Abstract:

BACKGROUND: Preliminary study has shown that the composite materials composed of magnesium-based materials and mineralized collagen have a good supporting effect on repairing the critical defects, which can improve the mechanical strength of mineralized collagen and premature collapse during bone healing to some extent. However, magnesium-based metals degrade fast in chloride-containing solutions (including human body fluids or plasma), and the effects of releasing magnesium ions on the proliferation and differentiation of osteoblasts are unknown.

OBJECTIVVE: To investigate the effects of magnesium ion combined with mineralized collagen on osteogenic differentiation of mouse preosteoblasts in vitro.

METHODS: Mineralized collagen extracts were prepared from complete medium with magnesium ion concentration of 0, 5, 10, and 20 mmol/L. Mouse preosteoblasts were cultured with four mineralized collagen extracts, respectively, which were divided into mineralized collagen group, and 5, 10 and 20 mmol/L Mg²⁺+mineralized collagen groups. The mouse preosteoblasts cultured in complete medium were used as control group. The cell morphology, proliferation, apoptosis, intracellular microfilament actin, and the activity of alkaline phosphatase and expression level of the osteogenic gene Runx2 after osteogenic differentiation were detected.

RESULTS AND CONCLUSION: (1) After 24 hours of culture, the cells in the mineralized collagen group, and 5 and 10 mmol/L Mg²⁺+ mineralized collagen groups adhered well, which showed no significant difference from the blank control group, and the elongated spindle cells with many synapses linked to the adjacent cells were observed. The cells in the 20 mmol/L Mg²⁺+mineralized collagen group showed obvious pyknosis. (2) After 1, 3 and 5 days of culture, the cell viability in the 10 mmol/L Mg²⁺+mineralized collagen group was significantly higher than that in the other four groups (P < 0.05). There was no significant difference among mineralized collagen, 5 mmol/L Mg²⁺+ mineralized collagen and blank control groups (P > 0.05). The cell viability in the 20 mmol/L Mg²⁺+mineralized collagen group was significantly lower than that in the mineralized collagen group (P < 0.05). (3) After 3 days of culture, DAPI staining showed that 20 mmol/L Mg²⁺+mineralized collagen group had obvious nuclear disintegration, the other four groups had no obvious nuclear disintegration. (4) After 24 hours of culture, phalloidin staining showed that except the blank control and 20 mmol/L Mg²⁺+mineralized collagen groups, the other three groups showed completely extended cell structure, and clear actin microfilaments, especially the 10 mmol/L Mg²⁺+mineralized collagen group. (5) After 7 days of osteogenic differentiation, except for 20 mmol/L Mg²⁺+mineralized collagen group, the activity of alkaline phosphatase and the expression level of Runx2 gene in the other three groups were significantly higher than those in the blank control group (P < 0.05), and those in the 10 mmol/L Mg²⁺+mineralized collagen group was significantly higher than those in the 5 mmol/L Mg²⁺+mineralized collagen and mineralized collagen groups (P < 0.05). (6) These results suggest that the combination of magnesium ion with mineralized collagen should be applied with appropriate concentration range of magnesium ion (≤ 10 mmol/L).

Key words: mineralized collagen artificial bone, nano-hydroxyapatite/collagen, mouse preosteoblasts, magnesium ions, mineralized collagen, viability of osteoblasts, bone repair materials, osteogenic differentiation, biocompatibility