RESUMEN
Objective To explore the feasibility and methods of preparing silk fibroin(SF)/ chitosan(CS)/nano 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/CS/nHA 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/CS/nHA 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.