关键词: 去细胞, 骨膜, 胶原, 生物相容性, 皮下包埋, Masson染色, 扫描电子显微术, 兔

Abstract:

Objective To prepare rabbit periosteal decellularized bioscaffold andprovide a natural bioscaffold for the treatment for bone defect or bone nonunion in bone tissue engineering. Methods Bilateral medial proximal tibia periosteum of healthy New Zealand rabbits was obtained. To prepare the scaffold, we used physical freeze thawing (-80℃, 24h), eluted detergent (triton-X 100, SDS) and enzyme digestion (DNA enzymes, RNA enzymes ). After decellularization process, normal periosteum and decellularized periosteum were examined by HE staining， 4’,6-diamidino-phenylindole(DAPI) staining, agarose gel electrophoresis and quantitative analysis of genomic DNA (n=5) to evaluate the residual cell components. The retention of main components of the extracellular matrix (collagen) was examined by Masson staining and the hydroxyproline measurement (n=6); Scanning electron microscopy (SEM) was used to observe the microstructure of the scaffold; The toxicity of leaching liquor from scaffolds was tested by CCK8 assay; The immunological rejection of the scaffold was evaluated by subcutaneous implantation (n=4). Results HE and DAPI staining showed that no cells remained in the scaffold. After separation, the visible DNA bands were not found in the agarose electrophoresis gel for the decellularized periosteum. The DNA quantitative analysis showed that more than 95% of periosteal cells were removed; Masson staining and hydroxyproline measurement revealed that the collagen of the extracellular matrix was preserved; SEM showed the loose three-dimensional network of the extracellular matrix. The CCK8 assays demonstrated that there was no significant difference of periosteal cells proliferation among different volume fractions of leaching liquor from scaffolds and the control group (normal medium ) (P＞0.05). The subcutaneous implantation showed no obvious immune response to the decellularized periosteum. Conclusion The decellularized periosteum obtained by the use of physical freeze thawing, eluted detergent and enzyme digestion methods was found to remove periosteal cells completely, while the extracellular matrix structure and the main components were well-preserved and the biocompatibility was excellent.

Key words: Decellularization, Periosteum, Collagen, Biocompatiblilty, Subcutaneous implantation, Masson staining, Scanning electron microscopy, Rabbit