Sharon Stevenson

The Effect of Regional Gene Therapy with Bone Morphogenetic Protein-2-Producing Bone-Marrow Cells on the Repair of Segmental Femoral Defects in Rats*

BACKGROUND Recombinant human bone morphogenetic proteins (rhBMPs) can induce bone formation, but the inability to identify an ideal delivery system limits their clinical application. We used ex vivo adenoviral gene transfer to create BMP-2-producing bone-marrow cells, which allow delivery of the BMP-2 to a specific anatomical site. The autologous BMP-2-producing bone-marrow cells then were used to heal a critical-sized femoral segmental defect in syngeneic rats. METHODS Femoral defects in five groups of rats were filled with 5 x 10(6) BMP-2-producing bone-marrow cells, created through adenoviral gene transfer (twenty-four femora, Group I); twenty micrograms of rhBMP-2 (sixteen femora, Group II); 5 x 10(6) beta-galactosidase-producing rat-bone-marrow cells, created through adenoviral gene transfer of the lacZ gene (twelve femora, Group III); 5 x 10(6) uninfected rat-bone-marrow cells (ten femora, Group IV); or guanidine hydrochloride-extracted demineralized bone matrix only (ten femora, Group V). Guanidine hydrochloride-extracted demineralized bone matrix served as a substrate in all experimental groups. Specimens that were removed two months postoperatively underwent histological and histomorphometric analysis as well as biomechanical testing. RESULTS Twenty-two of the twenty-four defects in Group I (BMP-2-producing bone-marrow cells) and all sixteen defects in Group II (rhBMP-2) had healed radiographically at two months postoperatively compared with only one of the thirty-two defects in the three control groups (beta-galactosidase-producing rat-bone-marrow cells, uninfected rat-bone-marrow cells, and guanidine hydrochloride-extracted demineralized bone matrix alone). Histological analysis of the specimens revealed that defects that had received BMP-2-producing bone-marrow cells (Group I) were filled with coarse trabecular bone at two months postoperatively, whereas in those that had received rhBMP-2 (Group II) the bone was thin and lace-like. Defects that had been treated with bone-marrow cells producing beta-galactosidase (Group III), uninfected bone-marrow cells (Group IV), or guanidine hydrochloride-extracted demineralized bone matrix only (Group V) demonstrated little or no bone formation. Histomorphometric analysis revealed a significantly greater total area of bone formation in the defects treated with the BMP-2-producing bone-marrow cells than in those treated with the rhBMP-2 (p = 0.036). Biomechanical testing demonstrated no significant differences, with the numbers available, between the healed femora that had received BMP-2-producing bone-marrow cells and the untreated (control) femora with respect to ultimate torque to failure or energy to failure. CONCLUSIONS This study demonstrated that BMP-2-producing bone-marrow cells created by means of adenoviral gene transfer produce sufficient protein to heal a segmental femoral defect. We also established the feasibility of ex vivo gene transfer with the use of biologically acute autologous short-term cultures of bone-marrow cells.

Bone morphogenetic protein-3 is a negative regulator of bone density

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily. Many BMPs are produced in bone and show osteogenic activity, suggesting that they may be determinants of bone mass. BMP3 was originally purified from bone as osteogenin, which induces osteogenic differentiation. Recombinant BMP3 (rhBMP3) has no biological activity, however, leaving its role in skeletal growth unclear. Here we show that BMP3 is an antagonist of osteogenic BMPs: BMP3 dorsalizes Xenopus laevis embryos, inhibits BMP2-mediated induction of Msx2 and blocks BMP2-mediated differentiation of osteoprogenitor cells into osteoblasts. These effects appear to be mediated through activin receptors. Finally, Bmp3−/− mice have twice as much trabecular bone as wild-type littermates, indicating that BMP3, the most abundant BMP in adult bone, is a negative determinant of bone density.

Factors affecting bone graft incorporation.

Successful graft incorporation requires that an appropriate match be made among the biologic activity of a bone graft, the condition of the perigraft environment, and the mechanical environment. The authors have studied, in a wide variety of animal models, the factors that affect the main components of bone graft incorporation: revascularization, new bone formation, and host-graft union. The principal determinant of the rate, pattern, and amount of revascularization is the presence or absence of a vascular pedicle. The nonvascularized bone graft is entirely dependent on the surrounding tissue for its revascularization, which results in a noticeable delay in vessel ingrowth. The principal determinant of the rate and amount of new bone formation on, in, or about a bone graft is the presence or absence of living, histocompatible, committed bone-forming cells. When living cells are not part of the graft at the time of implantation, the cells that form new bone are derived from host tissues, and new bone formation is delayed. The principal determinants of host-graft union are stability of the construct and contact between host bone and the graft. Factors that slow or inhibit all of these processes are reduction of the biologic activity of the graft by freezing or some other treatment, histocompatibility antigen disparities between donor and recipient, mechanical instability between the graft and the perigraft environment, and local and systemic interference with the biologic activity of the graft and surrounding tissue, for example, by irradiation or the administration of cisplatin. The task of the clinician who does a bone grafting procedure is to choose the right graft or combination of grafts for the biologic and mechanical environment into which the graft will be placed.

A semiquantitative scale for histologic grading of articular cartilage repair

This laboratory has developed a semiquantitative scale for grading the natural healing process of defects drilled into articular cartilage. The scale is composed of four parameters: percent filling of the defect, reconstitution of the osteochondral junction, matrix staining and cell morphology; it has a score range from 0 (best) to 14 (worst). The scale was used to evaluate the healing of defects drilled into rabbit knee articular cartilage at 2, 14, 30, 60 and 120 days after surgery. No statistically significant difference in the graded score was found between the two different defect sizes (2.7 and 1.5 mm). However, the differences in score observed between specimens from different sacrifice times were significant (p less than 0.01). Currently many investigators are manipulating cartilaginous lesions in an attempt to improve healing, and this scale will provide a means for quantitatively comparing results from control and experimental groups.

The influence of a hydroxyapatite and tricalcium-phosphate coating on bone growth into titanium fiber-metal implants.

A study was done in rabbits to determine the effect of a hydroxyapatite and tricalcium-phosphate coating on bone growth into titanium fiber-metal implants. Titanium fiber rods with a solid titanium core were implanted bilaterally into the distal aspect of the femora of fifty-five New Zealand White rabbits. One rod was uncoated and the other rod was surface-coated with hydroxyapatite and tricalcium phosphate by the plasma-spray technique. Thirty-five rabbits were labeled sequentially with fluorochromes; killed at one, two, three, four, six, twelve, or twenty-four weeks after the operation; and studied histologically and histomorphometrically. The implants in the remaining twenty rabbits were subjected to pull-out testing to determine the shear strength at the implant-bone interface at three, six, twelve, and twenty-four weeks after the operation. Histomorphometry revealed significant effects of the hydroxyapatite and tricalcium-phosphate coating. When whole-group means (which included all time-points) were compared, it was found that 44 per cent of the perimeter of the hydroxyapatite and tricalcium-phosphate-coated implants was covered with bone compared with 12 per cent of the perimeter of the uncoated implants. The percentage of the internal surface of the implant that was covered with bone was also significantly higher in the hydroxyapatite and tricalcium-phosphate-coated implants: 27 per cent of the internal surface of the coated implants was covered compared with 8 per cent in the uncoated implants. The amount of bone in the pores of the implants was also higher in the hydroxyapatite and tricalcium-phosphate-coated implants: 12 per cent of the available pore space in the hydroxyapatite and tricalcium-phosphate-coated implants was filled with bone compared with 4 per cent in the uncoated implants. Scanning electron microscopy of the implants, done in backscatter mode, demonstrated apposition of new bone directly on the hydroxyapatite and tricalcium-phosphate coating, with variable degrees (amounts) of hydroxyapatite and tricalcium-phosphate resorption and new-bone replacement over time. Bone was never directly apposed to uncoated titanium fiber-metal. The pull-out strength of the hydroxyapatite and tricalcium-phosphate-coated implants was consistently greater than that of the uncoated implants, at all time-periods.

The influence of surface-blasting on the incorporation of titanium-alloy implants in a rabbit intramedullary model.

The apposition of new bone to polished solid implants and to implants with surfaces that had been blasted with one of three methods of grit-blasting was studied in a rabbit intramedullary model to test the hypothesis that blasted implant surfaces support osseous integration. Intramedullary titanium-alloy (Ti-6Al-4V) plugs, press-fit into the distal aspect of the femoral canal, were implanted bilaterally in fifty-six rabbits. Four surface treatments were studied: polished (a surface roughness of 0.4 to 0.6 micrometer) and blasted with stainless-steel shot (a surface roughness of five to seven micrometers), with thirty-six-grit aluminum oxide (a surface roughness of five to seven micrometers), or with sixty-grit aluminum oxide (a surface roughness of three to five micrometers). Localized attachment of new bone to the surfaces of the blasted implants was present radiographically at twelve weeks. The total bone area was significantly affected by the level of the section (the diaphysis had a greater bone area than the proximal part of the metaphysis and the proximal part of the metaphysis had a greater bone area than the distal part of the metaphysis; p < 0.001) and the quadrant within each section (the posterior and anterior quadrants had greater bone area than the medial and lateral quadrants; p < 0.00001). The length of the bone-implant interface was significantly affected by the surface treatment (the length of the bone-implant interface for the implants that had been blasted with sixty-grit aluminum oxide was greater than the length for the polished implants; p = 0.02), the time after implantation (the interface was longer at six and twelve weeks than at three weeks; p < 0.00001), and the level of the section (the interface was longer at the diaphysis than at the proximal part of the metaphysis and longer at the proximal part of the metaphysis than at the distal part of the metaphysis; p = 0.004). Blasting of the surface of titanium-alloy implants did not have an effect on the area of bone formation around the implants, but it did significantly affect the area of bone formation on the implant and the shear strength at the bone-implant interface. The two effects were not necessarily parallel, as significantly less (p < 0.05) bone formed on implants that had been blasted with stainless-steel shot than on those blasted with aluminum grit, whereas their interface shear strengths were similar.

Enhancement of fracture healing with autogenous and allogeneic bone grafts.

The factors contributing to a delayed union or nonunion are many. In general they may be divided into three major categories: deficiencies in vascularity and angiogenesis, deficiencies in the robustness of the chondroosseous response, and deficiencies in stability, strain, or physical continuity. Frequently, deficiencies in more than one category are present, thus complicating the approach to therapy. For a bone grafts to enhance fracture healing, it must provide or stimulate that which is deficient. Autogenous fresh cancellous and cortical bone most frequently are used, but other common grafts include allogeneic frozen, freeze dried, or processed allogeneic cortical, corticocancellous and cancellous grafts, and demineralized bone matrix. These grafts have varying capacities to provide active bone formation, to induce bone formation by cells of the surrounding soft tissue, and to serve as a substrate for bone formation. However, the graft cannot exert its biologic activity in isolation, dependent as it is on the surrounding environment for cells to respond to its signals and, in some cases, for blood supply. The mechanical environment of the graft site is also important. Successful graft incorporation requires that an appropriate match must be made between the biologic activity of a bone graft, the condition of the perigraft environment, and the mechanical environment. The task of the clinician who performs a bone grafting procedure for the enhancement of fracture healing is to choose the right graft or combination of grafts for the biologic and mechanical environment into which the graft will be placed.

Critical biological determinants of incorporation of non-vascularized cortical bone grafts. Quantification of a complex process and structure

Our goal in this study was to evaluate the effects of and the interaction between the hypothesized principal determinants of the incorporation of grafts: antigenicity and treatment of the graft. We implanted fresh and frozen cortical bone grafts that were matched for both major and non-major histocompatibility complex antigens (syngeneic grafts), matched for major but not for non-major histocompatibility complex antigens (minor mismatch), and mismatched for both major and non-major histocompatibility complex antigens (major mismatch). We used a rat model with an eight-millimeter segmental defect in the femur. The construct was stabilized with a plastic plate, threaded Kirschner wires, and cerclage wires. We evaluated the grafts at one, two, and four months after implantation. We measured the immune response; assessed the incorporation of the graft with use of histological examination, biomechanical testing, and quantitative isotopic kinetics; and statistically analyzed the effects of and the interactions among three independent variables: time, the degree of matching for major histocompatibility complex antigens, and the treatment of the graft (whether it was fresh or frozen). These three independent variables had profound effects on the pattern, rate, and quality of the incorporation of the graft. Two-way and three-way interactions among these variables were also noted. Serial changes in every dependent variable were observed with time. Systemic antibody specific for donor antigens was measurable only in the serum of animals that had a major mismatch, but freezing markedly attenuated the systemic antibody response. Revascularization was profoundly affected by histocompatibility-antigen matching; the syngeneic grafts were revascularized more quickly and to a greater degree than the grafts with either a minor or a major mismatch. Freezing significantly (p < 0.001) reduced the revascularization of the syngeneic grafts but had no discernible effect on the grafts with a minor mismatch. CLINICAL RELEVANCE: The findings of this investigation are clinically important because they help to explain the unpredictability of incorporation of cortical bone grafts. The graft that is most commonly implanted clinically, the frozen (or processed) mismatched allograft, had the least predictable process of incorporation. However, our findings suggest that the process of incorporation may be manipulated; for example, by the addition or removal of cells and, indirectly, of cytokines.

Osseointegration of surface-blasted implants made of titanium alloy and cobalt-chromium alloy in a rabbit intramedullary model

The purpose of this study was to compare the osseointegration of surface-blasted Ti6A14V and CoCr implants in vivo. Ti6A14V and CoCr rods blasted with 710 microm A12O3 particles were bilaterally press-fit into the medullary space of distal femora of 24 rabbits. Evaluation was made radiographically, histologically, histomorphometrically (3, 6, and 12 weeks after implantation), and mechanically (12 weeks). Both Ti6A14V and CoCr implants demonstrated good biocompatibility radiographically and histologically. Toluidine blue-stained sections revealed an osteoconductive effect of the blasted surface, and fluorochrome labeling analysis showed active bone formation at the bone-implant interface at as late as 12 weeks for both specimens. CoCr showed significantly lower interfacial shear strength than Ti6A14V although the bone contact area with the implant surface was comparable and no intervening soft tissue at the bone-implant interface could be seen for either implant by scanning electron microscopy backscatter analysis. Unmineralized tissue (cartilage and osteoid) was observed more frequently on the CoCr surface than on the Ti6A14V surface. These data show less osseointegration of CoCr implants with this blasted surface for this short period, possibly due to a slight difference in surface roughness and some negative effects of CoCr on bone attachment.

The effect of osteogenin (a bone morphogenetic protein) on the formation of bone in orthotopic segmental defects in rats.

We studied the effects of partially purified, natural osteogenin, a bone morphogenetic protein, on the formation of bone in rats. An osteoperiosteal segmental defect, eight millimeters wide, in the middle of the femoral diaphysis was created bilaterally in thirty-six adult male Fischer rats and stabilized with a polyacetyl plate and threaded Kirschner wires. One defect was filled with a cylinder of 60 per cent hydroxyapatite and 40 per cent tricalcium phosphate ceramic (pore diameter, 250 to 400 micrometers) containing 100 micrograms of partially purified bovine osteogenin, and the contralateral defect was filled with a hydroxyapatite-tricalcium ceramic cylinder without osteogenin. Eighteen animals (six animals each at one, two, and four months after the operation) were studied histologically and histomorphometrically. The implants from eighteen additional animals (six animals each at one, two, and four months after the operation) were subjected to biomechanical testing. Histomorphometry revealed that the total area of bone, the area of bone outside of the implant, and the amount of bone within the pores of the implant were all significantly (p < or = 0.05) greater in the femora that had an implant with osteogenin than in those that had an implant without osteogenin at most time-periods. The presence of osteogenin had no significant effect on the biomechanical parameters measured in this study.