脱蛋白联合冷冻干燥法制备异种骨的实验研究

Experimental study on xenogeneic bone prepared with deproteinization and freeze-drying method

目的通过脱蛋白联合冷冻干燥的方法制备异种骨，探讨其能否在保持其生物力学特性的同时良好地消除异种骨的抗原性，以满足骨移植的需求。方法取牛股骨上端去除表面筋膜、结缔组织和皮质骨部分，制备成骨粒和圆柱形骨棒。将十二烷基苯磺酸钠（ABS）和脂肪醇聚氧乙烯醚硫酸钠（AES）以及蒸馏水以重量比13：7：80的比例配制复合表面活性剂。将牛骨粒或者骨棒与复合表面活性剂以重量比1：10的比例置于烧瓶中，超声振荡清洗，去除其抗原性，电镜下观察其结构。骨粒和骨棒经乙醇和乙醚脱水、脱脂，冷冻干燥。骨粒用作溶血试验和细胞毒性检测，骨棒进行新西兰大白兔的长期骨植入实验（4、12、26、52周）检测其生物相容性。乙醇抽吸法检测脱蛋白．冻干骨与未脱蛋白的单纯冻干骨的孔隙率。并且比较牛松质骨骨粒与脱蛋白．冻干松质骨骨粒的生物力学特性差异。结果电镜下观察，制备的异种骨材料为天然多孔结构，保留了骨组织的三维结构，骨小梁间有200～650I．tm骨髓腔。溶血率检测未超过5％，判定溶血率合格。细胞毒性检测为1级，极低细胞毒性，材料合格。长期骨植入实验表明，植入4周即有植入的骨棒与兔自身骨出现融合，未见严重炎症反应；52周时，植入骨已完全吸收。孔隙率检测表明，脱蛋白．冻干骨与未脱蛋白的单纯冻干骨相比无显著性差异，均可达到60％以上，符合骨移植材料的要求。脱蛋白．冻干骨以及未脱蛋白的单纯冻干骨的力学特性与新鲜松质骨块相比均有显著降低，但两者之间无显著性差异。结论通过脱蛋白联合冷冻干燥的方法制备的异种骨，能够在保持其生物力学特性的同时良好地消除抗原性：满足骨移植的需求。

Objective To study the mechanical property and antigenicity of bovine cancellous bone after deproteinization and freeze-drying, and to determine the possibility of applying it as the scaffold of bone transplantation. Methods Bone particles and sticks of cancellous bone from bovine furmer were made with deproteinization with the liquid of sodium dodecyl benzene sulfonate （ABS） and anionic surfactant sodium fatty alcohol ether sulfate （AES） and distilled water in a ratio of 13：7：80 （weight）. And sonic oscillation was used to further eliminate the residual protein in cancellous particles and sticks. The bone structures were observed under electron microscope. Hemolytic, cytotoxic and bone-implantation tests were examined to determine the histocompatibility of this kind of xenogeneic bone, and the mechanical property was also measured and compared with bovine cancellous particles. The porosity was examined with liquid discharge method. Results It showed that deproteinized cancellous bone maintained natural network pore system. No obvious cytotoxicity and hemolysis was observed. As found by bone-implantation test, bone sticks were fused with the peripheral bone of the rabbit only after implantatation of 4 weeks, and were completely absorbed after 52 weeks. The porosity was over 60%. Conclusion This kind of xenogeneic bone has good histocompatibility and mechanical property, and is a proper scaffold of bone transplantation with deproteinization and freeze-drying method.