新型组织工程椎间盘纤维环支架的制备与性能检测

摘要

背景:以组织工程技术修复退变椎间盘,目前的研究多集中在如何修复摘除的髓核组织,然而髓核组织工程方法无法完整重建椎间盘的结构和功能,所以相应的纤维环组织工程被认为是组织工程椎间盘治疗策略的主要限制因素之一.目的:制备天然猪脱细胞脱钙骨基质明胶,并验证其作为组织工程椎间盘纤维支架的可行性. 方法:取猪股骨近端松质骨,用环钻钻取直径10 mm、内径5 mm、厚3 mm的中空环状骨,高压水枪冲洗,进行脱脂、脱钙、脱细胞等相关处理,制成环状的支架材料.对材料进行大体、组织学、光镜、扫描电镜观察,并对支架的吸水率、孔隙率、生物力学参数等进行检测.分离培养犬骨髓间充质干细胞,采用MTT法分析支架浸提液毒性.结果与结论:支架为乳白色,中空环状,质地柔软的多孔结构.苏木精-伊红染色示组织无细胞结构残留.光镜及扫描电镜示支架孔隙均匀,孔隙相通,平均孔径为(401.4±13.1) μm,孔隙率为(62.12±1.52)%,吸水率为(409.77±11.34)%.生物力学结果示支架的弹性模量为(47.75±6.32) kPa.MTT法显示不同浓度支架浸提液与对照 DMEM 培养液吸光度值比较,差异无显著性意义(P ＞ 0.05),支架无细胞毒性.提示猪脱细胞脱钙骨基质明胶去细胞彻底,具有良好的孔径、孔隙率及生物力学强度,并且无毒,具备良好的生物相容性,可作为组织工程椎间盘纤维环支架材料.%BACKGROUND:Tissue engineering strategies to repair degenerated intervertebral disc are being developed to replace or regenerate the herniated nucleus pulposus. However, the tissue engineering of nucleus pulposus alone can not restore intact structure and function of intervertebral disc. Tissue engineering of annulus fibrosus is deemed as a major limiting factor for tissue engineered interve rtebral disc treatment strategy.OBJECTIVE:To fabricate a natural acellular demineralized bone matrix gelatin and to verify the feasibility of it as a scaffold for annulus fibrosus tissue engineering.METHODS:Pig proximal femoral cancellous bone rings (external diameter 10 mm, internal diameter 5 mm, thickness 3 mm)were fabricated, then dealed with high-pressure water washing, degreasing, decalcification, decellularization and other related treatments. The ring scaffolds were investigated by gross, histological, light microscopy and scanning electron microscopy observations, as well as porosity measurement, water absorption rate and biomechanical analysis. Dog bone marrow mesenchymal stem cells were isolated and cultured, MTT test was also done to assess cytotoxicity of the scaffolds.RESULTS AND CONCLUSION:The scaffold showed white, porous cancellous ring. The hematoxylin-eosin staining revealed that there were no cell fragments in the scaffolds. The light microscopy and scanning electron microscopy observations demonstrated that the novel porous scaffold had a good pore interconnectivity with (401.4±13.1) μm pore diameter,(62.12±1.52％) porosity and (409.77±11.34)％ water absorption rate. The biomechanical test showed that the longitudinal elastic modulus was (47.75±6.32) kPa. The intrinsic cytotoxicity assessment of novel scaffolds using MTT test showed that there were no significant differences in the absorbance value between scaffold leaching liquid ad control DMEM culture solution, thus the scaffolds do not exert any cytotoxic effect on cells. Novel acellular demineralized bone matrix gelatin scaffold had good pore diameter and porosity, appropriate biomechanical character, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative scaffold for annulus fibrosus tissue engineering.