构建韧带组织工程脱细胞支架的可行性研究

Construction of acellular tendon scaffold for ligament tissue engineering

背景:十二烷基磺酸钠脱细胞在脱细胞的同时对支架结构存在一定的损伤,降低了支架的生物学性能;胰酶等脱细胞方法较为温和,虽最大程度保留支架结构与生物学性能,但脱细胞效果并不彻底。目的:制备并分析脱细胞牛肌腱作为组织工程韧带支架的可行性。方法:取新鲜小牛跟腱,通过物理法去除肌腱表面腱膜、滑膜及软组织,制备50个跟腱,随机分为两组,均行冻干处理。实验组以Triton X-100行脱细胞处理,对照组不予脱细胞处理。两组均以PBS冲洗后室温干燥备用。行组织学、DNA含量、细胞增殖、生物力学(最大应力、弹性模量以及刚度)检测,比较两组的差异。结果:对照组和实验组的DNA残留量分别为(0.34±0.15)μg/mg和(0.7±0.03)μg/mg,实验组显著低于对照组,差异有统计学意义(P<0.05)。实验组与对照组支架DNA含量对比显示,支架脱细胞效果彻底,无显著的免疫原性。两组在组织形态结构上无明显差异。细胞增殖显示脱细胞支架对细胞增殖无明显影响。实验组的最大应力小于对照组,弹性模量无统计学差异,刚度试验显示两组无明显差异。结论:脱细胞支架在保留天然支架生物学性能的基础上可作为组织工程支架的选择。

Background：The cells can be removed completely by sodium dodecyl sulfate （SDS）,but the scaffold structure may be damaged and result in decreased biological characteristics.The scaffold structure and biological characteristics can be maximally preserved by pancreatic enzyme,while the cells couldn＆#39;t be removed completely because the pancreatic enzyme are relatively mild.Objective：To prepare and analyze the feasibility of acellular bovine tendon as scaffold of tissue engineered ligament.Methods：Fifty fresh bovine tendons were obtained.Their tendon membrane,synovial membrane and,soft tissue were removed by physical method.Then the tendons were randomly divided into two groups and the freeze-drying was used for them.The experimental group was decellullarised with Triton X-100,and control group was not decellullarised.All the cells in both groups were dried up at room temperature after PBS flushed.Then histological slices were performed.DNA content,cell proliferation,and biomechanics （maximum stress,elastic modulus and stiffness） were measured and compared between the two groups.Results：The residual amount of DNA was （0.34±0.15） μtg/mg and （0.7±0.03） μg/mg in the control group and experimental group,respectively （P〈0.05）.The significant difference of DNA residual amount between the two groups showed that the cells could be removed completely with no significant immunogenicity.There was no significant difference in morphological structure between the two groups.The decellularized scaffold did not affect the cell proliferation.The maximal stress of the experimental group was significantly lower than that of the control group,while no significant difference was found in the elastic modulus or the stiffness between the two groups.Conclusions：The acellular tendon can be used as the scaffold of tissue engineering ligament based on biological properties of natural scaffolds.