Introduction: Ethanol is one of risk factors associated with osteonecrosis, it has been demonstrated that ethanol induces adipogenesis, decreases osteogenesis in bone marrow stroma cells and produces intracellular lipid deposits, resulting in the death of osteocytes.

Materials and Methods: In this approach, we isolated human bone marrow stroma cells and triggered for different differentiations.

Results: These cells could be induced for osteogenesis, adipogenesis, and chondrogenesis. We also evaluated cell surface markers of isolated human bone marrow stromal cells that were found to express CD29, CD49d, CD62 CD90, CD105/SH2, SH3, CD133, and CD166, but not CD31, CD34, CD45, or CD56.

Discussion: We demonstrated that ethanol decreases the expression of osteogenic genes, but increases adipogenic genes expressions. Moreover, we found that ethanol decreases the beta-catenin-dependent canonical Wnt signaling pathway related gene expressions, including Wnt 3a and LRP5 genes. Interestingly, ethanol also diminishes the intra-nuclear translocation of β-catenin in human bone marrow stromal cells. Therefore, these results indicate that ethanol might decrease osteogenic gene expressions through Wnt signaling pathway.

Introduction: Studies have shown steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) suppress bone remodeling. Previous results have indicated that NSAIDs suppress proliferation and induce cell death in cultured osteoblasts and pluripotent stem cells (D1-cells), suggesting these effects might be one of the mechanisms contributing to their inhibitory effects on bone remodeling in vivo. On the other hand, our previous results indicated that dexamethasone treatment shifts the characteristics of osteogenesis into adipogenesis in D1-cells. However, the influences of NSAID on adipogenesis in pluripotent stem cells have rarely been investigated. In this study, we tested the adipogenesis of D1-cells upon long-term treatment of NSAIDs. NSAID influence on the osteocalcin expressions of D1-cells was also examined.

Materials and Methods: The effects of treatments with indomethacin, ketorolac, diclofenac and piroxicam (10⁻⁵ and 10⁻⁴ M) for 2, 4 6 or 8 days were evaluated. Lipid droplets in cultures were detected by oil red staining. Adipsin and osteocalcin mRNA expressions were examined by RT-PCR.

Results: In this study, 10⁻⁴M of NSAID treatment for 4–8 days induced adipogenesis in D1-cells, while shorter duration and lower concentration did not. Mild adipogenesis also occurred in cultures treated with 10⁻⁵M of indomethacin for 6 or 8 days, revealing the strongest effect among the 4 NSAIDs. Piroxicam revealed less effects on adipogenesis in D1-cells. However, despite 2-days of treatment with 10⁻⁵M indomethacin, NSAIDs did not affect the expression of osteocalcin either at 10⁻⁵–10⁻⁴M or during 2–8 days of treatments.

Conclusion: These results suggest that high dose and long term administration of NSAIDs may induce adipogenesis in pluripotent stem cells.

Introduction: Our previous study found that glucocorticoids shifted the properties of osteogenesis to adipogenesis in murine marrow stem cells. These effects may be one of the important mechanisms in the pathogenesis of osteonecrosis. Statins prevented these steroid effects. In this study, we investigated the effects of dexamethasone and lovastatin on the expressions of bone morphogenetic protein-2 (BMP2) in the bone marrow stroma cells cultured from osteonecrotic patients.

Materials and Methods: Bone marrow fluid aspiration from iliac crest was performed in osteonecrosis (ON) and non-ON patients after surgical treatment for their hip disorder. The mean age of the patients was 59 years in the ON group and 63 years in the non-ON group. Nucleated stroma cells were isolated from bone marrow fluid by percol separation. The third passage cultures were used for experiments. Drug treatments for cultures included dexamethasone (10⁻⁷M), lovastatin (10⁻⁶ M), and dexamethasone plus lovastatin for 4 days. BMP-2 mRNA expression was evaluated by RT-PCR. Different responses to drugs between the ON group and the non-ON group were compared.

Results: Bone marrow stroma cells of ON patients were found to be more susceptible to the suppressive effect of dexamethasone on BMP2 expression.

Discussion: Lovastatin stimulated the osteogenesis and reversed the steroid suppressive effect in bone marrow stroma cells in non-ON cases. However, this reverse effect was found to be mild in ON cases.

Introduction: Core binding factor 1 (Cbfa1) is one of the most important transcription factors that direct the osteogenesis of mesenchymal stem cells and osteoblastic functions. It is likely that the factors controlling Cbfa1 expression would trigger the early steps of osteoblast differentiation.

Materials and Methods: By using reporter gene assay for 4.5 kb Cbfa1 promoter, it was found that the first 320 bp of Cbfa1 promoter are active in D1 cells. Within this region, electromobility shift assays delineated a 6 bp of CACATG bound specifically by the proteins from D1 cell nuclear extract. Antibody super-shift and DNA-coupling magnetic bead pull-down assay indicated that the protein bound to this sequence is USF2. Site-specific mutagenesis revealed that this sequences contributed mainly to the activity of 320 bp Cbfa1 promoter.

Discussion: In conclusion, USF2 is the major regulator for the expression of Cbfa1 gene.