Basic Research On Preparation Of Biomimetic Biphasic Porous Scaffolds Based On Silk Fibroin,Gelatin,Chitosan And Hydroxyapatite For Repair Of Full-thickness Cartilage Defects

Objective: Cartilage tissue engineering(CTE)provides new ideas and approaches for the repair of full-thickness cartilage defects.The main purpose of this research is to develop a biomimetic biphasic porous scaffold that can meet the requirements of osteochondral special structural units and osteochondrogenic bidirectional differentiation,and to evaluate its feasibility for repairing full-thickness cartilage defects.Methods: Four groups of cartilage scaffolds and five groups of subchondral bone scaffolds were prepared based on gelatin(Gel),chitosan(CS),silk fibroin(SF)and hydroxyapatite(Hap),respectively.Suitable cartilage phase and subchondral bone phase scaffolds were screened by scanning electron microscopy,X-Ray Diffractometer(XRD),porosity,water absorption swelling and biodegradation rate studies and mechanical property studies,and then a bionic biphasic scaffold was developed by a combination of freeze-drying and freeze-thawing to meet the special structure of osteochondral units and the bi-directional osteogenic-chondrogenic differentiation The biocompatibility,cytotoxicity and ability to provide a suitable microenvironment for cartilage tissue regeneration were evaluated by adhesion rate,cell death staining,ghost pen cyclic peptide staining and CCK8 cell activity proliferation assay.Results: Among the cartilage scaffolds,the SF-CS-Gel scaffold had a suitable pore size of 176±53.68 μm and porosity of 80.15±2.57%,good water absorption and swelling rate of 1745 ± 19%,suitable biodegradation rate of 46.87 ± 3.25% and mechanical properties;among the subchondral bone scaffolds,the SF-CS-Gel-2% Hap scaffold had a pore size of212.89 ± 62.22 μ m size pore size and suitable porosity of 75.19±3.47%,good water absorption and swelling rate of 1019±31% and biodegradation rate of 37.09±1.41% and mechanical strength,Hap was deposited as crystalline clusters adhering to the pore wall of the composite scaffold,elemental analysis detected Ca/P ratio within the crystalline clusters of about 1.7,XRD patterns showed that at 25.7°,32.2°,33.0°,34.3°,40.3°,46.6° and49.6 °.The mimetic biphasic scaffold constructed on this basis had a mean pore size of156.06 ± 42.36 μm for the cartilage phase and 214.38 ± 65.82 μm for the subchondral bone phase,and showed good cell adhesion and proliferation during co-culture with chondrocytes,and most of the cells showed green fluorescence and rare dead cells after using live-dead cell staining,and ghost pen loop peptide staining showed that the skeletal proteins The arrangement was regular,with a tendency to aggregate,and the cell morphology in the3 D scaffold maintained the original morphological growth as in vivo.Conclusion: The new bionic biphasic porous scaffold can not only mimic the structural characteristics of articular cartilage,calcified cartilage and subchondral bone,but also has good biocompatibility,thus providing the best reticular skeleton and ECM for nutrient transport and cell attachment,proliferation and three-dimensional growth,offering a new option for the treatment of total cartilage defects.