中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (4): 560-566.doi: 10.12307/2022.092

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

天然丝素蛋白构建仿生取向微通道纤维环支架

何冠宇1,2,徐宝山2,杜立龙2,张同星2,霍振鑫1,2,申  力1,2   

  1. 1天津医科大学研究生院,天津市   300070;2天津市天津医院微创脊柱外科,天津市   300020
  • 收稿日期:2021-01-13 修回日期:2021-01-16 接受日期:2021-02-18 出版日期:2022-02-08 发布日期:2021-11-03
  • 通讯作者: 徐宝山,博士,主任医师,天津市天津医院微创脊柱外科,天津市 300020
  • 作者简介:何冠宇,男,1993年生,北京市人,汉族,天津医科大学在读硕士,主要从事脊柱外科、椎间盘组织工程椎间盘研究。
  • 基金资助:
    国家自然科学基金资助项目(82072491,31670983),项目负责人:徐宝山;国家自然科学基金资助项目(31900967),项目负责人:杜立龙;天津市自然科学基金项目(19JCQNJC09300),项目负责人:杜立龙

Biomimetic orientated microchannel annulus fibrosus scaffold constructed by silk fibroin

He Guanyu1, 2, Xu Baoshan2, Du Lilong2, Zhang Tongxing2, Huo Zhenxin1, 2, Shen Li1, 2   

  1. 1Graduate School of Tianjin Medical University, Tianjin 300070, China; 2Department of Minimally Invasive Spine Surgery, Tianjin Hospital, Tianjin 300020, China
  • Received:2021-01-13 Revised:2021-01-16 Accepted:2021-02-18 Online:2022-02-08 Published:2021-11-03
  • Contact: He Guanyu, Master candidate, Graduate School of Tianjin Medical University, Tianjin 300070, China; Department of Minimally Invasive Spine Surgery, Tianjin Hospital, Tianjin 300020, China
  • About author:He Guanyu, Master candidate, Graduate School of Tianjin Medical University, Tianjin 300070, China; Department of Minimally Invasive Spine Surgery, Tianjin Hospital, Tianjin 300020, China
  • Supported by:
    the National Natural Science Foundation of China, No. 82072491, 31670983 (to XBS), No. 31900967 (to DLL); Natural Science Foundation of Tianjin, No. 19JCQNJC09300 (to DLL)

摘要:

文题释义:
纤维环:是椎间盘保持其发挥负重和轴承作用的重要组成部分,纤维环由外、中、内三层纤维蛋白呈同心圆排列构成,层间纤维呈60°斜角叠交叉排列,这种特殊的结构保证了纤维环拥有较强的抗拉伸与压缩性,可有效避免髓核的疝出,对保持椎间盘稳定性起着至关重要的作用。
熔融纺丝技术:将高分子聚合物加热熔融成为一定黏度的纺丝熔体,利用纺丝泵连续均匀地挤压到喷丝头,通过喷丝头的细孔压出成为细丝流,然后在空气或水中使其降温凝固,通过牵伸成丝。熔融纺丝的主要工艺参数包括挤出温度、聚合物通过喷丝板各孔的质量流速、卷绕速度或落丝速度、纺丝线的冷却条件、喷丝孔形状和尺寸及间距、纺程长度等。
背景:纤维环是椎间盘重要的组成部分,是维持椎间盘力学及生理功能的关键,因此构建模拟天然纤维环结构的生物支架对于组织工程修复椎间盘至关重要。
目的:以天然丝素蛋白为原料构建取向微通道纤维环支架,评估其作为椎间盘纤维环组织工程支架的可行性。
方法:以聚己内酯为原料,通过熔融纺丝技术制备60°取向叠交叉模板,灌注浓度为15%的丝素蛋白溶液,液氮塑型后使用冷冻干燥机进行冷冻干燥,使用三氯甲烷洗脱沥滤聚己内酯材料,取向微通道纤维环支架制备完成;同时制备普通的丝素蛋白支架作为对照,表征支架的微观形貌与力学性能。将第3代兔纤维环细胞分别接种于两种支架上,通过死活染色及CCK-8实验分析细胞在支架上的存活能力与增殖能力。将两种支架分别植入大鼠皮下,4周后取出支架进行苏木精-伊红染色。
结果与结论:①体视显微镜与扫描电镜显示,取向微通道支架表面有大量规则通道排布且深入支架内部,对照支架表面只有少量裂缝;两种支架的孔径为(152.0±9.3) μm,取向微通道支架、对照支架的孔隙率分别为(89.0±3.3)%,(73.0±2.6)%;②微通道支架、对照支架的压缩模量分别为(2.65±0.11),(3.05±0.13) MPa,组间比较差异有显著性意义(P < 0.05);③死活染色与扫描电镜显示,细胞在两种支架上生长增殖良好,在对照支架表面黏附生长,在取向微通道支架可通过微通道迁移至支架内部且分泌大量外基质;CCK-8实验显示,取向微通道支架可促进细胞的增殖;④苏木精-伊红染色显示,取向微通道支架通道内可见大量细胞及细胞外基质成分,对照支架仅表面可见大量细胞及细胞外基质成分;⑤结果表明,取向微通道丝素支架能够模拟天然纤维环的结构,拥有良好的力学性能,细胞可沿通道生长入支架内部,具有良好的生物相容性,是构建纤维环支架合适的生物支架。

https://orcid.org/0000-0001-9781-0605 (何冠宇) 

中国组织工程研究杂志出版内容重点:生物材料;骨生物材料口腔生物材料纳米材料缓释材料材料相容性;组织工程

关键词: 组织工程, 纤维环, 椎间盘, 丝素蛋白, 微通道, 生物相容性, 生物支架材料, 椎间盘退变, 冷冻干燥, 组织再生修复

Abstract: BACKGROUND: The annulus fibrosus is an important part of the intervertebral disc, and is the key to maintain the mechanical and physiological functions of the intervertebral disc. Therefore, the construction of a biological scaffold that mimics the structure of the natural annulus fibrosus is essential for tissue engineering to repair the intervertebral disc.
OBJECTIVE: To construct biomimetic orientated microchannel and control scaffold with silk fibroin as raw material, and to evaluate its feasibility as a tissue engineered annulus fibrosus scaffold. 
METHODS: The polycaprolactone was used as raw material to construct 60° oriented fiber pattern by melt spinning technology, and filled with 15% silk fibroin solution, quick-freeze with liquid nitrogen, freeze-drying by lyophilizer. Polycaprolactone material was eluted by chloroform. The preparation of the orientated microchannel fibrous ring scaffold was completed; at the same time, a common silk fibroin scaffold was prepared as a control. The microscopic morphology and mechanical properties of the scaffold were characterized. The third-generation rabbit annulus cells were inoculated on the two scaffolds respectively. The viability and proliferation ability of the cells on the scaffold were analyzed by Live/Dead staining and CCK-8 assay. The two scaffolds were implanted subcutaneously in rats, and the scaffolds were removed 4 weeks later for hematoxylin-eosin staining.
RESULTS AND CONCLUSION: (1) Stereoscopic microscope and scanning electron microscope showed that there were a large number of regular channels arranged on the surface of the oriented microchannel scaffold and the channels penetrated deeply into the scaffold, and there were only a few cracks on the surface of the control scaffold. The diameter of two kinds of scaffolds was (152.0±9.3) μm. The porosity was (89.0±3.3)% in the oriented microchannel  group and (73.0±2.6)% in the control group. (2) The compression elastic modulus was (2.65±0.11) MPa in oriented microchannel group and (3.05±0.13) MPa in the control group (P < 0.05). (3) The Live/Dead staining and scanning electron microscope results showed that cells grow well on both scaffolds. In the control group, the scaffold cells only attached to the scaffold surface. The cells grew into the scaffolds along the microchannels and the extracellular matrix was secreted sufficiently. CCK-8 assay showed that the oriented microchannel scaffolds could promote cell proliferation. (4) Hematoxylin-eosin staining revealed that a lot of extracellular matrix components and cells were found in the oriented microchannel scaffolds. In the control group, a lot of cells and extracellular matrix only grew on surface of scaffold. (5) It is concluded that the oriented microchannel silk fibroin scaffold can simulate natural annulus fibrosus microstructure, which have good mechanical property; and the cells can along the channel grow into the scaffold, which has good biocompatibility. It is a suitable biological scaffold for the construction of annulus fibrosus scaffold.

Key words: tissue engineering, annulus fibrosus, intervertebral disc, silk fibroin, microchannel, biocompatibility, biological scaffold material, degenerative disc disease, freeze-drying, tissue repair

中图分类号: