Guy A. Howard

Ethanol inhibits human bone cell proliferation and function in vitro

The direct effects of ethanol on human bone cell proliferation and function were studied in vitro. Normal human osteoblasts from trabecular bone chips were prepared by collagenase digestion. Exposure of these osteoblasts to ethanol in concentrations of 0.05% to 1% for 22 hours induced a dose-dependent reduction in bone cell DNA synthesis as assessed by incorporation of 3H-thymidine. After 72 hours of ethanol exposure in concentrations of 0.01% to 1%, protein synthesis as measured by 3H-proline incorporation into trichbroacetic acid (TCA)-precipitable material was reduced in a dose-dependent manner. Human bone cell protein concentrations and alkaline phosphatase total activity were significantly reduced after exposure to 1% ethanol for 72 hours, but not with lower concentrations of ethanol. This reduction in osteoblast proliferation and activity may partially explain the development of osteopenia in humans consuming excessive amounts of ethanol.

Evidence for the coupling of bone formation to bone resorption in vitro

An in vitro organ culture system of embryonic chick tibiae was used to examine bone formation and bone resorption under conditions of stimulation with 10−12 M parathyroid extract. The bones were cultured in a serum-free medium to alleviate any potential interference by serum growth factors. Evidence is presented showing that this bone culture system responds to parathyroid extract with an increase in both bone resorption and bone formation. Although initially there was a decrease in formation, this was followed after 12–16 h by an increase in formation which was correlated with resorption (r = .96, p < .05). These results suggest that the increase in formation was some consequence of the increase in resorption, and as such are consistent with the interpretation that coupling can occur in vitro.

Extracts of bone contain a potent regulator of bone formation

We prepared aqueous extracts of whole femorae and tibiae of embryonic chicks. An amount of extract containing 25 microgram of protein resulted in a 500% increase in DNA synthesis in calvarial cell culture, and significant effects were detected with 5 microgram (55%). The time course for stimulation of DNA synthesis showed a peak occurring 16-20 h after addition of the extract. This matrix factor is nondialyzable, and fractionation on a column of Sephadex G-100 indicated a molecular weight of 60-80,000. At the maximum dose used, [3H]proline incorporation into total protein of calvarial cells was increased by 55%, and thus far, all fractions active in promoting DNA synthesis have been found to increase collagen synthesis in cultured chick tibiae. These data are consistent with an effect on osteoblasts as well as bone precursor cells. Extracts prepared from tibiae of 2-day-old chicks, from which the marrow had been removed, also stimulated DNA synthesis (280% increase), thus ruling out the possibility that the factor is a relatively nonspecific nitrogen from the hematopoietic cell line. We conclude that bone matrix contains a substance which could regulate bone formation in vitro by control of mitosis in osteogenic precursors and/or stimulation of osteoblast activity.

Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry.

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.

Regulation of DNA synthesis in chick calvaria cells by factors from bone organ culture.

Abstract Embryonic chick bones growing in organ culture release a substance into the culture medium which stimulates bone formation in previously untreated bones. This “conditioned” medium also enhances proliferation of monolayer cultures of chick calvaria cells in serum-free medium. The active principle is nondialyzable, indicating a molecular weight greater than 12,000 daltons. Dialysis also separates the mitogenic activity from a low-molecular-weight inhibitor. The amount of the mitogen found in conditioned medium increases as the rate of bone resorption increases in response to treatment with parathyroid hormone or 1,25-dihydroxyvitamin D3. Maximal stimulation of DNA synthesis in calvaria cells is evident with conditioned medium obtained 3 to 5 days after treatment of bone cultures with parathyroid hormone. The cells must be treated with the conditioned medium continuously for 20 hr in order to obtain peak enhancement of DNA synthesis; there is no detectable effect in the first 8 hr. In contrast, the inhibitor acts within 4 hr. The data suggest that the stimulatory factor acts to increase cell proliferation by promoting entry of cells into the S phase of mitosis. We conclude that this stimulator is a locally produced regulator of bone formation, probably acting via an increased proliferation in osteoblast precursor cells.

Serum-free culture of Japanese quail kidney cells. Regulation of vitamin D metabolism.

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 . 10(6) cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25--30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromatographically separated on Sephadex LH-20. Three have been identified as 1 alpha, 25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1 alpha, 24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1 alpha- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1 alpha-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25(OH)2D-3) synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.

Metabolism of 25-hydroxyvitamin D3 during photo-induced reproductive development in female Japanese quail.

Abstract Synchronous development of the reproductive tract in adult female Japanese quail has been achieved by manipulation of photoperiod length. This has made possible determination of changes in vitamin D metabolism associated directly with the onset of reproductive maturity. The distribution of polar metabolites of 25-hydroxy [ 3 H] vitamin D 3 ([ 3 H]-25-OH-D 3 ) was altered shortly before the onset of egg laying, such that the specific activity of the 25-OH-D 3 -1α-hydroxylase increased 390%, while the production of 24,25-dihydroxyvitamin D 3 [24,25(OH) 2 D 3 ] and an unidentified metabolite (metabolite IV) decreased 37 and 61% respectively. In addition, kidneys of reproductively mature female quail produced a metabolite, tentatively identified as 1,24,25-trihydroxyvitamin D 3 [1,24,25(OH) 3 D 3 ], which was not found in the kidneys of male quail or in those of female quail with immature oviducts. The concentration of oviduct nuclear estrogen receptor-estrogen complex increased to a level comparable to laying female quail very early in reproductive tract development. A 280% increase in kidney size occurred concurrently. In contrast, the altered renal metabolism of [ 3 H]-25-OH-D 3 did not occur until much later in development, suggesting that kidney growth and renal metabolism of vitamin D are independently regulated. It appears that the increased specific activity of the 25-OH-D 3 -1α-hydroxylase immediately prior to egg laying is important in the provision of calcium for shell formation. The close temporal relationship between the altered metabolism of [ 3 H]25-OH-D 3 and the onset of medullary bone formation suggests that a vitamin D metabolite may mediate the formation and utilization of medullary bone.

Bone mineralization and metabolism of3H-25-hydroxyvitamin D3 in thyroparathyroidectomized rats treated with parathyroid extract

SummaryThyroparathyroidectomized rats fed a diet containing 1.2% calcium, 0.55% phosphorus maintain normal serum levels of these ions. Treatment of such rats with parathyroid extract (PTE; 20 U/100 g twice daily; 10 days) has no statistically significant effect on rates of bone formation, matrix apposition, or osteoid maturation. Significant decreases in osteoid width and mineralizing front width, as well as a 60% increase in the rate of initial mineralization were observed in the PTE-treated group. Conversion of3H-25-hydroxyvitamin D3 to3H-1,25-dihydroxyvitamin D3 was 4-fold higher in the PTE-treated group than in the untreated animals. Increased formation of 1,25-dihydroxyvitamin D3 in response to treatment with PTE may play a major role in correcting the mineralizing defect resulting from thyroparathyroidectomy.

In Vivo Calvarial Bone Cell Responses to Dietary Perturbations and the Implications for Mineral Homeostasis

Abstract Calvariae from small animals have been an important source for in vitro studies of bone. However, few in vivo studies have been undertaken on quantitative cell changes in calvariae. In the present study of mineral perturbations, rats were first deprived of calcium. After 18 days endosteal osteoclasts and nuclei/osteoclast in the parietal bone had increased 120% (P < 0.001) and 26% (P < 0.001), respectively, the marrow space had increased 141% (P < 0.001), and the bone area experienced a 49% decrease (P < 0.001). This thinning and weakening of the calvaria was accompanied by a compensatory increase in the number of endosteal osteoblasts (297%, P < 0.001). These rats were then replenished with calcium, and after 14 days the number of endosteal osteoclasts had decreased to 86% (P < 0.001) below the control and the endosteal surface was almost completely covered by osteoblasts (866% above the control, P < 0.001). Bone area was increased by 51% (P < 0.01). Similarly, in calcium-deficient rats in the tibial diaphysis at the fibular junction, the number of endosteal osteoclasts and the medullary space increased 1606% (P < 0.001) and 63% (P < 0.001), respectively, which were accompanied by a 32% decrease (P < 0.001) in cortical bone area. After calcium replenishment, most endosteal osteoclasts in the tibial diaphysis disappeared from the endosteal surface and were replaced by osteoblasts (increased 487%, P < 0.001). These results indicate that changes in bone cell activity in response to calcium deficiency are similar in calvariae and long bones, and that mobilization of calcium from the calvaria during calcium deficiency occurs at the expense of the protective action of the calvaria. Therefore, long bones as well as membranous bones are apparently important for the maintenance of mineral homeostasis.

Effect of treatment with parathyroid extract on bone parameters and metabolism of 3H-25-hydroxyvitamin D3 in Hypocalcemic Thyroparathyroidectomized Rats

Abstract Thyroparathyroidectomized (TPTX) rats fed diets containing 0.1% calcium, 0.6% phosphorus (low calcium), or 0.6% calcium, 0.6% phosphorus (normal calcium) become hypocalcemic and hyperphosphatemic. Inhibitory effects of parathyroid extract (PTE; 20 Units/100 g twice daily, 8–9 days) on rates of bone formation, matrix apposition, and osteoid maturation were observed in TPTX rats fed the low calcium diet, but not in those fed the normal calcium diet. Conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 was increased in response to PTE only in TPTX rats fed the normal calcium diet. In this group increased formation of 1,25-dihydroxyvitamin D3 was associated with a significant reduction in the width of the mineralizing front. Our results indicate that serum calcium plays an important role in modulating the effect of PTE on metabolic processes in bone and kidney.