[Ⅱ型胶原与透明质酸构建组织工程软骨复合体三维纳米支架的体外实验研究]

Yang Zelong, Chen Zhu, Liu Kang, Bai Yiguang, Jiang Ting, Feng Daxiong, Feng Gang

Research Institute of Tissue Engineering and Stem Cell, Nanchong Central Hospital, Second Clinical College of North Sichuan Medical College, Nanchong Sichuan 637000, PR China.

Department of Plastic and Aesthetic Surgery, Nanchong Central Hospital, Second Clinical College of North Sichuan Medical College, Nanchong Sichuan 637000, PR China.

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Oct;27(10):1240-5.

OBJECTIVE

To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning.

METHODS

The 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.

RESULTS

The 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) degrees, 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.

CONCLUSION

The 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

目的

探讨通过静电纺丝技术构建含Ⅱ型胶原蛋白和透明质酸（HA）的组织工程软骨复合物三维纳米支架的可能性。

方法

采用静电纺丝技术，将Ⅱ型胶原蛋白与HA（质量比8∶1）溶解于三氟乙醇和水（体积比1∶1）的混合溶剂中，制备三维多孔纳米支架。通过光学显微镜和扫描电子显微镜（SEM）观察其形态。检测其孔隙率、吸水率、接触角及降解率。从1周龄日本大白兔获取软骨细胞，用0.25%胰蛋白酶消化30分钟，再用1%胶原酶过夜消化。将第2代软骨细胞接种于纳米支架上。采用细胞计数试剂盒8（CCK-8）评估细胞黏附及增殖情况。将细胞-支架复合物体外培养2周，通过组织学分析观察其生物学形态及细胞外基质（ECM）分泌情况。

结果

纳米支架的最佳静电纺丝条件为：静电纺丝溶液浓度10%、接收距离10 cm、纺丝注射速度5 mL/h。通过光学显微镜和SEM观察，支架直径均匀，孔隙良好。直径为300～600 nm，孔隙率为89.5%±25.0%。接触角为（35.6±3.4）°，24小时吸水率为1 120%±34%，表明其具有良好的亲水性。48天时降解率为42.24%±1.51%。CCK-8结果显示，细胞与支架的黏附率在12小时时为169.14%±11.26%，7天时细胞存活率为126.03%±4.54%。组织学及免疫组化染色结果显示，软骨细胞可在支架上良好生长并分泌ECM。共培养2周时可发现类似软骨陷窝结构，提示形成了透明软骨。