An experimental study in molecular biology for alcohol-induced osteonecrosis

Objective: Observe the effect of alcohol on the differentiation of marrow stromal cells(MSCs)into adipocytes to elucidate the pathogenesis of the alcohol-induced osteonecrosis of the femoral head.Methods: The primary MSCs from the femora of the mice were procured and cultured. The samples were isolated after adherent growth culture in vitro, and were treated with ethanol that acted as adipocyte differentiation inducer. The cells in culture were stained with Sudan Ⅲ.The number of adipocytes was counted under light microscope. The level of intracellular triglyceride,alkaline phosphatase(ALP)activity in the cells, and osteocalcin in culture media were determined. The expression level of 422 (aP2) and type-Ⅰ collagen mRNA's in the cells treated with 0.09 mol/L ethanol and without with ethanol was investigated by means of intact cell RNA dot blot hybridization. Results: The cells in culture were treated with increasing concentration, (0.03?0.09?0.15 mol/L), of ethanol for 21 days. The number of adipocytes increased with longer durations of exposure to ethanol and with higher concentrations of ethanol, which showed the time-and dose-dependent relation. There were lowest adipocytes in the control group. The level of triglyceride in the cells treated with 0.15mol/L of ethanol markedly increased, which was 3.9-fold higher than that in the control cells. ALP activity in the cells decreased with higher concentration of ethanol. The level of osteocalcin in culture media of the cells treated with 0.15mol/L of ethanol. The 422(aP2)mRNA contents in the experimental group, 7207.8±331.3, were markedly higher than that in the control group, 652.2±62.6. By contrast, the type-I collagen mRNA contents in the experimental group, 3567.3±300.9, were significantly lower than that in the control group, 7487.0±488.4. Conclusion: Alcohol could induce the differentiation of MSCs into adipocytes and inhibit osteogenic differentiation by regulating gene expression. This might lead to an increase in volume of fat marrow in the femoral head following long-term alcohol abuse, with a concomitant increase in intraosseous pressure, which could decrease in vascular perfusion, eventually resulting in ischemia in the marrow, and without sufficient repair of the necrotic bone. The final result is osteonecrosis of the femoral head. It may be a new pathogenesis of the alcohol-induced necrosis of the femoral head.