Experimental Study On Repair Of Rat Skull Defect With 3D Printing Nano-hydroxyapatite/Methylacrylylated Silk Fibroin Scaffold

Background: Repair of bone defects remains a major challenge in clinical practice,and its treatment requires bone grafts or bone replacement materials.Existing biomaterials have many limitations and cannot meet various needs of clinical treatment.To treat bone defects,the development of three-dimensional porous scaffolds has received considerable attention in bone engineering.A variety of biomaterials and fabrication methods have emerged for filling complex bone defects,aiming to fabricate patient-specific bioactive scaffolds with controlled microarchitecture.In recent years,3D bioprinting,as an emerging field of regenerative medicine,has experienced rapid development,a large number of tissue engineering scaffolds for preclinical and clinical applications have been constructed using new materials and innovative techniques.Objective: To construct nano-hydroxyapatite（n HA）/methylacrylylated silk fibroin（Sil MA）composite scaffold by photocurable 3D printing,and analyze the morphology and structure of the scaffold,detect the biocompatibility of the constructed scaffold and the osteogenic effect of the scaffold in vitro and in vivo.Methods: In this study,scanning electron microscopy（SEM）was used to detect the changes of the morphological structure of the composite scaffold with different contents of nano-hydroxyapatite,and Fourier transform infrared spectroscopy（FTIR）was used to detect the functional groups and chemical bonds in the composite scaffold.In vitro experiments,bone marrow mesenchymal stem cells from SD rats were co-cultured with scaffold extract.the cytotoxicity,cell proliferation,bone alkaline phosphatase activity and alizarin red staining of scaffolds were detected to determine the biocompatibility of scaffolds and the effect of promoting proliferation and osteogenesis of bone marrow mesenchymal stem cells.In vivo experiment,skull defect model was constructed in adult SD rats.After 4 weeks and 8 weeks of feeding,the bone formation effect of the scaffold in the skull defect was detected by animal microscopic CT.Results: In this study,the morphology and structure of the scaffold were analyzed.We found that the scaffold preserved the sheet structure of silk fibroin well.With the mixing of n HA,the surface of scaffold becomes rougher,the contact area of cells increases,and the adhesion ability of cells is enhanced.Meanwhile,the lamellar microstructure provides a better spatial microenvironment for cell migration and proliferation.FTIR results showed that scaffold retained the β-folded structure of silk fibroin completely,and without obvious impurities.The results of Live/Dead cell staining showed that the constructed scaffolds had no obvious cytotoxicity.CCK-8assay also showed that the scaffolds constructed had good biocompatibility.The osteogenic induction results show that the scaffold has good osteogenic induction ability.The scaffold has better osteogenic ability when the ratio of Sil MA:n HA is 1:0.5.After the scaffold was implanted in vivo,the results of Micro-CT and bone histometry in rats were consistent with those in vitro,and the rats in the 1:0.5 group had more osteogenesis at the bone defect site.Conclusion: In this study,a composite osteogenic scaffold was successfully constructed by photocuring 3D printing.The constructed n HA/ Sil MA biocomposite has good biocompatibility and good osteogenic function.At the same time,it can also promote the repair of bone defects in the in vivo microenvironment,and can be used as a potential bone defect filling material for bone regeneration applications.