Role of osteocytes in mediating bone mineralization during hyperhomocysteinemia

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   Figure 1

   Changes in femoral cortical bone geometry upon HHCY. (A) Gray scale segmentation of bone cylinder. Scale = 100 µm. (B and C) MicroCT50 data showing changes in relative bone volume (B) and cortical thickness (C) in cortical bones isolated at days 7, 15 and 30 after homocysteine induction (5 mg/100 g bwt i.p. for 30 days). (D) 3D rendering of proximal femoral cylinders by MicroCT50 (scale = 100 µm). (E) MicroCT50 data showing changes in closed pores of sizes (200–800 µm³) per unit volume of cortical bone. (F) Changes in total cortical porosity (%) by microCT analysis. (G) Changes in cortical tissue mineral density by MicroCT50. (H) Changes in mineral content after total ashing. (I) Changes in Ca and P levels in ashed bone. (J) Alizarin staining of cortical bone. Values are mean ± s.e.m.; n = 4. *Statistical significance with vehicle control; **statistical significance with HHCY day 7 bone. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-16-0562
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   Figure 2

   Changes in osteocyte population, osteocyte lacunar density and open pores in cortical bones during HHCY. (A) H&E-stained cortical bone sections showing changes in osteocyte numbers, scale = 200 µm. Av.Ot.N, average number of nucleated osteocytes; Av.Ot.Lac, average number of empty lacunae. Quantitative analysis of these parameters were done based on n = 100 unit measurements per sample. (B) MicroCT50 analysis showing changes in osteocyte lacunae pores (200–300 µm³). (C) Scanning electron microscopy microphotographs showing changes in vascular open pores, scale = 2 µm. (a) Changes in vascular pores at lower s.e.m. magnification. (b) Changes in vascular pores at higher s.e.m. magnification. (c) Changes in size of open blood vessels in cortical bone between vehicle and HHCY groups. Quantitative analysis of this parameter was done based on n = 20 unit measurements per sample. (d) Changes in the average number of open blood vessels in cortical bone between vehicle and HHCY groups. Quantitative analysis was done based on n = 100 unit measurements per sample. Values are mean ± s.e.m.; n = 4. *Statistical significance with vehicle control; **statistical significance with HHCY day 7 bone. A full colour version of this figure is available at http://dx.doi.org/10.1530/JOE-16-0562
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   Figure 3

   Changes in osteocyte markers during HHCY. (A) Changes in mRNA levels of genes by quantitative PCR. Data are shown as fold-changes with respect to vehicle control and mean of three independent PCR experiments done in triplicate normalized to expression of beta-actin. (B) Immunohistochemical staining of cortical bone sections. (a) Sclerostin, (b) Dmp1 and (c) cleaved caspase 3. Scale = 150 µm. (d) Histogram showing changes in average number of Sost⁺, Dmp1⁺ and cleaved caspase⁺ osteocyte lacunae population among groups. (e) Histogram showing mean intensity of Sost, Dmp1 and cleaved caspase 3 immunostaining per osteocyte. Quantitative analysis was done based on n = 50 unit measurements per sample. (C) Changes in carboxylated osteocalcin and sclerosin in serum by ELISA. Values are mean ± s.e.m.; n = 4. *Statistical significance with vehicle control; **statistical significance with HHCY day 7 bone.
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   Figure 4

   Changes in mechanical property of the bone after HHCY induction. (A) Ultimate load, (B) ultimate stress, (C) ultimate strain and (D) Youngs modulus. Values are mean ± s.e.m.; n = 4. *Statistical significance with vehicle control; **statistical significance with HHCY day 7 bone.

• © 2017 Society for Endocrinology