Ⅱ型胶原-透明质酸构建组织工程软骨复合三维纳米支架的体外实验研究

EXPERIMENTAL STUDY ON TISSUE ENGINEERED CARTILAGE COMPLEX THREE-DIMENSIONAL NANOSCAFFOLD WITH COLLAGEN TYPE Ⅱ AND HYALURONIC ACID IN VITRO

目的探讨Ⅱ型胶原-透明质酸（hyaluronic acid，HA）构建软骨组织工程复合三维纳米支架的可行性。 方法将Ⅱ型胶原和HA以8∶1（W∶W）比例溶于三氟乙醇和水按1∶1（V∶V）比例混合的溶剂中制成溶液，通过静电纺丝技术制备三维多孔纳米支架材料，通过光镜和扫描电镜观察其表面形貌，测定其孔隙率、吸水率、表面接触角和降解率。取1周龄日本大耳兔肋软骨，采用酶消化法制备软骨细胞。取第2代软骨细胞种植于支架上，用细胞计数试剂盒8（cell counting kit 8，CCK-8）评价其细胞黏附性及增殖情况；细胞-支架复合物体外连续培养2周后，行组织学和免疫组织化学染色观察其生物学形态及细胞外基质（extracellular matrix，ECM）分泌情况。 结果以最佳电纺液浓度为10%、接收距离为10 cm、纺丝注射速度为5 mL/h电纺获得的支架材料，光镜及扫描电镜观察示其孔隙均匀，纳米纤维直径均匀（300～600 nm），孔隙率为89.5% ± 25.0%，表面接触角为（35.6 ± 3.4）°，24 h内支架吸水率可达1 120% ± 34%，48 d内支架降解率可达42.24% ± 1.51%。CCK-8检测结果显示，培养12 h细胞在支架上黏附率可达169.14% ± 11.26%，培养7 d时细胞存活率可达126.03% ± 4.54%。组织学和免疫组织化学染色示，细胞在支架上黏附增殖情况良好，可分泌ECM；细胞在支架上培养2周后，有类似于软骨陷窝的结构形成。 结论以Ⅱ型胶原-HA制备的复合三维纳米支架材料具有良好的物理性能、生物学性能和促进细胞黏附及增殖的能力，是一种良好的组织工程软骨支架材 料。

ObjectiveTo explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid （HA） by electrospinning. MethodsThe three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA （8∶1, W∶W）, which was dissolved in mixed solvent of 3-trifluoroethanol and water （1∶1, V∶V）. The morphology were observed by light microscope and scanning electron microscope （SEM）. And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 （CCK-8）. The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix （ECM） secretion were observed by histological analysis. ResultsThe optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was （35.6 ± 3.4）°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. ConclusionThe collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.