N. Loveridge

Cell proliferation within the growth plate of long bones assessed by bromodeoxyuridine uptake and its relationship to glucose 6-phosphate dehydrogenase activity

Bromodeoxyuridine (BrdUrd) was incorporated into the DNA of proliferating chondrocytes of the tibiae and metatarsal growth plate of rats of differing ages. Immunodetection of the incorporated BrdUrd was achieved using a monoclonal antibody to BrdUrd and indirect immunofluorescence procedures. The labelling index within the growth plate was found to decrease with advancing age in the tibia and metatarsal. Glucose 6-phosphate dehydrogenase (G6PD) activity appeared to be related to chondrocyte proliferation as a significant correlation was observed between G6PD activity and the labelling index of the tibial chondrocytes. The results of this study indicate that G6PD activity is a possible marker for cartilage growth and also that the BrdUrd technique has several advantages over the conventional autoradiographic methods for the assessment of cell replication.

Bone mineral density and metabolism in premenopausal women taking l‐thyroxine replacement therapy

BACKGROUND AND OBJECTIVE Excess endogenous thyroxine causes bone loss, but the effects of exogenous thyroxine are disputed. We report on bone mass and metabolism in women taking l‐thyroxine therapy.

Bone mass and metabolism in women aged 45–55

OBJECTIVEu2003Changes in calcium homeostasis and bone mass around the climacteric are poorly understood. We examined relations between endocrine factors and indices of bone mass and metabolism in healthy women approaching the menopause.

Alterations in glycosaminoglycan concentration and sulfation during chondrocyte maturation

We have used antibodies to chondroitin 4- and 6-sulfate and keratan sulfate along with Alcian blue staining of sulfated proteoglycans to investigate changes in content and sulfation within the avian growth plate. In normal chicks, chondroitin 4- and 6-sulfate content were similar in the proliferating and transitional zones but in the hypertrophic zone, chondroitin 4- and 6-sulfate were slightly lower (13% and 18%, respectively) and keratan sulfate was markedly lower (58%). Compared with the proliferative zone, Alcian blue staining of sulfated glycosaminoglycans was markedly lower in both the transitional (46%) and hypertrophic (22%) zones. In tibial dyschondroplasia, where chondrocyte maturation is arrested at the transitional zone, there was no difference in the chondroitin 4- and 6-sulfate or keratan sulfate staining between the proliferative and transitional zones, which were similar to normal birds. With Alcian blue staining there was no difference in the intensity of the staining within the proliferating zone compared with normal birds but stainign in the transitional chondrocytes was markedly higher (39%). These results suggest that in the early steps of chondrocyte maturation there may be a decrease in the degree of glycosaminoglycan sulfation without any alteration in glycosaminoglycan concentration, and that further maturation may be accompanied by a change in the nature of the proteoglycans which may also affect the level of sulfation.

Effect of diets varying in nitrogen or phosphorus content on indicators of bone growth in lambs

Growing lambs were fed diets low in nitrogen and phosphorus (LNLP), low in nitrogen and high in phosphorus (LNHP), high in nitrogen and low in phosphorus (HNLP) or high in nitrogen and phosphorus (HNHP) and the effects on bone growth and on blood and urinary bone marker levels or excretion rates were monitored. Plasma calcium concentrations were higher, and phosphorus concentrations lower, in lambs fed the low phosphorus diets but there were no differences in plasma 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) concentrations. Lambs fed both low phosphorus diets (LNLP and HNLP) had lower plasma osteocalcin and higher bone‐specific alkaline phosphatase concentrations than those fed the high phosphorus diets. Urinary pyridinoline and deoxypyridinoline excretion were also affected by treatment, with their rates of excretion being highest in lambs fed the diet low in both nitrogen and phosphorus (LNLP). Lambs fed the low phosphorus diets were lighter in weight at slaughter and had lighter bones that were less well mineralized than those fed the high phosphorus diets. Reducing the nitrogen content of the diet appeared to have little effect on bone composition. These results suggest that bone markers that have proved useful in the diagnosis and treatment of bone disease are sensitive to variation in nutrient supply and may prove useful in early detection of nutrient deficiencies that affect bone growth.

Mitogenic action of insulin-like growth factor-I on human osteosarcoma MG-63 cells and rat osteoblasts maintained in situ: the role of glucose-6-phosphate dehydrogenase

The mechanisms involved in the mitogenic actions of insulin-like growth factor-I (IGF-I) on skeletal cells are at present unclear. We have investigated the role of glucose-6-phosphate dehydrogenase (G6PD) in this mechanism and provide strong evidence that stimulation of G6PD activity is required for the growth promoting activities of IGF-I. IGF-I (10 ng/ml) significantly elevated G6PD activity in MG-63 human osteosarcoma cells within 30 min which preceded the IGF-I induced DNA synthesis in these cells. Inhibition of G6PD activity by epiandrosterone decreased DNA synthesis in IGF-I stimulated MG-63 cells but this was partly overcome by the addition of a combination of the four deoxyribonucleosides. IGF-I did not cause a general increase in cell metabolism as succinate dehydrogenase and iso-citrate dehydrogenase activity were not altered. Although IGF-I caused a significant increase in lactate dehydrogenase activity this was not inhibited by epiandrosterone. The culture of metatarsals of 4-week-old rats with IGF-I (10 ng/ml) also stimulated G6PD activity in osteoblasts lining the metaphyseal trabeculae.

Endogenous mediators of growth

In the present review it is not possible to discuss the effects of the numerous endogenous mediators of growth. What we have attempted to do is to indicate the areas of controversy and the need for further research. In our view, four main questions arise. First, what are the relative contributions of the direct and indirect effects of GH? Indeed, if GH can produce all its effects by local production of IGF, is the original somatomedin hypothesis still tenable? Second, how is the biological activity of the IGF modified by the presence of binding proteins? Because of the role of binding proteins in modulating IGF bioactivity, care must be taken when interpreting results from immunoassays for IGF because this will only represent the concentration of IGF not the level of biological activity, a situation which is analogous to that which pertains with certain polypeptide hormones (for review, see Robertson et al. 1987). Third, how are the activities of the osteoblast and osteoclast coupled so that in the mature adult, bone formation and bone resorption are roughly equivalent? Understanding of this process will undoubtedly involve the elucidation of the roles and interactions between a number of locally acting growth factors and systemic hormones and will lead to the understanding of certain metabolic bone diseases such as osteoporosis. Last, how is the response to local stimuli such as mechanical stress transduced? This is again probably dependent on the activity of local growth factors but may also involve changes in the interactions between bone cells and their underlying matrix components (Skerry et al. 1988).

Micronutrients and longitudinal growth

The present review has concentrated on the control of longitudinal growth and the relative importance of certain micronutrients. By far the most significant of these is 1.25(OH)2D3 which is now being recognized, not only for its role in maintaining Ca homeostasis, but also its major role in chondrocyte differentiation within the growth plate.

Molybdenum-induced changes in the epiphyseal growth plate

SummaryMolybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.

Dextran Sulfate Promotes the Rapid Aggregation of Porcine Bone-Marrow Stromal Cells

Despite the fact that cells of the mammalian stromal compartment of bone marrow have been shown to contain multipotential stem cells when studied in diffusion chambers it is notable that the same range of possible phenotypes (e.g., chondrocytic) has not been induced in freshly isolated marrow stromal cells in vitro. To investigate the possible role of glycoconjugates on phenotype expression, the effects of chondroitin sulfate, dermatan sulfate, dextran 500, and dextran sulfate on the cell morphology and differentiation of confluent porcine bone-marrow stromal-cell monolayers were studied. Of these glycosaminoglycan molecules only dextran sulfate induced confluent porcine bone-marrow stromal-cell monolayers to retract into tight, circular cell aggregates. Retraction began within 6 h, was complete after 3-5 days, and was dose dependent. Subsequent removal of dextran sulfate from the culture medium resulted in a return to a monolayer culture. Aggregated cells were essentially nonmitotic but dye exclusion indicated high cell viability. Dexamethasone, ascorbate, and beta-glycerophosphate produced no morphological change within 6 days when administered alone, but increased proliferation and aggregation in dextran sulfate-treated cultures. Immunocytochemistry of monolayer cultures revealed positive staining for type I but not type II collagen and addition of dexamethasone, ascorbate, and beta-glycerophosphate increased type I collagen deposition. In contrast, the centers of dextran sulfate-induced aggregates were positive for type II collagen, whereas type I collagen was only present at the periphery of the aggregates. Further addition of dexamethasone, ascorbate, and beta-glycerophosphate had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)