Zhang Peng, Wang Wenliang
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Dec;27(12):1517-22.
To prepare the silk fibroin (SF)-chitosan (CS) scaffolds by adjusting the mass ratio between CS and SF, and test and compare the properties of the scaffolds at different mass ratios.
According to the mass ratios of 6:4 (group A), 6:8 (group B), and 6:16 (group C) between SF and CS, CS-SF scaffolds were prepared by freeze-drying method, respectively. The material properties, porosity, the dissolubility in hot water, the modulus elasticity, and the water absorption expansion rate were measured; the aperture size and shape of scaffolds were observed by scanning electron microscope (SEM). Density gradient centrifugation method was used to isolate the bone marrow mesenchymal stell cells (BMSCs) of 4-week-old male Sprague Dawley rats. The BMSCs at passage 3 were seeded onto 3 scaffolds respectively, and then the proliferation of cells on the scaffolds was detected by MTS method.
The results of fourier transform infrared spectroscopy proved that with the increased content of CS, the absorption peak of random coil/alpha helix structure (1 654 cm(-1) and 1540 cm(-1)) constantly decreased, but the absorption peak of corresponding to beta-fold structure (1628 cm(-1) and 1516 cm(-1)) increased. The porosity was 87.36% +/- 2.15% in group A, 77.82% +/- 1.37% in group B, and 72.22% +/- 1.37% in group C; the porosity of group A was significantly higher than that of groups B and C (P < 0.05), and the porosity of group B was significantly higher than that of group C (P < 0.05). The dissolubility in hot water was 0 in groups A and B, and was 3.12% +/- 1.26% in group C. The scaffolds had good viscoelasticity in 3 groups; the modulus elasticity of 3 groups were consistent with the range of normal articular cartilage (4-15 kPa); no significant difference was found among 3 groups (F=5.523, P=0.054). The water absorption expansion rate was 1 528.52% +/- 194.63% in group A, 1 078.22% +/- 100.52% in group B, and 1320.05% +/- 179.97% in group C; the rate of group A was significantly higher than that of group B (P=0.05), but there was no significant difference between groups A and C and between groups B and C (P > 0.05). SEM results showed the aperture size of group A was between 50-250 microm, with good connectivity of pores; however, groups B and C had structure disturbance, with non-uniform aperture size and poor connectivity of pores. The growth curve results showed the number of living cells of group A was significantly higher than that of groups B and C at 1, 3, 5, and 7 days (P < 0.05); and there were significant differences between groups B and C at 3, 5, and 7 days (P < 0.05).
The CS-SF scaffold at a mass ratio of 6:4 is applicable for cartilage tissue engineering.
通过调整壳聚糖（CS）与丝素蛋白（SF）的质量比制备丝素蛋白-壳聚糖（SF-CS）支架，并对不同质量比支架的性能进行测试和比较。
按照SF与CS质量比6:4（A组）、6:8（B组）、6:16（C组），分别采用冻干法制备CS-SF支架。测定材料性能、孔隙率、在热水中的溶解性、弹性模量及吸水膨胀率；通过扫描电子显微镜（SEM）观察支架的孔径大小及形态。采用密度梯度离心法分离4周龄雄性Sprague Dawley大鼠的骨髓间充质干细胞（BMSCs），将第3代BMSCs分别接种于3种支架上，然后采用MTS法检测细胞在支架上的增殖情况。
傅里叶变换红外光谱结果显示，随着CS含量增加，无规卷曲/α螺旋结构（1654 cm⁻¹和1540 cm⁻¹）的吸收峰不断降低，而对应β折叠结构（1628 cm⁻¹和1516 cm⁻¹）的吸收峰升高。A组孔隙率为87.36%±2.15%，B组为77.82%±1.37%，C组为72.22%±1.37%；A组孔隙率显著高于B组和C组（P<0.05），B组孔隙率显著高于C组（P<0.05）。A组和B组在热水中的溶解性为0，C组为3.12%±1.26%。3组支架均具有良好的黏弹性；3组弹性模量均在正常关节软骨范围（4～15 kPa）内；3组间差异无统计学意义（F=5.523，P=0.054）。A组吸水膨胀率为1528.52%±194.63%，B组为1078.22%±100.52%，C组为1320.05%±179.97%；A组膨胀率显著高于B组（P=0.05），A组与C组、B组与C组间差异无统计学意义（P>0.05）。SEM结果显示，A组孔径在50～250μm之间，孔隙连通性良好；而B组和C组结构紊乱，孔径大小不均，孔隙连通性差。生长曲线结果显示，在第1、3、5和7天，A组活细胞数量显著高于B组和C组（P<0.05）；在第3、5和7天，B组和C组间差异有统计学意义（P<0.05）。
质量比为6:4的CS-SF支架适用于软骨组织工程。
