外源性胶原对组织工程肌腱构建影响的体外形态学观察(英文)

In vitro morphological observation on the effect of exogenous collagen on the construction of tissue-engineered tendon

背景:I型胶原抗原性低,具有为细胞移行、增殖和分泌提供支架结构的特殊功能。目的:利用I型胶原观察转化人胚腱细胞与人工材料复合后的形态学变化,为体外构建组织工程化肌腱提供实验依据。设计:随机对照观察。单位:四川大学华西医院骨科。材料:实验于1998-07/09在华西医科大学移植免疫室完成。实验设立人发复合胶原组、碳纤维复合胶原组、聚羟基乙酸复合胶原组作为实验组,设立人发未复合胶原组、碳纤维未复合胶原组、聚羟基乙酸未复合胶原组作为对照组。人工材料:健康成人头发(长约30cm,80g左右),碳纤维编织带(兰州碳素厂提供),聚羟基乙酸纤维束(美国Johnson)。方法:①人发复合胶原组选9根健康成人头发,经处理后3根为一股编织成腱,长约0.8cm,直径约0.4cm,两端自身打结。②碳纤维复合胶原组将购买的碳纤维编织带分开,制成直径及长度与人发腱相同的人工材料,两端用5/0丝线捆扎。③聚羟基乙酸复合胶原组将3根聚羟基乙酸纤维束互相编织成束,直径及长度与人发腱相同,两端用5/0丝线捆扎。④各对照组编织支架材料过程与其对应实验组相同。⑤各组支架材料均制备5根。实验组支架材料60Co灭菌后,无菌条件下置于I型胶原溶液中0.5h,取出后室温干燥备用。对照组支架材料均不与I型胶原复合。⑥各组分别与细胞密度为3×106/mm3的转化人胚腱细胞培养基进行体外联合培养,于培养后不同时间点对细胞数量及形态进行倒置显微镜和扫描电镜观察。主要观察指标:①细胞生长情况及附着情况倒置显微镜观察结果。②细胞形态及与材料附着情况扫描电镜观察结果。结果:①细胞生长情况及附着情况倒置显微镜观察结果:培养2h后,各实验组均可见细胞向材料周围聚集,附着于材料的部分细胞呈圆形;培养3d后,附着于材料上的细胞开始增多,形态由圆形向椭圆形演变,可见圆形与椭圆形细胞“共存”现象;而各对照组培养2h和3d后,3种材料上细胞附着均较少。培养5d后,各实验组材料上细胞形态向梭形演变,细胞数量进一步增多,且碳纤维材料之间可观察到大量梭形细胞生长;而各对照组细胞形态大部分仍为圆形及椭圆形,细胞数量少于各自对应实验组。②细胞形态及与材料附着情况扫描电镜观察结果:体外培养5d后,各实验组材料表面腱细胞绝大部分呈梭形,同时腱细胞伸出突起,沿材料纵轴生长,其间有丝状及片状物质,相互交织。而对照组细胞明显较各自对应实验组附着少,细胞也呈梭形或圆形,但未见材料间的丝状及片状物质,也未见细胞伸出突起。结论:人发、碳纤维、聚羟基乙酸表面复合I型胶原后,有利于细胞附着,材料周围的细胞不仅数量增多,且出现圆形与椭圆形细胞“共存”现象,细胞形态向梭形演变,说明外源性胶原有助于细胞对材料的附着以及细胞自身的增殖。

BACKGROUND： Collagen type I possesses low antigenicity and provides special functions of scaffold structure for cell migration, proliferation and secretion. OBJECTIVE： To observe the morphological change after combination of transformed human embryonic tendon cells and artificial materials withcollagen type 1, so as to provide the experimental basis for constructing tissue-engineered muscle tendon in vitro. DESIGN： Randomized and controlled observation. SETTING： Department of Orthopaedics, West China Hospital, Siehuan University. MATERIALS： This experiment was carried out at the Department of Transplantation Immunology, West China University of Medical Sciences from July to September 1998. Human hair, carbon fiber and polyglycolic acid were combined with collagen and then set corresponding groups respectively. Simply hair, carbon fiber and polyglycolic acid control groups were set respectively. Artificial materials： Healthy adult hair （30 cm in length, 80 g in weight）, cation fiber knitted strip （Lanzhou carbon factory ）and polyglyolic acid fasciculi （Johnson , American） were used in this experiment. METHODS： ①In the group of human hair combined with collagen, nine healthy adult hairs were chosen . Three hairs as one bundle were knitted ＇into tendon with 0.8 cm in length and 0.4 cm in diameter and a knot was tied respectively at the two ends. ②In the carbon fiber combined with collagen group, the purchased carbon fiber knitted strip was separated and made into artificial materials with the same diameter andlength as those of human hair tendon.. Two ends of the artificial materials were packed with 5/0 silk thread. ③In the polyglycolic acid combined with collagen group, three polyglycolic acid fasciculi were weaved into bundle with the same diameter and length as those of human tendon, and two ends were packed with 5/0 silk thread.④The process of knitting scaffold materials was the same in control groups as in the correspending experimental groups. ⑤Five pieces were prepared in each scaffold material. After sterilized by ^60Co, the scaffold materials were put in collagen type I solution for 0.5 hour, then taken out and dried at room temperature for use in the experimental groups.⑥transformed human embryonic tendon cell, density for 3×10^6/mm^3, was used for combined culture in each group, and cell number and morphology were observed at different time points after culture under an inverted microscope and scanning electron microscope. MAIN OUTCOME MEASURES： ① Observation of cell growth and adherence under an inverted microscope ② Observation of cell morphology and material adherence under scanning electron microscope RESULTS： ① Observation of cell growth and adherence under an in- verted microscope ：In each experimental group, at two hours after culture, cells gathered around the materials, and some cells adhered to materials presented round; At three days after culture, the number of cells adhered to materials began to increase, morphology changed from round to oval, and the co-existence of round and oval cells was seen; While in each control group, at two and three hours after culture, few ceils adhered to the three materials. On day five after culture, in each experimental group, cell morphology turned into fusiform, the number of cells increased further and a great many fusiform cells grew among carbon fiber materials; while in the control group, most cells presented round and ellipse, and the number of cells was less as compared with the corresponding experimental group. ② Cell morphology and material adherence under scanning electron microscope： On day 5 after in vitro culture, most tendon cells on the surface of materials presented spindle-shape in each experimental group. Apophysis stretched out from the tendon cells, grew along longitudinal axon of the materials. Silk and piece-like substance appeared among the apophysis and weaved each other. While there were fewer ceils adhered in control group than in corresponding experimental group, cells also presented fusiform or round, but no silk or piece-like substance was found, also no apephysis appeared. CONCLUSION： Human hair, carbon fiber and pelyglycolic acid com- bined with collagen type I will facilitate cell adherence. Calls increase around the materials and co-existence of round and oval cells could be seen, and cells turned into fusiform, suggesting that exogenous collagen facilitate the cells to adhere onto the materials and proliferate.