Scott C. Miller

EXPRESSION AND SIGNAL TRANSDUCTION OF CALCIUM-SENSING RECEPTORS IN CARTILAGE AND BONE

We previously showed that Ca 21 -sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca 21 ] ([Ca 21 ]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (;140 ‐190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca 21 ]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca 21 ]o reduced PTH-induced cAMP formation. Raising [Ca 21 ]o also increased intracellular free [Ca 21 ]i n SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca 21 ]o may couple to signaling pathways important in skeletal metabolism. (Endocrinology 140: 5883‐5893, 1999)

The bone lining cell: a distinct phenotype?

SummaryIt is argued that the flat cells that line nonremodeling endosteal bone surfaces are a distinct phenotype. These cells have a distinct morphology and they most likely have important functional roles in skeletal physiology, metabolism, and remodeling. For these reasons this cell seems deserving of a proper name. The namebone lining cell seems to have gained some acceptance but there will be continued confusion as long as skeletologists use this same term to generically describe cells that line bone surfaces, regardless of their actual identity.

Calcium absorption and osseous organ-, tissue-, and envelope-specific changes following ovariectomy in rats

Because cancellous bone loss occurs following ovariectomy (OVX) in rats, this has become a popular model to explore therapeutic modalities for postmenopausal bone loss in humans. The purpose of this study was to determine intestinal calcium absorption in situ and organ-, tissue-, envelope-, and site-specific changes in osseous tissues at six weeks after OVX in rats using chemical, biochemical, absorptiometric, microradiographic, and morphometric methods. There were no changes in intestinal absorption of calcium, but duodenal weight per length was significantly increased in the OVX animals compared with age-matched, sham-operated controls. There was an increase in wet bone weight, but decreases in ash/dry bone weight, total bone Ca, and Ca per ash weight in the OVX animals. There were significant decreases in the OVX animals in metaphyseal bone mineral content, as determined by photon absorptiometry and metaphyseal cancellous bone volume. The perimeter to area ratio of the metaphyseal cancellous bone in the OVX animals was increased compared with controls. Endochondral growth rates were increased in the OVX animals, attributable to an increased growth plate hypertrophic cell size and rate of chondrocyte proliferation. In the OVX animals there was an increase in modeling in the formation mode of the periosteal surface at the tibio-fibular junction. Increased periosteal modeling in the formation mode was also observed in the body of the mandible, suggesting that the changes in periosteal bone formation are not strictly coupled with changes in endochondral growth. There was an increase in modeling in the resorption mode of the endocortical surface at the tibio-fibular junction in the OVX animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in bone mineral and bone formation rates during pregnancy and lactation in rats

Changes in bone chemistry, cortical bone morphometry, and periosteal and trabecular bone formation rates were determined during pregnancy and lactation in rats. Data were obtained using chemical, static morphometric, and fluorochrome-based histomorphometric methods in pregnant or lactating animals and compared with age-matched, unmated controls. There were significant increases in bone weight, ash weight, calcium content, and femoral cross-sectional area by late pregnancy and decreases in these same parameters during lactation. There were also decreases in bone mass and increases in surface:volume ratios of trabecular bone during lactation. There were increases in femoral periosteal and endosteal perimeters associated with the reproductive cycle. Bone formation and appositional rates at the periosteal and endosteal surfaces were elevated during pregnancy, particularly evident at midpregnancy. Periosteal bone formation rates declined during lactation, but trabecular bone formation rates increased. These results indicate that during pregnancy there are increases in bone formation rates contributing to the increases in skeletal mass. During lactation in rats, reductions in skeletal mass are accompanied by increases in bone turnover, particularly evident in trabecular bone.

Ethane-1-hydroxy-1, 1-diphosphonate (EHDP). Effects on growth and modeling of the rat tibia.

One hundred gram male Sprague-Dawley rats were divided into groups which were injected daily for 10 or 30 days with vehicle (control group), 0.2, 0.4, 2.0, or 10.0 mg ethane-1-hydroxy-1,1-diphosphonate (EHDP)/kg/day. The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation factors. Knowing these, information about hard tissue resorption was deduced.After ten or thirty days treatment with 2.0 or 10.0 mg EHDP/kg/day, there was an increase in percentage of hard tissue. This was due to a decrease in bone formation with a greater decrease in hard tissue resorption. Furthermore, after thirty days treatment with 0.2 and 0.4 mg EHDP/kg/day, there was an increase in percentage of hard tissue, which was due to a decrease in resorption with no change in formation.Ultrastructural studies on osteoclasts from EHDP-treated rats showed a general decrease in their vacuolization and amount of organelles as dosage of EHDP increased. Histologic findings suggest that EHDP is similar to fluoride in the way in which it depresses hard tissue resorption.

Effect of Estrogen Deficiency on Cancellous and Cortical Bone Structure and Strength of the Femoral Neck in Rats

Abstract. Eighty mature Sprague-Dawley rats were weight matched before ovariectomy (Ovx) or Sham surgery (Sham). Sham rats had free access to food and water throughout the experiment, whereas Ovx rats were kept on the pair-fed diet. Rats were euthanized at 4, 8, and 12 weeks after surgery, and had received fluorochrome bone markers at 9 and 2 days prior to euthanasia. In addition 10 rats were euthanized at the time of surgery serving as baseline controls. All rats were also scanned for body composition and bone mineral parameters by DEXA before surgery and euthanasia. Left proximal femurs (femoral necks) were used for bone histomorphometry, whereas right femurs were used for in vitro DEXA measurements and mechanical testing. Despite pair-feeding, ovariectomized rats had increased body weights and fat body mass, whereas the percent lean body mass steadily declined throughout the experiment. Mineral density of the whole femur and femoral neck was significantly higher in the Sham rats relative to Ovx animals. Ovariectomy reduced trabecular number and thickness, and increased trabecular separation and bone marrow space at the femoral midneck location. The structure of the remaining trabeculae was dramatically changed toward simpler struts as revealed by nodal analyses. Cortical thickness in Ovx rats was reduced because of the high endocortical resorption, which, in addition to cancellous bone resorption, resulted in fewer endocortico-trabecular connections. Femoral necks obtained from ovariectomized rats had reduced strength and were less stiff relative to controls. Because of the enormous clinical significance of the proximal femur for osteoporosis in humans, and the opportunity for studying bone BMD, mass, structure, and strength at the same skeletal location, the femoral neck appears superior to other skeletal sites routinely used for bone histomorphometry or mechanical testing in the Ovx rat model.

Mayak Worker Study: An Improved Biokinetic Model for Reconstructing Doses from Internally Deposited Plutonium

Abstract Leggett, R. W., Eckerman, K. F., Khokhryakov, V. F., Suslova, K. G., Krahenbuhl, M. P. and Miller, S. C. Mayak Worker Study: An Improved Biokinetic Model for Reconstructing Doses from Internally Deposited Plutonium. Radiat. Res. 164, 111–122 (2005). The plutonium production facility known as the Mayak Production Association was put into operation in June 1948. A high incidence of cancer in the Mayak workers has been related to the level of exposure to plutonium, but uncertainties in tissue doses have hampered development of dose–risk relationships. As part of an effort to improve dose estimates for these workers, the systemic biokinetic model for plutonium currently recommended by the International Commission on Radiological Protection (ICRP) has been modified to reflect recently developed data and facilitate interpretation of case-specific information. This paper describes the proposed model and discusses its implications for dose reconstruction for the Mayak workers.

Novel dexamethasone-HPMA copolymer conjugate and its potential application in treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown etiology. Effective treatment of this disorder has been hampered by the lack of availability of agents that selectively target affected joint tissue. We developed a novel pH-sensitive drug delivery system of dexamethasone (Dex) based on an N-(2-hydroxypropyl)methacrylamide copolymer (P-Dex) and have shown that the delivery system specifically accumulates in inflamed joints in an animal model of arthritis. We hypothesize that the arthrotropism of the delivery system and the local acidosis-mediated drug release provide superior therapeutic efficacy and potentially reduced side effects in RA treatment. The initial in vitro drug-release study confirmed that the Dex release is indeed dependent upon the environmental pH. At pH 5, 37°C, the conjugate shows the highest level of drug release. When administered systemically in an adjuvant-induced arthritis rat model, P-Dex offers superior and longer-lasting anti-inflammatory effects compared with systemically administered free Dex. In addition, greater bone and cartilage preservation was observed with the P-Dex treatment compared with free Dex treatment. Our data indicate that the differential effect of the conjugate is related to its selective accumulation, potential macrophage-mediated retention, and pH-sensitive drug release (extracellular and intracellular) in arthritic joints. This newly developed drug delivery system provides a unique method for selective targeting of glucocorticoids to inflamed joints which may potentially reduce systemic side effects.

AVAILABLE ANIMAL MODELS OF OSTEOPENIA - SMALL AND LARGE

Animal models of osteopenia are reviewed. Endocrine excess or deficiency conditions include ovariectomy, orchidectomy, glucocorticoid excess and other endocrine states. Seasonal and reproductive cycles are usually transient and include pregnancy and lactation, egg-laying, antler formation and hibernation. Dietary conditions include calcium deficiencies, phosphate excess and vitamin C and D deficiencies. Mechanical usage effects include skeletal underloading models. Aging is also associated with osteopenia in many species.

The comparative effects of dichloromethylene diphosphonate (Cl2MDP) and ethane-1-hydroxy-1,1-diphosphonate (EHDP) on growth and modeling of the rat tibia

SummaryMale rats weighing 100 g were assigned to groups and injected daily for 10 days with vehicle (control), 0.4, 2.0, 4.0, 10.0, or 20.0 mg/kg/day of ethane-1-hydroxy-1,1-diphosphonate (EHDP) or dichloromethylene diphosphonate (Cl2MDP). The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation parameters. From the data, information about hard tissue resorption was deduced.All doses of Cl2MDP and doses of 2.0 mg EHDP/kg/day and greater caused significant increases in percentage of hard tissues with Cl2MDP being more effective than similar doses of EHDP in decreasing bone resorption.Osteoclast population parameters were increased with all doses of both Cl2MDP and EHDP with Cl2MDP having a greater effect than similar doses of EHDP. Decreases in the proliferation of the osteoprogenitor pool parallel the decreases in osteoblasts and bone formation parameters. These decreases in osteoprogenitor pool proliferation do not account for the increases with diphosphonates in osteoclast population parameters.