10% of patients. 
Other common donor sites include the proximal and distal tibia, distal radius, greater trochanter, and medial femoral condyle. Postoperative pain and superficial hematomas are relatively common complications of harvest from these areas. Significant adverse events from proximal tibia graft harvesting include deep hematomas and infections, joint compromise or proximal tibial fracture, and permanent neurologic injury, occurring in 0.5% to 2% of patients.117,120,123,239 The distal radius harvesting site is associated with de Quervain’s tenosynovitis, superficial radial nerve injury and fracture through the donor site.256 
A relatively new technique developed in 2005 to overcome the donor-site morbidity associated with autologous bone-graft harvesting is the use of the reamer-irrigator-aspirator (RIA). This is a novel intramedullary (IM)-reaming device that is used to irrigate and aspirate the bone marrow canal. Initially developed to reduce IM pressure and fat embolism associated with reaming, its indications have also begun to include autologous bone marrow and graft harvesting.175,219,239 Overall, it appears to be a safe and effective method for autologous graft harvesting, leading to less persistent postoperative pain.33,175,219,239 Belthur et al.33 compared 41 patients who underwent either RIA of the femur or tibia, or iliac crest bone-graft harvest. The patients who underwent RIA had lower pain scores than the iliac crest group. The average graft volume was 40.3 mL (25 to 75 mL) from the RIA system. Porter et al 261 demonstrated the aspirate obtained via RIA contains many osteogenic growth factors, including FGF-2, IGF-1, and TGF-β1, as well as multiple mesenchymal progenitor cells. Complications have been reported with its use, including fractures at the donor site (Fig. 5-4).129,213,263 
Figure 5-4
Reamer Irrigator Aspirator (RIA) system and its complications.
 
A: Total pain scores reported by patients in the autograft and RIA groups. With RIA, the pain scores were substantially less immediately postoperatively and at long-term follow-up. B: The system consists of a reamer head, collection tube, drive shaft, and aspiration and irrigation ports. C: Anteroposterior and lateral radiographs demonstrate a perforation of the anterior femoral cortex from using the RIA. (Reprinted with permission from: Belthur MV, Conway JD, Jindal G, et al. Bone graft harvest using a new intramedullary system. Clinic Orthop Relat Res. 2008;466(12): 2973–2980).
A: Total pain scores reported by patients in the autograft and RIA groups. With RIA, the pain scores were substantially less immediately postoperatively and at long-term follow-up. B: The system consists of a reamer head, collection tube, drive shaft, and aspiration and irrigation ports. C: Anteroposterior and lateral radiographs demonstrate a perforation of the anterior femoral cortex from using the RIA. (Reprinted with permission from: Belthur MV, Conway JD, Jindal G, et al. Bone graft harvest using a new intramedullary system. Clinic Orthop Relat Res. 2008;466(12): 2973–2980).
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Figure 5-4
Reamer Irrigator Aspirator (RIA) system and its complications.
A: Total pain scores reported by patients in the autograft and RIA groups. With RIA, the pain scores were substantially less immediately postoperatively and at long-term follow-up. B: The system consists of a reamer head, collection tube, drive shaft, and aspiration and irrigation ports. C: Anteroposterior and lateral radiographs demonstrate a perforation of the anterior femoral cortex from using the RIA. (Reprinted with permission from: Belthur MV, Conway JD, Jindal G, et al. Bone graft harvest using a new intramedullary system. Clinic Orthop Relat Res. 2008;466(12): 2973–2980).
A: Total pain scores reported by patients in the autograft and RIA groups. With RIA, the pain scores were substantially less immediately postoperatively and at long-term follow-up. B: The system consists of a reamer head, collection tube, drive shaft, and aspiration and irrigation ports. C: Anteroposterior and lateral radiographs demonstrate a perforation of the anterior femoral cortex from using the RIA. (Reprinted with permission from: Belthur MV, Conway JD, Jindal G, et al. Bone graft harvest using a new intramedullary system. Clinic Orthop Relat Res. 2008;466(12): 2973–2980).
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In conclusion, autologous bone graft remains the gold standard in bone grafting due to its osteogenic, osteoinductive, and osteoconductive properties. However, it is associated with important complications that must be considered before proceeding with this technique. Use of the RIA is a relatively new technique that holds promise to reduce persistent postoperative pain associated with autograft harvesting. However, to date there have been insufficient studies to fully evaluate its efficacy and any associated adverse events. 

Allogeneic Bone

Due to the morbidity associated with harvesting autogenous bone graft and the limited quantity available when attempting to fill large defects, alternatives such as allogeneic bone graft have gained popularity and shown significant promise.71,153 The abundance of modern tissue banks and stringent measures to ensure safety have lead to the use of hundreds of thousands bone allografts each year.321 They account for approximately one-third of the bone grafts performed in the United States. Cortical allografts are harvested from a number of sites including the pelvis, ribs, and fibula. Allografts are frequently used in spinal surgery,87 joint arthroplasty,103,232 and upper- and lower-extremity arthrodesis, including total wrist arthrodesis.65 Despite their widespread use in these elective procedures, only recently have they been investigated for use in the repair of fresh fractures or nonunions. 
Many of the limitations of the efficacy of allogeneic bone as a bone-graft material are likely associated with its preparation. To decrease the risk of disease transmission, allograft bone is prepared and sterilized via freeze-drying, freezing, or irradiation. Freeze-drying, or lyophilization, via a removal of water and vacuum packing of the tissue significantly reduces immunogenicity.115,351 For example, it reduces the osteoblast expression of the major histocompatibility complex (MHC) class I antigen, blunting the host immune response usually modified by MHC antigens.115,154,351 Furthermore, Pelker et al.258 demonstrated that such treatment of the graft reduces its mechanical integrity, thereby diminishing its load-bearing properties. Irradiation has a similar effect on the mechanical strength, decreasing it in a dose-dependent fashion.169 In addition, both freeze-drying and irradiation reduce the osteoinductive potential of the allograft by inducing the death of its osteogenic cells. 
Although slower than with autografts, allograft incorporation occurs through a process similar to autogenous graft incorporation.131 The delayed incorporation is in part due to the paucity of donor progenitor cells, and partly due to an inhibitory host immune response to the allograft that inhibits osteoblastic differentiation.184 Mononuclear cells have been demonstrated to line newly developing blood vessels. Thus, a limited initial revascularization, creeping substitution and ultimate remodeling lead to a higher incidence of early fractures.55,106,309,319 Enneking and Mindell106 reported the histologic evaluation of 73 retrieved allografts, 24 (33%) of which were obtained at autopsy or after amputation. The investigators found new vessel penetration rarely exceeded a depth of 5 mm within the first 2 years, and new bone apposition occupied no more than 20% of the graft. The depth of penetration after 2 years was typically less than 10 mm, with necrotic tissue remaining in the central aspects of the allograft throughout the remodeling process. 
The biologic nature of the recipient host bed is a critical factor in facilitating allograft incorporation. Allograft bone incorporation occurs by sporadic appositional bone formation and is dependent on neoangiogenesis. A well-vascularized bed aids in the incorporation of the allograft through a combination of revascularization, osteoconduction, and remodeling.182 However, poor vascularization, as seen in some large defects, leads to a prolonged incorporation process and significant mechanical weakness. 
A technique first described by Wang and Weng341 to combat the relative inertness of cortical allografts involves placing harvested autologous iliac crest at the allograft–host bone interface. They treated 13 patients with femoral nonunions via open reduction and internal fixation combined with deep-frozen cortical allograft struts. Seven unicortical, five bicortical, and one tricortical allografts, with an average length of 10 cm, were used. Autogenous bone grafts were inserted into the defect between the allograft and host femur. All nonunions united at an average of 5 months. 

Demineralized Bone Matrix

Demineralized bone matrix (DBM) is produced by acid extraction of allograft bone.330 Although it contains type I collagen, noncollagenous proteins, and osteoinductive growth factors, such as BMPs and TGF-betas, similar to allografts it provides little structural support.214 However, the abundance of growth factors gives it more osteoinductive potential than allografts.110 Numerous DBM formulations exist based on refinements of the manufacturing processes. They are available as a freeze-dried powder, granules, a gel, a putty, or strips. Recently, various companies have combined DBM with copolymers that become firm when warmed to body temperature.184 Despite these many options, there is minimal clinical data to support each one’s efficacy. Furthermore, donor-to-donor variability in the osteoinductive capacity of DBM exists, resulting in the requirement by the American Association of Tissue Banks and the U.S. Food and Drug Administration (FDA) that each batch of DBM be obtained from a single human donor.19 Bae et al.18 examined 10 production lots of a single DBM product, demonstrating significant variations in the BMP-2 and BMP-7 content, both of which have a large impact on fusion rates (Fig. 5-5). 
Figure 5-5
The content of bone morphogenetic protein (BMP) 2 and 7 across 10 production lots of a single demineralized bone matrix (DBM) product.
 
A: The content of both BMP 2 and 7 in DBM varies significantly across multiple samples of DBM lots. The content of BMP 2 was positively correlated with BMP 7 (p < 0.0001), suggesting the osteoinductive capabilities of some products are significantly higher than others. B: The effective doses of BMP 2 and 7 in DBM as a predictive measure for fusion rates. The higher concentrations in the DBM lead to higher fusion rates. (Reprinted with permission from: Bae H, Zhao L, Zhu D, et al. Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg Am. 2010;92(2):427–435.)
A: The content of both BMP 2 and 7 in DBM varies significantly across multiple samples of DBM lots. The content of BMP 2 was positively correlated with BMP 7 (p < 0.0001), suggesting the osteoinductive capabilities of some products are significantly higher than others. B: The effective doses of BMP 2 and 7 in DBM as a predictive measure for fusion rates. The higher concentrations in the DBM lead to higher fusion rates. (Reprinted with permission from: Bae H, Zhao L, Zhu D, et al. Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg Am. 2010;92(2):427–435.)
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Figure 5-5
The content of bone morphogenetic protein (BMP) 2 and 7 across 10 production lots of a single demineralized bone matrix (DBM) product.
A: The content of both BMP 2 and 7 in DBM varies significantly across multiple samples of DBM lots. The content of BMP 2 was positively correlated with BMP 7 (p < 0.0001), suggesting the osteoinductive capabilities of some products are significantly higher than others. B: The effective doses of BMP 2 and 7 in DBM as a predictive measure for fusion rates. The higher concentrations in the DBM lead to higher fusion rates. (Reprinted with permission from: Bae H, Zhao L, Zhu D, et al. Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg Am. 2010;92(2):427–435.)
A: The content of both BMP 2 and 7 in DBM varies significantly across multiple samples of DBM lots. The content of BMP 2 was positively correlated with BMP 7 (p < 0.0001), suggesting the osteoinductive capabilities of some products are significantly higher than others. B: The effective doses of BMP 2 and 7 in DBM as a predictive measure for fusion rates. The higher concentrations in the DBM lead to higher fusion rates. (Reprinted with permission from: Bae H, Zhao L, Zhu D, et al. Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg Am. 2010;92(2):427–435.)
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Animal studies have demonstrated monocyte cell infiltration into the graft within 18 hours and cartilaginous differentiation of progenitor cells within the first week after DBM grafting. Cartilage mineralization ensues, followed by perivascular infiltration and the eventual formation of osteoblasts, leading to complete DBM resorption and bone formation.308 In humans, multiple case series have demonstrated DBM to be a viable alternative in both acute and nonunion fracture care, arthrodesis, and implant fixation. Ziran et al.352 followed 107 patients treated with DBM and cancellous allograft bone chips for the treatment of acute fractures with bone loss or atrophic nonunions, the majority (18 of 25) of which occurred in smokers. They found that 87 fractures healed at a mean of 32 months. Hatzokos et al.142 treated 43 patients with an average tibial bone defect of 9.49 cm via distraction osteogenesis, filling the defects with either closed compression, autograft, or DBM and bone marrow. Although the closed compression site had a prolonged consolidation time, there were no differences between the autograft and DBM regarding the docking site healing time. 
A prospective nonrandomized study comparing the use of autograft and human DBM (Grafton, Osteotech, Inc., Eatontown, NJ) in anterior cervical spine fusion found higher rates of pseudarthrosis and graft collapse with DBM, although the differences did not reach statistical significance.7 Ziran et al.353 retrospectively reviewed 41 patients with atrophic and oligotrophic nonunions treated with human DBM (AlloMatrix; Wright Medical Technologies, Memphis, TN). Postoperative complications were high, with 51% experiencing wound complications, of which 32% required operative debridement. Of the 41 treated patients, only 22 went on to heal the nonunion without the need for additional bone grafting. Bibbo and Patel38 studied the use of human DBM and calcium sulfate compound (AlloMatrix Wright Medical, Arlington, TN) combined with vancomycin for the treatment of calcaneal fractures. Their results demonstrated that fractures treated with AlloMatrix and vancomycin healed at a mean of 8.2 weeks, compared with 10.4 weeks being needed for those that were not grafted. Of note, while the study was not randomized, the fractures that received DBM and calcium sulfate represented more significant injuries in that they had substantial bone loss and included six open fractures (Gustilo grade I). Hierholzer et al.151 retrospectively reviewed the results of the treatment of 45 aseptic nonunions of the humerus treated with either autograft or DBM allograft (Grafton; Osteotech, Inc., Eatontown, NJ). The union rate in the 45 patients treated with autograft was 100%, which was similar to the 97% union rate in 33 patients treated with DBM. Donor-site pain was a significant problem in the patients treated with autograft, with 44% of the patients experiencing prolonged pain or paresthesias and one patient having an infection requiring operative debridement. 

Authors’ Preferred Method of Treatment

 
 
Autologous Cancellous Bone Graft
 

We prefer the autologous cancellous bone graft to be used for fractures associated with bone loss, nonunions, and small bone defects (e.g., a metaphyseal or mid-diaphyseal cyst that has undergone curettage). Due to its osteoconductive, osteoinductive, and osteogenic properties, autologous graft material has a well-established history of successful augmentation of fracture healing. Up to 12-cm diaphyseal defects can be treated with nonvascularized autografts, while those over 12 cm are successfully augmented with vascularized grafts. However, there are significant complications associated with autograft harvesting, including deep infections and hematomas, neurologic or vascular injury, iatrogenic fractures, nonunions, and persistent postoperative pain. Use of the RIA is a new harvesting technique that has the potential to overcome some of these morbidities, but has not been extensively studied enough for us to make a recommendation.

 

Regarding allogeneic bone, there is limited information on its use in fresh fractures or nonunions. In combination with autologous graft, we recommend the use of allograft to augment the volume of graft material in the treatment of fractures associated with large volume loss or nonunions. Incorporation of allogeneic strut grafts may also be enhanced by the use of autogenous cancellous bone at the junction with the host bone.

 

The efficacy of human DBM as a graft material remains unclear. Although widely available and known to contain BMP, we do not believe there is sufficient evidence demonstrating its efficacy when used alone in the treatment of fresh fractures or nonunions or in the reconstruction of bone defects. However, when used in conjunction with autologous cancellous bone, it has tremendous potential. We believe that DBM provides an osteogenic advantage and may enhance the ability of a fixed volume of autologous graft or bone marrow to be effective.

Bone-graft Substitutes

An ideal osteoconductive scaffold should have the appropriate three-dimensional structure to promote osteoconduction, as well as allow for osteointegration and invasion by cells and blood vessels. In addition, it should also be biocompatible and biodegradable with biomechanical properties similar to those of the surrounding bone. 

Calcium Phosphate Ceramics

The first clinical use of calcium phosphate ceramics for the repair of bony defects was reported by Albee in 19205 and repeated in several animal studies. Despite these early experiments, it was not until the 1970s that calcium phosphates, and in particular, hydroxyapatite (HA), were synthesized, characterized, and used clinically.165,225,277 Calcium phosphate ceramics are osteoconductive materials produced by a process called sintering in which mineral salts are heated to over 1,000°C. Sintering reduces the amount of carbonated apatite, an unstable and weakly soluble form of HA, producing a solid, porous substance. Part of the osteoconductive potential of calcium phosphate depends on porosity and pore size, with the optimal size being greater than 150 μm.79 Examples of calcium phosphate ceramics include HA, tricalcium phosphate (TCP), and calcium phosphate–collagen composite. 

Hydroxyapatite

Calcium phosphate ceramics can be divided into slowly and rapidly resorbing ceramics. This is important with regard to whether the compound will need to provide long-term structural support or is acting as a void filler that will be quickly replaced.108 HA is a slowly resorbing compound that is derived from several sources, both animal218 and synthetic.145,270 It is degraded by osteoclasts in 2 to 5 years.41 Interpore (Interpore International, Irvine, CA) is a coralline HA and was the first calcium phosphate–based bone-graft substitute approved by the FDA. A hydrothermal treatment process converts it from its native coral state to the more stable HA form with pore diameters of between 200 and 500 μm, a structure very similar to human trabecular bone. Bucholz et al.52 randomized 40 patients with tibial plateau fractures to be treated with either Interpore HA or autologous bone graft. After insertion of the graft, cortical fracture fragments were reduced, and a standard AO interfragmentary screw and plate fixation device was used to stabilize the reduction. With an average of 15.4 months for the autograft and 34.5 months for the Interpore-treated groups, radiographic and functional knee joint assessments revealed no differences between the two groups. However, attempts at using HA as a stand-alone implant for fixation in distal radius fractures did not show such promising results.167 Compared with Kapandji wiring, those fractures treated with only HA showed substantial loss of reduction at 6, 12, and 26 weeks. Clinical parameters were also decreased for the HA-treated patients with regard to decreased grip strength and palmar flexion. Another commercial HA, Apapore 60, has shown promise in impaction grafting for acetabular defects. McNamara et al.229 demonstrated a 100% clinical survival for acetabular total hip reconstructions at a mean of 5-year follow-up when using irradiated allograft bone combined with Apapore 60. Sixty percent showed radiographic signs of incorporation, while only 4% demonstrated cup migration prior to stabilization. 

Tricalcium Phosphate

TCP undergoes partial resorption and some of it may be converted to HA once implanted in the body. The composition of TCP is very similar to the calcium and phosphate phase of human bone. This, combined with its porous nature appears to facilitate incorporation with host bone in both animals and humans by 24 months.11,16 
Reports have demonstrated the efficacy of TCP as a bone-graft substitute. McAndrew et al.225 investigated the suitability of TCP to treat bony defects in a series of 43 patients with 33 acute fractures and 13 nonunions. Patients were followed for an average of 1 year. Healing was demonstrated in 90% of the fracture patients and 85% of those with nonunions. Radiographic analysis showed complete resorption of the TCP between 6 and 24 months after implantation. Anker et al.11 retrospectively reviewed 24 patients with 24 bone defects treated with TCP. Most of the defects were metaphyseal and located in the lower extremity. The average defect size was 43 cm3, and the patients were followed for an average of 10 months. Full weight bearing in patients with a lower-extremity defect occurred at a mean of 7 weeks, and radiographic follow-up showed that the graft had completely resorbed in all defects smaller than 43 cm at 6 months. 
Similar to HA, TCP has shown promise in acetabular grafting for revision hip surgery. Multiple biomechanical studies have demonstrated superior stability when HA/TCP composites are mixed with allograft bone when compared to allograft alone.43,334 BoneSave is a biphasic ceramic composed 80% of TCP and 20% of HA. Blom et al.39 examined the use of this composite combined with allograft bone for acetabular impaction grafting in revision total hip arthroplasty in 43 patients. At a mean of 2-year follow-up, there were no rerevisions and no radiographic evidence of implant migration. In a large animal spinal fusion study, Solchaga et al.303 reported on the ability of Augment (Biomimetic Therapeutics, Inc., Franklin, TN), a material combining recombinant PDGF and TCP, to facilitate double-level fusion via an interbody spacer. The fusion rates with this growth factor–TCP combination were equivalent to those of autograft controls. 

Calcium Phosphate Cements

Calcium phosphate cements (CPCs) can be used as bone-void fillers in the treatment of bony defects associated with acute fractures. Inorganic calcium and phosphate are combined to form an injectable paste that can be delivered into the fracture site. After injection the CPC hardens within minutes, achieving its maximum compressive strength after approximately 4 hours. This strength is comparable to intact cancellous bone. However, its strength is significantly diminished in torsion or shear and so it should be used as an adjunct, rather than as the primary method of fixation, in these cases.196 
Sanchez-Sotelo et al.284 conducted a randomized controlled study examining the use of a commercially available CPC, Norian Skeletal Replacement System (Norian SRS) (Norian Corporation, Cupertino, CA), in the treatment of distal radius fractures. One hundred and ten patients, who were between 50 and 85 years of age and who had sustained either an AO type A3 or C2 distal radius fracture, were followed for 12 months. Patients were prospectively randomized to receive either closed reduction with a short arm cast for 6 weeks or closed reduction and stabilization with Norian SRS and cast immobilization for 2 weeks. The results showed improved functional and radiographic outcomes in the patients treated with Norian SRS. In a subsequent randomized controlled study, Cassidy et al.66 compared the use of Norian SRS and closed reduction to closed reduction and the application of a cast or external fixation in 323 patients with fractures of the distal radius. Significant clinical differences were seen at 6 to 8 weeks postoperatively, with better grip strength, wrist and digit range of motion, and hand function and less swelling in the patients treated with Norian SRS. By 1 year, these differences had disappeared. 
In light of the promising results seen with distal radius fractures, Norian SRS has been used to treat other fractures. Schildhauer et al.288 reported its use in the treatment of complex calcaneal fractures. Thirty-six joint depression fractures were treated with Norian SRS after standard open reduction and internal fixation. Patients were allowed to bear weight fully as early as 3 weeks postoperatively. Results demonstrated no statistical difference in clinical outcome scores in patients who bore full weight before or after 6 weeks postoperatively, suggesting that this cement may permit early full weight bearing after surgical treatment of this fracture. Another study by Thordarson and Bollinger, examined the treatment of 15 patients with intra-articular calcaneal fractures using the same CPC technique, with the walls of the defect impacted with a curette and filled with SRS cement. All fractures had less than 2 mm of step-off on postoperative CT which also showed complete filling of the bony defect with cement. Despite early weight-bearing protocols at 3 and 6 weeks, there was no demonstrated loss of reduction at an average of 13-month follow-up.320 
CPC has also been used in the treatment of valgus-impacted proximal humerus fractures. Robinson and Page273 demonstrated complete healing without any signs of osteonecrosis or loss of reduction in 29 patients with severely valgus-impacted proximal humerus fractures treated with screws or buttress plates augmented with CPC in the subchondral space. Egol et al.94 examined the treatment of 92 patients with proximal humerus fractures treated via open reduction and internal fixation without augmentation, with cancellous bone chips, or with CPC. At 3-, 6-, and 12-month follow-ups, the CPC group had significantly less fracture settling and intra-articular screw penetration. 
Tibial plateau fractures can displace due to the bone void underneath the articular surface. In order to promote early weight bearing without loss of reduction, these fractures may undergo operative fixation with grafting of the bone void to withstand significant compressive forces. Although such forces are not able to be withstood by autograft until the fracture has completely healed, multiple biomechanical studies have demonstrated that CPC possesses the necessary compressive strength to enable early weight bearing.196,348 Furthermore, multiple animal studies have demonstrated its effectiveness in reducing tibial plateau subsidence at long-term follow-up after fracture fixation.196,343 
Lobenhoffer et al.210 used Norian SRS in the treatment of 26 tibial plateau fractures (OTA types B2, B3, and C3) followed for a mean of 19.7 months. Twenty-two fractures healed without any displacement or complications. Two cases required early wound revision secondary to sterile drainage, and two cases developed partial loss of fracture reduction between 4 and 8 weeks postoperatively requiring revision surgery. The high mechanical strength of the cement allowed earlier weight bearing after a mean postoperative period of 4.5 weeks. Similar results supporting the use of Norian SRS to fill metaphyseal defects in the treatment of displaced tibial plateau fractures have been reported by others.155,178,343 Simpson and Keating300 followed 13 tibial plateau fractures treated with either limited internal fixation and injectable Norian SRS or buttress plating and cancellous autograft. At 1-year follow-up, the mean subsidence of the autograft-treated group was 4 mm, while the SRS-treated group had only subsided 0.7 mm. Russell and Leighton280 performed a prospective, randomized multicenter examination of CPC versus autograft in acute, unstable tibial plateau fractures. There was a significantly higher rate of articular subsidence between 3- and 12-month follow-up in the autograft group compared to the CPC-treated fractures. 
Although most intertrochanteric fractures are stable after internal fixation, CPC has shown promise in those patterns that possess inherent instability. Elder et al.98 performed a biomechanical study of unstable intertrochanteric fractures with a detached lesser trochanter, comparing sliding hip screw alone to its combination with Norian SRS CPC. Fracture stiffness, stability, and ultimate strength were significantly increased by supplementation with CPC. Mattsson et al.221 performed a prospective, multicenter study of 112 unstable trochanteric fractures with detached posteromedial fragments augmented with Norian SRS CPC compared to controls without augmentation. The patients were allowed to undergo early weight bearing after surgery. Augmentation with CPC improved the patient’s postoperative pain, SF-36 lifestyle scores and ability to return to activities of daily living. A subset of these patients underwent a biomechanical analysis to show that CPC decreased fracture displacement and varus angulation.223 CPC has also shown promise in augmenting femoral neck fracture fixation by stabilizing the threads of cannulated screws. A biomechanical study showed improved stability and screw pull out strength in those augmented with CPC.307 However, this same group examined CPC augmentation of internal fixation of displaced femoral neck fractures and did not find any difference in the group augmented with CPC with regard to pain, quality of life, or reoperations.222 
Bajammal et al.21 conducted a meta-analysis of 14 randomized controlled trials that evaluated the use of CPC. The authors found that the use of CPC was associated with a lower incidence of pain compared with control subjects with no graft material used. They also found a 68% relative risk reduction in the loss of fracture reduction compared with fractures supplemented with autograft. Despite this, sterile serous drainage was reported in at least three of the papers.209,220,331 The exact cause for the sterile drainage is not known but may be related to local reaction to cement particles or loose bodies secondary to hematoma formation before complete curing of the cement. 

Calcium Sulfate

Calcium sulfate, or plaster of Paris, was first used as a bone filler in the early 1900s.316 It acts as an osteoconductive material, which completely resorbs within 6 to 12 weeks as newly formed bone remodels and restores anatomic features and structural properties.259 
Calcium sulfate alone and in combination with autologous bone graft has been shown to significantly augment fracture healing. In a prospective nonrandomized multicenter study, Kelly et al.180 treated 109 patients with bone defects with calcium sulfate pellets alone or mixed with unconcentrated bone marrow aspirate, demineralized bone, or autograft. After 6 months, the radiographic results showed that 99% of the pellets were resorbed and 88% of the defects were filled with trabeculated bone. Borrelli et al.45 treated 26 patients with persistent long-bone nonunions or osseous defects after an open fracture, with a mixture of autogenous iliac crest bone graft and medical-grade calcium sulfate. Twenty-two patients achieved healing after the primary surgery, while an additional two demonstrated union after a second procedure. Persistent nonunions were seen in two patients. Kim et al.186 filled bony voids left after the treatment of 56 patients with various bone tumors with either injectable calcium sulfate or DBM with 28 patients in each group. Successful incorporation and fusion was seen in 24 patients in each group, with times to complete healing of 17.3 versus 14.9 weeks in the calcium sulfate and DBM groups, respectively. Yu et al.350 treated 31 patients with tibial plateau fractures with an injectable calcium sulfate followed by early range of motion exercises. Although 3 were lost to follow-up, all 28 patients demonstrated complete fracture healing with radiographic evidence of cancellous bone incorporation by 6 months following surgery. 
Despite these encouraging results, Jepegnanam and von Schroeder166 reported on two cases of calcium sulfate failure following a corrective distal radius osteotomy for distal radius malunion. Both were elderly patients with poor underlying bone quality. The implant failures were thought to occur as the result of inadequate bone formation resulting from the resorption of the calcium sulfate graft. This highlights an important consideration for patients who lack inherent osteoinductive capabilities, such as is seen in many elderly patients. 
Calcium sulfate is also available as an injectable cement. The cement (CSC) possesses good biocompatibility, is rapidly incorporated and is resorbed by 70 days.179 In a prospective, randomized trial, McKee et al.228 treated 30 patients with chronic osteomyelitis and nonunions with either antibiotic impregnated polymethylmethacrylate (PMMA) or calcium sulfate cement to fill the bone void. The involved bones included femurs, tibias, humeri, and one ulna. Infection was eradicated in 86% of patients in both groups who were treated for osteomyelitis. Seven of 8 patients achieved healing of their nonunion in the calcium sulfate group, while 6 of 8 patients achieved union in the PMMA group. The patients receiving PMMA cement had a significantly higher rate of reoperations (p = 0.04). 

Authors’ Preferred Method of Treatment

 
 
Calcium-based Bone graft Substitutes and Calcium Phosphate–Based Cement
 

The calcium-based bone-graft substitutes are best used as bone void fillers, especially when supplemented with autologous bone. It is preferable to use them in parts of the skeleton where tensile strains are low or nonexistent, as their compressive strength is comparable to cancellous bone.126 Calcium sulfate, which is much more rapidly resorbed than the other calcium-based materials, must be used in parts of the skeleton where compressive strength is required for only short periods. These materials should not be used to bridge segmental diaphyseal defects or as onlay grafts where the majority of the surface is exposed to soft tissues.

 

Calcium phosphate–based cement has been tested in several randomized controlled clinical trials. Based on these data, its use to shorten the time in a cast during treatment of distal radius fractures or to shorten the time to loading such as weight bearing in the augmentation of tibial plateau, distal radius, proximal humerus, and calcaneal fractures is supported by clinical evidence, and this is a viable treatment options for these indications. Furthermore, it appears to lower postoperative pain levels when compared to no graft, as well as to decrease the risk of loss of fracture reduction when compared to autograft. It may be useful in other applications such as acetabular fractures and fractures of the hip, but sufficient evidence is not yet available for its use in these settings.

Enhancement of Fracture Healing with Biologic Therapies

Mesenchymal Stem Cells and Progenitor Cells

Adult MSCs and pluripotent progenitor cells are able to differentiate into multiple musculoskeletal cell lineages. The differentiation of these cells into mature osteoblasts is controlled by complex interactions involving systemic hormones, growth factors, and signaling molecules. In addition to differentiation, these factors are also critical in the regulation of cell growth and tissue repair. These molecules have autocrine, paracrine, or endocrine effects through actions on appropriate target cells. In addition to promoting cell differentiation, some have direct effects on cell adhesion, proliferation, and migration by modulating the synthesis of proteins, other growth factors, and receptors.170 
Fracture repair requires that cells be present or recruited to the site of injury to provide a source to differentiate into chondroblasts and osteoblasts during endochondral and intramembranous bone formation. In the musculoskeletal system, MSCs are able to differentiate into a variety of bone and soft tissue lineages.267 These cells play a critical role in fracture healing. However, in the elderly, the pool of available cells may be diminished, leading to delayed or possibly impaired fracture healing.139,311 
Adult MSCs obtained from bone marrow have been shown to be a source of autologous graft material. When Muschler et al.238 aspirated MSCs from the iliac crest, they noted that the mean prevalence of colony-forming units expressing alkaline phosphatase (CFU-APs), a marker of osteoblast progenitors, was 55 per 1 million nucleated cells. The investigators demonstrated an age-related decline in the number of progenitor cells for both men and women. When considered as graft material, these investigators showed that the volume of aspirate used for grafting can affect the number of CFU-APs. Although the numbers of CFU-APs increase as the aspirate volume increases, so does contamination of the sample by peripheral blood. For example, increasing the aspirate volume from 1 to 4 mL caused an approximate 50% decrease in the final concentration of CFU-APs.237 These and other findings have resulted in the search for alternatives to standard autogenous bone marrow grafting, including the use of allogeneic MSCs and expansion of autogenous cells in vitro. 
Early reports in patients with the use of unconcentrated bone marrow showed promising results.150 Healey et al.143 treated eight patients with nine nonunions after lower-extremity sarcoma resections using injections of freshly harvested, unconcentrated autologous bone marrow. The results showed that five of nine constructs had achieved union, with new bone formation evident in seven of the patients. Later, Hernigou et al.147 aspirated autologous bone marrow from the iliac crest of patients and concentrated the progenitor cells via centrifuge. They injected 189 patients with the MSC concentrate during core decompression for avascular necrosis of the femoral head and found an increasing cell concentrate lead to improved overall results. In a later study, Hernigou et al.149 studied percutaneous injection of concentrated autologous bone marrow aspirated from the iliac crest in 60 patients with established nonunions of the tibia. Analysis of the patients at 6 months found that bony union had occurred in 53 of the patients as determined by clinical and radiographic criteria. A retrospective analysis of the composition of the graft found that osteogenic progenitor cell concentration was significantly lower (<1,000 cells/cm3) in the seven patients who failed to achieve union in comparison to the 53 who did heal. In light of these findings, the authors recommended the use of greater than 1,000 progenitors/cm3 in the treatment of tibial nonunions. 
In addition to adult stem cells, it has been hypothesized that embryonic stem cells are deposited during embryogenesis in various organs, including bone marrow, and may persist in these locations into adulthood as pluripotent stem cells.122,193 These cells have the capability to both respond to a normal repair process in the body and participate in the repair of soft tissue and bone. Examples of such cells include very small embryonic-like (VSEL) cells, multipotent adult progenitor cells (MAPCs), MSCs, and marrow-isolated adult multilineage inducible (MIAMI) cells.266 Although there is little known about the use of these cells for skeletal grafting, there is currently great interest in gaining a better understanding of their potential because the use of embryonic stems cells in other organ systems has yielded impressive results.268 Undale et al.327 compared the induction of fracture repair by embryonic and bone marrow–derived stem cells in a rat nonunion model. Following fixation, fractures supplemented with adult bone marrow stromal cells demonstrated higher torque and stiffness than those supplemented with embryonic stem cells. However both of the groups treated with cells induced more radiographic evidence of fracture healing than the control group. 
The ability to derive progenitor cells from adult cells offers a promising source of stem cells. Kim et al.185 isolated pluripotent stem cells from human adipose tissue and assessed their ability to induce osteogenic differentiation and produce osteoid matrix when supplemented with DBM in critical-sized calvarial defects. The adipocyte-derived stem cells were able to be induced into osteoprogenitor phenotypes, and when supplemented with DBM, demonstrated enhanced bone healing when compared to DBM alone. 

Bone Morphogenetic Proteins

Since the discovery of the osteoinductive properties of BMP,328 these proteins have been defined to play an important role in osteogenic development and bone repair.72,170,271 BMPs are a group of noncollagenous glycoproteins that belong to the TGF-β superfamily. They exert their effects by autocrine and paracrine mechanisms. Fifteen different BMPs have been identified in humans and their genes have been cloned.78 The two that have received approval for patient treatment by the FDA are BMP-2 and BMP-7 (also called OP-1). However, while BMP-2 has received full premarket approval for the treatment of open tibia fractures, OP-1 has only received a Humanitarian Device Exemption for the treatment of recalcitrant nonunions of long bones. This approval limits the sales of OP-1 to only 4,000 units per year and requires that the surgeon obtain Institutional Review Board approval in order to use it. The studies that led to these approvals are discussed below. 
After promising results in bone development and regeneration in animal studies, as well as spine and other joint fusion studies, recent investigations have established the key role that BMPs play in fracture healing. Kloen et al.188 established that human fracture callus contains multiple BMPs and their receptors. Tissue was obtained from the fracture site of malunions in five patients undergoing a corrective osteotomy. Immunohistochemical analysis was performed and results demonstrated consistent positive staining for all BMPs and BMP receptors, most intense for BMP-3 andBMP-7. More recently, Tsuji et al.324 demonstrated the importance of BMP-2 in the fracture repair cascade. Tibia fractures were produced in transgenic mice in which BMP-2 was deleted in a limb-specific manner, before the onset of skeletal development. Mice heterozygous for this mutation were shown to have impaired healing during the earliest stages of repair with reduced periosteal reaction and decreased formation of other BMPs involved in the repair process (e.g., BMP-4 and BMP-7). However, in mice homozygous for this mutation, fracture healing was completely abolished. This study demonstrated that BMP-2 is essential for fracture healing.324 
Several clinical investigations have tested the use of recombinant human (rh)BMPs (BMPs synthesized by recombinant gene technology using human BMP DNA) in the treatment of fractures and nonunions. In a large prospective randomized partially blinded, multicenter study, Friedlaender et al.116 assessed the efficacy of rhBMP-7 (OP-1) versus iliac crest bone graft in the treatment of 122 patients with 124 tibial nonunions treated with IM nail fixation. Clinical assessment at 9 months indicated equivalent rates of union (81% of patients treated with BMP-7 and 85% of the 61 control autograft patients), while radiographic union was seen in 75% and 84% of these patients, respectively. As these results showed equivalent efficacy between OP-1 and autogenous bone graft, the authors concluded that OP-1 was a safe and effective alternative to bone graft in the treatment of tibial nonunions (Fig. 5-6). 
Figure 5-6
Sequential radiographs of a tibial nonunion treated with recombinant human OP-1 immediately postoperatively and at 9 months and 24 months after intramedullary nailing.
 
Note the bridging callus and subsequent tibial union. (Reprinted with permission from: Friedlaender GE, Perry CR, Cole JD, et al. Osteogenic protein 1 (bone morphogenetic protein 7) in the treatment of tibial nonunions. J Bone Joint Surg Am. 2001;83:S151–S158).
Note the bridging callus and subsequent tibial union. (Reprinted with permission from: Friedlaender GE, Perry CR, Cole JD, et al. Osteogenic protein 1 (bone morphogenetic protein 7) in the treatment of tibial nonunions. J Bone Joint Surg Am. 2001;83:S151–S158).
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Figure 5-6
Sequential radiographs of a tibial nonunion treated with recombinant human OP-1 immediately postoperatively and at 9 months and 24 months after intramedullary nailing.
Note the bridging callus and subsequent tibial union. (Reprinted with permission from: Friedlaender GE, Perry CR, Cole JD, et al. Osteogenic protein 1 (bone morphogenetic protein 7) in the treatment of tibial nonunions. J Bone Joint Surg Am. 2001;83:S151–S158).
Note the bridging callus and subsequent tibial union. (Reprinted with permission from: Friedlaender GE, Perry CR, Cole JD, et al. Osteogenic protein 1 (bone morphogenetic protein 7) in the treatment of tibial nonunions. J Bone Joint Surg Am. 2001;83:S151–S158).
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Bong et al.44 prospectively followed 23 patients with humeral nonunions treated with plate and screw or IM nail fixation in conjunction with various combinations of autograft, allograft, or DBM, in conjunction with rhOP-1. While all patients had healed at an average of 144.3 days, they concluded that OP-1 used in conjunction with allograft and/or DBM was equivalent to autograft for the treatment of humeral nonunions. These findings were supported by a prospective cohort study performed by Dimitriou et al.85 of 25 patients with 26 upper and lower-extremity nonunions that were treated with OP-1. Autologous bone grafting was also used in 10 of the 26 fractures. Twenty-four of 26 nonunions went on to union at an average of 4.2 months, with the 2 cases of persistent nonunion occurring in prior open fractures complicated by infection. Another prospective trial evaluated 45 patients with humeral, femoral, or tibial aseptic atrophic nonunions who underwent autologous bone grafting combined with BMP-7. Union was achieved in 100% of the nonunions, with the median time to union of 5 months and profoundly improved long-term pain and functional scores.127 
Another TGF-β family member, BMP-2, has shown promise in the treatment of acute fractures in several human studies. The BMP-2 Evaluation in Surgery for Tibial Trauma (BESTT) Study Group reported on a large prospective randomized controlled multicenter trial evaluating the effects of rhBMP-2 in the treatment of open tibial fractures.135 Four hundred and fifty patients with these injuries were randomized to receive irrigation and debridement followed by treatment either with IM nail fixation alone or IM fixation plus an implant containing either 0.75-mg/kg or 1.5-mg/kg rhBMP-2. The implant was placed over the fracture site at the time of wound closure. After 1 year, there were fewer secondary interventions (returns to the operating room for additional treatment) in the group treated with 1.5-mg/kg rhBMP-2. In addition, those patients treated with 1.5-mg/kg rhBMP-2 had accelerated times to union, improved wound healing, and reduced infection rates (Fig. 5-7). A subgroup analysis was performed on this cohort by Swiontkowski et al.,313 who analyzed 113 patients with either type IIIA or type IIIB open fractures and included only patients who received placebo (65 patients) or 1.5 mg/ml of rhBMP-2 (66 patients). The results showed that the treatment group required significantly fewer bone grafts to achieve union and had a lower incidence of infection. Another subgroup analysis by Jones et al.171 compared the treatment of 30 patients with tibial shaft fractures and significant associated cortical defects with IM fixation augmented with either autograft or allograft and rhBMP-2. There were comparable rates of union and functional outcome scores, while the blood loss and operative time was significantly less in the rhBMP-2 group. 
Figure 5-7
Radiographs of a patient who had sustained an open fracture of the left tibia (Gustilo and Anderson type IIIB) and was treated with an unreamed intramedullary nail and 1.50 mg/mL recombinant human BMP-2.
 
The fracture was considered to be clinically healed by 20 weeks and healed radiographically by 26 weeks. (Reprinted with permission from: Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein 2 for the treatment of open tibial fractures. A prospective, controlled, randomized study of 450 patients. J Bone Joint Surg Am. 2002;84:2123–2134).
The fracture was considered to be clinically healed by 20 weeks and healed radiographically by 26 weeks. (Reprinted with permission from: Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein 2 for the treatment of open tibial fractures. A prospective, controlled, randomized study of 450 patients. J Bone Joint Surg Am. 2002;84:2123–2134).
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Figure 5-7
Radiographs of a patient who had sustained an open fracture of the left tibia (Gustilo and Anderson type IIIB) and was treated with an unreamed intramedullary nail and 1.50 mg/mL recombinant human BMP-2.
The fracture was considered to be clinically healed by 20 weeks and healed radiographically by 26 weeks. (Reprinted with permission from: Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein 2 for the treatment of open tibial fractures. A prospective, controlled, randomized study of 450 patients. J Bone Joint Surg Am. 2002;84:2123–2134).
The fracture was considered to be clinically healed by 20 weeks and healed radiographically by 26 weeks. (Reprinted with permission from: Govender S, Csimma C, Genant HK, et al. Recombinant human bone morphogenetic protein 2 for the treatment of open tibial fractures. A prospective, controlled, randomized study of 450 patients. J Bone Joint Surg Am. 2002;84:2123–2134).
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Despite numerous studies demonstrating the positive effects of BMPs in animal and human models of fracture and nonunion healing, results of the use of BMPs in many other clinical trials or reviews have been less impressive. For example, Aro et al.12 performed a randomized control trial of 277 patients with Gustilo and Anderson141 grade IIIB open tibial shaft fractures treated with either IM nail fixation alone or augmented with a collagen sponge containing 1.5 mg/ml of rhBMP-2. Initially there was a slight difference in healing at 13 weeks, with 60% of fractures in the rhBMP-2 group compared to 48% in the control group being healed; however, the healing rates had become equivalent at 20 weeks at 68% and 67%, respectively. Furthermore, 30% of those in the rhBMP-2 group underwent another procedure compared to 57% in the control group, but this and other adverse events differences were not statistically significant. One possible explanation for the conflicting results between studies might be related to the differential response of mesenchymal progenitor cells to BMPs. Diefenderfer et al.84 cultured bone marrow cells from patients undergoing hip replacement with or without dexamethasone and treated with BMPs. The results demonstrated no significant osteogenic response to BMP-2, BMP-4, or BMP-7, unless the cells were pretreated with dexamethasone. Moreover, even when the cells were pretreated, the osteogenic response to BMPs was only about 50% of mouse bone marrow stromal cell cultures. Thus, the response to BMPs may differ between species, and even between individual patients. 
Finally, BMP-2 must be administered in children with caution. There are no well-established dosing parameters, thus potentially exposing them to unwanted side effects not seen in the adult population. For example, Ritting et al.272 reported on a case of a significantly enhanced inflammatory response and subsequent bone resorption in response to BMP-2 treatment of an ulnar nonunion in a child. 

Wnt Proteins

The Wnt signaling pathway is critical in the regulation of osteogenesis and bone formation. The Wnt ligands are a secreted group of proteins that act by binding to the LRP 5/6 receptors. This leads to activation of a complex composed of multiple factors, including GSK3, that causes B-catenin translocation into the nucleus to induce the production of osteogenic differentiation-inducing factors.1,54 The Wnt signaling cascade is responsible for the osteogenic effects of parathyroid hormone (PTH).337 
The Wnt signaling cascade is regulated by the secreted proteins Dkk1 and sclerostin, which competitively bind to the LRP 5/6 receptors to inhibit bone formation (Fig. 5-8)337 Animals deficient in sclerostin, or to a lesser extent Dkk1, exhibit increased bone mass and bone formation.204,205,227,269 Antibodies to these Wnt inhibitors have been shown to enhance bone formation. Animal studies have shown antibodies to both Dkk1 and sclerostin increase bone mass, cortical and trabecular bone formation, and bone mineral density.18,207 Furthermore, cultured cells from human nonunions were shown to have increased levels of Dkk1.20 These antibodies have shown promise in fracture healing. Komatsu et al.191 demonstrated mice with closed femur fractures treated with Dkk1 antibodies had increased fracture callus volume, bone mineral density and overall strength. Ominsky et al.251 found that rats with closed femur fractures and monkeys with fibular osteotomies treated with sclerostin antibodies had increased callus size, bone mass, and fracture strength. Other studies have demonstrated increased bone formation and enhanced proximal tibial metaphyseal fracture healing by antibodies to Dkk1 and sclerostin.2,3,227,333 Both of these proteins are currently being evaluated in phase two clinical trials regarding fracture and nonunion healing. 
Figure 5-8
Parathyroid hormone induces fracture healing via modulation of the Wnt pathway.
 
Total callus formation and chondrogenesis induction were increased in fractures in rats which were treated with daily injections of PTH (1–34) (A) Radiographs examining callus formation in the femurs of rats 2 and 3 weeks after injury. B: Safranin O staining of fracture slices 5 and 10 days after injury. Chondrogenic cells are stained red. C, D, E, and F. MicroCT analysis of callus and bone volume and mineral density. G: PTH exerts its effects through the Wnt pathway, inducing the osteogenic and hypertrophic chondrogenic differentiation of progenitor cells, while inhibiting the adipogenic lineage. These effects are modulated by the Wnt inhibitor, Sclerostin, which is secreted by osteocytes in a feedback loop. (Reprinted with permission from: Kakar S, Einhorn TA, Vora S, et al. Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures. J Bone Miner Res. 2007;22(12):1903–1912; and from: Wagner E R, Zhu G, Zhang BQ, et al. The therapeutic potential of the Wnt signaling pathway in bone disorders. Curr Mol Pharmacol. 2011;4(1):14–25.)
Total callus formation and chondrogenesis induction were increased in fractures in rats which were treated with daily injections of PTH (1–34) (A) Radiographs examining callus formation in the femurs of rats 2 and 3 weeks after injury. B: Safranin O staining of fracture slices 5 and 10 days after injury. Chondrogenic cells are stained red. C, D, E, and F. MicroCT analysis of callus and bone volume and mineral density. G: PTH exerts its effects through the Wnt pathway, inducing the osteogenic and hypertrophic chondrogenic differentiation of progenitor cells, while inhibiting the adipogenic lineage. These effects are modulated by the Wnt inhibitor, Sclerostin, which is secreted by osteocytes in a feedback loop. (Reprinted with permission from: Kakar S, Einhorn TA, Vora S, et al. Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures. J Bone Miner Res. 2007;22(12):1903–1912; and from: Wagner E R, Zhu G, Zhang BQ, et al. The therapeutic potential of the Wnt signaling pathway in bone disorders. Curr Mol Pharmacol. 2011;4(1):14–25.)
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Figure 5-8
Parathyroid hormone induces fracture healing via modulation of the Wnt pathway.
Total callus formation and chondrogenesis induction were increased in fractures in rats which were treated with daily injections of PTH (1–34) (A) Radiographs examining callus formation in the femurs of rats 2 and 3 weeks after injury. B: Safranin O staining of fracture slices 5 and 10 days after injury. Chondrogenic cells are stained red. C, D, E, and F. MicroCT analysis of callus and bone volume and mineral density. G: PTH exerts its effects through the Wnt pathway, inducing the osteogenic and hypertrophic chondrogenic differentiation of progenitor cells, while inhibiting the adipogenic lineage. These effects are modulated by the Wnt inhibitor, Sclerostin, which is secreted by osteocytes in a feedback loop. (Reprinted with permission from: Kakar S, Einhorn TA, Vora S, et al. Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures. J Bone Miner Res. 2007;22(12):1903–1912; and from: Wagner E R, Zhu G, Zhang BQ, et al. The therapeutic potential of the Wnt signaling pathway in bone disorders. Curr Mol Pharmacol. 2011;4(1):14–25.)
Total callus formation and chondrogenesis induction were increased in fractures in rats which were treated with daily injections of PTH (1–34) (A) Radiographs examining callus formation in the femurs of rats 2 and 3 weeks after injury. B: Safranin O staining of fracture slices 5 and 10 days after injury. Chondrogenic cells are stained red. C, D, E, and F. MicroCT analysis of callus and bone volume and mineral density. G: PTH exerts its effects through the Wnt pathway, inducing the osteogenic and hypertrophic chondrogenic differentiation of progenitor cells, while inhibiting the adipogenic lineage. These effects are modulated by the Wnt inhibitor, Sclerostin, which is secreted by osteocytes in a feedback loop. (Reprinted with permission from: Kakar S, Einhorn TA, Vora S, et al. Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures. J Bone Miner Res. 2007;22(12):1903–1912; and from: Wagner E R, Zhu G, Zhang BQ, et al. The therapeutic potential of the Wnt signaling pathway in bone disorders. Curr Mol Pharmacol. 2011;4(1):14–25.)
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Other Peptide Signaling Molecules

Transforming Growth Factor-β

TGF-β has a similar structure and function to the BMPs. It is known to influence a number of cell processes including the stimulation of MSC growth and differentiation, and the enhancement of collagen and other extracellular matrix protein synthesis. It also functions as a chemotactic factor for fibroblast and macrophage recruitment.183 
Several studies have found dose-dependent effects of TGF-β improve fracture healing. Lind et al.209 analyzed the use of TGF-β in rabbits with tibial defects treated with plate fixation, demonstrating that those treated with the low dose had improved stiffness, while both low and high doses increased callus formation. Critchlow et al.77 established in a tibial defect healing model in rabbits that a low dose of TGF-β had an insignificant effect on callus development, whereas the higher dose resulted in a larger callus. 
Although TGF-β appears to only have a modest, dose-dependent influence on fracture repair, a recombinant fusion protein with TGF-β, containing a collagen-binding domain, has been shown to induce osteogenic differentiation of bone marrow cells in rats.8 Becerra et al.29 presented a case report of a 69-year-old man with a proximal tibial defect from resection of long-standing osteomyelitis. Bone marrow cells were cultured in the presence of the TGF-β fusion protein and then placed in the tibial defect in conjunction with an HA carrier. Imaging at 90 days was consistent with new bone formation including bridging callus, and biopsy samples taken at 8 weeks showed new bone formation. 

Vascular Endothelial Growth Factor

Angiogenesis is required for bone healing to occur, permitting cells to receive nutrients and oxygen. Early in the fracture repair process, vascular endothelial growth factor (VEGF) has been shown to be upregulated.310 Eckardt et al.92 tested the ability of rhVEGF to heal critical-sized defects in rabbits when compared to autograft controls. Biomechanical testing of the treated bones found that the ultimate strength and stiffness were significantly greater in the rhVEGF-treated animals than in controls and equivalent to autograft treatment. Micro-computed tomographic analysis showed abundant callus in both the rhVEGF-and autograft-treated groups, but absent callus in the control groups. 
As noted earlier, remodeling of allograft bone is a slow process that occurs through creeping substitution. Surface healing can leave large central areas of necrotic bone that contributes to the 25% to 35% failure rate with this type of grafting.36,212 Ito et al.163 found that VEGF and receptor activator of nuclear factor–κB ligand (RANKL) were downregulated during allograft healing. They developed a method by which RANKL and VEGF were combined with a viral vector and attached to the surface of allografts. Theses allografts were then used in a mouse fracture model, where histologic analysis at 4 weeks showed periosteal resorption with new bone formation and medullary neovascularization that was not seen in untreated controls. These preliminary results demonstrate a novel way to increase allograft healing and warrant further study. 

Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF), also known as FGF-2, belongs to a class of growth factors that have an affinity for heparin.164,187 It is one of the most potent stimulators of angiogenesis, partially through its influence on endothelial cell migration and upregulation of integrin expression.187 Its mitogenic effects on osteoblasts, chondrocytes, and fibroblasts play a critical role during growth, wound healing, and fracture repair.158,162 
During fracture repair, FGFs differ in their temporal and spatial expression.279 In the early stages, FGF-1 and FGF-2 are localized to the proliferating periosteum. This expression is then limited to osteoblasts during intramembranous bone formation and to the chondrocytes and osteoblasts during endochondral bone formation. In light of their active involvement during fracture repair, investigators have studied the potential therapeutic roles of FGFs in bone formation. Chen et al.68 demonstrated FGF-2 injections into tibial shaft fractures in rabbits increased bone density and fracture callus volume, with increased cellular proliferation markers. Kawaguchi et al.177 performed a recent randomized control trial comparing 70 patients with tibial shaft fractures treated with IM nailing who were augmented with a gelatin hydrogel alone, or in combination with 0.8 mg or 2.4 mg of rhFGF-2 in the fracture site. The high-dose group demonstrated significantly higher rates of radiographic union compared to the placebo, and 0 compared to 4 nonunions, respectively. There were no differences in long-term weight bearing or functional outcomes measures. At this time, the status of the FGFs in the enhancement of fracture healing in patients is relatively unknown, but it appears to have a satisfactory safety profile. 

Platelet-derived Growth Factor

PDGF is a large polypeptide that consists of two chains that share 60% amino acid sequence homology.304 Its potential role in bone healing is related to its mitogenic and chemotactic properties for osteoblasts.60,63 A positive effect of PDGF on fracture healing was demonstrated in a rabbit tibial osteotomy model in which the fractures were injected with either 80 μg of PDGF in a collagen carrier or collagen alone.242 Results showed an increase in callus formation, but no effects on the mechanical properties of the calluses compared with controls. A more recent study by Hollinger et al.152 in a geriatric, osteoporotic rat model found significant gains in mechanical strength in fractures treated with PDGF combined with an injectable beta-TCP–collagen matrix. At 5 weeks after the initial injury, the torsion to failure in the PDGF-treated tibias was comparable to that of the uninjured extremity, while control and untreated fractures remained unhealed. These preclinical data and encouraging results from clinical studies of PDGF treatment of dental implants244 and diabetic foot ulcers345 suggest a potential role for PDGF in skeletal trauma. 

Prostaglandin Modulators

Prostaglandins (PGs) comprise a group of unsaturated fatty acids that include PGE and PGF. These are known to have osteogenic effects when implanted into skeletal sites203,253 or infused systemically.325 The release of arachidonate from alkyl-arachidonyl phospholcholine produces the precursor of several proangiogenic and proinflammatory mediators. These factors are important in the early phases of bone formation, as they serve as osteogenic differentiation mediators of progenitor stem cells. Thus, inhibition of this cascade by drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) in the acute phase of fracture healing or bone formation can block inflammatory cell recruitment and stem cell differentiation.233 
Arachidonic acid is converted to several types of PGs by two known PG synthases (cyclooxygenases): COX-1 or the inducible COX-2. These bind to one of four EP receptors, EP1, EP2, EP3, or EP4. In a study of rabbit tibial fractures, Dekel et al.80 demonstrated that PGE2 caused a dose-dependent stimulation of callus formation and an increase in total bone mineral content. Its effects were also shown to be greatest during the latter stages of fracture healing, suggesting that the primary effect may be to stimulate osteoblasts and osteoprogenitor cells as opposed to undifferentiated MSCs. The inhibition of lipoxygenase, an enzyme that converts arachidonic acid to leukotrienes, may enhance bone healing. Cottrell and O’Connor76 administered 5-lipoxygenase inhibitors to rats with closed femoral fractures and noted callus proliferation and mineralization rate, as well as completion via endochondral ossification to be significantly enhanced. In addition, mice lacking 5-lipoxygenase have significantly enhanced fracture healing.217 

Nonsteroidal Anti-Inflammatory Drugs

NSAIDs are effective and commonly used analgesics. They act by binding to and blocking the activity of the COX enzymes, which results in a suppression of PG synthesis.195 These drugs can either selectively target COX-2 or nonselectively target both COX-1 and COX-2. Targeting the COX-2 enzyme with either selective or nonselective inhibitors leads to an inhibition of PGE2 and PGF thereby preventing their ability to activate osteoblasts.35,233 Although the role of PGs in acute fracture healing is well established, the clinical evidence that inhibition of this pathway has a significant adverse effect has yet to be established. 
Both selective and nonselective NSAIDs have been shown to delay acute fracture healing and increase the risk of nonunions.50,195,236,269,312,344 For example, Simon et al. treated closed rat femoral fractures with varying concentrations and durations of the selective COX-2 inhibitor celecoxib.236,297 They found early administration of celecoxib reduced the mechanical properties of the callus and increased the proportion of nonunions. Conversely, treatment with celecoxib preoperatively or 14 days postprocedure did not affect fracture healing. Other studies have confirmed this finding, demonstrating a decrease in mineralized callus and inhibition of haversian remodeling secondary to NSAID administration.195,269,312,344 Another study in rats found NSAIDs administered during the acute phases of fracture healing caused a significant decrease in bone density, as well as a reduction in the bone’s overall strength and stiffness.335 COX-2 knockout mice were also found to have suppressed intrinsic capabilities in fracture healing.336 In rabbits, multiple models have demonstrated COX-2 inhibitors impair fracture healing, bone ingrowth, and callus formation.86,101,140,236 
There are a number of studies that provide conflicting data to the negative effects of NSAIDs on fracture healing.30,133,176,211,262 Goodman et al.133 implanted cylindrical titanium chambers called osseointegration centers into rabbit tibiae and administered rofecoxib (COX-2 inhibitor) for the first 2 weeks of fracture healing, weeks 5 and 6 of the repair process or continuously for 6 weeks postoperatively. These bone harvest devices contain an inner and out chamber that is able to measure the rate of bone ingrowth. The authors noted no difference in bone ingrowth or osseointegration for the initial or final 2 weeks, but did demonstrate less ingrowth if treated continuously for 6 weeks. Karachalios et al.176 compared the administration of prednisolone, indomethacin, meloxicam, or rofecoxib for 5 days after right ulna mid-diaphyseal osteotomy. Although radiographic and biomechanical parameters were lower in the prednisolone, indomethacin, and meloxicam groups, the highly selective COX-2 inhibitor rofecoxib did not demonstrate a significant difference in fracture healing from the control group. Similarly, Long et al.211 demonstrated that the rate of spinal fusions in rabbits was negatively affected by the nonselective COX inhibitor indomethacin, but not the COX-2 inhibitor celecoxib. 
These conflicting results may be due to the effects of differing dosage regimes and subsequent local bioavailability of the NSAID. Bo et al.40 examined the effect of treating closed, nonimmobilized femoral shaft fractures in rats with indomethacin at varying concentrations. They demonstrated the suppression of fracture healing when indomethacin was administered at doses >2 mg/kg/day. Many other studies have confirmed this effect from high-dose regimens of both selective and nonselective COX inhibitors.86,101,102,125 However, similar to the timing of NSAID administration, there appears to be controversy regarding the dose-dependent effects as many rodent trials have failed to demonstrate any significant suppression in fracture healing.4,86,233 It also appears that after NSAID discontinuation, the PGE2 levels gradually return to normal and any effect on fracture healing diminishes. Gerstenfeld et al.124 administered either ketorolac, valdecoxib, or a control dose to rats in a fracture-healing model for either 7 or 21 days. Although there was a trend for a higher rate of nonunions after the 7 and 21 days protocols, when each was discontinued for 14 days, the rate of nonunions and overall levels of PGE2 were comparable to the controls. 
Considerable controversy exists as to the effect of NSAIDs in patients with acute fractures.95 Giannoudis et al.128 retrospectively examined the effect of NSAIDs in 32 patients with femoral nonunion compared to 67 patients with united femoral shaft fractures. The primary NSAIDs taken were ibuprofen and diclofenac. A larger portion of patients with fracture nonunions had taken the NSAIDs (62.4% vs. 13.4%) for a longer period of time (average of 21.2 weeks vs. 1 week). Burd et al.57 examined 112 patients with acetabular fractures who also had concomitant long-bone fractures that were randomized to receive radiation therapy, high-dose indomethacin 25 mg three times a day for 6 weeks or no prophylaxis against heterotropic ossification. Those who received indomethacin had a significantly higher rate of nonunions of the long bones than those in either of the other groups (p = 0.004). Another study reviewed a cohort of 9,995 patients with humeral shaft fractures from the Medicare Database, and found that prescription of NSAID used within 90 days after the fracture was correlated to an increase in the incidence of nonunions.37 In addition to the dose-dependent effects from the above studies, the authors noted an increase in nonunions among patients who took NSAIDs within the 61 to 90 days time period after the injury and not in the 1- to 30- or 31- to 60-day time periods. 
Although there appears to be a trend toward a decreased rate of fracture healing from high-dose NSAID administration in animal studies, this relationship has not been demonstrated in clinical studies. Given the paucity of large prospective studies and inherent deficiencies with the existing retrospective body of literature, the exact role of NSAIDs during fracture healing remains unclear. Furthermore, the inhibitory effect of NSAIDs appears to be reversible and PGE levels return to normal about 1 to 2 weeks after discontinuation of NSAID use. Therefore, as noted by Kurmis et al.,195 NSAID use as an analgesic appears to be safe in short durations (10 to 14 days) after fractures or spinal fusions, as long as they are stopped within a couple weeks. 

Authors’ Preferred Method of Treatment

 
 
Bone Morphogenetic Proteins
 

We recommend the use of OP-1 (BMP-7) for the treatment of recalcitrant nonunions of long bones and BMP-2 for the treatment of open tibia fractures and those with large cortical defects. Some of other molecules, including Wnt pathway modulators and FGF-2 have shown promising preclinical and early clinical study results, but there is not enough evidence to recommend for or against their use at this time.

 

Regarding freshly harvested bone marrow, we believe there is insufficient evidence to support its routine use in traumatic or reconstructive orthopedic surgery. Alternatively, multiple small aspirations from the iliac crest, with centrifuge-mediated concentration (bone marrow aspirate concentrate) has been able to optimize the concentration of the osteoprogenitor cells.148,150 The senior author (TAE) has used this technique with success in several cases of long-bone nonunions.

 

While the role of NSAIDs in animal models appears to be well established, there is lack of scientifically rigorous clinical data for or against its effects in the acute phases of fracture healing. In addition, if there is an effect of NSAIDs on fracture healing, it appears to be dose-dependent and reversible, as it disappears after 7 to 10 days once the NSAID has been stopped. Therefore, we believe NSAIDs are safe to be used as an analgesic in short durations (10 to 14 days) after fractures or spinal fusions. However, we would recommend caution of their use in patients with comorbidities, such as smoking, glucocorticoid use, and diabetes.

Systemic Enhancement of Fracture Healing

Parathyroid Hormone

Calcium and phosphate homeostasis is a complex process that involves multiple signaling pathways and organ systems. The largest storage site of calcium and phosphate is the skeletal system, and the release of these ions is largely regulated by the coordinated stimulation and suppression of osteoblasts and osteoclasts. PTH is a major regulator of mineral homeostasis, exerting its effects by binding to a receptor on osteoblasts.274,275 PTH is an 84-amino acid peptide that is produced in response to depressed serum calcium levels. Its major effects are in the kidneys, where it regulates phosphate diuresis and 1,25-dihydroxyvitamin D synthesis with its subsequent enhancement of gastrointestinal calcium and phosphate absorption.264 The actions of PTH on bone metabolism can be both stimulatory and inhibitory. It has been found that continuous release of PTH leads to an increase in osteoclast numbers and activity,206 while intermittent exposure results in increased bone formation in both rats and humans through osteoblast and osteoprogenitor cell recruitment.82,243 This occurs through the activation of the Wnt signaling pathway within osteogenic differentiation.261 
Teriparatide (PTH [1–34]) is currently an FDA-approved treatment for osteoporosis. Intermittent injections increase bone formation on all aspects of the bone, including the cortical and cancellous matrices.54,114 Clinical trials using PTH (1–34) have shown an increase in bone mass in osteoporotic men and an increase in bone mineral density and a reduction of vertebral and other osteoporotic related fractures in postmenopausal women.82,243 Neer et al.243 assessed the efficacy of intermittent PTH (1–34) to improve bone mineral density in a clinical trial involving 1,673 postmenopausal women with prior nontraumatic vertebral fractures. Results demonstrated that PTH increased bone mineral density and reduced the risk of fracture. In addition, Saag et al.282 demonstrated that teriparatide is more effective at treating glucocorticoid-induced osteoporosis and reducing vertebral fracture risk than the bisphosphonates raloxifene and alendronate. 
Based on this anabolic effect of PTH on the skeleton, several animal studies have been conducted examining the effects of PTH on the repair of bone. All have demonstrated an enhancement of fracture healing when doses were given intermittently.240,241 Kakar et al.174 examined daily systemic PTH injections in the treatment of femoral shaft fractures in mice. The PTH injections increased callus volume and density, with a greater induction of chondrogenesis and chondrocyte hypertrophy, carried out through an induction of the canonical Wnt pathway (see Fig. 5-8). Manabe et al.216 studied PTH in 17 female cynomolgus monkeys who underwent a femoral osteotomy with plate fixation, augmented with either low-dose (0.77 μg/kg) or high-dose (7.5 μg/kg) PTH or placebo, given twice weekly for 3 weeks. All groups healed by 26 weeks, with a larger callus size but a lower callus density in the control animals. The ultimate stress and elastic moduli of the healing osteotomy were significantly higher in PTH-treated animals. Alkhiary et al.6 reported on the use of PTH (1–34) in the treatment of experimental femur fractures in Sprague-Dawley rats. Animals were treated with either 5- or 30-μg/kg/day PTH (1–34) for a total of 35 days beginning at the time of fracture creation. There were significant increases in strength and bone mineral content for the 30-μg/kg group as early as 3 weeks, and these differences were sustained at 85 days. 
A clinical trial on the use of PTH (1–34) in the treatment of fractures of the distal radius in humans showed that the time to fracture healing was shorter in the PTH-treated patients.6 Aspenberg et al.17 performed a randomized double-blind placebo controlled trial of 102 postmenopausal women with distal radius fractures treated with teriparatide beginning within 10 days of the fracture and continuing for 8 weeks. Although a 40 mcg daily dosage did not significantly enhance fracture-healing times, it was noted that a lower dose of 20 mcg did shorten the time to complete fracture healing, although it was not quite statistically significant. Other case series have shown improved fracture healing from teriparatide treatment in patients with pelvic fragility fractures, as well as nonunions of the humeral shaft and sternum.69,257 In a randomized control trial, Peichl et al.257 treated 65 elderly osteoporotic patients with pelvic fragility fractures with either daily injections of PTH (1–84) or a placebo. These treatments reduced the time to fracture healing to 7.8 weeks, as compared to 12.6 weeks for the controls. These findings suggest that PTH (1–34), as well as other PTH fragments, may have a role in the clinical treatment of fractures, especially in osteoporotic bones. 

Growth Hormone and Insulin-like Growth Factor I

Growth hormone (GH) and insulin-like growth factors (IGFs) play an important role in skeletal development and remodeling. GH is currently used clinically to treat patients with short stature249 because it stimulates endochondral ossification, periosteal bone formation, and linear growth. It mediates these effects through the IGF system including the ligands, receptors, IGF-binding proteins (IGFBPs), IGFBP proteases and activators, and inhibitors of IGFBP proteases. Through these mediators, it is able to induce osteogenic differentiation and upregulate bone formation. 
Two IGFs have been identified: IGF-I (somatomedin C) and IGF-II. Although IGF-II is the most abundant growth factor in bone, IGF-I has the greater potency for promoting growth and has been localized in healing fractures of humans.10,61,62 IGF-I and IGF-II promote bone matrix formation (type I collagen and noncollagenous matrix proteins) by fully differentiated osteoblasts, inhibit collagen degradation, and promote osteoblast maturation and replication.64 Expression of the IGF-I increases with expression of GH,265 and it is likely responsible for the anabolic effects of GH. 
Several studies have reported moderate enhancement of skeletal repair using either GH9,22,190 or IGF-I.318 Mazziotti et al.224 found increased spinal deformities in patients with a GH deficiency, and a reduced fracture risk after GH therapy administration. A recent randomized clinical trial was presented by Raschke et al.265 in which 406 patients with tibia fractures were treated daily for 26 weeks with either placebo or gradually increasing concentrations of GH in an attempt to avoid adverse events, such as water retention, typically caused by GH. Although there was no difference in radiographic union rates in the open fractures, the relative risk for healing a closed fracture was greatest in the group treated with the highest dose of GH (60 μg/day; relative risk [RR], 1.44; 95% confidence interval [CI], 1.01 to 2.05; p = 0.045). While patients treated with 60-μg/day GH were able to bear full weight earlier, there was a higher number of adverse events: 58% in the 60-μg/day GH-treated group compared with 35% in controls. These adverse events included arthralgias, edema, and, to a lesser extent, wound infection. Two other prospective randomized controlled trials demonstrated that recombinant GH administration enabled an earlier return to prefracture activity and overall function in elderly patients with hip fractures or those undergoing total hip arthroplasties.333,342 When considering GH treatment for fractures or other musculoskeletal pathologies, one must consider the risk associated with high or even moderate dose GH administration, as a higher mortality has been shown in critically ill patients who received GH treatments.314 

Statins

Statins, HMG-CoA reductase inhibitors, are lipid-lowering drugs that block cholesterol synthesis through the inhibition of mevalonic acid production. The conversion of HMG-CoA to mevalonic acid occurs early in the pathway and also inhibits the production of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Small GTP-binding proteins such as Rho and Ras require GGPP and FPP, respectively, for translocation to the plasma membrane.197 Inhibition of this process by statins may block osteoclast maturation and subsequent bone catabolism.295 In addition, studies have shown that statins stimulate the BMP-2 promoter in osteoblasts leading to enhanced bone formation.234 
In mice, Skoglund and Aspenberg301 showed that daily injections of simvastatin had no effect on fracture healing, while a continuous systemic infusion and continuous local delivery improved the force to failure by 160% and 170%, respectively. However, Chissas et al.70 treated 54 rabbits with low- or high-dose simvastatin, or a control group. The high-dose simvastatin reduced callus formation and decreased the fracture gap compared to the low dose or control groups. Local delivery of statins, such as with hydrogels or nanoparticle beads, has shown promise in enhancing fracture repair. Garrett et al.121 used poly(lactic-coglycolide acid) nanoparticle beads containing various concentrations of lovastatin to augment femur fracture repair in rats. Their results showed at 4 weeks that doses of 1 and 1.5 μg/day significantly accelerated fracture healing as measured by the size of the fracture gap and biomechanical strength. Furthermore, Fukui et al.119 found simvastatin conjugated gelatin hydrogel increased the healing of a femoral nonunion in rats. The success of local delivery also demonstrates that the effects of statins on fracture healing is likely a locally mediated induction, independent of the systemic cholesterol lowering effects of the drug. 
In a recent meta-analysis of multiple cohorts and reviews, Bauer et al.27 found that statins reduce fracture risk in the hip and to a lesser extent in the spine. This finding is likely due to the increased bone mineral density as the result of statin therapy, as demonstrated by Edwards et al.93 in postmenopausal women. However, there remains a paucity of good prospective controlled trials analyzing the true effects of statins on fracture healing. 

Bisphosphonates and Osteoclast Inhibitors

Bisphosphonates are commonly used for the treatment of osteoporosis. They act by binding to HA and inhibiting osteoclast-mediated bone resorption by inducing osteoclast apoptosis. Bone remodeling is subsequently inhibited, increasing the bone mineral density. 
The role of bisphosphonates in fracture prevention and repair is less clear. Multiple animal studies have demonstrated bisphosphonates to enhance callus formation and bone mineral density; however, the remodeling and mineralization phases are delayed or completely inhibited.54,202,226 Huang et al.157 demonstrated a 50% delay in spinal fusion healing in rats treated with bisphosphonates. Furthermore, unlike the effects seen when administering PTH, Sloan et al.302 found bisphosphonates to suppress fracture healing by a rate of 44% compared to the control. Interestingly in patients, Munns et al.235 examined the effects of pamidronate therapy on pediatric patients with osteogenesis imperfecta. They noted a significant delay in long-bone osteotomy healing, but minimal delays in acute fracture healing. Rozental et al.278 reviewed 196 patients who sustained a distal radius fracture, with 43 who were currently on bisphosphonate therapy at the time of injury. The fractures in the patients, treated both with operative and nonoperative management, who were taking bisphosphonates demonstrated a longer time to union. 
One potential solution to the pitfalls seen with using bisphosphonates for fracture prevention or treatment could be to augment it with an anabolic agent, such as a BMP. Schindeler et al.289 demonstrated significant increases in number and rates of unions in neurofibromatosis-1 (NF-1) deficient mice with tibial pseudarthrosis treated with both a BMP and bisphosphonate. 
Another antiresorptive agent that has recently been approved for osteoporosis is the monoclonal antibody denosumab. This antibody binds to osteoblast-produced RANK ligand, preventing its association with the RANK receptors on osteoclasts. Although there have not been any clinical studies on fracture healing, this antibody has been shown to have a similar effect to bisphosphonates, by increasing callus formation and delaying remodeling.15 However, unlike bisphosphonates, Denosumab increased bone mineral density within the callus, not just bone mineral content. 

Authors’ Preferred Method of Treatment

 
 

Although the above compounds all show some promise for the systemic enhancement of fracture healing, their lack of FDA approval would require off-label use in the setting of fracture treatment. Because of this, the authors cannot recommend their use at this time.

Physical Enhancement of Skeletal Repair

The mechanical environment has a direct impact on fracture healing. Direct mechanical perturbation and biophysical modalities such as electrical and ultrasound stimulation have been shown to affect fracture healing. To enhance fracture repair by these mechanical measures, it is necessary to develop a fundamental understanding of the ways by which the mechanical environment impacts cellular and molecular signaling. 

Mechanical and Biophysical Stimulation

Mechanical forces play a crucial role in the healing process. Sarmiento et al.286 found that early weight bearing accelerates the fracture-healing process. Early weight bearing in rats after standard nonrigid IM femoral fracture fixation demonstrated improved histologic, radiographic, and mechanical parameters of fracture healing. The authors attributed these findings to early mobilization facilitating the maturation of callus tissue produced by endochondral ossification. 
The degree of stability at the fracture site has a direct impact on the repair process. Lewallen et al.200 demonstrated compression plating–enhanced bone formation 120 days after injury when compared to external fixation. Those treated with the external fixators had significantly less intracortical and endosteal new bone formation, with more bone porosity compared to those treated with compression plates. Compression plating increased fixation stiffness in almost all modes. Thus, the authors concluded that the rigidity of the fixation is critical in early fracture remodeling. Furthermore, several investigators have attempted to show micromotion as seen with the compression plating actually modulates fracture healing. For example, in a prospective randomized clinical trial, Kenwright et al.181 used a pneumatic pump to deliver a small cyclic amount of axial displacement in order to compare the effects of controlled micromotion on tibial diaphyseal fracture healing in patients treated with external fixation. The controlled micromotion significantly enhanced both clinical and mechanical healing compared with only rigid fixation, with no increase in complication rates. 

Distraction Osteogenesis

Limb lengthening was first described by Codivilla74 in 1904 for the treatment of limb length discrepancies. It was not until the work of Ilizarov et al.160,161 50 years later that the technique of distraction osteogenesis gained popularity as a method for enhancing bone regeneration in both orthopedic and maxillofacial operations. 
Distraction osteogenesis is divided into three phases, latency, distraction, and consolidation. The latency phase begins immediately after the osteotomy and is related to the robust inflammatory response and recruitment of molecules involved in the early phases of fracture repair. The distraction phase occurs when the longitudinal stresses creating the gap lead to a central fibrous zone with chondrocytes and fibroblast progenitors along with columns of early mineralization.14 This histologically resembles endochondral ossification in the early phases, but gradually becomes intramembranous ossification in the later stages.14,107,283 Once the distraction phase ceases, the consolidation phase takes over with extensive bone matrix and osteoid production by osteoblasts. 
Through the controlled distraction of bone fragments, the expression of various growth factors ensues, including those involved in angiogenesis. Pacicca et al.252 demonstrated the expression of several of these molecules localized to the leading edge of the distraction gap. The greatest levels were seen during the active phase, consistent with the apposition of new bone matrix. Others have shown that robust angiogenesis and progenitor recruitment, under VEGF control, occurs during the active and consolidation phase.199 
Several investigators have used the technique of distraction osteogenesis to stimulate new bone formation in the clinical setting. Kocaoglu et al.189 treated 16 patients with hypertrophic nonunions with the Ilizarov distraction method. All patients had at least 1 cm of shortening, three patients had a deformity in one plane, and the remainder had a deformity in two planes. Distraction was begun on the first postoperative day at the rate of 0.25 mm/day and was left in place until at least three of four cortices showed bridging callus. All nonunions had healed at an average follow-up of 38.1 months, with correction of all preoperative length inequalities and limb angulation to normal alignment. A similar study of 17 patients with tibial nonunions associated with bone loss found an average treatment time of 8 months, with functional results being reported as excellent in 15 and good in 2.292 
Open fractures have also been managed successfully with distraction osteogenesis. Sen et al.293 managed 24 patients with Gustilo and Anderson141 grade III open tibia fractures with compression–distraction osteogenesis using the Ilizarov-type circular external fixator. After an average of 30-month follow-up, results were excellent in 21 and good in 3 patients. Functional assessment scores were excellent in 19, good in 4, and fair in 1 patient. 
However, not all studies have shown acceptable bone formation as the result of distraction osteogenesis. Many factors have been associated with increased fracture risk after the removal of the external fixators, including age, location of lengthening, and smoking.13,159,299 Fracture rates can be as high as 8% to 9% after fixator removal and initiation of weight bearing.299 Thus, there may be a need to augment the healing and bone formation with additional modalities, such as low-intensity pulsed ultrasound (LIPUS), pulsed electromagnetic field (PEMF), extracorporeal shockwave therapy (ESWT), or osteogenic-inducing agents. 

Electrical Stimulation

Electrical potentials were first described in mechanically loaded bone by Fukada and Yasuda118 in 1957. With this discovery, investigators began to study the influence that electrical current might have on the healing of bone. In 1971, Friedenberg et al.113 found that the healing of nonunions could be affected by the use of direct current. Within 5 years, more than 119 articles had been published highlighting the use of electrical stimulation on bone growth and repair.48 Its effects are thought to be carried out by stimulating the local production of osteogenic and mitogenic growth factors, including BMP-2 and BMP-4, as well as TGF-β.67 These induce osteogenesis and recruit osteoprogenitors to facilitate bone formation. 
There are currently three methods for the electrical stimulation of bone healing: (i) constant direct current (DC) stimulation with the use of percutaneous or implanted electrodes (invasive), (ii) capacitive coupling (noninvasive), or (iii) time-varying inductive coupling produced by a magnetic field (noninvasive; also known as PEMF stimulation). DC stimulation uses stainless steel cathodes placed in the tissues near the fracture site, stimulating new bone formation according to the level of applied current, with a threshold level above which cellular necrosis may occur.112 With pulsed electromagnetic stimulation, externally applied coils produce an alternating current, leading to time-varying magnetic and electrical fields within the bone. In capacitively coupled electric fields (CCEFs), an electrical field is induced in bone through the use of an external capacitor—that is, two electrically charged metal plates placed on either side of a limb.109 
Electrical stimulation has primarily been used in orthopedics for the treatment of nonunions. Brighton et al.49 found DC for the treatment of 178 nonunions in 175 patients resulted in an 84% union rate regardless of the presence of metallic internal fixation devices. Interestingly, the investigators found that even in the presence of osteomyelitis the healing rate was nearly 75%. Although initially half of the patients received a lower dose of 10 μA, poor healing was noticed after 12 weeks and all patients were switched to receive the higher dose of 20 μA. When this study was expanded to include other centers, an additional 58 of 89 nonunions achieved similar results. Treatment failures were attributed to inadequate electricity, the presence of a synovial pseudarthrosis or infection, and dislodgment of the electrodes. Complications were minor with the exception of patients with previous osteomyelitis. The authors concluded that given proper electrical parameters and cast immobilization, a rate of bone union comparable to that seen with bone-graft surgery could be achieved. 
Scott and King290 reported similar results in a prospective, double-blind trial using capacitive coupling in patients with established nonunions. In a population of 21 patients, healing was achieved in 60% of the patients who received electrical stimulation. Patients managed with the placebo unit showed a complete lack of bone formation. 
Bassett et al.26 reported on the use of PEMF in the treatment of 127 nonunited tibial diaphyseal fractures treated with long-leg plaster cast immobilization. Patients were treated with nonweight-bearing ambulation and a total of 10 hours of PEMF stimulation daily. Bony union occurred in 87% of the patients and was independent of patient age or sex, the number of previous operations, and the presence of infection or metal fixation. Later, Sharrard296 conducted a double-blind, multicenter trial of the use of PEMFs in patients who had developed a delayed union of a tibial fracture. Forty-five tibial fractures that had not united for more than 16 weeks but less than 32 weeks were treated with immobilization in a plaster cast that incorporated the PEMF coils, with activation in only 20 of the 45 units. There was radiographic evidence of union in nine of the fractures that had been subjected to electromagnetic stimulation compared with only three of the fractures in the control group. Simonis et al.298 performed a similar double-blind trial in 34 patients with tibial nonunions treated with an oblique fibular osteotomy and external fixator. The union rate for those treated with electrical stimulation was 89%, compared 50% in the control group. 
Despite the promising results seen in patients with nonunions and delayed unions, the application of this technology to the treatment of fresh fractures has not been clearly defined. Although some studies have shown that PEMFs favorably influence fracture healing in experimental animals112 and osteotomies in patients,46,215 other studies have failed to demonstrate clinically significant effects.26 Beck et al.31 found no difference in healing time in 44 patients who were randomly assigned to either CCEF or placebo. 
Mollon et al.230 performed a meta-analysis of 11 randomized control trials evaluating the efficacy of electrical stimulation in fracture healing. The authors found electrical stimulation had a nonsignificant benefit in delayed unions or fracture nonunions (p = 0.15), as well as in callus formation in femoral intertrochanteric osteotomies. However, there was minimal to no benefit in limb-lengthening, nonoperative management of Colles fractures, tibial stress fractures, or operations for pseudarthrosis. Thus, while some studies have shown a potential benefit for this technology, there are methodologic limitations in the current literature without any unified consensus and therefore we are unable to determine the impact of electrical stimulation on fracture healing. 

Ultrasound Stimulation

LIPUS has been shown to promote fracture repair and increase the mechanical strength of fracture callus in both animal260,347 and clinical144,192 studies. LIPUS increases the quantity of osteoprogenitors recruited to the fracture site in animal studies,194 thus acting as an osteoinduction modulator. 
In a prospective randomized double-blind trial, Heckman et al.144 examined the use of LIPUS as an adjunct to conventional treatment with a cast in 67 patients with closed or open Gustilo and Anderson141 grade I tibial shaft fractures. Thirty-three fractures were treated with the active device and 34 with the placebo. Using clinical and radiographic criteria, the authors noted that there was a statistically significant decrease in the time to union (86 ± 5.8 days in the LIPUS treatment group vs. 114 ± 10.4 days in the control group) and in the time to overall healing (96 ± 4.9 days in the ultrasound treatment group vs. 154 ± 13.7 days in the controls). There were no issues with patient compliance in the treatment group and no serious complications reported with its use. 
In a subsequent multicenter prospective randomized double-blind study, Kristiansen et al.192 evaluated the efficacy of LIPUS in the treatment of dorsally angulated distal radius fractures that had been treated with closed reduction and a cast. The time to union was significantly shorter for the fractures that were treated with LIPUS compared with the controls (61 ± 3 days vs. 98 ± 5 days). The authors further noted that treatment with LIPUS was associated with significantly less loss of reduction (20% ± 6% vs. 43% ± 8%) as determined by the degree of volar angulation as well as with a significant decrease in the mean time until the loss of reduction ceased (12 ± 4 days vs. 25 ± 4 days). 
There are several known risks factors for delayed or nonunion, and one of the most common is tobacco use. Cook et al.75 studied LIPUS for the treatment of acute tibial and distal radius fractures in smokers. Healing time in this patient population is typically delayed, with tibial and distal radius fractures requiring 175 ± 27 days and 98 ± 30 days, respectively, to achieve bony union. Treatment with LIPUS was able to reduce this time to 103 ± 8.3 days in the tibial fracture group and 48 ± 5.1 days in the patients with distal radius fractures. Treatment with LIPUS also substantially reduced the incidence of delayed union in tibias in smokers and nonsmokers. These results are important because they suggest that LIPUS can override some of the detrimental effects that smoking has on fracture healing. Rutten et al.281 prospectively analyzed 71 cases of tibial nonunion and found that treatment with LIPUS resulted in a healing rate of 73%, and that this was significantly higher than the rate of spontaneous healing. Within the subgroups analyzed, the rate of healing in smokers and nonsmokers was not found to be statistically significantly different. Multiple other studies have found LIPUS to be a successful treatment for nonunions, with union rates between 75% and 86% with important confounders including time from initial surgery, BMI, and smoking habits (Fig. 5-9).146,168,246,276 
Figure 5-9
Sequential radiographs of a patient who sustained a grade II distal tibia fracture that underwent irrigation and debridement with placement of an external fixator.
 
A: At 4 months, the external fixator was removed and the patient was believed to have a delayed union. Daily treatments with Exogen (Smith and Nephew, Memphis, TN) ultrasound stimulation was started. B: At 1 month of treatment, the patient progressed to partial weight bearing. C: At 2 months, radiographs showed continued progression of healing. D: At 6 months, the patient was bearing full weight without pain. (Courtesy Paul Tornetta III, MD.)
A: At 4 months, the external fixator was removed and the patient was believed to have a delayed union. Daily treatments with Exogen (Smith and Nephew, Memphis, TN) ultrasound stimulation was started. B: At 1 month of treatment, the patient progressed to partial weight bearing. C: At 2 months, radiographs showed continued progression of healing. D: At 6 months, the patient was bearing full weight without pain. (Courtesy Paul Tornetta III, MD.)
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Figure 5-9
Sequential radiographs of a patient who sustained a grade II distal tibia fracture that underwent irrigation and debridement with placement of an external fixator.
A: At 4 months, the external fixator was removed and the patient was believed to have a delayed union. Daily treatments with Exogen (Smith and Nephew, Memphis, TN) ultrasound stimulation was started. B: At 1 month of treatment, the patient progressed to partial weight bearing. C: At 2 months, radiographs showed continued progression of healing. D: At 6 months, the patient was bearing full weight without pain. (Courtesy Paul Tornetta III, MD.)
A: At 4 months, the external fixator was removed and the patient was believed to have a delayed union. Daily treatments with Exogen (Smith and Nephew, Memphis, TN) ultrasound stimulation was started. B: At 1 month of treatment, the patient progressed to partial weight bearing. C: At 2 months, radiographs showed continued progression of healing. D: At 6 months, the patient was bearing full weight without pain. (Courtesy Paul Tornetta III, MD.)
View Original | Slide (.ppt)
X
In contrast to these findings, the effects of LIPUS on fracture healing may be affected by the presence of fixation devices. Emami et al.100 noted that ultrasound did not appear to have a stimulatory role on tibial fracture repair in a prospective randomized controlled double-blinded study to evaluate its effects in patients with fresh tibial fractures who were treated with a reamed and statically locked IM rod. Patients all underwent treatment with an ultrasound device for 20 minutes daily for 75 days without knowing whether it was active. LIPUS did not shorten the healing time compared to the inactive control. However, when combined with distraction osteogenesis, it does appear to improve healing time. In a randomized control trial of distraction osteogenesis alone or combined with LIPUS, Dudda et al.89 found most healing indices were not significantly different, although LIPUS significantly reduced the fixator gestation period. 
In a meta-analysis performed by Bashardoust Tajali et al.,25 23 prospective or cohort studies were identified examining LIPUS in acute fractures and nonunions. They found the time to fracture healing was significantly reduced in acute fractures by LIPUS. This was determined by an increase in periosteal reaction or density as seen on radiographs, as well as an earlier improvement in clinical outcome measures. Although they also found six out of seven studies of nonunions to have an increased rate of fracture healing stimulated by LIPUS, there was a paucity of uniform outcome measures and no definitive conclusions could be drawn. Another meta-analysis was performed by Griffin et al.136 examining LIPUS for the treatment of acute fractures. Twelve randomized control trials were examined. They did not find a significant improvement with LIPUS in reducing the time to union in the initial treatment of the acute fractures. However, there were minimal complications associated with this therapy. 

Extracorporeal Shock Wave Therapy

Extracorporeal shock wave therapy (ESWT) involves the production single high amplitude sound waves producing tension and forces on a focused area. This stimulates bone formation by increasing local and systemic inflammatory and osteogenic growth factors. The shock wave translates into a formation of membrane hyperpolarization and growth factor production. Wang et al.340 found systemic concentrations of TGF-β1, VEGF, BMP-2, and nitric oxide after treatments of ESWT. These are in part stimulated by the creation of free radicals that induce this osteogenic response.339 
Several studies have examined the effects of ESWT on nonunions. A prospective randomized control trial performed by Cacchio et al.59 treated 126 patients with long-bone hypertrophic nonunion with either ESWT with an energy flux density of 0.4 or 0.7 mJ/mm(2), or surgery. At 6 months, 70% to 71% of the fractures had healed in the ESWT groups compared to 73% in the surgical group. However, at both 3 and 6 months after treatment, the ESWT groups had significantly improved pain and functional outcome scores compared to the surgical group. There was no difference by 1 year. In a retrospective review by Elster et al.99 of 129 tibial nonunions treated with ESWT, 80% demonstrated complete healing with the average time to healing completion of 4 to 5 months. Multiple other studies have shown similar efficacies of ESWT in the treatment of nonunions.287,332,346 Wang et al.338 treated 72 patients with nonunions of long-bone fractures, and demonstrated an 80% union rate at 1-year follow-up. ESWT worked well for hypertrophic nonunions but was less optimal for atrophic nonunions. Although there are promising initial results in the treatment of nonunions, there have not been enough clinical studies to evaluate the efficacy of this relatively new technology. 

Authors’ Preferred Method of Treatment

 
 
Distraction Osteogenesis, Electrical Stimulation, Ultrasound Stimulation
 

The use of controlled micromotion to enhance fracture healing, as described by Easley et al.90 has not been widely used and we have no experience with this method. The use of distraction osteogenesis for the treatment of nonunions for surgeons experienced in this technique is appropriate, but there is an established risk of fracture after the removal of external fixators.

 

There are data to support the use of electrical stimulation for the treatment of nonunions and delayed unions. DC, capacitive coupling, and PEMFs have all shown the potential to stimulate the healing of nonunions. PEMFs can also be used for the treatment of delayed unions. However, methodologic limitations and high between-study heterogeneity leave the impact of electromagnetic stimulation on fracture healing uncertain. There is no evidence that electrical stimulation of any type enhances the healing of fresh fractures.

 

Ultrasound stimulation can be used for the treatment of fresh closed fractures of the distal radius and tibia when treated in a cast or external fixation device. We have also had good results in the treatment of tibia fractures that show delayed union. While this therapy might not reduce the rate of reoperations, it appears to influence fracture-healing time and union rates. Until there is evidence to support the use of LIPUS in patients treated with fixation devices apart from distraction osteogenesis, we do not recommend the use of ultrasound in the treatment of fractures of patients who have undergone an operation in which fixation devices have been implanted.

 

ESWT is a relatively new technology whose potential in fracture healing does not have enough evidence to evaluate for or against its use.

Conclusions and Future Directions

The repair of fractures is a predictable event for most skeletal injuries. There are, however, instances when fracture repair is delayed or fails to occur. With improved understanding of the intracellular and extracellular pathways involved in bone healing, our ability to successfully augment this repair process continuously evolves. 
Currently, the ability to promote fracture healing is limited. Accepted options include returning to the operating room to perform an open procedure supplemented with some type of physiologic or synthetic graft, rhOP-1 or rhBMP-2, electrical or mechanical stimulation or the use of LIPUS. Systemic treatments, such as the use of statins or hormones, are still in the development stages. Current advances, including improved methods for obtaining autogenous and allogenic MSCs, development of delivery mechanisms for gene therapy, and improvements in synthetic bone-graft materials, may enhance the ability to improve fracture repair in the future. 

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