海马神经干细胞与胶原蛋白海绵及明胶海绵的生物相容性

背景:在应用组织工程修复周围神经的研究中,载体的选择至关重要。理想的载体应与细胞外基质类似,与活体细胞有良好的生物相容性。目的:评价豚鼠海马神经干细胞与胶原蛋白海绵和明胶海绵的生物相容性,探讨它们作为周围神经组织工程载体材料的可行性。设计:随机对照实验。单位:郑州大学第一附属医院耳鼻咽喉科,郑州大学基础医学院解剖教研室。材料:实验于2005-07/2005-12在郑州大学基础医学院解剖教研室神经生物学研究室完成。健康新生(<24h)杂色豚鼠12只,清洁级,雌雄不限,体质量50-70g,由郑州大学医学院实验动物中心提供。方法:新生(<24h)豚鼠,10g/L水合氯醛腹腔麻醉,体积分数为0.75乙醇消毒,无菌操作分离出海马组织。体外无血清培养海马神经干细胞,传至第2代,将浓度调整为1×1010L-1后分别与胶原蛋白海绵、明胶海绵联合体外培养,一周后取材,进行细胞计数,倒置相差显微镜及扫描电镜观察,并测定两种材料与细胞的吸附率。主要观察指标:①观察神经干细胞在胶原蛋白海绵和明胶海绵上的生长、黏附情况,并测定其细胞总数与载体材料的吸附率。②免疫细胞化学染色分化细胞形态观察。结果:①神经干细胞可以在胶原蛋白海绵和明胶海绵上生长,逐渐黏附,胶原蛋白海绵细胞吸附率高于明胶海绵(37.17%和14.87%,χ2=4.819,P<0.05)。②对照组、胶原蛋白海绵组、明胶海绵组细胞总数差异无显著性犤(53.17±3.5)×104,(53.25±2.6)×104,(52.04±4.05)×104,F=0.233,P>0.05犦。结论:胶原蛋白及明胶两种材料,尤其是胶原蛋白,具有良好的神经干细胞相容性,可以作为周围神经组织工程的支架材料应用于临床。

Biocompatibility of hippocampal neural stem cells with collagen and gelatin sponge

BACKGROUND: The selection of carrier plays an essential role in the research of applying tissue-engineering to fix the peripheral nerves. An ideal carrier would be one that is similar to extracellular matrix and that it has biocompatibility with in vivo cells.OBJECTIVE: To evaluate the biocompatibility of hippocampal neural stem cells with collagen and gelatin sponge in vitro and to probe into the feasibility of using the materials as biomaterial scaffold in peripheral nerve tissue engineering.DESIGN: A randomized and controlled trial.SETTING: Department of Otorhinolaryngology of the First Affiliated Hospital of Zhengzhou University; Department of Anatomy of the School of Basic Medical Sciences of Zhengzhou University.MATERIALS: The experiment was carried out from July to December 2005 at the Laboratory of Neurobiology of the Department of Anatomy of the School College of Basic Medical Sciences of Zhengzhou University.Twelve New born (＜ 24 hours) clean grade guinea pigs of either gender with a body mass of 50-70 g, were provided by the Experimental Animals Center of Zhengzhou University School of Medicine.METHODS: The new born (＜ 24 hours) guinea pigs were anesthetized intraperitoneally with 10 g/L chloral hydrate and sterilized in 0.75 volume fraction of alcohol. Hippocampal tissue was resected from the brain under a surgical microscope. Hippocampus neural stem cells were cultured in vitro. The cultured cells of two generations were suspended at a density of 1 ×1010L-1 and respectively combined with collagen and gelatin sponge.The number of cells was counted and histological changes were observed under an inverted phase microscope and scanning electron microscope after 7 days, and the adhesion rate of the two materials to the cells were measured.MAIN OUTCOME MEASURES: ①Growth of the neural stem cells and their adhesion to the collagen and gelatin sponge were observed and the total number of the cells and adhesion rate with carrier were measured. ②The morphological changes of differentiation cells by immunocytochemical staining.RESULTS: ①Hippocampus neural stem cells could grew on the collagen and gelatin sponge and attached to them gradually. The adsorption rate of collagen was higher than thai of gelatin sponge (37.17 % and 14.87 %,x2=4.819,P ＜ 0.05). ② There was no significant difference in the total number of the cells in the control group, collagen group and gelatin sponge group [(53.17±3.5)×104,(53.25±2.6)×104, (52.04±4.05)×104,F=0.233,P ＞ 0.05].CONCLUSION: Two biomaterials (collagen and glutin sponge), especially collagen, have good biocompatibility with hippocampus neural stem cells from the guinea pigs and can be used safely as scaffold materials in peripheral nerve tissue engineering.