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The current landscape of growth factors in orthopaedic trauma is dominated by the BMPs, as they are the most extensively studied growth factors in clinical applications pertaining to orthopaedic trauma. Despite this, their application and indications for use in trauma remain controversial. This article highlights a number of selected growth factors, other than BMPs, that are currently being investigated in orthopaedic trauma applications. These growth factors have shown significant promise in preclinical and early clinical investigation.

Ongoing research at the molecular level has expanded our understanding of the physiological processes that regulate the complex phenomena of fracture healing and bone regeneration. A number of key molecules have been identified and shown to facilitate the progression of healing from one stage to another, leading to an uneventful outcome. Among these candidate molecules, bone morphogenetic proteins (BMPs) possess potent osteoinductive properties. They interact with osteoprogenitor cells, regulating both mitogenesis and differentiation potential. Since the discovery of BMPs, a number of experimental and clinical trials have supported their safety and efficacy of their use in therapy. Nonetheless, at times their efficacy falls short of expectations. Several factors have been identified as contributing to this result. It is anticipated that, as our knowledge expands and we understand better the complex pathways and cascades of molecular events attributable to BMPs, the application of these molecules in the clinical setting will continue to increase and to show more favourable outcomes., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Bone morphogenetic proteins play a key role in the regulation of the three major phases of fracture healing; the inflammatory response, the chondrogenic stage and the osteogenic stage. BMP-2 and 4 show a rapid response during the initial inflammatory stage, and BMP-2 has been shown to be essential for fracture healing to occur. BMP-2 and 4 are also present in the chondrogenic stage, although expression levels of BMP-3b (GDF10) and BMP-6 seemingly have specific peaks at day 7, suggesting a crucial role in this process. BMP-3, 4 and 5 are also abundantly present during this stage. In the osteogenic stage studies have shown that BMPs 1, 2, 3, 3b, 4, 5, 6, 7 and 8A have high levels of expression. It is important to note that of these, BMP-3 and 4 have a peak in their expression and BMP-7 and 8A are almost exclusively expressed during day 14 and 21. The expression of the different BMPs in these stages is believed to be orchestrated by autoregulatory activation between the BMPs, negative feed back loops and external antagonists. Alterations of the BMP signaling pathway, either by direct targeting of the BMPs or by their inhibitors has been shown to interfere with embryologic skeletal development and a post natal regenerative repair following skeletal trauma., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Bone healing is a predictable process that has a high rate of success. For some patients, and in certain clinical settings, this process can be delayed or completely inhibited. This leads to significant morbidity and may also result in time lost from work, costs related to prolonged medical treatment, and continued pain at the site of nonunion or failed spinal fusion. Several growth factors, specifically BMP-2 and BMP-7, have been approved in several countries for specific indications. The use of these products and potential complications of their use are reviewed.

Heterotopic ossification occurring after the use of commercially available bone morphogenetic proteins has not been widely reported. We describe four cases of heterotopic ossification in patients treated with either recombinant bone morphogenetic protein 2 or recombinant bone morphogenetic protein 7. We found that while some patients were asymptomatic, heterotopic ossification which had occurred around a joint often required operative excision with good results.

Forty years after the discovery by Marshal R. Urist of a substance in bone matrix that has inductive properties for the development of bone and cartilage, there are now 15 individual human bone morphogenetic proteins (BMPs) that possess varying degrees of inductive activities. Two of these, BMP-2 and BMP-7, have become the subject of extensive research aimed at developing therapeutic strategies for the restoration and treatment of skeletal conditions. This has led to three different therapeutic preparations, each for a distinct clinical application. Non-union, open tibial fractures and spinal fusions are the three conditions for which there is clinical approval for use of BMPs. This article reviews the evidence supporting the therapeutic applications of BMPs as they are presently available and suggests future applications based on current research. Among the future directions discussed are percutaneous injections, protein carriers, advances in gene transfer technology and the use of BMPs to engineer the regeneration of skeletal parts.

The present review evaluates the existing scientific proofs of this supplementary role of the BMPs and summarises its clinical implications. Bone regeneration is a process consisting of bone formation and bone resorption, two different but closely coupling pathways, which in most circumstances proceed simultaneously. Plenty of evidence has also characterised the bone morphogenetic proteins (BMPs) as inducing factors of bone formation. However, there is also evidence that these multifunctioning proteins affect bone resorption and the osteoclast homeostasis utilising various pathways. The present review evaluates the existing scientific evidence of this supplementary role of the BMPs, and summarises its clinical implications.

Previous studies have demonstrated the ability of direct adenoviral BMP-2 (Ad.BMP-2) gene delivery to enhance bone repair. Nevertheless, in studies using a rat segmental defect model, it has not proved possible to achieve reliably full osseous union in all animals. To address this issue, we evaluated the effect of vector dose on healing. Critical-size defects were created in the right femora of 27 Sprague-Dawley rats. The defects received a single, intralesional, percutaneous injection of 2.7 x 10(7) (low dose), 2.7 x 10(8) (medium dose), or 2.7 x 10(9) (high dose) plaque-forming units of Ad.BMP-2. After 8 weeks, femora were evaluated by X-ray, dual-energy X-ray absorptiometry, microcomputed tomography (microCT), and histology. The high dose of vector bridged 100%, the medium dose 11%, and the low dose 25% of the defects, as evaluated by X-ray and microCT imaging. Bone mineral content and bone volume of the defects receiving the high dose of vector were significantly higher than those of both groups receiving lower doses. Histologically, defects treated with the high dose were filled by trabecular bone and small amounts of cartilage, whereas large areas of fibrous tissue and cartilage remained in the defects receiving lower doses. However, the newly formed bone lacked the structural organization of native bone, suggesting that further maturation is necessary.

The purpose of this study was to establish a reliable model of metaphyseal bone healing and to use this model to investigate the effect of recombinant human osteogenic protein 1 (rhOP-1; BMP-7) and parathyroid hormone fragment (PTH 1-34) on healing. A wedge-shaped osteotomy was created in the distal tibia of 16-week-old female New Zealand White rabbits (n = 20) and was bridged with a custom-made external fixator. Five experimental groups of four animals each were investigated. In groups 1-4 the osteotomy gap was filled with tricalcium phosphate (TCP), and the gap was left unfilled in group 5 ("normal healing"). In group 1, 200 microg OP-1 was mixed in with the TCP. Groups 2 and 3 received daily subcutaneous injections of 10 and 40 microg/kg PTH, respectively, beginning on postoperative day 1. Radiographs were taken weekly. Following sacrifice on postoperative day 28, peripheral quantitative computed tomography (pQCT), histology, and mechanical testing (axial compression and torsion) were performed. Only one animal failed to complete the full 4-week time course, and no infections were encountered. Bone healing occurred in all animals. OP-1 stimulated bone formation locally, while the lower dose of PTH enhanced bone formation systemically (p < 0.05). Tibiae treated with OP-1 exhibited higher torsional strength (p = 0.04) than those in the normal healing group. These results indicate that a reliable and reproducible surgical model of metaphyseal healing has been established. In addition, differences in systemic versus local effects of PTH and OP-1 in accelerating metaphyseal fracture healing were found.

The direct, local, administration of adenovirus carrying human BMP-2 cDNA (Ad.BMP-2) heals critical-sized femoral bone defects in rabbit and rat models. However, the outcome is suboptimal and the technology needs to provide a more reliable and uniform outcome. To this end, we investigated whether the timing of Ad.BMP-2 administration influenced the formation of mineralized tissue within the defect. Critical-sized defects were created in the femora of 28 Sprague-Dawley rats. Animals were injected intralesionally with a single, percutaneous injection of Ad.BMP-2 (4 x 10(8) plaque-forming units) either intraoperatively (day 0) or 24 h (day 1), 5 days or 10 days after surgery. The femora were evaluated 8 weeks after surgery by X-ray, microcomputed tomography, dual-energy X-ray absorptiometry and biomechanical testing. The incidence of radiological union was markedly increased when administration of Ad.BMP-2 was delayed until days 5 and 10, at which point 86% of the defects healed. These time points also provided greater bone mineral content within the defect site and improved the average mechanical strength of the healed bone. Thus, delaying the injection of Ad.BMP-2 until 5 or 10 days after surgery enables a greater percentage of critical-sized, segmental defects to achieve radiological union, producing a repair tissue with enhanced mineralization and greater mechanical strength.

The expression patterns of (bone morphogenetic proteins) BMPs during fracture repair and pre-natal bone development suggest that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) in culture provide a well recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs and to characterize their interactions. MSCs were harvested from the bone marrow of tibiae and femora of 8 to 10-week-old male C57/B6 mice and prepared by standard methods. Osteogenic differentiation was assessed by histological assays, alkaline phosphatase enzyme activity and assays for the expression of multiple mRNAs for BMPs and osteogenic development. The role of autogenously expressed BMPs in controlling the osteogenic differentiation of marrow stromal cells in vitro was assessed in both gain-of-function and loss-of-function experiments. Gain of function experiments were carried out in the presence of exogenously added BMP-2 or -7 and loss-of-function experiments were carried out by BMP antagonism with noggin and BMP-2 antibody blockade. Osteogenic differentiation was concurrent with and proportional to increases in the expression of BMPs-2, -3, -4, -5, -6 and -8A. BMP antagonism with either noggin or BMP-2 antibody blockade inhibited osteogenic differentiation by 50% to 80%, respectively, and reduced the expression of endogenous levels of BMPs-2, -3, -5 and -8A. In contrast, antagonism induced the expression of BMP-4 and -6. The addition of rhBMP-2 or -7 enhanced osteogenic differentiation and produced a reciprocal expression profile in the endogenous BMPs expression as compared to BMP antagonism. BMP antagonism could be rescued through the competitive addition of rhBMP-2. These studies demonstrated that osteogenic differentiation was regulated by a complex network of multiple BMPs that showed selective increased and decreased expression during differentiation. They further demonstrated that BMP-2 was a central regulator in this network.

Background: Healing of segmental bone defects can be induced experimentally with genetically modified osteoprogenitor cells, an ex vivo strategy that requires two operative interventions and substantial cost. Direct transfer of osteogenic genes offers an alternative, clinically expeditious, cost-effective approach. We evaluated its potential in a well-established, critical-size, rat femoral defect model., Methods: A critical-size defect was created in the right femur of forty-eight skeletally mature Sprague-Dawley rats. After twenty-four hours, each defect received a single, intralesional, percutaneous injection of adenovirus carrying bone morphogenetic protein-2 (Ad.BMP-2) or luciferase cDNA (Ad.luc) or it remained untreated. Healing was monitored with weekly radiographs. At eight weeks, the rats were killed and the femora were evaluated with dual-energy x-ray absorptiometry, micro-computed tomography, histological analysis, histomorphometry, and torsional mechanical testing., Results: Radiographically, 75% of the Ad.BMP-2-treated femora showed osseous union. Bone mineral content was similar between the Ad.BMP-2-treated femora (0.045 +/- 0.020 g) and the contralateral, intact femora (0.047 +/- 0.003 g). Histologically, 50% of the Ad.BMP-2-treated defects were bridged by lamellar, trabecular bone; the other 50% contained islands of cartilage. The control (Ad.luc-treated) defects were filled with fibrous tissue. Histomorphometry demonstrated a large difference in osteogenesis between the Ad.BMP-2 group (mean bone area, 3.25 +/- 0.67 mm(2)) and the controls (mean bone area, 0.65 +/- 0.67 mm(2)). By eight weeks, the Ad.BMP-2-treated femora had approximately one-fourth of the strength (mean, 0.07 +/- 0.04 Nm) and stiffness (mean, 0.5 +/- 0.4 Nm/rad) of the contralateral femora (0.3 +/- 0.08 Nm and 2.0 +/- 0.5 Nm/rad, respectively)., Conclusions: A single, percutaneous, intralesional injection of Ad.BMP-2 induces healing of critical-size femoral bone defects in rats within eight weeks. At this time, the repair tissue is predominantly trabecular bone, has normal bone mineral content, and has gained mechanical strength.

Recently, several prospective randomized clinical trials have resulted in the publication of critical studies on the efficacy of recombinant human morphogenetic proteins BMP-2 (rhBMP-2) and BMP-7 (OP-1) in spinal fusion and fracture healing. The clinical use of BMPs is now in its infancy and understanding the mechanism and the appropriate application of these proteins is necessary for all practicing orthopedic surgeons. This article will revisit some of the early studies using rhBMPs and review the current literature on their role in fracture healing.

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism., (Copyright 2003 Wiley-Liss, Inc.)

Background: Recombinant human bone morphogenetic protein-2 (rhBMP-2), surgically implanted with a matrix material, has been shown to induce bone formation and enhance fracture repair. The purpose of this investigation was to test the hypothesis that a single, local, percutaneous injection of rhBMP-2 would accelerate fracture-healing in a standard rat femoral fracture model., Methods: Fractures were created, following intramedullary pinning, in the femora of 144 male Sprague-Dawley rats. The animals were divided into three groups of forty-eight each. Six hours after the fracture, one group received an injection of 80 micro g of rhBMP-2 in 25 micro L of buffer vehicle, one received an injection of 25 micro L of buffer vehicle alone, and one did not receive an injection. Twelve animals from each of these three groups were killed at one, two, three, and four weeks after treatment, and the femora were harvested for torsional biomechanical testing. An additional cohort of seventy-two animals, in which a fracture was also created, was divided into the same three treatment groups; six animals from each of these groups was killed at one, two, three, and four weeks; and the femora were processed for qualitative histological analysis., Results: Torsional biomechanical testing indicated that the stiffness of the rhBMP-2-treated fractures was twice that of both control groups at the two, three, and four-week time-points. The strength of the rhBMP-2-treated fractures was 34% greater than that of the buffer-treated controls (p = 0.03) at three weeks and, at four weeks, was 60% and 77% greater than that of the buffer-treated controls and that of the untreated controls, respectively (p < 0.005). At four weeks, the stiffness and strength of the rhBMP-2-treated fractures were equal to those of the intact contralateral femora, whereas the buffer-treated and untreated fractures were significantly weaker than the intact femora. At two and three weeks, large areas of bone formation, typically spanning the fracture, were observed histologically in the rhBMP-2-treated sites. In contrast, the control fractures exhibited primarily soft cartilaginous callus at these time-points. By four weeks, remodeling of the hard callus and recorticalization were observed in the rhBMP-2-treated fracture sites, whereas cartilage and/or soft tissue was still present in the control fracture sites., Conclusions: These data demonstrate that a single, local, percutaneous injection of rhBMP-2 accelerates fracture repair in this rat femoral fracture model. This effect appears to result from a combination of the induction of bone formation at the fracture site and acceleration of the rate at which the fracture callus matures.

Cartilage formation always precedes that of bone during endochondral skeletal development. To determine if chondrocytes provide inductive signals for osteogenesis, C3H10T(1/2) mesenchymal stem cells were co-cultured in membrane separated transwell culture chambers with chondrocytes, osteoblasts, or fibroblasts. Osteogenesis, as assessed by the expression of osteocalcin mRNAs, was strongly induced in the C3H10T(1/2) cells co-cultured with chondrocytes but not induced by co-culture with either osteoblasts or fibroblasts. Interestingly, while only osteogenic differentiation was observed in the C3H10T(1/2) cells co-cultured with chondrocytes, bone morphogenetic protein (BMP)-7 treatment induced an ordered endochondral progression of skeletal cell differentiation in which chondrogenic differentiation preceded osteogenesis by 2 to 4 days. A nutrient enriched growth environment enhanced osteogenic differentiation induced by either co-culture or BMP-7 treatment 2- to 5-fold. Nutrient enhanced osteogenic differentiation was associated with an activation of the retinoblastoma-mediated signal transduction pathways. In summary, these results show that osteogenesis is selectively induced by morphogenetic signals produced by chondrocytes and that a nutrient rich environment enhances both BMP-7- and co-culture-induced osteogenic differentiation.

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T1/2 mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T1/2 mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T1/2 cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T1/2 cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T1/2 cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T1/2 cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.

Basic and clinical research have shown the efficacy of various cellular mediators (bone morphogenetic proteins, interleukins, angiogenic growth factors) in healing bone defects. The potential application of these growth and differentiation factors to other musculoskeletal conditions, including osteonecrosis of the femoral head, only recently has been explored. Osteonecrosis is a disease of unknown pathogenesis that usually progresses to hip joint destruction necessitating total hip arthroplasty. The pathology involves ischemic events followed by death of bone and marrow elements. A process of repair then is initiated, but unless the lesion is small (less than 15% of the femoral head involved), this repair process is usually ineffective. The net result is weakening of subchondral bone with subsequent collapse of the articular surface. Because the results of hip arthroplasty in patients with osteonecrosis are relatively poor, much focus has been on modalities aimed at femoral head preservation. The surgical alternatives may include core decompression, osteotomy, nonvascularized, and vascularized bone grafting, which might be enhanced with the use of growth and differentiation factors. At least three of these factors are potential candidates as therapeutic modalities: cytokines (such as interleukins, tumor necrosis factors, and signaling molecules such as fibroblast growth factors, platelet derived growth factors, insulinlike growth factors, and transforming growth factor betas), bone morphogenetic proteins, and angiogenic factors. Despite considerable effort, evaluation of these growth and differentiation factors has been hampered by the lack of an animal model that adequately simulates the pathology of osteonecrosis in humans. Therefore, investigators have attempted to model certain aspects of the disease process. Recently, several investigators have attempted to mimic osteonecrosis in the femoral head of large mammals by combinations of devascularization, freezing, osteotomy of the femoral neck, or creation of a head defect. Results from some of these studies have confirmed the potential for growth and differentiation factors to effect more rapid healing and filling of defects with biomechanically competent and viable bone. The application of this therapy shows promise, and clinical studies on efficacy and safety are ongoing.