Rosiglitazone induces adipogenesis of both marrow and periosteum derived mesenchymal stem cells during endochondral fracture healing

Abstract

Background: Type 2 diabetes mellitus (T2DM) afflicts about six percent of the global population, and these patients suffer from a two-fold increased fracture risk. Thiazolidinediones (TZDs), including rosiglitazone, are commonly used medications in T2DM because they have a low incidence of monotherapy failure. It is known that rosiglitazone is associated with secondary osteoporosis, further increasing the fracture risk in an already susceptible population. However, it is not yet understood how rosiglitazone impacts endochondral bone healing after fracture. The aim of this study is to elucidate how rosiglitazone treatment impacts endochondral fracture healing, and how rosiglitazone influences the differentiation of skeletal stem and progenitor cells from the bone marrow and the periosteum.

Methods: An in-vivo mouse femur fracture model was employed to evaluate differences in fracture healing between mice treated with and without rosiglitazone chow. Fracture healing was assessed with histology and micro computed tomography (μCT). In-vitro assays utilized isolated mouse bone marrow stromal cells and periosteal cells to investigate how rosiglitazone impacts the osteogenic capability and adipogenicity of these cells.

Results: The in-vivo mouse femur fracture model showed that fracture callus in mice treated with rosiglitazone had significantly more adipose content than those of control mice that did not receive rosiglitazone. In addition, μCT analysis showed that rosiglitazone treated mice had significantly greater bone volume, but overall greater porosity when compared to control mice. In-vitro experimentation showed significantly less osteogenesis and more adipogenesis in bone marrow derived progenitor cells that were cultured in osteogenic media. In addition, rosiglitazone treatment alone caused significant increases in adipogenesis in both bone marrow and periosteum derived cells.

Conclusion: Rosiglitazone impairs endochondral fracture healing in mice by increasing adipogenesis and decreasing osteogenesis of both bone marrow and periosteum derived skeletal progenitor cells.

Figure 1:

Weights (grams) of mice provided with a standard diet or standard diet supplemented with rosiglitazone (N=12 in each group). Recording of weights began on the day the mice were provided their respective diets. *, p<0.05; **, p<0.01.

Figure 2:

Stitched images demonstrating pentachrome staining of histological slices sampled from the fracture callus present POD28 after a femoral defect in a mouse provided with either a standard diet or rosiglitazone supplemented diet. Scale = 500μm.

Figure 3:

Fracture callus μCT analyses in mice provided with a standard diet (black) or rosiglitazone supplemented diet (grey). *, p<0.05; **, p<0.01; ***, P<0.001. BV, bone volume; TV, trabeculae volume; TB.Th, trabeculae thickness; Tb.N, trabeculae number; Tb.S, trabeculae spacing.

Figure 4A and 4B:

Quantification via absorbance of alizarin red staining (4A) and Oil-Red-O staining (4B) of mouse BMSCs cultured in osteogenic differentiation media with or without rosiglitazone. *, p<0.05; **, p<0.01. GM, growth media; OM, osteogenic media; Rosi, rosiglitazone.

Figure 5:

Quantitative PCR of mouse BMSCs cultured in OM with or without rosiglitazone. Example experiments completed in technical triplicates are shown.; ***, P<0.001. GM, growth media; OM, osteogenic media; Rosi, rosiglitazone.

Figure 6A and 6B:

Quantification (6A) and imaging (6B) of Oil-Red-O staining of mouse BMSCs cultured with or without rosiglitazone. *, p<0.05. GM, growth media; Rosi, rosiglitazone.

Figure 7:

Quantitative PCR of mouse BMSCs cultured with or without rosiglitazone. Example experiments completed in technical triplicates are shown. **, p<0.01. GM, growth media; Rosi, rosiglitazone.

Figure 8A and 8B:

Quantification (8A) and imaging (8B) of Oil-Red-O staining of mouse periosteal cells cultured with or without rosiglitazone. ***, p<0.001. GM, growth media; Rosi, rosiglitazone.

Figure 9:

Quantitative PCR of mouse periosteal cells cultured with or without rosiglitazone. Example experiments completed in technical triplicates are shown. *, p<0.05; ***, p<0.001.