An Experimental Study Into The Cyclosporine-impregnated Allograft Bone

BACKGROUNDRepairing of bone defects, especially massive bone defects, has been a tough problem to surgeons since a long time ago. There have been mainly three kinds of repairing materials, such as autogtaft, allograft and synthetic bones. Because of the superiority of allograft bones in terms of osteogenesis, mechanical properties and harvesting unlimitation, allograft bones have been extensively used in spite of their immunogenicity and risk of disease transmission. Deep-freezing, freeze-drying and gamma irradiation are most commonly used preparation methods for allograft bones dealing with immunogenicity and risk of disease transmission. But they have deleterious effects on bone healing and mechanical properties of allograft bones. And until now, clinically, the outcomes of massive bone defects repaired by allograft bones have not been satisfying. Therefore, we attempted to substitute a new way of preparing allograft bones, cyclosporine impregnation and Low-temperature-plasma Sterilization, for deep-freezing, freeze-drying and gamma irradiation. OBJECTIVETo initially test the feasibility and superiority of preparing allograft bones by cyclosporine impregnation and Low-temperature-plasma Sterilization, compared with deep-freezing, freeze-drying and gamma irradiation, in immunosuppression, bone-healing promotion and mechanical reservation. Meanwhile, to initially test the safety of this new preparation method.METHODSThe segmental defects in right radii of rabbits were repaired with cyclosporine-impregnated allograft bones (CABs, study) and deep-frozen/freeze-dried irradiated allograft bones (D/FIAB, control). Comparisons of immunologic (peripheral blood T lymphocyte subset analysis and CD₂₅ immunohistochemistric stain), bone-healing (X-ray analysis and quantitative histologic analysis), mechanical (four-point bending test) and safety(cytotoxicity, hepatotoxicity and nephrotoxicity) tests were conducted at different time points.RESULTSAll of operative incisions in the study and control groups were found primary healing, no red swelling, effusion or diapyesis recorded.Immunohistochemistric analysis of CD₂₅ in the study and control groups indicated that, at postoperative weeks 1 and 4, the density of positive material in the two groups was similar, while at postoperative week 16, the density in the control group was found higher than that in the study group. There were significant differences of percentages of CD₄⁺T lymphocytes in peripheral blood of the study group(F=2.941, P=0.067) and the control group(F=23.425, P＜0.001) between different time points(F=11.980, P＜0.001). The percentages of the control group remained decreased during the first 1 and 4 weeks after surgery, then reached the peak at postoperative week 16. There was no significant difference of the percentages between the two groups(F=0.984,P=0.345). At preoperative day 1(P=0.984) and postoperative week 1(P=0.702), there was no significant difference of the percentages between the two groups. At postoperative week 4, the percentages in the study group were significantly higher than that in the control group(t=6.906,P＜0.001), at postoperative week 16, the percentages in the control group were significantly higher than that in the study group(t=-3.583,P=0.003).There were significant differences of percentages of CD₈⁺T lymphocytes in peripheral blood of the study group(F=1.860, P=0.180) and the control group(F=3.733, P=0.035) between different time points(F=4.586,p=0.009). There was no significant difference of the percentages between the two groups(F=3.658,P=0.085). At preoperative day 1, postoperative weeks 1 and 16 respectively, there was no significant difference of the percentages between the two groups(P＞0.05). At postoperative week 4, the percentages in the study group were significantly higher than that in the control group(t=2.413,P=0.033).There was no significant difference of ratios of CD₄⁺T/CD₈⁺T lymphocytes in peripheral blood of the study group(F=0.960, P=0.437) and the control group(F=4.402, P=0.076) between different time points(F=2.397, p=0.130). There was no significant difference of the ratios between the two groups(F=1.944, P=0.193). At preoperative day 1, postoperative weeks 1 and 4 respectively, there was no significant difference of the ratios between the two groups(P＞0.05). At postoperative week 16, the ratios in the control group were significantly higher than that in the study group(t=-2.533, P=0.030).There were significant differences of X-ray assessment scores in the study group(F=211.000, P＜0.001) and the control group(F=175.689, P＜0.001) between different time points(F=386.159, P＜0.001). The scores of the two groups were found minimal at postoperative week 1, then remained risen during following 3 and 15 weeks. The score of the study group was significantly higher than that of the control group(F=5.671,P=0.032). There was no significant difference between the two groups at postoperative week 1(P=0.475) and postoperative week 4(P=0.052). At postoperative week 16, there were significant difference between the two groups(t=4.056,P=0.001 ).New bone formation occurred earlier during healing of cortical bone defects filled with CABs compared with those filled with D/FIABs. At postoperative weeks 1 and 4, there were plenty of new born typeâ… collagens in defects of the study group while less in the control group. By postoperative week 16, in the study group most collagens had become mature and bone lamellas had formed, while in the control group, there had been few mature collagens and bone lamellas formed.There were significant differences of areas of bone-formation in proximal part of graft in the study group(F=14.081, P=0.010) and the control group(F=15.445, P=0.001) between different time points(F=28.237, P＜0.001). The areas of bone-formation in proximal part of graft in the two groups were found minimal at postoperative week 1, then remained risen during following 3 and 15 weeks. The areas of bone-formation in proximal part of graft in the study group were significantly larger than that in the control group(F=89.023, P＜0.001). At postoperative weeks 1, 4 and 16 respectively, the areas of bone-formation in proximal part of graft in the study group were significantly larger than that in the control group(P＜0.05).There were significant differences of areas of bone-formation in distal part of graft in the study group(F=24.401, P＜0.001) and the control group(F=15.515, P=0.001) between different time points(F=33.009, P＜0.001). The areas were found minimal at postoperative week 1, then remained risen during following 3 and 15 weeks. The areas in the study group were significantly larger than that in the control group(F=27.864, P＜0.001). There was no significant difference of the areas between the two groups at postoperative week 16(t=1.715, P=0.146), and at postoperative weeks 1 and 4 respectively, the areas in the study group were significantly larger than that in the control group(P＜0.05).There were significant differences of areas of bone-formation in central part of graft in the study group(F=35.887, P＜0.01) and the control group(F=7.981, P＜0.01) between different time points(F=20.769, P＜0.001). The areas were found minimal at postoperative week 1, then remained risen during following 3 and 15 weeks. The areas in the study group were significantly larger than that in the control group(F=43.922, P＜0.001). At postoperative weeks 1, 4 and 16 respectively, the areas in the study group were significantly larger than that in the control group(P＜0.05).The result of four-point bending test indicated that, the ultimate bending loading of specimens in the study group was significantly higher than that in the control group(t=2.257, P=0.048).After 7day incubation, BMSCs in prosperity around a cyclosporine-impregnated allograft bone were shown under an optical microscope. And BMSCs with satisfactory shape and adhesiveness on the surface of a CAB were shown using a scanning electron microscope. Sediments on the surface of cells demonstrated vigorous exocytosis of BMSCs.No obvious complication caused by cyclosporine, such as restlessness, trembling, vomiting, diarrhea or gum hypertrophy, was recorded in the study and control groups throughout the observation.There were significant differences of OD values of BMSCs between different time points(F=21.714, P=0.001). Concentrationâ… group, F=21.808, P＜0.001; Concentrationâ…¡group, F=7.057, P=0.006; Concentrationâ…¢group, F=10.109, P=0.002; Concentrationâ…£group, F=14.262, P＜0.001; Concentrationâ…¤group, F=87.650, P＜0.001; Blank Group, F=419.880, P＜0.001; 75% Alcohol Group, F=1157.776, P＜0.001. There were significant differences of OD values of BMSCs between different groups(F=278.126, P＜0.001). The 3rd day, F=27.558, P＜0.001; the 5th day, F=177.827, P＜0.001; the 7th day, F=24.401, P＜0.001; the 9th day, F=6.961, P=0.001.The BMSC growth curves indicated that high concentration of cyclosporine topically administrated had depressant effect on growth of BMSCs. On the whole, the higher the concentration was, the more obvious the depressant effect was. However, the growth tendency of BMSCs in the highest concentration group(5mg/ml) was in the middle of the tendency in the relatively lower concentration groups(5μg/ml, 50μg/ml and 500μg/ml) and the tendency in the lowest concentration groups(Blank, 75% Alcohol, 0.05μg/ml and 0.5μg/ml).Histologic analysis indicated livers and kidneys in the two groups remained normal at postoperative day 7.There was no significant difference of serum GPT concentrations in the study group(F=1.907, P=0.199) and the control group(F=1.179, P=0.371) between different time points(F=3.019, P=0.057). There was no significant difference of the concentrations between the two groups(F=0.025, P=0.880). At preoperative day 1(P=0.808), postoperative day 1(P=0.721), postoperative day 7(P=0.608) and postoperative day 30(P=0.913), respectively, there was no significant difference of the concentrations between the two groups.There were significant differences of serum Cr concentrations in the study group(F=6.305, P=0.083) and the control group(F=1.740, P=0.228) between different time points(F=7.092, P=0.002). There was no significant difference of the concentrations between the two groups(F=0.117, P=0.744). At preoperative day 1(P=0.725), postoperative day 1(P=0.811), postoperative day 7(P=0.695) and postoperative day 30(P=0.320), respectively, there was no significant difference of the concentrations between the two groups.CONCLUSIONThese results indicate that, compared with deep-freezing, freeze-drying and gamma irradiation, preparing allograft bones by cyclosporine impregnation and Low-temperature-plasma Sterilization might be a better method of bone allograft preparation in terms of immunosuppression, bone healing promotion and mechanical reservation without severe side effects.