仿生圆周取向微米纤维支架构建组织工程椎间盘纤维环

Biomimetic circumferential oriented polycaprolactone microfiber scaffold for annulus fibrosus tissue engineering

目的探讨仿生圆周取向微米纤维支架作为组织工程椎间盘支架的可行性。方法以聚己内酯溶液为原料,采用湿法纺丝技术模拟天然纤维环结构,制备三维圆周取向的微米纤维支架。通过体视显微镜观察支架宏观结构;扫描电镜、Micro-CT对支架微观结构、孔隙率、纤维直径进行测定;兔纤维环细胞复合微纤维支架体外培养14d,运用扫描电镜、Live/dead染色评价支架的细胞生物相容性;DiI标记细胞膜和鬼笔环肽染色观察细胞在支架上的长入和生长情况;H&E染色、番红O染色、Ⅰ型免疫组化和免疫荧光染色对细胞在支架上的行为进行组织学评价。结果体视显微镜显示,纤维环支架呈类似天然纤维环的圆环结构;扫描电镜和Micro-CT显示,支架呈3D圆周取向纤维结构,纤维之间无粘连。纤维环支架的孔隙率为69.33%±6.67%,纤维直径为(16.13±2.77)μm。扫描电镜和鬼笔环肽染色见细胞呈长梭形并沿纤维方向铺展,DiI标记细胞膜显示培养14 d后纤维环细胞长入支架内部。组织学染色显示纤维环细胞和分泌的细胞外基质均可沿微米纤维呈取向性分布。结论以聚己内酯为材料,采用湿纺成形法制备的微米纤维支架可诱导纤维环细胞取向性生长,并诱导细胞外基质取向分布,是构建组织工程椎间盘的理想支架载体。

Objective To investigate oriented polycaprolactone （PCL） microfiber scaffold for intervertebral disc （IVD） tissue engineering and verify the feasibility as a potential candidate for IVD tissue engineering. Methods A biomimetic cireumferentially oriented mierofiber scaffold was prepared by wet spinning method using PCL solution. The scaffold was investigated by stereomicroseope. The scanning electron microscopy （SEM） and micro-CT were used to determine the mierostructure, porosity and fiber diameter of the scaffolds. The rabbit annulus fibrosus （AF） cells were isolated from IVD and seeded into the microfiber scaffold. The cell-scaffold complex was cultured for 14 days in vitro. The biocompatibility of the scaffold was evaluated by SEM and Live/dead staining. The cell attachment and infiltration were evaluated by phalloidin staining and DiI fluorescence labeling. The histology analyses of the cell-scaffold were examined by H＆E staining, Safranin O staining, and type Ⅰ immunohistochemistry and immunofluorescence staining. Results The stereomicroscope images showed that the AF scaffold presented a ring structure similar to the native AF tissue. SEM and micro-CT revealed that the AF scaffold was 3D circumferentially oriented fiber structure without adhesion. The scaffold had a porosity of 69.33%±6.67%, and the fiber diameter was （16.13±2.77） μm. SEM and phalloidin staining revealed that the AF cells spread along the fiber direction. The DiI-labeled images revealed that the AF cells grew into the inside of the scaffold after 14 days of culture. Histological staining showed that the AF cells grew inside the scaffold and spread along the microfiber direction and secreted AF-related extracellular matrix, which also oriented along the microfiber direction. Conclusion The biomimetic circumferential oriented PCL microfiber scaffold has high porosity and good biocompatibility, and could guide oriented growth of AF cells and orient deposition of AF-related ECMs. They are ideal scaffolds for the application of regeneration of IVD.