Objective To explore the feasibility and methods of preparing silk fibroin(SF)/ chitosan(CS)ano hydroxyapatite(nHA)composite osteochondral scaffolds with a gradient pore size structure.Methods We prepared an SF solution,a CS solution and an nHA suspension,all with a 2％ concentration,and mixed them with equal proportions.The mixture was used to prepare SF/CSHA composite osteochondral scaffolds with a pore size gradient through a centrifugal freeze drying and chemical cross linking method.The porosity,hot water loss rate,water swelling rate and mechanical properties of the scaffold were measured,and the dissolution curve and stress strain curve were drawn.The internal structure and morphology of the scaffold were observed by scanning electron microscopy(SEM)and the sizes of pores in the scaffold were measured.Results The porosity of the scaffold was(91.30± 3.35)％;The five week hot water loss rate was(16.57± 3.18)％;And the water swelling rate was (3218.53 ± 84.37)％.Mechanical test results demonstrated a good compression performance of the scaffold.SEM showed that the internal pores of the scaffolds were honeycomb structured with communicating passages;The density of pores gradually increased with decreasing pore sizes from the top to the bottom(pore sizes at four different levels:(141.11± 11.85)μm,(119.94± 9.05)μm,(93.10 ± 14.98) μm,and (79.95 ± 8.65)μm,respectively,F =22.973,P =0.000).Scaffold cytotoxicity test results indicated no significant difference between A values of the extract group and of the negative control group at any time point(t24 h =0.520,P =0.610;t48 h =0.665,P =0.515;t72 h =0.439,P =0.666),and all RGR values were greater than 100％.Conclusions SF/CSHA composite osteochondral scaffolds with a gradient pore size structure can be prepared with a centrifugal-freeze drying and chemical cross-linking method.Scaffolds prepared this way have a three-dimensional structure,a gradient pore size structure,high porosity,strong water absorption,suitable degradation rates and good compressive resistance.Besides,the good cell compatibility and low cytotoxicity of the scaffolds satisfy the requirements for osteochondral tissue engineering materials.%目的 探索丝素蛋白(SF)、壳聚糖(CS)和纳米羟基磷灰石(nHA)制备骨、软骨梯度孔径支架方法及可行性. 方法 制备浓度为2％的SF和CS溶液及nHA悬液,三者等比例混合,采用离心-冷冻干燥法及化学交联法制备SF/CSHA骨、软骨梯度孔径支架,检测支架孔隙率、热水溶失率、吸水膨胀率及力学性能,绘制溶失曲线及应力应变曲线,采用扫描电镜(SEM)观察支架内部结构及形态并测量支架孔径大小. 结果 SF/CSHA骨、软骨梯度孔径支架孔隙率为(91.30±3.35)％,5周后热水溶失率为(16.57±3.18)％,吸水膨胀率为(3218.53±84.37)％,力学检测结果显示支架抗压性能良好.SEM显示支架内部孔隙呈蜂窝状、相互交通,孔隙分布从上到下逐渐密集,孔径大小从上至下逐渐减小,分别为(141.11±11.85)μm、(119.94±9.05)μm、(93.10±14.98)μm、(79.95±8.65)μm,各层互相比较差异有统计学意义(F=22.973,P=0.000).支架细胞毒性检测结果显示各个时间段浸提液组A值与阴性对照组相比差异均无统计学意义(t24h=0.520,P=0.610;t48h =0.665,P=0.515;t72h=0.439,P=0.666),细胞相对增殖率(RGR)值均大于100％. 结论 利用离心冷冻干燥法及化学交联法可初步制备SF/CSHA骨、软骨梯度孔径支架.支架呈三维立体结构、渐进的梯度式孔径、高孔隙率、极强的吸水性、适宜的降解速率及良好的抗压性能,支架无明显细胞毒性,细胞相容性良好,基本符合骨组织工程材料的要求.
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