The histological findings at the fracture sites supported the biomechanical evidence of acceleration of fracture repair by rhBMP-2. Substantial differences in the rhBMP-2-treated and control sites were already visible by one week after treatment. The fracture calluses in the rhBMP-2-treated limbs were generally larger than those in the controls ( Fig. 2 ). Cartilaginous callus formation was visible in the rhBMP-2-treated fracture sites ( Fig. 3 ), whereas no cartilage formation was yet visible in the control fractures. Periosteal bone formation was present proximal and distal to the fracture site in both the treated and the control specimens. However, in the rhBMP-2-treated limbs, large areas of endochondral bone formation were present superior to this reactive bone formation ( Fig. 4 ). The bone in these supraperiosteal areas was highly vascular. At two weeks, the control callus showed periosteal bone formation at the edges, with substantial regions of cartilage formation at the fracture site and some fibrous tissue invasion in the center. In contrast, the rhBMP-2-treated sites typically contained a large area of trabecular bone formation, often bridging the defect. While trabecular bone spanning the fracture was often observed, cartilage and/or loose connective tissue typically still intervened between the cortical ends. In addition, this large area of bone formation in the rhBMP-2-treated femora was often asymmetrical (i.e., present on only one side of the fracture), suggesting localization of the rhBMP-2 effect at its injection site ( Fig. 2 , weeks 2 and 3). The callus was routinely larger in the rhBMP-2-treated fractures, with larger areas of cartilaginous and/or osseous callus.
At three weeks, progression of endochondral ossification of the callus was observed in the control samples, although substantial cartilage and fibrous tissue remained at the fracture site. In contrast, the rhBMP-2-treated fractures contained osseous callus, now well-integrated with the cortical bone and spanning both periosteal and endosteal surfaces. By four weeks, remodeling of the osseous callus was already visible in the rhBMP-2-treated sites. Both cortices were typically bridged, and remodeling of the trabecular osseous callus into cortical bone was evident. The control samples demonstrated increased amounts of osseous callus at four weeks, but the fracture sites were still interrupted by areas of cartilage, with little visible bone-remodeling.
There was no evidence of malignant transformation in any of the cell types in the calluses in the rhBMP-2-treated groups. Histological examination for malignant transformation was not performed at other skeletal sites.
This study demonstrated that a percutaneous injection of rhBMP-2 substantially accelerates and enhances the fracture-healing process in a rat model. Torsional biomechanical testing showed that, after only two weeks of healing, the rhBMP-2-treated fractures had more than twice the stiffness of the control specimens (treated with buffer only or untreated). A significant increase in strength was observed subsequently, at the three-week time-point. A study by White et al. 20 suggested that fracture-healing can be divided into four stages on the basis of biomechanical properties. Fracture sites have low stiffness and low strength in the first stage, high stiffness and low strength in the second stage, and high stiffness and high strength in the third stage. In the fourth stage, stiffness and strength are sufficiently high so that if the bone is refractured, the fracture line propagates to areas of previously unfractured, intact bone. Our finding that rhBMP-2 treatment leads to increased stiffness followed by increased strength suggests that the healing sequence follows the pattern described by White et al. but proceeds at an accelerated rate. In addition, failure of the rhBMP-2-treated bones during biomechanical testing transitioned from failure through the fracture site during the first three weeks to failure through the fracture site and the intact bone in many of the animals at four weeks. In a study that demonstrated acceleration of healing by operative implantation of rhBMP-2 with a collagen sponge in a rabbit ulnar osteotomy model, stiffness and strength increased concurrently 13. In the rabbit model, the ulnar osteotomy was stabilized by the radius, whereas in the current study an intramedullary pin was used to stabilize the fractured femur. Thus, the observed difference in the biomechanical healing patterns may be due to differences in the stability of the fractures. Alternatively, there may be a difference associated with the healing of an osteotomy site as opposed to a fracture produced by blunt trauma.
One limitation of our study is that the untreated fractures never healed within the time course of the investigation. Thus, it is difficult to quantitate the magnitude of the acceleration of fracture-healing by rhBMP-2. However, previous experiments 19 and extrapolation of the biomechanical data ( Fig. 1 ) indicate that it is likely that the control fractures would have healed at approximately five to six weeks after the fracture. In contrast, the rhBMP-2-treated fractures healed between three and four weeks after the fracture. Thus, the time to healing was decreased by 20% to 50%. This finding is in keeping with the 33% acceleration of healing observed following implantation of rhBMP-2 in an absorbable collagen sponge in the rabbit ulnar osteotomy model 13.
Our histological evaluation of the time-course of healing suggests that rhBMP-2 may augment fracture repair by means of several interrelated mechanisms. First, the bone-inductive effect of rhBMP-2 is evident. Areas of bone formation were observed within the fracture callus as early as seven days after injection of the rhBMP-2. This endochondral bone formation is spatially distinct from the periosteal bone formation observed in both the treated and the control fractures at this early time-point. In addition, substantial areas of trabecular bone spanned the rhBMP-2-treated fractures at two to three weeks. Thus, percutaneous injection of rhBMP-2 produces the same effect as open placement of autogenous bone graft: it creates a large mass of bone at the fracture site, which enhances the ability of the fracture to heal. Second, when large areas of cartilaginous callus were present in the control defects (at two to three weeks), substantially less cartilage and correspondingly larger quantities of bone were present in the rhBMP-2-treated fractures. This suggests that rhBMP-2 accelerates endochondral bone formation and causes more rapid conversion of cartilaginous callus to bone. In support of this hypothesis, in vitro studies have demonstrated that rhBMP-2 can cause not only differentiation of mesenchymal cells into chondrocytes and osteoblasts, but also conversion of cells with the chondroblastic phenotype into those with an osteoblastic phenotype 21. Although these findings suggest direct effects on cellular function and acceleration of a normal physiological process, there was no evidence of malignant transformation in any of the calluses stimulated by rhBMP-2.
The increase in torsional biomechanical stiffness and strength correlated with the histological observation of increasing amounts of bone bridging the fracture site. For example, at three weeks, the rhBMP-2-treated limbs had large amounts of trabecular bone spanning the fracture in contrast to the control limbs, in which soft tissue remained interposed; thus, the biomechanical properties were enhanced in the rhBMP-2-treated limbs. However, stiffness and strength did not return to normal levels (those in the unfractured limbs) before the osseous callus had remodeled and the cortical structure had been reestablished.
BMPs are typically applied in a carrier or matrix material. A carrier has several functions, including providing a format for surgical delivery of the osteoinductive protein, maintaining the BMP at the site of application for sufficient time for the bone-inductive process to occur, and perhaps providing an environment in which bone formation can take place 22. In the present study, application of rhBMP-2 without a matrix clearly accelerated fracture repair in the rats. In addition, the presence of large areas of bone formation within the fracture callus as early as one to two weeks after treatment indicates that the rhBMP-2 had a bone-inductive capacity even without a carrier application system. A similar observation was made in a study of rhBMP-7 (OP-1), in which rhBMP-7 in an aqueous buffer improved healing in a goat tibial fracture model but rhBMP-7 combined with a collagenous matrix did not 23. The preliminary studies described in the Materials and Methods section of the present report suggested that the use of a carrier decreased the ability of rhBMP-2 to enhance fracture repair in this particular model. On the other hand, acceleration of healing by rhBMP-2 combined with an injectable collagenous carrier has been observed in a rabbit model 24. It is possible that the presence of particular types of matrix material may physically disrupt the formation of the fracture callus in the rapidly healing rat model.
Studies of local retention have indicated that, when rhBMP-2 is delivered in aqueous buffer, it is detectable at the injection site for approximately seven days 25. The use of carrier systems can extend this residence time to several weeks. While aqueous rhBMP-2 without a carrier was effective in accelerating fracture repair in this rodent model, fracture repair in the rat is rapid compared with that in primate models and that in the clinical situation 26. Thus, one must be cautious about extending these observations to situations in which healing is slower. As the BMP is applied only once, it is likely that application of BMP in an aqueous form may not result in a sufficiently long residence time for the osteoinductive protein to provide optimal bone induction and augmentation of fracture repair in the wide range of potential clinical situations.
Bioactive factors other than BMPs have also been evaluated to assess their abilities to accelerate fracture repair in animal models 27. Although transforming growth factor-β (TGF-β) is not osteoinductive, it has been found to stimulate osteogenesis when injected subperiosteally. This effect is likely a result of proliferation of bone cells and/or an increase in their synthesis of bone extracellular matrix 28. In studies of rats and rabbits, a single administration of TGF-β did not augment fracture repair (and, at the highest dose, appeared to inhibit it); multiple or continuous administration appeared to be necessary for a positive effect 29-31. The practical difficulties associated with this type of treatment regimen appear to have limited the clinical utility of TGF-β for this application.
Fibroblast growth factor-2 (FGF-2) has also been evaluated to assess its effects on bone in several settings, including fracture repair 32-34. A single application of FGF-2 results in an increase in the size of the fracture callus, with a subsequent increase in biomechanical strength, in dog and nonhuman primate models 35-37. The magnitude of the acceleratory effect is difficult to discern from these studies, however, as single-time end points were used. FGF-2 most likely stimulates proliferation of the mesenchymal precursor cells within the periosteum, which then increases the volume of the fracture callus. Thus, FGF-2 would likely have to be administered early in the fracture-repair process and at a site with adequate periosteum. It is also possible that FGF-2 exerts an angiogenic effect that results in increased callus size. It is unclear if this effect is dependent on the timing of the administration after the fracture.
Platelet-derived growth factor (PDGF) is a general mitogen for a variety of cell types. A preliminary study of rabbits indicated that application of PDGF accelerated the healing of tibial osteotomy sites 38. However, the lack of subsequent in vivo studies suggests that the positive effect of PDGF on fracture-healing is limited.
Unlike the above growth factors, rhBMP-2 is a differentiation factor that causes differentiation of mesenchymal progenitor cells into bone and cartilage-forming cells. Thus, in addition to augmenting the ongoing fracture-repair process, it has the capacity to form new volumes of bone at the site of its application.
In summary, using a well-established model of fracture repair in the rat, we showed that a single percutaneous injection of rhBMP-2 accelerates healing. If these findings can be translated into clinical applications, the impact of such an advance could be substantial. Not only might this technology obviate the need for harvesting autologous bone in certain settings in which bone graft is now needed, but the availability and ease of administration of an injectable bone-inductive compound could be used to shorten the time to healing and restore skeletal function in patients in whom normal fracture-healing is anticipated.
Note: The authors thank Howard Seeherman for insightful comments on the manuscript.
Investigation performed at Boston University Medical Center, Boston, Massachusetts, Mount Sinai Medical Center, New York, New York, and Wyeth Research, Cambridge, Massachusetts
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Wyeth (formerly Genetics Institute). In addition, one or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (Wyeth). Also, a commercial entity (Wyeth) paid or directed, or agreed to pay or direct, benefits to a research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.
A commentary is available with the electronic versions of this article, on our web site (http://www.jbjs.org) and on our quarterly CD-ROM (call our subscription department, at 781-449-9780, to order the CD-ROM).
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