Fig 2: Expression of TLCD3B in callus tissue during fracture repair in C57BL/6 mice. Top row: AB-OG-HE trichrome staining of undecalcified methylmethacrylate-embedded callus tissues harvested at Days (D) 10, 12, 15, 18 and 21 postosteotomy. Squares represent areas for fluorescent imaging and 4× zoom in rows B–D. Scale bar = 1 mm. Representative images from three samples per group. AB-OG-HE = Alcian Blue-Orange G-Hematoxylin.

Fig 3: Quantification of TLCD3B-positive cells and mineralized versus unmineralized area in Days 10 and 12 callus tissue from inbred mice supplemented with 24,25(OH)2D3 or LacCer. Ratio of TLCD3B-positive cells per DAPI-stained in Day 10 (A) and Day 12 (B) postfracture callus tissue sections. Cells were counted with ImageJ within four regions of interest of 200 × 265 μm per image, for at least two images per sample, then averaged. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, one-way ANOVA followed by Dunnett's post-test. Ratio of mineralized versus unmineralized cartilage in Day 10 (C) and Day 12 (D) callus tissue sections. Mineralized versus unmineralized callus tissue areas were measured with ImageJ within two regions of interest per sample comprising each half the callus, then averaged and expressed as a ratio. Three (Day 10) to four (Day 12) samples per group. 24,25D3 = 24,25(OH)2D3; LacCer = lactosylceramide; Veh = vehicle.