新生牛跟腱胶原蛋白海绵与细胞的生物相容性

Biocompatibility of collagen sponge made from new bovine tendons and cells

目的:采用新生牛跟腱制备生物医用胶原蛋白海绵,通过分别接种Vero细胞、天祝白牦牛胚胎皮肤原代细胞和岷县黑紫羔羊睾丸原代细胞至胶原蛋白海绵组织支架上,观察新生牛跟腱胶原蛋白海绵与3种细胞的生物相容性。方法:实验于2006-02/05在西北民族大学生命科学与工程学院生物工程与技术国家民委重点实验室完成。①用新生牛跟腱经冰醋酸、胃蛋白酶等消化,经盐析、透析及冻干等处理后,制备成胶原蛋白海绵。②在六孔板中,将Vero细胞、天祝白牦牛胚胎皮肤原代细胞和岷县黑紫羔羊睾丸原代细胞分别接种于经紫外线、臭氧灭菌后的胶原蛋白海绵上,经37℃体积分数为0.05的CO2恒温培养;并用另一组细胞在凯氏瓶中培养作对照实验。用奥林帕斯倒置相差显微镜及JVC摄像系统观察、拍摄与记录细胞生长情况,并于培养11d,用考马斯亮蓝和苏木精-伊红染色,证实相差显微镜观察的结果。结果:①胶原蛋白海绵制备结果:用新生牛跟腱制备得到具有一定孔隙度的胶原蛋白海绵,经紫外线、臭氧灭菌后可进行细胞培养。②3种细胞在胶原蛋白海绵上和六孔板底的生长情况:3种细胞接种后5h,在胶原蛋白海绵周围的六孔板孔底可看到Vero细胞已贴壁、伸展,个别细胞有分裂现象,在胶原蛋白海绵表面,隐约可见圆形细胞排列。Vero细胞和天祝白牦牛胚胎皮肤原代f2细胞接种72h、岷县黑裘皮羔羊睾丸原代f2细胞接种48h时,孔底细胞已经铺满单层,但与对照孔比较,生长速度较慢。②3种细胞用考马斯亮蓝细胞骨架染色和苏木精-伊红染色的结果:培养到第11天,3种来自不同动物、不同组织的细胞接种的胶原蛋白海绵支架孔中均有大量细胞良好生长,胶原海绵外观变得挺拔、透明、有韧性。结论:新生牛跟腱胶原蛋白海绵,对Vero细胞、天祝白牦牛胚胎皮肤原代细胞和岷县黑紫羔羊睾丸原代细胞无毒性,3种细胞均能在其上良好生长,但在体内是否会引起免疫排斥反应,还有待进一步研究。新生牛跟腱有望成为医用胶原蛋白海绵产品新的原材料。

AIM： To prepare collagen sponge with new bovine tendons by respectively inoculating Vero cell, primary embryo skin cell of Tianzhu White Yak and lamb testicle cell of Minxian black fur sheep on the tissue scaffold of collagen sponge, and observe the biocompatibility of collagen sponge with three different cells. METHODS ： The experiment was conducted in the Key Laboratory of Bioengineering of State Ethical Committee, Life Science and Engineering College of Northwest University for Nationalities between February and May 2006. ① New bovine tendons was digested by glacial acetic acid and pepsinum firstly, and then salting-out, dialysis and freeze drying ware performed to prepare collagen sponge. ② In a 6-mesh board, the Vero cell, primary embryo skin cell of Tianzhu White Yak and lamb testicle cell of Minxian black fur sheep ware respectively inoculated into the collagen sponge, which is treated by ultraviolet and sterilized by ozone. Samples ware cultured in 5% CO2 at a constant temperature of 37 %, while another board of cells ware cultured in Kjeldahl bottle for control. Olympus inverted phase contrast microscope and JVC video system ware adopted to observe, shoot and record the cell growth, and Coomassie brilliant blue as wall as HE staining ware conducted at 11 days after culture to identify the observation under the contrast phase microscope. RESULTS： ① Preparation of collagen sponge： The collagen sponge with certain porosity was prepared by new bovine tendons, which was then cultured after being sterilized by ultraviolet and ozone. ② The growth of 3 cells in collagen sponge and 6-mesh board： Five days after inoculation of 3 cells, there ware adherence and expansion of Vero cells at the bottom of board, and some of the cells split. On the surface of collagen sponge, a round cell arrangement could be seen indistinctly. 72 hours after inoculation of Vero cell and primary embryo skin cell f2 of Tianzhu White Yak and 48 hours after inoculation of lamb testicle cell f2 of Minxian black fur sheep, the monolayer was full of porocytes, while the growth velocity was slower in comparison with that in the control group. ③ Coomassie brilliant blue and HE staining of three kinds of cells： On the 11^th day of cultivation, there ware large amount of cells wall grew in the pores of collagen sponge scaffold that inoculated with 3 different cells, and the collagen sponge turned to be eminent, transparent and tenacious. CONCLUSION： The collagen sponge made from new bovine tendon is avirulent to the Vero cell, primary embryo skin cell of Tianzhu White Yak and primary lamb testicle cell of Minxian black fur sheep, and all these cells can better grow in it, while whether the immunological rejection in vivo is still in need of further investigation. New bovine tendons might be a new source for collagen sponge of medical use.