冻干去抗原羊松质骨支架的生物相容性评价

Biocompatibility of freeze-dried antigen-extracted sheep cancellous bone scaffolds

背景:异种骨具有与人类骨类似的天然多孔结构,在治疗骨缺损时可引导骨组织再生,但植入过程中也会引起不同程度的免疫反应。目的:采用冻干法制备去抗原羊脊椎松质骨支架材料,并评价其生物相容性。方法:取羊脊椎松质骨,制备两组去抗原异种骨支架,化学组经H2O2、甲醇/氯仿混合液等化学试剂处理;冻干组将经化学处理的羊松质骨在-80℃冰箱中低温冷冻4周,真空干燥,60Co照射消毒。①细胞毒性实验:分别采用化学组材料浸提液、冻干组材料浸提液与DMEM/F12培养基培养羊骨髓间充质干细胞。②热源实验、急性毒性实验:从兔耳缘静脉分别注射化学组材料浸提液、冻干组材料浸提液与生理盐水。③皮内刺激实验:分别在兔脊柱背部皮下注射化学组材料浸提液、冻干组材料浸提液、生理盐水及乙醇。结果与结论:冻干组支架无细胞毒性、急性毒性及热源反应,皮内刺激实验阴性;化学组支架有细胞毒性及轻微急性毒性反应,有致热源作用及轻度皮肤刺激性。结果表明经过化学处理羊脊椎松质骨支架的生物相容性相对较差,而化学处理配合低温冷冻、真空干燥及60Co照射的羊脊椎松质骨生物相容性较好,基本能达到骨组织工程支架材料的要求。

BACKGROUND：Xenogeneic bone has a natural porous structure that is similar to human bone. In the treatment of bone defects, the porous structure is helpful to guide bone regeneration, but different degrees of immune responses wil be caused during the implantation process. OBJECTIVE：To prepare a freeze-dried antigen-extracted sheep cancelous bone scaffold and to evaluate its biocompatibility. METHODS： The sheep vertebral cancelous bone was colected to prepare two kinds of antigen-extracted heterologous bone scaffolds that were treated with chemical methods as chemical group and treated with chemical methods＋cryopreservation at a-80℃ refrigerator for 4 weeks＋drying in vacuum apparatus＋60 RESULTS AND CONCLUSION：Freeze-dried bone had no cytotoxicity, no acute toxicity and heat reaction, and was negative for the intracutaneous stimulation test. The scaffold in the chemical group had cytotoxicity and mild irradiation as freeze-dried bone group. （1）Cytotoxicity test： Bone marrow mesenchymal stem cels isolated from sheep were cultured in extracts of the chemical group, free-dried bone group and Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12. （2） Heat reaction and acute toxicity tests： Extracts from the chemical group, freeze-dried bone group and normal saline were respectively injected into the ear vein of rabbits. （3） Intracutaneous stimulation test： Extracts from the chemical group, freeze-dried bone group and normal saline were respectively injected subcutaneously into the back of rabbits. Co＆amp;nbsp;acute toxicity reaction, sent heat source and had mild irritation. Results show that after freeze drying processing, the sheep vertebral cancelous bone has good biocompatibility, can meet the requirements of bone tissue engineering, but the bone that through chemical processing exhibits a relatively poor biocompatibility that cannot achieve the safety standard of biological scaffold materials.