We monitored the responses of multipotent human marrow cells treated with dexamethasone and ethanol to evaluate the pathologic change of ON by monitoring gene expressions related to osteogenesis and adipogenesis and that of Wnt signaling-related genes. To confirm the posttranscriptional effects of ethanol on adipogenesis and the Wnt signaling pathway, we analyzed the protein expression of β-catenin and PPARγ by Western blot analysis. β-catenin, the pivotal protein in the Wnt signaling pathway, was also observed by confocal microscopy for intranuclear translocation.
We enrolled 13 patients with ethanol-induced ON of the femoral head and nine patients without ON. Patients with impaired renal or liver function, patients receiving hormone therapy, and those with malignancy or diabetes mellitus were excluded. Age, gender, and body mass index were similar between groups. The non-ON group included three patients who had THA for dysplastic arthritis of the hip and six patients who had internal fixation for a fresh fracture at the acetabulum, pelvis, or femoral shaft within 2 days after injury. Frequent alcohol consumption of more than 400 mL ethanol per week was reported for all patients with alcohol-induced ON.
We previously reported the detailed procedures of HMC culture [4
]. Cells from 22 patients were cultured and tested separately. The cells were separated by a Percoll™ gradient (Amersham Pharmacia, Piscataway, NJ); the nucleated stroma cells were then collected for primary cell culture [13
]. The third passage of culture was used for experiments. Donor cells (104
) were seeded on a plate and when they reached 80% confluence were treated with either 10 or 30 mmol/L ethanol (Sigma, St Louis, MO) or 100 nmol/L dexamethasone. For immunohistochemistry assay, the cells were pretreated with 25 mmol/L LiCl (Sigma) as provocative treatment and then further treated with and without ethanol. Messenger RNA expression of all target genes was evaluated by semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) and quantitative real-time PCR after treatment for 3 days. Total protein was isolated for Western blotting after treatment for 3 days. All independent experiments containing at least three tests were repeated at least twice (Table ).
All 22 cell lines were tested for the surface markers and the ability of osteogenic, adipogenic, and chondrogenic differentiation. The surface markers in all cell lines are compatible with those of mesenchymal stem cells. All cell lines showed good osteogenic, chondrogenic, and adipogenic differentiation after proper induction as previously reported [19
We examined the mRNA expression of the Wnt signaling ligand, Wnt 3a; the Wnt protein antagonist, SFRP2; membrane coreceptor, LRP5; and osteogenic-related genes, including BMP2, Runx2, and osteocalcin. The adipogenic-related genes, including PPARγ and adipsin, were examined using RT-PCR and quantitative real-time PCR. For each gene, the quantitative RT-PCR experiments were performed with at least three independent batches of cDNAs. Changes (x
-fold) in gene expression level were calculated by the 2-ΔΔct
]. Analysis of variance was performed using Excel software (Microsoft Corp, Cupertino, CA) as in previous studies [4
]. HMCs were isolated from 22 patients (13 ON and nine non-ON cases; non-ON cases included six trauma and three osteoarthritis cases). In total, we have RT-PCR data from seven patients, real-time PCR data from 22 patients, Western blot data from eight patients, and β-catenin intranuclear translocation data from six patients. All independent experiments containing at least three tests were repeated at least twice (Table ).
We performed Western blots on cell extracts in eight different cell lines, including four patients with ON and four patients without ON, separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and blotted onto Hybond-C membrane (Amersham Pharmacia). The membranes were blocked by 5% nonfat milk and probed with β-catenin, PPARγ, and β-actin. Blots were incubated with a horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized by the enhanced chemiluminescence system (Amersham Biosciences). The optical densities of the resolved bands were then semiquantified using Image-Pro Plus® analysis software (Media Cybernetics, Bethesda, MD). All independent experiments containing at least three tests were repeated at least twice (Table ).
To further understand whether ethanol suppresses the canonic Wnt signaling pathway, human marrow cells from six different cell lines, three from patients with ON and three from patients without ON, were treated with LiCl, an inhibitor of GSK-3β, to activate β-catenin. LiCl treatment was used as a provocative treatment in the intranuclear translocation of β-catenin by immunofluorescence analysis. Human marrow cells were cultured on glass coverslips. After drug treatments, the cells were fixed and permeated with 0.2% Triton X-100 (Sigma) and then blocked. Cells were incubated with anti-β catenin antibody for 1 hour and goat anti-mouse IgG coupled to FITC for 40 minutes. Meanwhile, the cells were counterstained with DAPI (Sigma) to highlight the nuclei. All images were observed with a Fluoview FV 500 (Olympus, Tokyo, Japan) confocal microscope and processed by a Fluoview FV 500 analysis system (Olympus). All independent experiments containing at least three tests were repeated at least twice (Table ).
In the first hypothesis, data from RT-PCR, real-time PCR, and Western blotting were presented as mean ± standard deviation and evaluated by one-way analysis of variance (ANOVA) and Post Hoc test by Scheffe’s method. The independent variables in the test of RT-PCR, real-time PCR, and Western blotting are dexamethasone and ethanol. A p value less than 0.05 was considered significant. In the second hypothesis, the intranuclear translocation of β-catenin was not evaluated statistically because of the difficulty in the quantitation of β-catenin on the image. The variables in the test of intranuclear translocation of β-catenin were LiCl and ethanol.