压缩载荷下软骨细胞/复合支架的三维培养

林祥龙; 高丽兰; 李瑞欣; 程威; 张杨; 张春秋; 张西正

摘要

BACKGROUND: The silk fibroin/type II collagen composite scaffold has been prepared by low-temperature bio-3D printing technology in the previous study and the scaffold has good mechanical properties. Studies have shown that mechanical stimulation is beneficial to bone remodeling, and gradient loading strain is beneficial to the activation of osteoblasts and osteoclasts. OBJECTIVE: To co-culture silk fibroin/type II collagen composite scaffolds with chondrocytes under compression loading, to observe the proliferation of cells, and to observe the preliminary repair effect of silk fibroin/type II collagen composite scaffold on cartilage defects. METHODS: The silk fibroin/type II collagen composite scaffold was prepared by low-temperature 3D printing to detect the porosity of the scaffold. The passage 3 mouse chondrocytes ADTC-5 were inoculated on the silk fibroin/type II collagen composite scaffold and cultured under static culture and mechanical load respectively. (1) Static culture: blank scaffold was set as control, and cell proliferation was detected by MTT assay at 1, 3, 5, 7, 10, 14 days of inoculation. (2) Culture under mechanical load: blank scaffold was set as control. At 1 day after inoculation, 0％, 1％, 5％, 10％, 15％, 20％ compressive strains were applied to the cell-scaffold complex, and continued to load for 3 days. Cell proliferation was detected by MTT assay, and the distribution, adhesion and morphology of the cells on the scaffold were observed by scanning electron microscopy and hematoxylin-eosin staining. A cartilage defect of 3.5 mm in diameter was made in the bilateral knee joint of New Zealand rabbits. The silk fibroin/type II collagen composite scaffold was implanted onto the left side, and no material was implanted onto the right side. The repair site was observed at 8 weeks after surgery. RESULTS AND CONCLUSION: (1) The porosity of the scaffold was (89.3±3.26)％, which was conducive to cell attachment. (2) After 5 days of static culture, the chondrocytes proliferated well on the surface of the composite scaffold. Under 0％, 1％, 5％, 10％, 15％, 20％ compressive strains, the cell proliferation on the scaffold first increased and then decreased, wherein the cell proliferation was highest under 10％ compressive strain, and lowest under 20％ compressive strain. (4) Under the scanning electron microscopy, the chondrocytes in the 0％ load group were distributed in the surface of the scaffold with irregularities, the cell morphology was obvious, and the cell protrusions were fully extended. There were few or no chondrocytes on the contact surface of the 10％ load group, and more cells distributed on the lateral and internal surfaces of the first layer, but the cell morphology was flat with obvious protrusions. (5) Hematoxylin-eosin staining showed that the chondrocytes in the 0％ load group were concentrated on the surface of the scaffold, and there were almost no cells in the pores, while the chondrocytes in the 10％ load group were distributed in the scaffold pores. (6) There was still a circular defect model with no scaffold implantation, and no obvious repair appeared; similar hyaline cartilage appeared in the defect after scaffold implantation, but there was no adhesion to the surrounding defected cartilage, and the new hyaline cartilage was independent. Overall, the adsorption, proliferation and growth of chondrocytes on the silk fibroin-type II collagen scaffolds is better when the compressive strain is 10％, and the composite scaffold can be used as a repair material for cartilage defects.%背景:前期研究利用低温生物3D打印技术制备了丝素蛋白/Ⅱ型胶原蛋白复合支架,并证明其具有良好的力学性能;研究表明,力学刺激有利于骨骼重塑,并且梯度变化的加载应变有利于成骨细胞和破骨细胞的活化.目的:在压缩应变下,将丝素蛋白/Ⅱ型胶原蛋白复合支架与软骨细胞共培养,观察细胞增殖变化,并观察丝素蛋白/Ⅱ型胶原蛋白复合支架修复软骨缺损的效果.方法:采用低温3D打印技术制备丝素蛋白/Ⅱ型胶原蛋白复合支架,检测支架的孔隙率.将第3代小鼠软骨细胞ADTC-5接种于丝素蛋白/Ⅱ型胶原蛋白复合支架上,分别进行静态培养与力学载荷下培养:①静态培养:设置空白支架为对照,接种1,3,5,7,10,14 d,采用MTT法检测细胞增殖;②力学载荷下培养:设置空白支架为对照,接种1 d后,对细胞-支架复合物分别施加0％,1％,5％,10％,15％,20％的压缩应变,持续加载3 d,采用MTT法检测细胞增殖,扫描电镜和苏木精-伊红染色观察支架上的细胞分布、黏附和形态.在新西兰兔双侧膝关节制作直径3.5 mm的软骨缺损,左侧植入丝素蛋白/Ⅱ型胶原蛋白复合支架,右侧不植入材料,术后8周观察修复部位.结果与结论:①支架孔隙率为(89.3±3.26)％,有利于细胞附着;②静态培养5 d后,软骨细胞在复合支架表面增殖良好;③施加0％,1％,5％,10％,15％,20％压缩应变组支架上的细胞增殖先升高后降低,其中施加10％压缩应变组细胞增殖效果最显著,施加20％压缩应变组增殖效果最低;④扫描电镜可见,施加0％压缩应变组软骨细胞多分布在支架表面有凹凸的地方,细胞形态明显,细胞触角伸展充分;施加10％压缩应变组支架受力接触表面上的软骨细胞极少,甚至没有,支架上首层侧面和内部表面细胞分布较多,细胞形态多为扁平状,触角明显;⑤苏木精-伊红染色显示,施加0％压缩应变组软骨细胞集中分布在支架表面,孔隙中几乎没有细胞;施加10％压缩应变组软骨细胞分布在支架孔隙内;⑥未植入支架的缺损处仍为圆形缺损模型,没有明显修复;植入支架的缺损处出现了类似透明软骨,但与周边缺损软骨没有发生粘连结合,新生的类透明软骨独立存在;⑦结果表明,在10％压缩应变作用下,软骨细胞在丝素蛋白/Ⅱ型胶原蛋白复合支架上增殖良好;丝素蛋白/Ⅱ型胶原蛋白复合支架可用于修复软骨缺损.

压缩载荷下软骨细胞/复合支架的三维培养

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