3D-printed nanohydroxyapatite/methylacrylylated silk fibroin scaffold for repairing rat skull defects

Abstract

The repair of bone defects remains a major challenge in the clinic, and treatment requires bone grafts or bone replacement materials. Existing biomaterials have many limitations and cannot meet the various needs of clinical applications. To treat bone defects, we constructed a nanohydroxyapatite (nHA)/methylacrylylated silk fibroin (MASF) composite biological scaffold using photocurable 3D printing technology. In this study, scanning electron microscopy (SEM) was used to detect the changes in the morphological structure of the composite scaffold with different contents of nanohydroxyapatite, and FTIR was used to detect the functional groups and chemical bonds in the composite scaffold to determine the specific components of the scaffold. In in vitro experiments, bone marrow mesenchymal stem cells from SD rats were cocultured with scaffolds soaking solution, and the cytotoxicity, cell proliferation, Western blot analysis, Quantitative real-time PCR analysis, 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 vitro. In the in vivo experiment, the skull defect was constructed by adult SD rats, and the scaffold was implanted into the skull defect site. After 4 weeks and 8 weeks of culture, the specific osteogenic effect of the scaffold in the skull defect site was detected by animal micro-CT, hematoxylin and eosin (HE) staining and Masson's staining. Through the analysis of the morphological structure of the scaffold, we found that the frame supported good retention of the lamellar structure of silk fibroin, when mixed with nHA, the surface of the stent was rougher, the cell contact area increased, and cell adhesion and lamellar microstructure for cell migration and proliferation of the microenvironment provided a better space. FTIR results showed that the scaffold completely retained the β -folded structure of silk fibroin, and the scaffold composite was present without obvious impurities. The staining results of live/dead cells showed that the constructed scaffolds had no significant cytotoxicity, and thw CCK-8 assay also showed that the constructed scaffolds had good biocompatibility. The results of osteogenic induction showed that the scaffold had good osteogenic induction ability. Moreover, the results also showed that the scaffold with a MASF: nHA ratio of 1: 0.5 (SFH) showed better osteogenic ability. The micro-CT and bone histometric results were consistent with the in vitro results after stent implantation, and there was more bone formation at the bone defect site in the SFH group.This research used photocurable 3D printing technology to successfully build an osteogenesis bracket. The results show that the constructed nHA/MASF biological composite material, has good biocompatibility and good osteogenesis function. At the same time, in the microenvironment, the material can also promote bone defect repair and can potentially be used as a bone defect filling material for bone regeneration applications.