Turner, Russell T.

Abstract The effects of castration on bone histomorphometry and mineral homeostasis were compared in male and female rats. Measurements were performed 4 weeks after sham operation or gonadectomy. Orchiectomy produced increases in serum calcium and decreases in serum testosterone and androstenedione, whereas ovariectomy produced decreases in serum estradiol and testosterone. Orchiectomy did not alter static bone histomorphometric measurements of the tibial diaphysis, whereas ovariectomy increased cross-sectional and medullary areas, lowered endosteal tetracycline-labeled surface length, and markedly increased endosteal nonlabeled surface length. Orchiectomy decreased mean periosteal bone formation rate and mean periosteal bone apposition rate, whereas ovariectomy increased both measurements. Orchiectomy and ovariectomy markedly diminished trabecular area and trabecular surface length at the tibial metaphysis. Orchiectomy did not alter the number of osteoclasts per mm trabecular surface or the percentage of trabecular surface covered by osteoclasts, whereas ovariectomy increased both measurements. These findings indicate that gonadal hormones produce separate and distinct effects on bone metabolism as determined by histomorphometry in male and female rats.

Abstract The effects of fluoride at concentrations of 2.0 and 4.5 mM in drinking water on growth rate, vitamin D, water and mineral metabolism, bone histomorphometry, and osteoinduction of demineralized allogenic bone matrix (DABM) were compared in the rat. Whereas fluoride did not influence fluid intake or growth rate at the lower concentration, it increased fluid intake and inhibited growth rate at the higher concentration. Fluoride produced dose-related increases in serum fluoride and alkaline phosphatase but did not alter serum 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D. Serum calcium and phosphate were reduced by fluoride at concentrations of 2.0 mM but not 4.5 mM. Cancellous bone fractional area was increased by fluoride at 2.0 mM and was reduced by fluoride at 4.5 mM. Fluoride had no effect on cancellous bone surface length or the percentage surface lined by osteoblasts and osteoclasts. Fluoride increased medullary area and decreased the endosteal bone formation rate. Fluoride increased periosteal bone formation and apposition rates at concentrations of 2.0 mM but not 4.5 mM. Fluoride inhibited mineralization in DABM implants, and at the higher concentration, fluoride increased the formation of new bone matrix. These results indicate that in the rat, fluoride increases cortical and trabecular bone at therapeutic doses and reduces trabecular bone at toxic doses. The serum concentration of flouride at therapeutic doses in the rat is similar to that in patients with osteoporosis who are on treatment with fluoride. In the rat, there is a narrow range between toxic and therapeutic doses.

Abstract Periosteal cells were isolated from tibiae of adult male rats after collagenase treatment. Northern blot analysis of total cytoplasmic RNA extracted from the isolated periosteal cells was positive for expression of genes encoding the osteoblast marker proteins osteocalcin (BGP) and pre-pro-α2(I) chain of type 1 procollagen. The isolated periosteal cells were incubated with 1 nM [3H]testosterone ([3H]T) for up to 240 minutes and the reaction products separated by high-performance liquid chromatography. [3H]5α-dihydrotestosterone ([3H]DHT) was not detected in extracts of periosteal cell incubations. In contrast, [3H]DHT was produced in a time-dependent manner by cells from seminal vesicles. These results suggest that testosterone 5α-reductase activity is not expressed by osteoblasts in rat tibial periosteum and that the anabolic effects of androgens in this tissue are not mediated by locally produced DHT.

Abstract This report describes histomorphometric evidence for an important role of androgens in maintaining cancellous bone balance at a remodeling site in vivo. Rats were orchiectomized (ORX) at 7 weeks of age and received either androgens or vehicle 1 week later (testosterone, 1-dehydrotestosterone, or 5-dihydrotestosterone) by subcutaneous pellet, producing controlled release of the drug for 3 weeks. Intact male rats and untreated ORX animals served as controls. After 4 weeks untreated ORX resulted in undetectable serum testosterone levels and marked atrophy of seminal vesicles compared with intact controls. Histomorphometry revealed severe cancellous osteopenia in the secondary spongiosa of the proximal tibial metaphysis. The length of bone surface lined by apparently “active” osteoblasts and number of osteoclasts per length of cancellous bone surface were increased following ORX. Testosterone treatment at 5 mg (per 21 days) produced subphysiologic serum testosterone levels. In contrast, 10 and 25 mg pellets resulted in serum testosterone levels comparable to those in intact rats. Testosterone treatment of ORX rats prevented the bone effects of ORX, and the degree of protection was dose dependent. 1-Dehydro- and 5-dihydrotestosterones displayed a bone-protective effect similar to that of testosterone. The results demonstrate that gonadal insufficiency results in a cancellous osteopenia that is preventable by testosterone treatment. Further, because a similar protective action was achieved using the nonaromatizable androgen 5-dihydrotestosterone, the results suggest that this bone-sparing effect is mediated by androgen rather than by metabolism of the androgen to an estrogen.

Abstract Gallium nitrate lowers the serum calcium in patients with hypercalcemia caused by malignancy and is available for clinical use. The mechanism for the hypocalcemic action is unknown, however. The present studies were undertaken to determine the effects of gallium on bone metabolism. Normal male rats were implanted subcutaneously with mineralized allogeneic bone matrix. Histomorphometry of the implants and of tibiae was determined after three doses of tetracycline administered at intervals of 1 week. Gallium as nitrate was administered daily by intraperitoneal injection at doses of 0.9, 1.8, and 3.6 mg elemental gallium per kg body weight for 21 days in one study and at 3.5 mg/kg for 33 days in a second study. All the gallium-treated rats gained weight. Rats given gallium at doses of 3.5 mg/kg or more grew at a lower rate than untreated controls (-7 and -10% at doses of 3.5 and 3.6 mg/kg, respectively; p < 0.05). At a dose of 0.9 mg/kg, gallium did not inhibit bone resorption or lower serum calcium but inhibited bone formation by 32% and bone apposition by 36% at the endosteal surface of the tibia. At a dose of 1.8 mg/kg, gallium produced modest hypocalcemia, prevented a rise in circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D], inhibited bone resorption in implants, and inhibited bone formation by 19% and bone apposition by 18%. At a dose of 3.5 mg/kg, gallium lowered the serum calcium and serum 1,25-(OH)2D, inhibited growth, and accentuated the antiresorptive and antiformative effects seen at the two lower doses. The results indicate that gallium inhibits bone resorption, produces hypocalcemia, alters vitamin D metabolism, and inhibits bone formation at doses below those that inhibit bone resorption. The mechanism for hypocalcemia may be by direct action on bone and could involve an indirect effect on calcium metabolism mediated by changes in the metabolism of vitamin D.

AbstractThe effects of prostaglandin E2 (PGE2) given in controlled-release pellets or by daily sc injection for 21 days on mineral homeostasis and bone histomorphometry were compared in 7-week-old female rats. Sham operation and ovariectomy were performed at the beginning of the studies. In experiment 1, 7.5 mg PGE2 or drug-free, controlled-release pellets were implanted sc at the back of the neck on day 7. In experiment 2, 3 mg/kg body weight of PGE2 or vehicle was injected sc daily beginning on day 7. The animals were sacrificed on day 28 of the two experiments, and the tibiae were removed for histomorphometric analysis of the diaphysis and metaphysis. When administered by pellets in experiment 1, PGE2 lowered serum 1,25-dihydroxyvitamin D and did not influence weight gain, serum calcium, phosphorus, or magnesium, cross-sectional or medullary areas, periosteal bone formation and apposition rates, endosteal bone formation and apposition rates, or endosteal tetracycline-labeled perimeter. PGE2 lowered cancellous bone area and cancellous bone perimeter in both the sham-operated and ovariectomized rats. In contrast, when administered by sc injection in experiment 2, PGE2 reduced weight gain, increased serum magnesium, increased cortical area, and reduced medullary area without changing cross-sectional area, increased periosteal bone formation and apposition rates and endosteal bone and apposition rates, did not alter endosteal tetracycline-labeled perimeter, and increased cancellous bone area and cancellous bone perimeter in both sham-operated and ovariectomized animals. PGE2 produced local inflammation when given by pellets, and the serum concentration of 13,14-dihydro-15-ketoprostaglandin E2, the major metabolite of PGE2, increased when PGE2 was given by sc injection but not when administered by pellets. Thus, PGE2 given sc by controlled-release pellets (1) produces local inflammation and systemic bone loss without increasing PGE2 systemically and (2) provides a model for inflammation-induced loss of cancellous bone. The results also indicate that the pellet is not a valid means for the delivery of PGE2 to the general circulation.

Abstract Calcein was given to growing male rats to label mineralizing bone, and groups of animals were sacrificed from 6 h to 12 days later. Fluorochrome-labeled perimeter and osteoblast perimeter were determined in a growth-adjusted sampling site in the secondary spongiosa of the proximal tibial metaphysis. The best correspondence between calcein-labeled perimeter and osteoblast-lined perimeter was during the initial 24 h following administration of the label. This association decreased progressively thereafter because of (1) cessation of bone formation on fluorochrome-labeled surfaces and (2) initiation of new foci of bone formation. The time-dependent decline in osteoblast-lined trabeculae with adjacent calcein label exceeded the increase in osteoblast perimeter on nonfluorochrome-labeled trabeculae, indicating that there was a net decrease in osteoblast number. Fluorochrome-labeled perimeter and bone area showed parallel decreases with time after labeling, as a result of bone resorption. Interestingly, the decrease in cancellous bone was caused exclusively by a reduction in trabecular number. There was no change in trabecular thickness. The findings suggest that the decreased osteoblast number and progressive bone resorption resulted in complete erosion of trabeculae most distal to the growth plate. As a result of the nearly equal growth and destruction of trabeculae, there was no change in the length of the cancellous metaphysis with time. It is concluded that cancellous bone turnover in growing rats represents a maturation process that differs fundamentally from bone turnover in adults.

Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with β-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism.