Cyclosporin A and tissue antigen matching in bone transplantation. Fibular allografts studied in the dog.

We studied the mechanical, metabolic, and histologic properties of short-term nonvascularized cortical bone grafts in a canine fibular graft model. Sham operated nonvascularized autotransplanted and allotransplanted bones were compared. The allografts were performed between dog leukocyte antigen (DLA) class I and II matched; DLA class I and II mismatched; and cyclosporin A (CsA) treated, DLA class I and II mismatched animals. Cyclosporin was given for 1 month, and all the animals were followed for 3 months after surgery. Mechanical properties were investigated using standard torsional tests, metabolic kinetics were assessed using isotopic prelabeling techniques, and histomorphometric analysis of cross-sectional area properties and sequential fluorochrome labels were performed. Autografts were mechanically stronger and stiffer than all the types of allograft. CsA-treated, DLA-mismatched allografts performed better than matched allografts. These in turn were stronger than non-CsA-treated, mismatched allografts, which underwent nearly complete resorption. These relationships were preserved in the metabolic and histologic analyses. In this short-term animal study, although DLA matching resulted in a slight improvement in graft outcome, mismatched grafts in dogs receiving a short course of cyclosporin A fared even better.

Biology of cancellous bone grafts.

Despite 30 years of experimental bone grafting research, the fresh cancellous bone graft remains the most osteogenic and reliable bone grafting material. Recent experimental data suggest that modification of the graft-host interaction by antigen matching or immune manipulation may allow increasingly successful use of allografts.

The long-term fate of fresh and frozen orthotopic bone allografts in genetically defined rats.

Fresh and frozen orthotopic iliac crest bone grafts in rats were studied histologically for determination of the long-term effects of histocompatibility matching and the freezing process on orthotopic bone graft incorporation. Grafts exchanged between groups of inbred rats, syngeneic or differing with respect to major or minor histocompatibility loci, were studied histologically at 20, 30, 40, 50, and 150 days after bone transplantation. A numerical histologic scoring system was developed and used by three observers for evaluation of coded hematoxylin and eosin sections. All frozen graft groups had the same fate regardless of histocompatibility relations between donors and recipients, and all grafts were inferior to fresh syngeneic grafts. Both fresh allograft groups received similar scores and initially at 20 and 30 days had scores similar to those of the fresh syngeneic groups. In the later intervals, however, the fresh allografts were inferior to the fresh syngeneic grafts and similar to the frozen groups. This is consistent with an older model describing two distinct phases of osteogenesis. In the long term, frozen syngeneic and fresh and frozen allografts across major and minor histocompatibility barriers were comparable, but all were significantly inferior to fresh syngeneic bone grafts.

Effects on bone of vascular interruption. Turnover and morphology in isotope-prelabelled rats.

The effects of bone devascularization were evaluated histologically and metabolically in rats prelabelled with 45Ca, 3H-tetracycline and 3H-proline by quantifying cortical bone resorption and formation. The interruption of blood supply to bone without invading its integrity resulted in a marked increase in bone turnover (resorption and formation) during the first and second months. The stimulated increase in bone resorption and formation did not affect the resultant mass of collagen and calcium. Thus, the increase in bone resorption was compensated by an equivalent increase in bone formation.

Improved acceptance of frozen bone allografts in genetically mismatched dogs by immunosuppression.

UNLABELLED: We studied the role of immunosuppressive therapy in improving the incorporation of frozen bone allografts exchanged across strong transplantation barriers in a canine cancellous ulnar segmental replacement model. Dogs receiving frozen bone from donors with major histocompatibility differences received one of three different immunosuppressive treatments. In two groups, azathioprine and prednisolone were administered for either twenty-eight or fifty-six days; anti-lymphocyte globulin was added for another twenty-eight-day group in a third regimen. Frozen bone was evaluated radiographically and histologically by criteria that quantified the biological characteristics of the bone itself and union between the graft and host at thirteen and twenty-six weeks after grafting. Graft incorporation in these animals was compared with graft acceptance in a similar group of untreated animals and in untreated animals in which bone was exchanged across weak transplantation barriers. Complications of immunosuppression included wound drainage, infection, weight loss, and falling white-blood-cell counts. Seven of the original thirty-seven animals died as a direct result of these complications. After twenty-six weeks the grafts in the recipients receiving immunosuppression appeared radiographically and histologically indistinguishable from those in the untreated, genetically closely matched group and from autografts. They were significantly better incorporated than identical allografts placed in untreated, genetically disparate recipients. There was no difference in the effectiveness of any of the immunosuppressive programs. CLINICAL RELEVANCE: Immunosuppression improves the biological outcome of otherwise poorly performing frozen bone allografts in dogs. This finding suggests that treatments that modify the immunological response of the host without major side effects may be useful clinically in improving the success of massive frozen bone allografts.

The effect of histocompatibility matching on canine frozen bone allografts.

The value of histocompatibility matching in frozen bone allografts was studied in a canine cancellous ulnar segmental-replacement model. Frozen bone that was exchanged across strong and weak transplantation barriers was evaluated histologically and radiographically at thirteen and twenty-six weeks after grafting. Histological grading criteria quantified the type of union at each end of the graft and the degree of remodeling of the marrow, spongiosa, and compacta. Radiographic grading criteria included the presence of union at each end of the graft and the degree of remodeling of the graft segment. In vitro studies for serum antibody and cell-mediated immunity were carried out by isotopic cytotoxicity methods at seven intervals during the twenty-six-week study period. Histologically, the strong-barrier allografts had fewer osseous unions and less reorganization of spongiosa and marrow when compared with autograft controls at both thirteen and twenty-six weeks. Radiographically, the strong-barrier allografts at thirteen weeks had fewer unions and marked resorption of grafts material when compared with autograft controls. There were no differences between weak transplantation-barrier grafts and control autografts radiographically or histologically at thirteen and twenty-six weeks after grafting. Frozen bone allografts did not elicit detectable serum antibody or lymphocytes that were cytotoxic for donor cells.

The material properties of immature bone.

The age-related material properties of developing immature canine bone were determined for the femora, tibiae, humeri, radii, and ulnae in animals from 1 wk of age to maturity. These properties included bone geometry changes, material tissue properties, and qualitative and quantitative morphological evaluations. All bones exhibited a two-phase growth cycle, an initial rapid phase (20 wk) followed by a substantially slower growth to maturation (48 wk). All properties showed age-related changes except bone tissue strain to failure.

Comparison of whole calvarial bones and long bones during early growth in rats. II. Turnover of calcified and uncalcified collagen masses.

The increase of total collagen and its destruction were compared for whole calvaria and long bones from young growing rats prelabeled in utero with 3H-L-proline. Rats were compared from birth to 16 weeks of age. Long bones and calvaria were isolated as intact anatomical units for autoradiography or separated by collagenase into calified and uncalcified collagens. Autoradiography using 14C-L-proline demonstrated eccentric modeling of bone collagen. With growth the mass of calcified collagen (bone) increased rapidly in calvaria and long bones. A similar increase in the mass of uncalcified collagen (mainly cartilage) occured in the long bones; a very small increase occurred in the fibrous tissue of calvaria. Total and specific radioactivities of collagens at each age were compared to that present at birth. With growth remodeling an almost complete loss of pre-existing radioactive collagen occurred from uncalcified fibrous tissue of calvaria as compared to a smaller but substantial loss from the uncalcified cartilage of long bones. A marked loss of calcifed collagen occurred in long bones as compared to a smaller loss from calvarial bones. The istopic data indicate a large turnover of fibrous tissue (type I collagen) with growth remodeling as compared to a smaller turnover of bone (calcified, type I collagen) and cartilage (typc I collagen). The turnover rate of skeletal collagens depends upon whether the collagen is calcified or not, and not upon the type of collagen.

Contribution of collagen and mineral to the elastic-plastic properties of bone.

Tension testing of wet bovine haversian cortical bone demonstrated marked plastic behavior. Progressive surface decalcification of this bone with dilute hydrochloric acid resulted in progressive decreases in the tension yield point and the ultimate stress with no change in the yield strain or ultimate strain unless decalcification was complete. The slope of the plastic region remained identical throughout decalcification. These findings are consistent with an elastic-perfectly plastic model for the mineral phase of bone tissue in which the mineral contributes the major portion of the tension yield strength. The slope or stiffness of the plastic region of the stress-strain curve is a function only of the properties of collagen, which itself plays a minor role in the tension yield strength of bone.

Comparison of whole calvarial bones and long bones during early growth in rats. Histology and collagen composition.

The distribution of ossified collagen (bone) and uncalcified collagen (fibrous tissue and cartilage) was compared histologically for rat and dog calvaria at birth. The relative amount of bone and uncalcified collagen was quantitated morphologically for rat calvaria during the first four weeks of rapid growth. Whereas dog calvaria are essentially ossified at birth, rat calvaria at birth consist mostly of fibrous tissue but rapidly become ossified with growth. Bacterial collagenase was used to separate uncalcified collagen from calcified collagen of whole membranous bones (frontal and parietal) and long bones (femur and humerus) at birth from man, monkey, dog, guinea pig, rabbit and rat. By this means quantitative changes in the relative fractions of the two forms of collagen were determined during the first eight weeks of postnatal growth for each type of rat bone. Quantitative biochemical data on whole rat bones (calvarium, femur, humerus) confirmed measurements based on histology which showed that at birth rat calvaria are mostly uncalcified as compared to other species whose bones are mostly ossified at birth. With growth rat membranous bones ossify more rapidly than long bones.