Figure 1.
HFD-fed mice are obese and glucose intolerant.
(A) Body weights of HFD-fed and control lean diet-fed mice at various time points after surgical fracture. Bars represent means ± SEM (n≥5). ***p<0.001 using a two-way, unpaired ANOVA with a Bonferroni post-test. (B) Glucose tolerance testing (GTT) was performed immediately prior to fracture on lean and HFD-fed mice. Plotted data points represent means ± SEM (n = 5). (C) Net area under the curve (AUC) was calculated for each mouse that underwent GTT (n = 5). *p<0.05 compared to respective lean diet controls using an unpaired, two-tailed Student's t test. Bars represent means ± SEM (n = 5).
Figure 2.
Callus neovascularization in lean- and HFD-fed mice is similar.
μCT scans of fractured tibiae that were perfused with lead chromate paint after decalcification were performed in lean and HFD-fed mice at 10 and 14 days post-fracture. Representative three-dimensional reconstructions are presented in panel (A). Vascular volume (B) and vascular volume normalized to callus bone volume (C) was quantified from μCT data. Bars represent means ± SEM (n≥5). Scale bar (white line) = 1 mm.
Figure 3.
μCT imaging reveals a trend toward decreased bone volume in HFD-fed mice.
μCT scans were performed on tibiae at 7, 10, 14, 21, 28, and 35 days post-fracture. Representative three-dimensional reconstructions, from groups of 5–7 tibiae, show bone volume within the external fracture callus. Scale bar (white line) = 1 mm. Fracture sites are denoted with yellow arrows.
Figure 4.
Quantitative analysis confirms decreased and delayed accrual of peak bone volume in fracture callus of HFD-fed mice.
Using μCT scans that were used create the reconstructions shown Fig. 3, bone volume was quantified from lean- and HFD-fed mice at 7, 10, 14, 21, 28, and 35 days post-fracture. Bars represent means ± SEM (n≥5). *p<0.05 compared to time-matched lean diet controls using two-way, unpaired ANOVA with Bonferroni post-tests.
Figure 5.
Woven bone content is decreased at later stages of fracture healing in HFD-fed mice.
(A) Representative Alcian Blue Hematoxylin/Orange G stained histologic sections of fracture callus in lean- and HFD-fed mice at 7, 10, 14, 21, 28, and 35 days post-fracture. Quantification of cartilage (B), woven bone (C), and stromal cell areas (D), expressed as percentages of total callus area, were determined using a point-counting histomorphometric method. Bars represent means ± SEM (n≥5). ***p<0.001 compared to time-matched controls using two-way, unpaired ANOVA with Bonferroni post-tests. The black size marker in the lower right panel = 1 mm.
Figure 6.
Adiposity is increased and osteoblast-occupied bone surface is decreased within the external fracture callus during late stage healing in HFD-fed mice.
Immunohistochemical staining for perilipin was performed to confirm the presence of adipocytes within the fracture callus. Representative sections from day 21 post-fracture demonstrate an increased number of adipocytes within the fracture callus of the HFD-fed mouse (B) compared to the lean diet control (A). Higher magnifications of selected regions from lean and HFD-fed calluses illustrate specificity of perilipin staining, with several typical perilipin-positive adipocytes denoted with red arrows. (C) Adipocyte area as a percentage of total callus area and percent of osteoblast occupied bone surface were determined using histomorphometric methods in lean- and HFD-fed mice at the indicated time points. Bars represent the means ± SEM (n≥5). **p<0.01 and ***p<0.001 compared to time-matched controls using an unpaired, two-way ANOVA with Bonferroni post-test. The black size marker in the low magnification images (left panels of A and B) = 1 mm. The black size marker for the high magnification images (right panels of A and B) = 100 µm.
Figure 7.
PPARγ expression is increased in fracture callus of HFD-fed mice.
Sections were subjected to immunohistochemical staining for PPARγ and representative fracture calluses at day 14 show increased PPARγ expression in the fracture callus of HFD-fed mice (B) compared to lean controls (A), particularly in cells adjacent to trabecular bone elements (marked by blue arrows in callus from lean-fed mice and red arrows in callus from HFD-fed mice). The black size marker (panel B) = 20 µm.
Figure 8.
Healed tibiae from HFD-fed mice are biomechanically weaker.
(A) Surgically fractured right tibiae from lean- and HFD-fed mice (n≥8) were subjected to biomechanical torsional testing at days 14 and 35 post-fracture, with a significant decrease in strength observed in the HFD-fed group at day 35. Bars represent the means ± SEM (n≥8). **p<0.01 compared to time-matched controls using an unpaired, two-way ANOVA with Bonferroni post-tests.