Chinese Journal of Tissue Engineering Research ›› 2024, Vol. 28 ›› Issue (3): 366-372.doi: 10.12307/2023.881
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Gu Mingxi1, Wang Changcheng2, Tian Fengde2, An Ning2, Hao Ruihu2, Guo Lin2
Received:
2022-04-19
Accepted:
2022-11-30
Online:
2024-01-28
Published:
2023-07-08
Contact:
Guo Lin, Chief physician, Professor, MD, Master’s supervisor, Second Department of Orthopedics, Zhongshan Hospital of Dalian University, Dalian 116001, Liaoning Province, China
About author:
Gu Mingxi, Master physician, Shenzhen Hospital of Peking University, Shenzhen 518000, Guangdong Province, China
CLC Number:
Gu Mingxi, Wang Changcheng, Tian Fengde, An Ning, Hao Ruihu, Guo Lin. Preparation and in vitro evaluation of a three-dimensional porous cartilage scaffold made of silk fibroin/gelatin/chitosan[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(3): 366-372.
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2.1.3 各组支架X射线衍射晶型分析 通过X射线衍射对各组支架进行了结构分析,4组支架的X射线衍射图谱如图4所示。明胶/壳聚糖支架的X射线衍射图谱的特征峰主要集中在2θ=7.7°,12°,18°和22°处,丝素蛋白/壳聚糖支架的X射线衍射图谱显示其特征峰集中在2θ=12°和22°。丝素蛋白/明胶支架的X射线衍射图谱显示其特征峰主要集中在2θ=7.7°和20.7°,丝素蛋白/明胶/壳聚糖支架在2θ=21°附近显示出一个宽大主峰,对应于壳聚糖、明胶和丝素蛋白的主反射的重叠。由于明胶与丝素蛋白及壳聚糖链的相互作用影响了其重组,明胶在2θ=7.7°时的低角反射特性已经消失,壳聚糖在2θ=18°时的低角反射峰消失,但是在2θ=12°时特征峰仍然存在。"
2.1.6 各组支架生物降解率 使用溶菌酶对4组支架进行了体外降解实验,如图7所示,4组支架的生物降解率比较差异有显著性意义(P < 0.05)。明胶/壳聚糖支架的降解率最高,为(53.96±2.62)%;丝素蛋白/壳聚糖支架和丝素蛋白/明胶/壳聚糖支架的降解率较低,分别为(30.15±2.71)%和(46.87±3.25)%;丝素蛋白/明胶支架的降解最为缓慢,降解率为(30.15±5.49)%。明胶/壳聚糖支架降解最快,在37 ℃环境下经过溶菌酶28 d培养后降解度可达50%以上,组织工程支架要求生物降解与软骨组织形成互相匹配才能用于修复软骨损伤,此次研究表明明胶/壳聚糖支架降解太快,明显不适合作为组织工程支架。"
原代软骨细胞形态大多为梭形和多角形,细胞在贴壁2 d后开始很好地附着在细胞培养瓶表面并开始增殖,并在7-9 d后达到90%的汇合状态。当细胞生长达到80%-90%的汇合密度时,对细胞进行胰蛋白酶消化传代。传代细胞表现出相似的形态,绝大数细胞表现为多角形,表面多树突状突起,随着培养时间增加,树突状突起伸展为细长触丝。使用甲苯胺蓝对提取的原代细胞进行染色,细胞核被染成紫蓝色,胞核偏向一侧,未见肥大样细胞,符合软骨细胞的特征。 2.2.2 软骨组织支架上的细胞黏附率 理想的生物支架可以改善细胞黏附、细胞分化和与周围天然组织的整合[10]。此次实验观察了软骨细胞在丝素蛋白/明胶/壳聚糖支架上接种2,4,6 h后的黏附率,如图11所示,软骨细胞能与支架良好黏附,随着共培养时间的延长,细胞黏附率逐渐增大,4-6 h后达到最大,表明丝素蛋白/明胶/壳聚糖支架很好的保持材料优良的生物活性。"
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