Effects of short-term cyclosporin-A on biomechanical properties of intact and fractured bone in the rat.

Cyclosporin-A is a new immunosuppressant drug being used widely for solid organ transplantation. The systemic effects of Cyclosporin-A on the skeletal system have not previously been defined. This study examined the effects of Cyclosporin-A on biomechanical properties of intact bone and fracture repair in a rat model. Closed midshaft femoral fractures were produced over a preplaced intramedullary pin. Cyclosporin-A, dissolved in olive oil and administered by gavage, was begun the day following surgery and continued for 14 consecutive days at a dose known to prolong solid organ allograft survival in rats (7 mg/kg/day), and a second group of control animals received comparable volumes of plain olive oil under identical circumstances. Groups of both experimental and control animals were killed at 4, 8, 12, or 16 weeks following fracture, after which both intact and fractured femurs were tested to failure in torsion. As judged by torque, angular deformation, stiffness, and energy absorption, Cyclosporin-A did not significantly alter the biomechanical properties of fracture repair or intact bone turnover. Based on current biomechanical data, we can find no reason to discourage the short-term clinical use of Cyclosporin-A in patients with or without fractures. Histomorphometric studies are required to accurately assess the biologic effects of this drug on bone biology and to complete the analysis of skeletal toxicity.

Postfracture irradiation effects on the biomechanical and histologic parameters of fracture healing.

The effects of single-dose local irradiation on the biomechanical properties of closed femoral fractures were studied in 75 mature Sprague-Dawley rats. Ten days after fracture, the rats were irradiated with 900 rads at 250 kV to the entire fractured femur. At 2, 3, 4, 8, and 16 weeks after fracture, both fractured and contralateral intact femurs were recovered and evaluated biomechanically by testing to failure in torsion. Results were compared with those from a similar study involving fractures irradiated 3 days after fracture as well as nonirradiated control fractures. Fracture healing progressed faster when irradiation was delayed 10 days than when delayed 3 days, and control fractures healed more rapidly than after either delay. In the 10-day delay group, fractures showed greater strength than did those in the 3-day delay group at 8 weeks, but the strength of irradiated fractures in both groups was similarly depressed at 16 weeks, with a maximum torque well below that of control fractures. These results suggest that delaying radiation exposure of a fracture may mitigate short-term deleterious effects on fracture repair, but that long-term results may be similar to those associated with expeditious irradiation.

The influence of ibuprofen on fracture repair: biomechanical, biochemical, histologic, and histomorphometric parameters in rats.

Ibuprofen is a widely used cyclo-oxygenase inhibitor in clinical practice. It has been demonstrated by others to have an inhibitory effect on fracture repair in animals. In the present study, we were unable to demonstrate any significant alterations in fracture biomechanics as measured by torsion testing and fracture stage in mature Sprague-Dawley rats treated with 30 mg/kg/day oral dose of ibuprofen, starting 3 days following fracture, over a 12-week time interval. Fracture histology and serum osteocalcin levels were no different in treated animals than control animals. Furthermore, histomorphometric parameters of bone remodeling, including bone volume and bone formation rate in the intact tail vertebrae of these animals with unilateral femur fractures, were no different between treated and control animals.

Effects of prefracture irradiation on the biomechanical parameters of fracture healing.

This study examined the effects on the biomechanical parameters of fracture healing of a single dose of 900 rad (the approximate single-dose equivalent of 2,500 rad in 10 divided doses), given 1 day prior to closed fracture of the femur. The femurs were recovered at 2, 3, 4, 8, and 16 weeks after fracture and were mounted and tested to failure in torsion; the results were compared with those in nonirradiated controls from a previously published study. Prefracture irradiation delayed the progressive increase in biomechanical parameters of fracture healing. The delay was statistically significant up to 8 weeks after fracture. At 4 weeks, the normalized torque was 44% that of intact bone in the treated group compared with 75% for the control group. Sixteen weeks after fracture, the biomechanical and histological parameters of fracture healing of the irradiated femurs were no different from those of the nonirradiated controls. Within the treated group, the irradiated fractures remained significantly weaker than their contralateral intact bone at all time intervals, with a torque of only 79% that of intact bone at 16 weeks. Thus, femoral fractures in rats healed (or regained substantial strength) following palliative doses of radiation delivered 1 day prior to injury, but the repair process was delayed compared with that of nonirradiated controls.

Effects of freezing and freeze-drying on the biomechanical properties of rat bone.

The effects of various preservation techniques on the biomechanical properties of bone have been investigated in a rat model. Freezing of the specimens to -20 degrees C, -70 degrees C, and -196 degrees C did not adversely affect the strength of long bones tested in torsion or of vertebral bodies tested in compression. Freeze-drying did not markedly affect the compression properties of the vertebral specimens; however, it did produce a significant deleterious reduction in the torsional strength of the long bones.

The effect of radiation on the fracture repair process. A biomechanical evaluation of a closed fracture in a rat model.

The effects of a single dose of irradiation on the biomechanical parameters of the fracture healing process were studied in a rat model. Intramedullary pinning was performed before production of a closed femoral midshaft fracture. The experimental group was exposed to 900 rad 3 days after fracture and was compared with a control group with a similar fracture that received no irradiation. Animals were killed at intervals ranging from 2-16 weeks after surgery and the bones were tested until failure in torsion. In the irradiated groups, a delay of 4 weeks was noted in the biomechanical parameters associated with fracture healing (torque to failure, torsional stiffness, angle to failure, and biomechanical stage). Despite this delay in the normal temporal progression, the staging and stiffness approached normal controls within an 8-week period. However, the torque to failure remained below normal levels at the conclusion of this study. These results differ from a previous study using an open fracture model.

Allograft incorporation: a biomechanical evaluation in a rat model.

Allogeneic bone from Sprague-Dawley rat femurs was subjected to levels of freezing and/or irradiation that are known to have produced changes in the associated immune responses to these grafts. This bone was transplanted into an experimentally created gap in the femur of Lewis rats. The subsequent healing of the transplants in the Lewis rats was studied at 2, 4, 8, and 16 weeks after transplantation using torsion testing to failure. There was no clear advantageous biologic response in the union of the grafted material accompanying the alterations in immunologic response as measured by biomechanical testing of the proximal osteosynthesis site in torsion. The torsional strength of all of these groups remained lower than that of intact bone. Furthermore, none of the frozen and/or irradiated allografts exceeded the strength of the fresh allograft at a statistically significant level.

Radiation-induced alterations of fracture healing biomechanics.

The effects of irradiation on the normal temporal progression of the physical properties of healing fractures were studied in a rat model. Fractures were surgically produced in the femur, stabilized with an intramedullary pin, and irradiated. One group of rats was exposed to 2,500 rads in divided doses over 2 weeks, beginning 3 days after fracture, and compared to a control group with fractures which were not irradiated. Animals were sacrificed at periodic intervals and the bones were tested to failure in torsion. The torque, stiffness, and energy increased and the angle decreased for the nonirradiated specimens in the expected fashion. This progression was deleteriously altered in the irradiated femurs.

Three-dimensional load-displacement curves due to forces on the cervical spine.

Load-displacement curves were measured for six types of pure force loading of the cervical spine specimens obtained from fresh human cadavers. A new measuring and mounting technique was developed that yielded data for all of the functional spinal units for each specimen tested. All five of the coupled, as well as the main, load-displacement curves were studied. For anterior and posterior shear loadings, the main resulting motions were translation in that direction (1.6 +/- 0.3 and 1.9 +/- 0.3 mm), and the major coupled motions were flexion and extension (3.6 degrees +/- 1.2 degrees and 6.3 degrees +/- 1.2 degrees). The main motions with right and left lateral shear loadings were translations laterally (1.4 +/- 0.3 and 1.6 +/- 0.3 mm), and the major coupled motions were axial rotations (1.5 degrees +/- 0.6 degrees and 2.3 degrees +/- 0.6 degrees) and not lateral bending. For compression and distraction loadings, the main motions were translations in that direction (0.7 +/- 0.3 and 1.1 +/- 0.3 mm), and the major coupled motions were flexion and extension (2.0 degrees +/- 1.0 degrees and 2.8 degrees +/- 1.0 degrees) and lateral bending (1.4 degrees +/- 0.3 degrees and 1.9 degrees +/- 0.3 degrees). The neutral zones for anterior and posterior shear forces were 1.6 +/- 0.2 mm of translation and 5.8 degrees +/- 1.3 degrees of rotation, for lateral shear force 1.4 +/- 0.3 mm and 2.0 degrees +/- 0.5 degrees, and for compression/distraction 0.6 +/- 0.1 mm and 2.8 degrees +/- 0.9 degrees.

Chemotherapy-induced alterations in the biomechanics of rat bone.

The effects of methotrexate and doxorubicin (Adriamycin) on intact and fractured bone were studied in a rat model. Methotrexate, 0.1 mg/kg i.p., was administered five times a week, or doxorubicin, 1 mg/kg i.v., was given once a week. Animals were killed and evaluated at 4, 8, 12, and 16 weeks after surgical production of a unilateral femoral osteotomy and initiation of the chemotherapeutic program. Both intact and fractured bones were tested to failure in rapid torsion, and the torque, angular deformation, stiffness, and energy were measured. There was a significant alteration in the strength of the healing osteotomies in doxorubicin- and methotrexate-treated animals compared with the control group. The torsional strength of intact bone was not significantly altered by either drug protocol over the period of this observation.

Induction of specific T-cell responsiveness to allogeneic bone.

The capacity of fresh murine allogeneic bone to induce a specific immune response in vitro was studied. T-cells stimulated by allogeneic bone in vitro were collected and were characterized for state of activation, cell-surface phenotype, and antigen specificity. The stimulating antigens were determined by genetic mapping with use of recombinant inbred strains of mice and by blocking of mixed lymphocyte cultures with use of neutralizing antibodies. Purified T-cells were cultured alone or with allogeneic or syngeneic bone. In some experiments, the bone marrow was removed before in vitro culture. Responding cells were recovered after a secondary exposure to the stimulating bone. Primed cells were used immediately or cell-lines were developed. The data demonstrated that (1) allogeneic bone activated T-cells and induced their proliferation; (2) bone-induced proliferation of T-cells was specific for antigens that map to the major histocompatibility complex of the bone donor; (3) within the major histocompatibility complex, the antigens responsible for proliferation of T-cells were apparently class-I and class-II determinants; (4) removal of bone-marrow cells had no effect on the ability of that bone to stimulate alloreactivity; and (5) all of the alloreactive T-cells had the cell-surface phenotype Thy-+ CD8+ CD4-.

A virally induced osteosarcoma in rats. A model for immunological studies of human osteosarcoma.

Inoculation of Moloney sarcoma virus into the medullary canal of the tibia in newborn Wistar-Lewis rats resulted in an initially localized osteosarcoma which usually metastasized to the lung and resulted in the death of the animal within four to five weeks. Tumor cells were grown in tissue culture and used as target cells in the assay of lymphocyte-mediated cytotoxicity using a microcytotoxicity and a radioisotope labeling method. Lymphocyte-mediated cytotoxicity was demonstrated throughout the course of the clinical disease as well as in a small number of animals which showed spontaneous regression of their tumors. Serum factors which could "block" or augment the cellular response were also identified. This model resembles the spontaneous osteosarcoma of humans in many respects and may be useful for studies of the human disease.

Studies on the antigenicity of bone. I. Freeze-dried and deep-frozen bone allografts in rabbits.

The antigenicity of deep-frozen and freeze-dried cortical and corticocancellous bone allografts placed orthotopically in rabbits was studied using a sensitive microcytotoxicity assay. Target cells were phytohemagglutinin-P-stimulated, 51chromium-labeled peripheral blood lymphocytes from the bone donors (Dutch belted rabbits), and sera or peripheral blood lymphocytes from the graft recipients (New Zealand white rabbits) were used as effectors of cytotoxicity. Fresh allografts and deep-frozen corticocancellous bone evoked detectable humoral and cell-mediated immunity,, whereas freeze-dried cortical bone allografts failed to sensitize the recipients and were the least antigenic of the allografts examined.

Studies on the antigenicity of bone. II. Donor-specific anti-HLA antibodies in human recipients of freeze-dried allografts.

UNLABELLED: Freeze-dried bone allografts from donors of known tissue type evoked donor-specific anti-HLA antibodies in nine of forty-three patients (forty-four procedures). Eight of the nine sensitized patients were followed roentgenographically for an average of twenty-three months and were known to have had a satisfactory resolution of the benign process for which the graft was employed. The ninth patient was doing well when lost to follow-up four months after receiving a bone allograft. CLINICAL RELEVANCE: The questions of whether immune responses to bone allografts occur in humans and, if so, adversely influence the incorporation of the graft can be answered at this time only by demonstrating the presence and frequency of such responses and how they correlate with clinical events. A large variety of techniques can be used to demonstrate immune responses. The technique that we used revealed that a small group of patients became sensitized to HLA antigens that were known to be present in the allograft donor but none of the recipients had evidence of adverse effects caused by the graft, as judged non-invasively.

Effects of chemotherapeutic agents on bone. I. Short-term methotrexate and doxorubicin (adriamycin) treatment in a rat model.

UNLABELLED: Wistar-Lewis rats received therapeutic doses of doxorubicin or methotrexate daily for five days, were pulse-labeled with a fluorescent compound (calcein) on the seventh and thirteenth days, and were killed fourteen days after initiation of the protocol. Proximal tail vertebrae were then evaluated histomorphometrically, and the effects of the chemotherapeutic agents on trabecular bone were quantitated with respect to changes in total bone mass, new-bone formation, and resorption. Short-term administration of methotrexate caused a 26.9 per cent reduction in net trabecular bone volume and doxorubicin, an 11.5 per cent decrease. Both drugs significantly and profoundly diminished bone-formation rates by nearly 60 per cent. The toxic effect on osteoblasts was also reflected in reduced volume and thickness of osteoid, but the total numbers of osteoblasts and the per cent of trabecular surface covered by bone-forming cells were not affected. The numbers of osteoclasts and the extent of their activity were not clearly different from those in untreated rats, but rates of resorption were not determined. The effects of chronic treatment with these chemotherapeutic agents on intact, fractured, and transplanted bone and the biomechanical significance of these changes has not yet been evaluated. CLINICAL RELEVANCE: Chemotherapeutic agents have an adverse effect on normal physiological bone turnover, especially osteoblastic activity, and would also be expected to alter fracture-healing and bone-allograft incorporation by these same mechanisms. Knowledge of these changes and efforts to favorably affect the remodeling cycle must address these specific defects before bone allografts can be reliably used and fracture-healing can be improved concomitant with chemotherapy.

Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions.

BACKGROUND: The role of bone morphogenetic proteins (BMPs) in osseous repair has been demonstrated in numerous animal models. Recombinant human osteogenic protein-1 (rhOP-1 or BMP-7) has now been produced and was evaluated in a clinical trial conducted under a Food and Drug Administration approved Investigational Device Exemption to establish both the safety and efficacy of this BMP in the treatment of tibial nonunions. The study also compared the clinical and radiographic results with this osteogenic molecule and those achieved with fresh autogenous bone. MATERIALS AND METHODS: One hundred and twenty-two patients (with 124 tibial nonunions) were enrolled in a controlled, prospective, randomized, partially blinded, multi-center clinical trial between February, 1992, and August, 1996, and were followed at frequent intervals over 24 months. Each patient was treated by insertion of an intramedullary rod, accompanied by rhOP-1 in a type I collagen carrier or by fresh bone autograft. Assessment criteria included the severity of pain at the fracture site, the ability to walk with full weight-bearing, the need for surgical re-treatment of the nonunion during the course of this study, plain radiographic evaluation of healing, and physician satisfaction with the clinical course. In addition, adverse events were recorded, and sera were screened for antibodies to OP-1 and type-I collagen at each outpatient visit. RESULTS: At 9 months following the operative procedures (the primary end-point of this study), 81% of the OP-1-treated nonunions (n = 63) and 85% of those receiving autogenous bone (n = 61) were judged by clinical criteria to have been treated successfully (p = 0.524). By radiographic criteria, at this same time point, 75% of those in the OP-1-treated group and 84% of the autograft-treated patients had healed fractures (p = 0.218). These clinical results continued at similar levels of success throughout 2 years of observation, and there was no statistically significant difference in outcome between the two groups of patients at this point (p = 0.939). All patients experienced adverse events. Forty-four percent of patients in each treatment group had serious events, none of which were related to their bone grafts. More than 20% of patients treated with autografts had chronic donor site pain following the procedure. CONCLUSIONS: rhOP-1 (BMP-7), implanted with a type I collagen carrier, was a safe and effective treatment for tibial nonunions. This molecule provided clinical and radiographic results comparable with those achieved with bone autograft, without donor site morbidity.

Clinical evaluation of freeze-dried bone allografts in periodontal osseous defects--part II.

Freeze-dried crushed cortical bone allografts were implanted into widemouthed three-wall, two-wall, one-wall, combination, and furcation defects. One hundred eighty-nine sites were reentered in 97 patients and of these 60% had osseous regeneration of greater than 50%. A total of 231 sites were evaluated for pocket elimination, of which 63% demonstrated greater than 50% pocket reduction. This study presented additional evidence indicating that freeze-dried bone allografts have definite potential as grafting material in certain periodontal osseous defects. Information from additional cases is being tabulated as it becomes available and will supplement the current data.

2000 Young Investigator Research Award winner. Evaluation of OP-1 as a graft substitute for intertransverse process lumbar fusion.

STUDY DESIGN: An established rabbit intertransverse process lumbar fusion model was used to evaluate osteogenic protein (OP)-1 as a potential graft substitute. OBJECTIVES: To determine whether OP-1 is effective in producing intertransverse process lumbar fusion in a rabbit model. SUMMARY OF BACKGROUND DATA: Autogenous iliac crest bone is the gold standard in grafting material for inducing intertransverse process fusion. However, bone graft substitutes are being considered as supplementary or alternative means to achieve such fusion with less morbidity. Relatively little research has been undertaken to investigate the efficacy of OP-1 in this role. METHODS: Single-level intertransverse process lumbar fusions were performed at L5-L6 of 31 New Zealand White rabbits. These were divided into three study groups: autograft, carrier alone, and carrier with OP-1. The animals were killed 5 weeks after surgery. Resultant fusion masses were evaluated by manual palpation, radiography, biomechanical multidirectional flexibility testing, and histology. RESULTS: Seven rabbits (23%) were excluded because of complications. Of the remaining 24 rabbits, 5 (63%) of the 8 in the autograft group had fusion detected by manual palpation, none (0%) of the 8 in the carrier-alone group had fusion, and all 8 (100%) in the OP-1 group had fusion. Radiographs were 55% sensitive and 92% specific for determining fusion. Biomechanical testing results correlated well with those of manual palpation. Histologically, autograft specimens were predominantly fibrocartilage, OP-1 specimens were predominantly maturing bone, and carrier-alone specimens did not show significant bone formation. CONCLUSIONS: OP-1 was found to reliably induce solid intertransverse process fusion in a rabbit model at 5 weeks.

Osteogenic protein-1 overcomes the inhibitory effect of nicotine on posterolateral lumbar fusion.

STUDY DESIGN: An established rabbit posterolateral lumbar fusion model was used to evaluate the ability of osteogenic protein-1 to overcome the inhibitory effect of nicotine. OBJECTIVE: To determine whether osteogenic protein-1 should be considered as a bone graft alternative for the patient who smokes. SUMMARY OF BACKGROUND DATA: Smoking interferes with the success of posterolateral lumbar fusion. This inhibitory effect has been attributed to nicotine and confirmed in a New Zealand white rabbit model. Osteoinductive protein-1 has been shown to induce posterolateral spine fusion reliably in the rabbit model. The effectiveness with which osteogenic protein-1 induces fusion in the presence of nicotine has not been studied previously. METHODS: Single-level posterolateral intertransverse process fusions were performed at L5-L6 in 18 New Zealand white rabbits. Either autograft or osteogenic protein-1 was used as grafting material. Nicotine was administered via subcutaneous mini-osmotic pumps. The animals were killed 5 weeks after surgery, and the resulting fusion masses were studied. RESULTS: Three rabbits (17%) were excluded because of complications. By manual palpation, two of the eight nicotine-exposed autograft rabbits (25%) and all of the nicotine-exposed osteogenic protein-1 rabbits (100%) were found to be fused. These results correlated well with those obtained from biomechanical testing. Histologically, the fusion zones of the nicotine-exposed autograft rabbits were distinctly less mature than the fusion masses of the nicotine-exposed osteogenic protein-1 rabbits. CONCLUSION: Osteoinductive protein-1 was able to overcome the inhibitory effects of nicotine in a rabbit posterolateral spine fusion model, and to induce bony fusion reliably at 5 weeks.

Biomechanical aspects of bone autografts and allografts.

The structural requirements of skeletal reconstruction should be included in the consideration of an appropriate type of bone graft, whether autogenous or allogeneic tissues are chosen. This analysis should include the nature of any fixation devices to be used, as well as characteristics of the host and donor bone. The mechanical properties and biomechanical response of the graft must be balanced against the type and magnitude of the load to which the graft will be subject. For example, the information presented here suggests that, from a biomechanical perspective, frozen bone would be better suited than freeze-dried bone when the graft is subject to large torsional loads or else the graft must be appropriately protected during incorporation by adequate internal fixation or external bracing. In a situation that is primarily subjected to compressive loads, however, freeze-dried grafts would be just as biomechanically sound. Thus, an understanding of the normal biomechanics of the anatomic region to be reconstructed is crucial. The mechanical properties of the graft are affected by preservation, storage, and sterilization. Incorporation and remodeling of the graft further alter its properties. These properties are, in turn, influenced by the host immune response as well as the local biomechanical environment. The influence of each of these factors is predictable. Obviously, there are numerous considerations in choosing approaches to skeletal reconstruction other than the mechanical issues discussed here. However, an understanding of the mechanical properties involved will help in optimizing the clinical success of these choices.