Morphological assessment of bone mineralization in tibial metaphyses of ascorbic acid-deficient ODS rats

Abstract

Osteogenic disorder shionogi (ODS) rats carry a hereditary defect in ascorbic acid synthesis, mimicking human scurvy when fed with an ascorbic acid-deficient (aa-def) diet. As aa-def ODS rats were shown to feature disordered bone formation, we have examined the bone mineralization in this rat model. A fibrous tissue layer surrounding the trabeculae of tibial metaphyses was found in aa-def ODS rats, and this layer showed intense alkaline phosphatase activity and proliferating cell nuclear antigen-immunopositivity. Many osteoblasts detached from the bone surfaces and were characterized by round-shaped rough endoplasmic reticulum (rER), suggesting accumulation of malformed collagen inside the rER. Accordingly, fine, fragile fibrillar collagenous structures without evident striation were found in aa-def bones, which may result from misassembling of the triple helices of collagenous α-chains. Despite a marked reduction in bone formation, ascorbic acid deprivation seemed to have no effect on mineralization: while reduced in number, normal matrix vesicles and mineralized nodules could be seen in aa-def bones. Fine needle-like mineral crystals extended from these mineralized nodules, and were apparently bound to collagenous fibrillar structures. In summary, collagen mineralization seems unaffected by ascorbic acid deficiency in spite of the fine, fragile collagenous fibrils identified in the bones of our animal model. Ascorbic acid, i.e., vitamin C, is essential for the hydroxylation of proline and lysine in the α-chains that occurs in the rough endoplasmic reticulum (rER) during the process of collagen synthesis. Shortage of ascorbic acid may disrupt the process of crosslinking through which collagenous α-chains are bundled so as to form the stable helical structure known as superhelix (9, 28). Unlike humans, wild-type rats can synthesize ascorbic acid; osteogenic disorder shionogi (ODS) rats, however, carry a hereditary defect in ascorbic acid synthesis caused by the absence of l-gulonolactone oxidase, which catalyzes the conversion of l-gulono-lactone into ascorbic acid (18,19). ODS rats, when fed with an ascorbic acidfree diet, show low body weight, small craniofacial size, low concentrations of plasma alkaline phosphatase (ALPase) and defective bone formation (23,35,30,36). Accordingly, the hydroxyproline level in collagen synthesis (20) was also decreased (18). Since type I collagen accounts for approximately 90% of the organic fraction of bone (15), the deficiency of ascorbic acid in ODS rats results in reduced bone formation accompanied by deleterious effects in bone mechanical properties (14,30,36).

Ascorbic acid insufficiency induces the severe defect on bone formation via the down-regulation of osteocalcin production

“…For example, the osteogenic disorder Shionogi rat is unable to synthesize ascorbic acid due to the lack of L-gulono-γ-lactone oxidase, reflecting the situation in humans and Gulo -/- mice. The accumulation of malformed collagen inside the osteoblast rough endoplasmic reticulum in these rats in response to conditions of dietary ascorbic acid insufficiency, has been shown to result in detachment of osteoblasts [19]. In addition, reductions in bone hydroxyproline content and osteocalcin mRNA expression [20, 21], as well as in bone and serum ALP activity [21], have been reported in ascorbic acid-insufficient guinea pigs.…”

Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization

“…We also examined mineralization on osteogenic disorder shionogi (ODS) rats, which carry a hereditary defect in ascorbic acid synthesis [70]. Fragile, fibrillar collagenous structures without evident striation were found in ODS rat bones, which may be a result of misassembling of the triple helices of collagenous α-chains secondary to ascorbic acid deprivation.…”

Biological application of focus ion beam-scanning electron microscopy (FIB-SEM) to the imaging of cartilaginous fibrils and osteoblastic cytoplasmic processes

“…Bone and cartilage are abundant with collagen fibrils (8,20,21), which facilitate the deposition of uranyl acetate and lead aspartate. Therefore, it was necessary to extend block staining for a longer period, as well as to repeat the washing procedures to obtain FIB-SEM images of enough resolution so that proper comparison with TEM images was possible.…”

“…Specimens were subjected to block staining as described below, and were dehydrated with ascending concentrations of acetone before embedding in epoxy resin (Taab, Berkshire, UK) (20). Semi-thin sections were obtained from the surfaces of the embedded specimens and observed under a light microscope after staining with toluidine blue (Merck, Darmstadt, Germany) to determine the area to be analyzed through FIB-SEM (JIB-4600F, JEOL Ltd., Tokyo, Japan).…”