2022, Vol. 26 ›› Issue (21): 3338-3344

Effect of hydroxyapatite three-dimensional scaffolds with different apertures on the biological properties of MC3T3-E1 cells

Yang Jiaojiao1, Hu Ming1, Li Yan2, Xia Delin1   

Abstract: BACKGROUND: The aperture of bone tissue engineering scaffold is one of the important indexes to evaluate its biological performance, and seeking the best combination of aperture and mechanical properties has always been an urgent problem to be solved in bone tissue engineering.
OBJECTIVE: To prepare three-dimensional scaffolds with different aperture gradients and to study their effects on the biological behavior of MC3T3-E1 cells.
METHODS: The three-dimensional nano-hydroxyapatite scaffolds with uniform pore size were prepared by mixing wax spheres of specific diameter (300, 500, 800 μm) with hydroxyapatite as pore-forming agent using the modified particle leaching method, which were recorded as the D300, D500, and D800 scaffolds, respectively. The wax spheres of three diameters were piled up in order from small to large. The pore gradient nano-hydroxyapatite three-dimensional scaffold was prepared by the modified particle leaching method, which was recorded as the D-Gradient scaffold. The porosity of scaffolds of the four groups was measured. MC3T3-E1 cells were inoculated on scaffolds of the four groups. The cells cultured separately were used as the blank control group. Cell proliferation activity, alkaline phosphatase activity, cell adhesion, cytoskeleton rearrangement and the transcription level of osteogenic differentiation marker factors were detected.
RESULTS AND CONCLUSION: (1) The porosity of scaffolds of the four groups was 60%-80%. (2) After 24 hours of inoculation, the scaffolds in each group could promote the proliferation of MC3T3-E1 cells. The proliferation index was: D-Gradient group > D500 group > D800 group > D300 group > blank control group. The scaffolds in the D300 group, D500 group, and D-Gradient group promoted the secretion of alkaline phosphatase in MC3T3-E1 cells. The amount of alkaline phosphatase secretion was: D300 group > D500 group > D-Gradient group > D800 group ≈ blank control group. The scaffolds in the D300 group, D500 group, and D-Gradient group promoted the expression of osteopontin and osteocalcin mRNA in MC3T3-E1 cells. The expression levels of the two genes from high to low were: D300 group > D500 group > D-Gradient group > D800 group ≈ blank control group. (3) Immunofluorescence staining after 48 hours of inoculation showed that compared with the blank control group, MC3T3-E1 cells on the four scaffolds showed better adhesion and locomotor activity. The fluorescence intensity of focal adhesion actin from high to low was: D-Gradient group > D500 group > D800C group > D300 group > blank control group. (4) It is concluded that the aperture gradient scaffolds promote the growth, proliferation, adhesion, and osteogenic differentiation of MC3T3-E1 cells.
Key words: hydroxyapatite, gradient scaffold, aperture, porosity, osteogenesis, proliferation index, focal adhesion, osteopontin, osteocalcin