Original ArticlesTrabecular bone turnover, bone marrow cell development, and gene expression of bone matrix proteins after low calcium feeding in rats
Introduction
Several in vivo experimental animal models, such as ovariectomy,20, 48, 53 parathyroidectomy,7 thyroparathyroidectomy,52 orchidectomy,53 tail suspended,26 and sciatic neurectomy,43 have been used to evaluate the pathogenesis of bone diseases and to develop an effective therapy. These experimental models have shown that a relatively long period is necessary to observe obvious bone loss.43, 48, 57 Thus, it is desirable to establish a new animal model that could induce short-term bone loss by a simple method.
Low-calcium feeding has been accepted as a method of increasing bone resorption and as one of the experimental models to reduce bone mass.46, 49 The low-calcium diet was often combined with ovariectomy and immobilization for enhancing more bone resorption.47, 55 These combined models were used to study the mechanism of bone resorption and to assess the efficacy of the drugs that prevent bone loss due to metabolic bone diseases.2, 28 The process of bone resorption induced by the low-calcium diet was known to be mediated by secondary hyperparathyroidism,39, 50 but the following process of bone resorption and the mechanisms leading to bone loss have not yet been fully elucidated.
It is generally believed that bone marrow contains progenitor cells both of osteoblasts and osteoclasts. Trabecular bone shows a readier access to bone marrow cells than cortical bone;37 therefore, the microenvironment for progenitor cells in bone marrow plays an important role in bone remodeling at the trabecular bone surface. To analyze the change in cell populations in bone marrow, a bone marrow cell culture system was usually used.15, 22, 43, 44 Under supplement with dexamethasone, osteoprogenitor cells in bone marrow are stimulated to differentiate to osteoblasts and produce a bone-like extracellular matrix that is then mineralized.22, 24, 44 It has been believed that bone nodule formation could be used to assess the number of osteogenic precursors present in the bone marrow.22, 27, 29 It is also indicated that rodent bone marrow cells develop into osteoclast-like multinucleated cells stimulated by various inducers, such as 1,25(OH)2D3, parathyroid hormone (PTH), and prostaglandin E2.16, 44 Using an ex vivo technique for the bone marrow cell culture, the alteration of the bone marrow cell population occurred in ovariectomized rats,44 immobilized mice,22, 43 and bisphosphonate-treated rats.15 Therefore, the change in bone marrow cell development in vitro is closely related to bone formation, resorption, and trabecular bone turnover in vivo. This technique gives us important information about cell development in the bone marrow and implications for the local regulations of bone turnover and bone remodeling under the low-calcium feeding in this study.
To estimate the activity of osteoblasts in bone remodeling, the labeling of bone using a fluorescent agent, such as tetracycline, has been accepted as a useful method, and labeling with several fluorochromes is used to measure the dynamic parameters in bone histomorphometry. However, this method could not be applied in this experiment, because the experimental period was too short to observe a distinct labeling pattern. Instead of the dynamic parameters of bone formation, the method of molecular biology was used to clarify the osteoblastic activity in our experiment.19, 20 Because it has been believed that the gene expression of matrix proteins is closely associated with bone turnover activity in vivo, the analysis of the bone matrix gene expression may be a good indication of bone remodeling in the present study.
To clarify the possible mechanisms that initiate bone remodeling and subsequent bone loss from trabecular bone induced by the low-calcium diet, we performed experiments to investigate the short-term effects of diet on the bone histology and evaluated the changes in osteoclasts, osteoblasts, and their progenitor cells using histomorphometrical analysis, a bone marrow cell culture system, and the gene expression of bone matrix proteins.
Section snippets
Experimental protocol
Male Wistar rats, 33 days old, were used in this study. Each of the rats was housed singly in a metabolic cage (Metabolic Type MCST; Sugiyamagen Co., Ltd., Tokyo, Japan) and fed on a semisynthetic diet using the pair feeding technique.1 The normal-calcium diet contained 0.5% calcium and 0.35% phosphorous. The low-calcium diet contained 0.05% calcium and 0.35% phosphorous. The diet was given to each rat from 5 to 6 p.m. daily, and 10 IU of vitamin D3 (Wako Pure Chemical Industries, Osaka, Japan)
Body weight, bone length, and biological parameters
During the experimental period, body weight and length of the tibia in the low-calcium group were not different compared with those of the normal-calcium group (Table 1). The level of serum calcium decreased and phosphate increased only at 12 h after changing the diet to a low-calcium one. Thereafter, the concentration of serum calcium and phosphate returned to the level of the normal-calcium group. The serum PTH level was temporarily increased after 12 h in the low-calcium diet group; this
Discussion
In this study, the change in the body weight and the longitudinal bone growth was not significant between the normal-calcium and low-calcium groups. However, the BMD of tibia in trabecular bone was significantly decreased in the low-calcium group. This means that bone resorption appeared in the low-calcium group without any influence by other nutritional elements except calcium using the pair feeding technique during the experimental period.
The levels of serum calcium and phosphate tended to
Acknowledgements
The authors are grateful to Dr. H. Kondo, Dr. H. Mukouyama, Dr. N. Arai, and Dr. W. R. Duarte (Tokyo Medical and Dental University) for their continuous support during the course of this work. This work was supported in part by a Research Grant from the Japan Society for the Promotion of Science (RFTF96I00205), by a Grant-in-Aid for Scientific Research (No. 09470400, 08045061, 09557163, 10145208) from the Ministry of Education in Japan, and by Japan Space Forum (Phase Ib, 69).
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