京尼平交联羊膜生物支架的生物物理特性

Biophysical characteristics of genipin-crosslinked amniotic membrane bio-scaffold

目的探讨京尼平交联羊膜作为生物支架的生物特性及可行性。方法获取新鲜脐带，采用贴壁法分离培养人脐带间充质干细胞（hUCMSCs）。采用流式细胞技术测定细胞表面标志物PE-CD34、PE-CD45、PE-CD90、FITC-105和FITC-Oct-4单克隆抗体及鉴定细胞。取第3代hUCMSCs进行成脂、成骨诱导培养，分别用茜素红和油红O染色鉴定。无菌条件下取健康剖宫产羊膜，将修剪的组织块固定于硝酸纤维素膜并按照京尼平质量浓度、交联温度和时间的不同分为12个组，分析质量分数0.5%、1%京尼平在37 ℃、45 ℃条件下交联羊膜24、36和48 h后羊膜的最大拉伸位移和最大载荷。将hUCMSCs分为hUCMSCs组、新鲜羊膜组（孔板中铺上新鲜羊膜后加入hUCMSCs进行培养）、交联羊膜组、交联羊膜＋明胶组和明胶组，分别按照分组在培养孔中铺上相应物质对hUCMSCs进行培养，采用羟脯氨酸试剂盒检测各组交联羊膜中羟脯氨酸含量。于培养后第3天采用MTT法检测各组细胞增生值（吸光度，A值）以评估交联羊膜的生物相容性。结果与0.5%京尼平交联羊膜相比，1%京尼平交联羊膜最大拉伸位移减小[（8.31±0.43）mm与（4.49±0.37）mm]，差异有统计学意义（t＝6.785，P〈0.05）。0.5%京尼平在37 ℃和45 ℃条件下交联24 h羊膜最大拉伸位移分别为（9.89±1.09）mm和（5.39±0.59）mm，差异有统计学意义（t＝6.389，P〈0.05）；0.5%京尼平在37 ℃条件下交联24、36和48 h羊膜最大拉伸位移逐渐减小，但差异无统计学意义（P〉0.05）。与0.5%京尼平交联羊膜相比，1%京尼平交联羊膜最大载荷增大[（3.94±0.31）N与（5.19±0.27）N]，差异有统计学意义（t＝3.034，P〈0.05）。0.5%京尼平在45 ℃条件下交联24 h羊膜载荷明显高于37 ℃条件下，差异有统计学意义（t＝5.528，P〈0.05）。1%京尼平交联羊膜、0.5%京尼平交联羊膜和新鲜羊膜中羟脯氨酸质量分数分别为（1.28±0.36）、（2.03±0.49）和（2.11±0.10）mg/g，1%京尼平交联羊膜中羟脯氨酸质量分数明显低于0.5%京尼平交联羊膜和新鲜羊膜，差异均有统计学意义（均P〈0.05）。新鲜羊膜组、明胶组和hUCMSCs组培养3 d后细胞增生值均明显低于交联羊膜组和交联羊膜＋明胶组，差异均有统计学意义（均P〈0.05），交联羊膜组与交联羊膜＋明胶组间细胞增生值的差异无统计学意义（P〉0.05）。结论不同温度、不同交联时间和不同质量分数京尼平均影响交联羊膜的力学特性，交联羊膜的细胞毒性小，可塑性更好，有望作为人工角膜的载体支架。

ObjectiveTo investigate the characteristics and feasibility of genipin-crosslinked amniotic membrane（AM） as bio-scaffold.MethodsHuman umbilical cord mesenchymal stem cells （hUCMSCs） were isolated from fresh umbilical cord and cultured by adherent method.The expressions of PE-CD34, PE-CD45, PE-CD90, FITC-105 and FITC-Oct-4, the markers of hUCMSCs, were detected by flow cytometry.Alizarin red and oil red O staining were performed to identify the cells after adipogenesis and osteogenesis induction on the third-generation cells.Human AMs were treated at 37 ℃ and 45 ℃ by 0.5% and 1% genipin solution for 24, 36 and 48 hours respectively, and the mechanical properties of AM in each group were measured and compared.The hUCMSCs were divided into only hUCMSCs culture group, fresh AM group, crosslinked AM group, gelatin group and crosslinked AM＋ gelatin group, and the cells were cultured in the corresponding medium.The content of hydroxyproline among the groups was detected with hydroxyproline kit, and proliferation of the cells （absorbance） was assayed by MTT method to evaluate the biological compatibility of crosslinked AM.ResultsThe maximum tensile displacement of the crosslinked-AM by 0.5% and 1% genipin was （8.31±0.43）mm and （4.49±0.37）mm respectively, and those after crosslinked with 0.5% genipin under the 37 ℃ and 45 ℃ for 24 hours was （9.89±1.09）mm and （5.39±0.59）mm, respectively, showing a significant difference between them （t=6.389, P〈0.05）. The maximum tensile displacement of the crosslinked-AM was gradually decreased as the lapse of crosslinking time, and an insignificant difference was found among 24, 36 and 48 hours after 0.5% genipin treatment under the 37 ℃ （P〉0.05）. The loading force of the crosslinked-AM was significantly higher in the 1% genipin treated group than that in the 0.5% genipin treated group （P〈0.05）, and the loading force of the AM was significantly increased in 45 ℃, 0.5% genipin, 24 hours crosslinked group compared with the 37 ℃, 0.5% genipin, 24 hours crosslinked group （t=5.528, P〈0.05）. The content of hydroxyproline in the AM was （1.28±0.36）, （2.03±0.49） and （2.11±0.10）mg/g in the 1% genipin crosslinked AM group, 0.5% genipin crosslinked AM group and fresh AM group, respectively, and the content of hydroxyproline in the AM in the 1 % genipin group was significantly lower than that in the 0.5% genipin group in the fresh AM group （both at P〈0.05）. The proliferative values of the hUCMSCs were significantly lower in the only hUCMSCs culture group, fresh AM group and gelatin group were significantly reduced in comparison with the crosslinked AM group and crosslinked AM＋ gelatin group （all at P〈0.05）. There was no significant difference in the proliferative values of the hUCMSCs between crosslinked AM group and crosslinked AM＋ gelatin group （P〉0.05）.ConclusionsDifferent crosslinked temprature, crosslinking period and concentration of genipin impact the mechanical properties of AM.Crosslinked AM with genipin is feasible as a carrier scaffold of artificial cornea because of less tissue toxicity and better plasticity.