Alfacalcidol enhances collagen quality in ovariectomized rat bones

Abstract

The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D3 : ALF) on bone collagen employing an ovariectomized rat model. Thirty-five 16-week-old female Sprague-Dawley rats were divided into five groups: SHAM (sham-operated + vehicle), OVX (ovariectomy + vehicle), and three ALF-treated groups, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/kg/day ALF, and ovariectomy + 0.2 µg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized; however, with the ALF treatment, it was improved in a dose-dependent manner. Contents of collagen and pyridinoline cross-link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF-treated groups. The total aldehyde, that is, a sum of free and those involved cross-links, in the highest dose of ALF was significantly higher than the rest of the groups (p < 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF-treated groups compared with OVX (p < 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy-induced osteoporotic bones, which likely contributes to the improvement of bone quality.

Keywords: alfacalcidol; collagen; collagen cross-link; ovariectomized rats; vitamin D.

Figure 1

Representative PSR staining of proximal metaphysis of tibiae in SHAM (A), OVX (B), 0.022 µg ALF (C), 0.067 µg ALF (D), and 0.2 µg ALF (E). Bone collagen matrix stained yellow to red in SHAM (Fig. 1A) and green to yellow in OVX (Fig. 1B). In the ALF-treated groups (Fig. 1C–E), the color changed from green to yellow and red in a dose dependent-manner. The collagen matrices at the 0.2 µg ALF group (Fig. 1E) appeared to be well organized.

Figure 2

Immunohistochemical analysis for LOX. Representative distribution of LOX in the cortical bone 1.0–2.0 mm distal below the tibial growth plate and 200 µm thickness from bone marrow side of SHAM (A), OVX (B), 0.022 µg ALF (C), 0.067 µg ALF (D), and 0.2 µg ALF (E). Arrowheads indicate the areas of strong LOX expression in osteocyte cells. Magnification: 40×. The bar represents 200 µm. (F) Quantitative analysis of LOX expression (*p < 0.05). The levels of LOX expression in tibiae in the ALF-treated groups were increased when compared with those in OVX. The intensity of extracellular LOX staining also appeared to be significantly higher in the ALF-treated groups (Fig. 2C–E) when compared with that in OVX (Fig. 2B).