京尼平交联羊膜的制备及其体外性能研究

Preparation and in vitro characterization of genipin-crosslinked human amniotic membrane

目的初步探讨新型交联剂京尼平对脱细胞羊膜性能的影响及意义,并在体外对其细胞相容性进行研究。方法用京尼平溶液交联羊膜30 min,使用光镜和扫描电镜进行形态学观察,在电子万能试验机上进行生物力学测试,以新鲜羊膜作对照,得出抗拉强度、断裂伸长率、弹性模量等,同时测定京尼平交联羊膜前后,羊膜组织的厚度及抗酶消化能力;以BALB/c 3T3小鼠成纤维细胞为实验细胞,采用MTT法在体外评估该材料的细胞毒性。结果京尼平交联后的羊膜呈蓝色,保持了天然组织构架的完整,纤维连接更为致密。交联羊膜组的抗拉强度为(1.489±0.405)MPa,断裂伸长率为(25.337±5.137)%,弹性模量为(0.004 7±0.002 1)GPa,与新鲜羊膜[(0.704±0.139)MPa,(15.488±6.021)%,(0.002 9±0.011 8)GPa]相比,三者均明显增加(P<0.05)。新鲜羊膜在第1天时已完全降解,交联羊膜至第4周观察结束时仍未完全降解,降解率为(74.62±3.81)%。该材料的相对增殖度较高,细胞毒性分级在0～1级。结论经新型生物交联剂京尼平交联的脱细胞羊膜组织材料具有良好的生物力学特性且无细胞毒性,是一种较为理想的眼表治疗的替代材料和组织工程角膜的支架材料。

Objective To investigate the characteristics and cytocompatibility of human-amniotic membrane（HAM） fixed with genipin in vitro and to provide a proper biomaterial for ocular surface reconstru-ction.Methods The de-epithelialized HAM were treated by 0.5% genipin for 30 min,then examined with naked-eye,light microscopy and scanning electron microscopy（SEM）.Untreated HAM served as control.Subsequently,the biomechanics tests were also performed for the tensile strength,elongation at break,and elastic modulus of the treated and untreated HAM.Their thickness and collagenase digestion were measured and observed on the treated and untreated HAM.The cytotoxicity of the genipin-fixed materials was evaluated in vitro by MTT assay using a mouse-derived established cell line of BALB/c 3T3 fibroblasts as test cells.Results The genipin-crosslinked HAM was blue,with very-well-preserved collagen fiber.The tensile strength,elongation at break,and elastic modulus were（1.489±0.405）MPa,（25.337±5.137）%,and（0.004 7±0.002 1） GPa respectively in the treated HAM,significantly higher than the fresh control HAM [（0.704±0.139）MPa,（15.488±6.021）%,（0.002 9±0.011 8）GPa,respectively P0.05].Genipin-crosslinked HAM had increased resistance to collagenase digestion,significantly better than the fresh control HAM.The former was still existed until the end of the experiment（4 weeks） with a degradation rate of（74.62±3.81）%,while the later HAM was degradated completely on the first day.The relative growth rate（RGR） of genipin-fixed HAM was high,and the cytotoxicity of this material was grade 0 to 1.Conclusion The genipin-fixed HAM is a promising material for ocular surface reconstruction,which can be used as a tissue engineering material for ocular surface treatment.