关键词: 壳聚糖, 矿化胶原, 小鼠成骨前体细胞, 多孔支架, 成骨分化, 生物相容性

Abstract: BACKGROUND: Chitosan material has good biocompatibility, biodegradability and low immunogenicity, but its activity is poor; mineralized collagen material has good biocompatibility, but its mechanical properties are insufficient, so their combination may show better mechanical properties and osteogenic activity.
OBJECTIVE: To construct a chitosan/mineralized collagen porous scaffold with different compositions and to show surface characteristics and biocompatibility.
METHODS: The porous scaffolds with different mass ratios (2:1, 1:1) of chitosan and mineralized collagen were prepared by freeze-drying. Scanning electron microscope, energy dispersive spectrometer, X-ray diffraction, and Fourier infrared Spectrum were used to show the surface morphology, element composition, and structure of porous scaffolds in each group. The mouse osteoblast precursor cells were directly inoculated on the surface of the scaffolds of three groups, and the cell adhesion was observed by scanning electron microscope. The blank control and three groups of material extracts were co-cultured with mouse osteogenic precursor cells. CCK-8 assay, phalloidin staining, and alkaline phosphatase analysis were applied to analyze the effect of materials on proliferation and differentiation of mouse osteogenic precursor cells.  
RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that there were uniform pores on the surface of chitosan composite materials. The interface of chitosan/mineralized collagen porous scaffold was tightly bonded. Simultaneously, as the content of chitosan in the scaffold decreased, the agglomeration of mineralized collagen particles in the scaffold became more obvious. Energy dispersive spectrometer displayed that elements on the surface of the simple chitosan scaffold were mainly carbon, nitrogen and oxygen. The calcium and phosphorous elements on the surface of the scaffold material gradually increased after mineralized collagen was added. X-ray diffraction and Fourier infrared Spectrum exhibited that chitosan and mineralized collagen were physically combined. (2) Scanning electron microscopy showed that the cells adhered and spread normally on the surface of the three groups of scaffold materials. Among them, the number of cells attached to the surface of the simple chitosan scaffold was less. (3) CCK-8 assay displayed that compared with the simple chitosan porous scaffold extract, two ratios of chitosan/mineralized collagen porous scaffold extract could promote the proliferation of mouse osteogenic precursor cells. Cytoskeleton staining showed that two ratios of chitosan/mineralized collagen porous scaffold had high cell density of the extract liquid group; the cell filament feet were connected to each other; and the expression of microfilament actin was clear. (4) The alkaline phosphatase activity test demonstrated that compared with the simple chitosan porous scaffold extract, the two ratios of chitosan/mineralized collagen porous scaffold extract can promote osteogenic differentiation of mouse osteogenic precursor cells. (5) It is concluded that chitosan/mineralized collagen porous scaffold can effectively promote proliferation and differentiation of mouse osteoblast precursor cells. 

Key words: chitosan, mineralized collagen, mouse osteogenic precursor cells, porous scaffolds, osteogenic differentiation, biocompatibility