探讨京尼平-壳聚糖牦牛跟腱胶原蛋白支架的构建及其细胞毒性和组织相容性。

以牦牛跟腱为材料，用酶-酸法进行粗提，结合高效液相色谱进行胶原蛋白的分离、纯化，加入交联剂构建京尼平-壳聚糖牦牛跟腱胶原蛋白支架及京尼平-壳聚糖标准I型胶原蛋白支架。将L929小鼠胚胎成纤维细胞接种于浇注2种支架的孔板上，分别培养1、3、5、7、9、11 d，倒置显微镜下观察细胞形态，细胞计数试剂盒-8进行细胞增殖/毒性实验，计算细胞活力。将2种蛋白支架材料分别移植于同一只大鼠脊柱2侧深筋膜层，分别于移植后3、5、7、9 d取材，10%中性福尔马林液固定，石蜡包埋、切片，苏木精-伊红染色，显微镜下观察胶原降解情况，血管长入情况。数据比较采用t检验。

培养1、3、5、7、9、11 d，接种于浇注京尼平-壳聚糖牦牛跟腱胶原蛋白支架与京尼平-壳聚糖标准I型胶原蛋白支架的孔板的L929小鼠胚胎成纤维细胞细胞活力分别为(3.14±0.04)%、(2.17±0.13)%、(2.60±0.11)%、(1.78±0.02)%、(1.64±0.32)%、(1.12±0.02)%，(2.96±0.07)%、(2.08±0.10)%、(2.68±0.14)%、(1.75±0.02)%、(1.84±0.01)%、(1.14±0.01)%，比较差异均无统计学意义(t＝－2.15、－1.31、1.22、－1.27、1.51、0.54, P＝0.06、0.22、0.25、0.25、0.16、0.63)。活体组织相容性实验，大鼠全部存活，手术切口愈合良好，未见急性炎症和局部积液、积脓改变。移植3、5、7、9 d取材，京尼平-壳聚糖牦牛跟腱胶原蛋白支架和京尼平-壳聚糖标准I型胶原蛋白支架均仍未降解，局部组织长入经交联的支架中，支架内见炎性细胞增殖，出现血红细胞及新生小血管。

京尼平-壳聚糖牦牛跟腱胶原蛋白支架生物安全性良好，具有作为组织工程支架的潜力。

To investigate the construction, cytotoxicity and histocompatibility of genipin-chitosan collagen scaffold for yak achilles tendon.

The tendon of yak was used as material for crude extraction by enzyme-acid method, and collagen was separated and purified by high performance liquid chromatography. Crosslinking agent was added to construct genipin-chitosan collagen scaffold for yak achilles tendon and genipin-chitosan standard type I collagen scaffold. L929 mouse embryonic fibroblasts were inoculated on the orifice plates of the two kinds of scaffolds and cultured for 1, 3, 5, 7, 9 and 11 days, respectively. Cell morphology was observed under inverted microscope. Cell count kit-8 was used for cell proliferation/toxicity test and cell viability was calculated. The two kinds of protein scaffolds were transplanted into the deep fascia layer on the two sides of the spine of the same rat respectively, and samples were taken on 3, 5, 7 and 9 days after transplantation, fixed with 10% neutral formalin solution, embedded with paraffin, sectioned, stained with hematoxylin and eosin. The degradation of collagen and the growth of blood vessels were observed under the microscope. Data were processed with t test.

Cultured for 1, 3, 5, 7, 9, 11 days, the cell viability of L1929 mouse embryonic fibroblasts, which were inaculated into the genipin-chitosan collagen scaffold for yak achilles tendon and genipin-chitosan standard type I collagen scaffold, were respectively (3.14±0.04) %, (2.17±0.13) %, (2.60±0.11) %, (1.78±0.02) %, (1.64±0.32) %, (1.12±0.02) %; (2.96±0.07) %, (2.08±0.10) %, (2.68±0.14) %, (1.75±0.02) %, (1.84±0.01) %, (1.14±0.01) %, the differences were not statistically significant (t=-2.15, -1.31, 1.22, -1.27, 1.51, 0.54; P=0.06, 0.22, 0.25, 0.25, 0.16, 0.63). In vivo histocompatibility test, all the animals survived, the surgical incision healed well, there were no changes of acute inflammation, local effusion and abscess. After 3, 5, 7 and 9 days of transplantation, the genipin-chitosan collagen scaffold for yak achilles tendon and genipin-chitosan standard type I collagen scaffolds were not degraded, and local tissues grew into the cross-linked scaffolds. Inflammatory cell proliferation and red blood cells and new small blood vessels were observed in the scaffolds.

The genipin-chitosan collagen scaffold for yak achilles tendon has good biosafety and potential as tissue engineering scaffold.