PMC

Tungsten inhibits osteogenesis of MSCs in vitro. MSC cultures were transitioned to OM medium and were either left untreated (OM) or treated with 15 μg/ml tungsten (OM +W) throughout osteoblast differentiation. A, RNA expression analysis of osteoblast gene markers (Runx2, Sp7, and Bglap) were analyzed by qRT-PCR at day 10 of differentiation. Graph shows the mean fold change in gene expression ± SEM, normalized to naïve gene expression for each gene. Data normalized to housekeeping gene Rn18s. ***P ≤ .001 1-way ANOVA. Graph is a representative experiment of 3 biological replicates each performed in four technical replicates. B, ALP activity was measured in MSC cultures on day 12 of differentiation. Graph shows the mean level of ALP activity (absorbance) ± SEM for naïve, OM, and OM +W samples. ***P ≤ .001 1-way ANOVA. Graph is a representative experiment of 3 biological replicates each performed in triplicate. C, Calcium mineralization was measured by Alizarin Red staining in MSC cultures at day 12 of differentiation. Graph shows the mean level of Alizarin Red dye (absorbance) ± SEM for naïve, OM, and OM +W samples. Graph is a representative experiment of 3 biological replicates each performed in triplicate. ***P ≤ .001 1-way ANOVA.

Tungsten-enhanced adipogenesis is influenced by sex and age of mice. A, Tungsten concentration in humeri bones measured by ICP-MS. Graph shows the mean ± SEM tungsten concentration in humeri bones in young (5 weeks) male and female, and adult (9 months) male and female mice, given tap water or 15 ppm in drinking water for 4 weeks (n ≥ 4). ***P ≤.001 1-way ANOVA. B, Analysis of perilipin staining by IHC in sections of femur bone tissue from control and tungsten-exposed animals (young male and female; middle-aged male and female mice). Graphs show the mean number of perilipin⁺ adipocytes/animal, average size of each adipocyte/animal, and the total perilipin⁺ adipocyte area/animal (n ≥ 7). Representative images of perilipin⁺ adipocytes in control and tungsten-exposed femur sections, 20×. *P ≤ .05 unpaired student t test, 1-tailed comparisons between control and tungsten-exposed animals per group.

Tungsten enhances rosiglitazone-induced adipogenesis of MSCs in vitro. MSC cultures were transitioned to OM medium and were either left untreated (OM) or treated with 15 μg/ml tungsten (OM +W), 1 nM rosiglitazone (OM +Rosi), or the combination of tungsten and rosiglitazone (OM +Rosi +W) throughout adipocyte differentiation. A, RNA expression analysis of adipocyte gene markers (Pparg, Fabp4, Plin1, and Adipoq) were analyzed by qRT-PCR at day 7 of differentiation. Graph shows the mean fold change in gene expression ± SEM, normalized to naïve gene expression for each gene. Data normalized to housekeeping gene Rn18s. ***P ≤ .001 1-way ANOVA. Graph is a representative experiment of 3 biological replicates each performed in four technical replicates. B, Adipocyte quantification in MSC cultures was analyzed by counting the number of adipocytes in 3 representative images (10×) of Oil Red O stained cells per sample at day 9 of differentiation. Graph shows the number of adipocytes ± SEM. Graph is a representative experiment of 3 biological replicates each performed in triplicate. ***P ≤ .001 1-way ANOVA. C, Representative images (10×) of Oil Red O stained MSC cultures at day 9 of differentiation.

Tungsten does not inhibit osteogenesis in vivo A, RNA expression analysis of osteogenic gene markers Runx2, Sp7, and Bglap in total bone marrow isolated from control and tungsten-exposed animals, analyzed by qRT-PCR. Graph shows mean fold change in gene expression normalized to one control sample (n = 4). Data normalized to housekeeping genes Gapdh and m36B4. B, Analysis of ALP activity in sections of tibia bone tissue from control and tungsten-exposed animals. Graphs show the average ALP staining thickness (μm)/animal and the number of positive pixels/analyzed region area (μm²)/animal (n ≥ 7). Representative images of ALP staining in cortical (left) and trabecular (right) bone regions from control and tungsten-exposed tibia sections, 20×. C, Ex vivo cultures were isolated from control and tungsten-exposed mice and differentiated in the absence of tungsten in vitro. MSC cultures were transitioned to OM medium throughout differentiation (Control OM or Tungsten OM). RNA expression analysis of osteogenic gene markers (Runx2, Sp7, and Bglap) in ex vivo MSC cultures analyzed by qRT-PCR at day 10 of differentiation. Graph shows the mean fold change in gene expression ± SEM (n = 3), normalized to naïve gene expression for each gene. D, ALP activity was measured in ex vivo MSC cultures on day 12 of differentiation. Graph shows the mean level of ALP activity (absorbance) ± SEM for naïve, Control OM, and Tungsten OM samples (n = 6). E, Calcium mineralization was measured by Alizarin Red staining in ex vivo MSC cultures at day 12 of differentiation. Graph shows the mean level of Alizarin Red dye (absorbance) ± SEM for naïve, Control OM, and Tungsten OM samples (n = 6).

Tungsten promotes adipogenesis in the bones of mice. Male C57BL/6 mice (5 weeks) were given tap water (control) or 15 ppm tungsten in drinking water for 4 weeks. A, RNA expression analysis of adipogenic gene markers Pparg, Fabp4, Adipoq in total bone marrow isolated from control and tungsten exposed animals, analyzed by qRT-PCR. Graph shows mean fold change in gene expression normalized to one control sample (n = 4). Data normalized to housekeeping genes Gapdh and m36B4. *P ≤ .5 unpaired student t test, 2-tailed. B, Analysis of perilipin staining by IHC in sections of femur bone tissue from control and tungsten exposed animals. Graphs show the mean number of perilipin⁺ adipocytes/animal, average size of each adipocyte/animal, and the total perilipin⁺ adipocyte area/animal (n ≥ 7). Representative images of perilipin⁺ adipocytes in control and tungsten-exposed femur sections, 20×. *P ≤ .05, unpaired student t-test, one-tailed. C, Ex vivo MSC cultures were derived from control and tungsten-exposed mice and differentiated in the absence of tungsten in vitro. Cultures were transitioned to osteoinductive medium and were either left untreated (Control OM or Tungsten OM) or treated with 1 nM rosiglitazone (Control OM + Rosi or Tungsten OM +Rosi) throughout adipocyte differentiation. RNA expression analysis of adipogcyte gene markers (Pparg, Fabp4, Plin1, and Adipoq) in ex vivo MSC cultures analyzed by qRT-PCR at day 7 of differentiation. Graph shows the mean fold change in gene expression ± SEM (n = 3), normalized to naïve gene expression for each gene. Table shows the mean fold change in gene expression ± SEM (n = 3) for Control and Tungsten samples cultured in OM media alone, normalized to naïve gene expression for each gene. Graphs **P ≤ .01 1-way ANOVA. Table *P ≤ .05, **P ≤ .01 unpaired student t test, 1-tailed. D, Adipocyte quantification in ex vivo MSC cultures was analyzed by counting the number of adipocytes in 3 representative images (10×) of Oil Red O stained cells per sample at day 9 of differentiation. Graph shows the number of adipocytes ± SEM (n = 6). *P ≤ .05 1-way ANOVA.