Biomedical subjects

D J Baylink

Publications and source records attributed to D J Baylink.

At least 235 records · Page 13Linked to original sources

PubMed1989

Stimulation of cellular alkaline phosphatase activity and its messenger RNA level in a human osteosarcoma cell line by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) significantly stimulated cellular alkaline phosphatase activity in a human osteoblastic osteosarcoma cell line (TE-85 cells) in serum-free medium with 0.1% bovine serum albumin as the hormone carrier in a dose- and a time-dependent manner. The extent of the maximal stimulation was greater and the minimal dose that was required for stimulation was lower than those previously reported for TE-85 cells in the presence of serum. The magnitude of the stimulation of alkaline phosphatase activity by 1,25(OH)2D3 varied with the cell density. Daily changes of conditioned medium, as compared with no medium changes, significantly reduced the magnitude of the stimulation, suggesting that endogenous factors secreted into culture medium could play an enhancing role. Finally, application of Northern blot analysis using an oligodeoxynucleotide probe corresponding to a unique sequence of the human bone/liver/kidney alkaline phosphatase cDNA coding region revealed that 1,25(OH)2D3 increased the alkaline phosphatase mRNA level, suggesting that the increase in alkaline phosphatase activity was a result of either an increase in the rate of transcription or an increase in message stability.

Alkaline Phosphatase↗

PubMed1989

A proposed mechanism of the mitogenic action of fluoride on bone cells: inhibition of the activity of an osteoblastic acid phosphatase.

Fluoride (F) is a potent inhibitor of osteoblastic acid phosphatase activity with an apparent Ki value (10 to 100 mumol/L) that corresponds to F concentrations that increase bone cell proliferation and bone formation in vivo and in vitro. This high sensitivity of acid phosphatase to F inhibition appeared to be specific for skeletal tissues. Mitogenic concentrations of F did not increase cellular cAMP levels but significantly stimulated net protein phosphorylation in intact calvarial cells and in isolated calvarial membranes. These concentrations of F also stimulated net membrane-mediated phosphorylation of angiotensin II (which contains tyrosyl but no seryl or threonyl residues), suggesting that some of the F-stimulated protein phosphorylations could occur on tyrosyl residues. F had no apparent effect on thiophosphorylation of membrane proteins, suggesting that the F-stimulated net protein phosphorylation in bone cells was probably not mediated via activation of protein kinases. Orthovanadate or molybdate at concentrations that inhibit bone acid phosphatase activity also stimulated bone cell proliferation, supporting the idea that inhibition of bone acid phosphatase would lead to stimulation of bone cell proliferation. Mitogenic concentrations of F potentiated the mitogenic activities of insulin, EGF, and IGF-1 (ie, growth factors the receptors of which are tyrosyl kinases) to a greater extent than they potentiated the action of basic FGF (a growth factor that does not appear to stimulate tyrosyl protein phosphorylation). Based on these findings, a model is proposed for the biochemical mechanism of the osteogenic action of F in which F stimulates bone cell proliferation by a direct inhibition of an osteoblastic acid phosphatase/phosphotyrosyl protein phosphatase activity, which in turn increases overall cellular tyrosyl phosphorylation, resulting in a subsequent stimulation of bone cell proliferation.

Acid Phosphatase↗

PubMed1989

Efficacy of long-term fluoride and calcium therapy in correcting the deficit of spinal bone density in osteoporosis.

Long-term fluoride therapy for osteoporosis has been shown to increase the thickness of vertebral trabeculae as seen on spinal radiographs. To determine if this qualitative finding represents a measurable increase in spinal bone density, quantitative computed tomography was utilized to measure trabecular vertebral body density (TVBD) in the lumbar spine of 18 female osteoporotic patients, all of whom had been treated with sodium fluoride, 77 +/- 13 mg/day (mean +/- SD), and calcium, 1000 mg/day, for 57 +/- 24 months. TVBD in these fluoride treated osteoporotic patients (132 +/- 82 mg/cm3) was found to be significantly greater than mean TVBD for an age-matched group of untreated female osteoporotic patients (51 +/- 21 mg/cm3, n = 89, p less than 0.001). The value for TVBD in the long-term fluoride treated osteoporotics was not only similar to previously published values for TVBD (104 +/- 30 mg cm3) in normal females of similar age, but was also above the calculated TVBD "fracture threshold" of 100 mg/cm3 for females. Only one of the 18 fluoride treated osteoporotics continued to have spinal fractures during therapy, accounting for 4 fractures per 87.2 patient years of observation, a value which is significantly lower than the published incidence of 76 fractures per 91 patient years for untreated osteoporotic patients (p less than 0.001). Together, these findings demonstrate that long-term fluoride and calcium therapy for osteoporosis increases TVBD in the majority of patients within a reasonable time frame and significantly reduces the risk for spinal fractures.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗

PubMed1989

The effect of prolonged fluoride therapy for osteoporosis: bone composition and histology.

To examine the long-term effects of fluoride therapy in osteoporosis, we obtained iliac crest biopsies from 11 osteoporotic patients 6 to 12 years after they had started fluoride therapy. Although basal biopsies were not obtained, nine subjects had been biopsied 4 years prior to the second biopsy. In addition, 4 subjects had stopped fluoride therapy prior to the second biopsy. Biopsy samples were divided and analyzed: (a) histomorphometrically for bone formation and mineralization; and (b) for mineral content. Parameters of bone formation were increased in the first biopsy of all patients; they remained elevated in the second biopsy of subjects still receiving fluoride, but decreased to normal values in subjects who stopped fluoride therapy. Parameters of mineralization (i.e., osteoid width and osteocytic osteoid) were elevated in the first biopsy, but had decreased in the second biopsy whether fluoride was stopped or not. There was no woven bone in these biopsies. Bone mineral content, whether measured as density or by summation of the individual ions (% mineral), was higher than normal in all subjects, whether or not they were still receiving fluoride. These results suggest that prolonged fluoride therapy of osteoporosis continues to stimulate bone formation, but does not cause a progressive mineralization defect. Mineral content is acutely increased following fluoride therapy, and persists after therapy is discontinued.

Alkaline Phosphatase↗

PubMed1989

Estrogens in the prevention of osteoporosis in postmenopausal women.

Menopause-related bone loss leaves a woman at high risk for fractures. Estrogen use by postmenopausal women, especially when started within 3 years of the last menstrual period, prevents bone loss and reduces the risk of osteoporotic fractures. Estrogens do not restore lost bone. Withdrawal of estrogen therapy is followed by significant bone loss, thus suggesting that long-term therapy is needed. Concomitant progesterone therapy does not impair estrogen's bone-preserving activity.

Bone Diseases, Metabolic↗

PubMed1989

Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: a potential local regulator of IGF action.

Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C4 reverse-phase, HPLC CN reverse-phase, and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known BPs. (iii) In-IGF-BP exhibited a single band with a molecular mass of 25 kDa under reducing conditions on sodium dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of 125I-labeled IGF-I or 125I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increased synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, we conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.

Amino Acid Sequence↗

PubMed1989

Androgens directly stimulate proliferation of bone cells in vitro.

This report describes the first observation of a direct mitogenic effect of androgens on isolated osteoblastic cells in serum-free culture. [3H]thymidine incorporation into DNA and cell counts were used as measures of cell proliferation. The percentage of cells that stained for alkaline phosphatase was used as a measure of differentiation. Dihydrotestosterone (DHT) enhanced mouse osteoblastic cell proliferation in a dose dependent manner over a wide range of doses (10(-8) to 10(-11) molar), and was maximally active at 10(-9) M. DHT also stimulated proliferation in human osteoblast cell cultures and in cultures of the human osteosarcoma cell line, TE89. Testosterone, fluoxymesterone (a synthetic androgenic steroid) and methenolone (an anabolic steroid) were also mitogenic in the mouse bone cell system. The mitogenic effect of DHT on bone cells was inhibited by antiandrogens (hydroxyflutamide and cyproterone acetate) which compete for binding to the androgen receptor. In addition to effects on cell proliferation, DHT increased the percentage of alkaline phosphatase (ALP) positive cells in all three bone cell systems tested, and this effect was inhibited by antiandrogens. We conclude that androgens can stimulate human and murine osteoblastic cell proliferation in vitro, and induce expression of the osteoblast-line differentiation marker ALP, presumably by an androgen receptor mediated mechanism.

Alkaline Phosphatase↗

PubMed1989

Beta 2-microglobulin is not a bone cell mitogen.

During the purification of skeletal growth factor/insulin-like growth factor-II and transforming growth factor-beta (TGF beta) from EDTA extracts of bovine bone matrix significant mitogenic activity for cultured osteoblast (Ob)-like cells eluted in fractions that contained a nearly homogeneous peptide with a mol wt of about 14,000. This peptide has been purified to apparent homogeneity and identified as bovine beta 2-microglobulin (beta 2M) by amino-terminal amino acid sequencing. During the final purification of beta 2M by CN reverse phase HPLC the mitogenic activity for bone cells separated from the beta 2M protein peak. In spite of this the apparently homogeneous beta 2M preparation retained some mitogenic activity. The ED50 of the bone-derived beta 2M (4,890 +/- 462 ng/ml) was several orders of magnitude (2 x 10(3) to 1.4 x 10(5) times) greater than the ED50 of simultaneously assayed purified growth factors and was no different from the ED50 of the crude EDTA matrix extract (3,350 +/- 890 ng/ml). The beta 2M accounted for less than 0.002% of the total mitogenic activity for Ob-like cells present in the extracted matrix proteins. The following lines of evidence suggest that the mitogenic activity of bone matrix-derived beta 2M (BMD-beta 2M) is due to contamination of the BMD-beta 2M with TGF beta rather than an intrinsic property of beta 2M: 1) the coelution of TGF beta through four successive purification procedures and purification of TGF beta from adjoining fractions from C4 reverse phase HPLC; 2) the abolition of biological activity of BMD-beta 2M and TGF beta with reducing agents; 3) the lack of additive stimulation of [3H] methylthymidine incorporation into bone cells when beta 2M was added to maximally active concentrations of purified TGF beta; 4) the reduction of mitogenic activity when the BMD-beta 2M was incubated with anti-TGF beta; and 5) the inhibition of mink lung epithelial proliferation by the beta 2M preparation. Based on these findings we conclude that although beta 2M is present in bovine bone matrix extracts, it is not a mitogen for Ob-like cells.

Amino Acid Sequence↗

PubMed1989

Castrated men exhibit bone loss: effect of calcitonin treatment on biochemical indices of bone remodeling.

To test the hypothesis that the reduction in gonadal function can lead to bone mass loss, a group of 12 men who had undergone bilateral orchidectomy at the age of 28.2 +/- 6.8 yr was evaluated. A progressive loss of the lumbar bone density was observed as a function of time after orchidectomy. Both the biochemical indices of bone resorption (urinary hydroxyproline/creatinine ratio and plasma tartrate-resistant acid phosphatase) and bone formation (serum osteocalcin and bone isoenzyme of alkaline phosphatase) were significantly increased in the patients compared with healthy controls. A positive correlation was found between urinary hydroxyproline excretion and percent change in spinal bone mineral density per yr. Because of this increase in the biochemically indicated bone resorption, nine of the patients were studied again after 1-3 yr and were thereafter treated with intranasal calcitonin. Urinary hydroxyproline excretion normalized after 3 months of treatment, and a significant decrease, but not to normal levels, was also observed in the mean values for the other biochemical indices of bone remodeling. Thus, testosterone deficiency, like estrogen deficiency, is associated with accelerated bone loss. The increase in osteoresorption was partially corrected by calcitonin treatment.

Adult↗

PubMed1988

Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-II.

Human skeletal growth factor (human SGF) extracted from human bone has been purified to homogeneity by hydroxyapatite chromatography and gel filtration under dissociative conditions followed by FPLC heparin-Sepharose affinity chromatography and reverse phase HPLC. Human SGF was homogeneous except that in each preparation about 30% of SGF molecules lacked the N-terminal alanine. 75% of the human SGF sequence has been determined. The amino acid sequences of the N-terminal 20 amino acids and of several tryptic fragments were identical to the corresponding sequences of human insulin-like growth factor-II (IGF-II) purified from serum. However, since the C-peptide (variable region) of human SGF has not yet been sequenced, we cannot conclude that SGF is identical to IGF-II. Comparison of the amino acid sequence of human SGF with that of IGF-II variants that have been described in the literature revealed that human SGF is not one of the known IGF-II variants. IGF-I was also found in human bone extract but was several-fold less abundant than SGF/IGF-II. The relative abundance of SGF/IGF-II and IGF-I in bone corresponded to the relative rates of production of these two mitogens by human bone cells in vitro. Regarding the physiological significance of IGF-II in bone, previous studies on the biological actions of SGF in vitro suggest that this growth factor can have both paracrine and autocrine functions on cells of the osteoblast line. In addition, we have proposed the concept that SGF is a mediator of the coupling of bone formation to bone resorption, an important bone volume regulatory mechanism. In as much as SGF is very similar (if not identical) to IGF-II, it seems likely that these proposed regulatory functions of SGF in bone are attributable to IGF-II.

Amino Acid Sequence↗

PubMed1988

Cobalamin and osteoblast-specific proteins.

Cobalamin deficiency has well-known hematologic and neurologic effects, but little is known about its other effects. We therefore studied the effect of cobalamin on osteoblast-related proteins. We found that mean (+/- 1 SD) levels of skeletal alkaline phosphatase in the blood were lower in 12 cobalamin-deficient patients (3.89 +/- 2.19 units per liter) than in 5 nondeficient and 5 iron-deficient control subjects (7.55 +/- 3.99 units per liter). The degree of the megaloblastic anemia correlated with the reduction in skeletal alkaline phosphatase levels (r = 0.67, P less than 0.01). With cobalamin therapy, levels of skeletal alkaline phosphatase rose in 11 of the 12 cobalamin-deficient subjects but not in the controls. The cobalamin-deficient patients also had significantly lower osteocalcin levels than the control subjects (1.11 +/- 0.77 vs. 1.84 +/- 0.49 nmol per liter). During cobalamin therapy, these levels rose in the cobalamin-deficient patients but not in the controls. In contrast to the levels of osteoblast-related proteins, hepatic alkaline phosphatase levels were similar in the patients and controls and were usually unaffected by cobalamin therapy. In vitro studies of calvarial cells from chicken embryos showed that their alkaline phosphatase content was cobalamin-dependent, thus supporting our in vivo observations in humans. Our findings suggest that osteoblast activity depends on cobalamin and that bone metabolism is affected by cobalamin deficiency, but we do not yet know whether cobalamin deficiency produces clinically important bone disease.

Adult↗

PubMed1988

Comparison of the biological actions of TGF beta-1 and TGF beta-2: differential activity in endothelial cells.

Beta transforming growth factor (TGF beta) has multiple in vitro biological effects including stimulation or inhibition of proliferation of specific cell types. A second major form of TGF beta, TGF beta-2, has recently been isolated from porcine platelets, from bovine bone matrix, and from several other sources. The two forms of TGF beta are biologically equipotent with the exception that TGF beta-2 was much less active than TGF beta-1 for inhibition of proliferation of a rat pleuripotent hematopoietic stem cell line. During the purification of beta TGF from bone, we obtained two fraction pools that differed in their ability to inhibit 3H-thymidine incorporation into aortic endothelial cells (AEC). We therefore compared highly purified TGF beta-1 and TGF beta-2 isolated from porcine platelets for inhibition of DNA synthesis in mink lung epithelial cells (MvILu), and in AEC, and for stimulation of 3H-thymidine incorporation in calvarial bone cells (CBC) in 3 experiments. TGF beta-1 and TGF beta-2 inhibited cell proliferation in MvILu with no significant differences in the ED50 (31 +/- 8 pg/ml vs 23 +/- 7). TGF beta-2 was much less potent than TGF beta-1 in inhibiting DNA synthesis in AEC (6310 +/- 985 pg/ml vs 101 +/- 34). The reduced specific activity of TGF beta-2 was also observed in adrenal capillary endothelial cells. Both beta-1 and beta-2 stimulated proliferation of CBC (ED50 26 +/- 2 pg/ml vs 10 +/- 4). We also examined the specificity of the MvILu and AEC inhibition assays. Epidermal growth factor (EGF), platelet derived growth factor (PDGF), acidic and basic fibroblast growth factors (FGF), skeletal growth factor (SGF)/insulin-like growth factor-II (IGF-II), and insulin-like growth factor-I (IGF-I) did not inhibit DNA synthesis in either assay system. However, when the growth factors were added to maximal inhibiting concentrations of TGF beta-1, both acidic and basic FGF significantly reduced TGF beta-1 inhibition in AEC. We conclude that (1) inhibition of DNA synthesis in endothelial cells is relatively specific for TGF beta-1, (2) inhibition of DNA synthesis in MvILu is a sensitive and specific assay for generic TGF beta activity but does not distinguish beta-1 from beta-2, (3) the relative inhibition of DNA synthesis in MvILu and AEC may provide a means to quantitatively estimate TGF beta-1 and TGF beta-2, and (4) both TGF beta-1 and TGF beta-2 are potent mitogens for chicken embryonic calvarial bone cells.

Animals↗

PubMed1988

In vitro evidence that local and systemic skeletal effectors can regulate 3[H]-thymidine incorporation in chick calvarial cell cultures and modulate the stimulatory actions(s) of embryonic chick bone extract.

These investigations were intended to determine whether local and systemic skeletal effectors--3'5'-cyclic adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)2D), calcitonin, and NaF--could regulate 3[H]-thymidine incorporation (i.e., into DNA) in serum-free, monolayer cultures of embryonic chick calvarial cells, and/or modulate the activity of embryonic chick bone extracts to increase 3[H]-thymidine incorporation. In the absence of added bone extract, we found that calcitonin (0.1 U/ml), NaF (100 microM) and low-dose PTH (0.1 nM) stimulated 3[H]-thymidine incorporation, P less than .05 for each; isobutylmethylxanthine (IBMX--1 mM), 1,25OHD (10 nM), and high-dose PTH (10 nM) decreased 3[H]-thymidine incorporation; and PGE2 (1 microM) had no effect. The stimulatory actions of calcitonin, fluoride, and low-dose PTH were inductive, and the inhibitory actions of IBMX and 1,25(OH)2D were acute. PTH had complex time-dependent actions on 3[H]-thymidine incorporation, being inhibitory after 4-8 hours of exposure and stimulatory after 20-24 hours (P less than .001 for each). The effects of calcitonin, fluoride, and low-dose PTH to increase 3[H]-thymidine incorporation were greater in calvarial cell cultures enriched for undifferentiated osteoprogenitor cells than in cultures enriched for differentiated osteoblastlike cells. PTH inhibited 3[H]-thymidine incorporation in the latter (i.e., osteoblastlike) cultures (P less than .005). The inhibitory actions of IBMX and 1,25(OH)2D were independent of cell differentiation. Additional studies further revealed that these local and systemic skeletal effectors could also modulate the activity of embryonic chick bone extracts to increase 3[H]-thymidine incorporation in calvarial cell cultures. We found that calcitonin, fluoride, and low-dose PTH enhanced the effect of the extracts to increase 3[H]-thymidine incorporation (P less than .001 for each). These activations were noncompetitive, indicating (1) mechanistic differences between the stimulatory actions of the effectors and the chick bone extract (i.e., different rate-limiting steps for the effects of each on 3[H]-thymidine incorporation); and (2) that neither calcitonin, fluoride, nor 0.1 nM PTH altered the apparent affinity of the cells for stimulatory activity(s) in the extract. High-dose PTH was a noncompetitive inhibitor with respect to bone extract activity, indicating that the effect of 10 nM PTH to decrease 3[H]-thymidine incorporation was mechanistically distinct from the effect of the bone extract to increase 3[H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Relationship between trabecular vertebral body density and fractures: a quantitative definition of spinal osteoporosis.

To evaluate the relationship between vertebral fractures and trabecular vertebral body density (TVBD), as measured by computed tomography (CT), we evaluated 110 female and 38 male patients referred consecutively to our clinic for an osteoporosis evaluation. Number of fractures per patient and TVBD was negatively correlated in both males and females (r = -.64, P less than .001 and r = -.69, P less than .001, respectively). Based on this relationship and that between percent of patients with fracture and TVBD, we devised four different approaches to calculate the fracture threshold. (1) Because the x-axis intercept of this regression line represents the TVBD value at zero fractures, this intercept can be considered the fracture threshold, which was 103 mg/cm3 for females and 132 mg/cm3 for males. (2) Breakpoint analysis of the relationship between the number of vertebral fractures per patient v TVBD gave a fracture threshold value of 98 mg/cm3 for females, but for males we were unable to compute a threshold value because of the small sample size. The percentage of patients with fractures was also negatively correlated with TVBD for males and females (r = -.98, P less than .001, and r = -.94, P less than .001, respectively). (3) the x-axis intercept of this relationship, which represents the fracture threshold, was 123 mg/cm3 for males and 101 mg/cm3 for females. (4) The fracture threshold, calculated as the mean TVBD + 2 SD for patients with fracture(s), was 120 and 92 mg/cm3 for males and females, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1988

Development of a new radioimmunoassay for human osteocalcin: evidence for a midmolecule epitope.

Osteocalcin is a vitamin K-dependent bone-specific protein that can be found circulating in the serum. The circulating levels of osteocalcin have been shown to be an index of bone turnover. Existing radioimmunoassays for osteocalcin have been shown to be specific for C-terminal epitopes, a region that is identical in the human and bovine osteocalcin. There are, however, five amino acids different in the N-terminal region of the molecule. We describe here an immunoassay for a midmolecule epitope of osteocalcin using osteocalcin purified from human femoral head bone powder. Antibody specificity was determined using tryptic digests and a synthetic fragment of human osteocalcin. This assay has only a partial crossreactivity with bovine osteocalcin. This is the first report of an assay against a midmolecular epitope of osteocalcin involving a region in which the human and bovine osteocalcins differ. Osteocalcin levels determined by this assay have a significant correlation with both the total serum alkaline phosphatase and the serum skeletal alkaline phosphatase levels in normal adult human serum and, to a greater degree, in sera of patients with conditions associated with increased bone turnover (Paget's disease, hyperparathyroidism, and newborn sera). These correlations are greater than those previously reported for C-terminal assays, suggesting the possibility that different regions of the molecule may elicit different information concerning bone turnover.

Adult↗

PubMed1988

Evidence that fluoride-stimulated 3[H]-thymidine incorporation in embryonic chick calvarial cell cultures is dependent on the presence of a bone cell mitogen, sensitive to changes in the phosphate concentration, and modulated by systemic skeletal effectors.

In previous studies we have shown that clinically effective concentrations of fluoride (5 to 30 mumol/L) could also have direct effects in vitro on skeletal tissues to increase embryonic chick bone formation and bone cell proliferation (3[H]-thymidine incorporation into DNA). From these observations, we hypothesized that fluoride-stimulated bone formation might be mediated by a direct effect of fluoride to increase bone cell proliferation. The current studies were intended to investigate the mechanism of fluoride-stimulated 3[H]-thymidine incorporation, in chick calvarial cell cultures, by assessing mitogenic interactions between fluoride and inorganic phosphate, bone-derived growth factors, and systemic skeletal effectors. With respect to fluoride-phosphate interactions, the results of our studies indicate that the effect of fluoride was dependent on the phosphate concentration in the medium. Fluoride did not increase 3[H]-thymidine incorporation in BGJb medium containing 1 mmol/L (total) phosphate; but, in 1.6 mmol/L phosphate medium, fluoride caused a dose-dependent increase in 3[H]-thymidine incorporation, between 1 and 20 mumol/L (P less than .001). The action of fluoride was also dependent on the presence of a bone cell mitogen. Fluoride increased 3[H]-thymidine incorporation when added to calvarial cell cultures in the cell-conditioned medium, but had no effect in unconditioned (ie, fresh) medium. The action of fluoride could be restored by adding an exogenous growth factor (ie, concentrated cell-conditioned medium, bone-derived growth factors, or a systemic bone cell mitogen) to the unconditioned culture medium, P less than .05 for each effector.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Bone repletion in vitro: evidence for a locally regulated bone repair response to PTH treatment.

Normally bone formation and resorption are balanced by coupling, but in some conditions such as dietary Ca deficiency, bone resorption exceeds formation, resulting in bone loss (termed bone depletion in previous animal studies). When conditions causing depletion return to normal, a compensatory decrease in resorption and increase in formation occurs, leading to replacement of the lost bone. To test if this recovery process, termed bone repletion, might be locally regulated as in coupling we determined whether cellular and metabolic changes associated with repletion in vivo would occur in vitro in neonatal mouse calvaria. To increase resorption and decrease formation, serum-free cultures were treated with parathyroid hormone (10 nM bovine PTH1-84). Although formation was inhibited ([3H]proline incorporation into [3H]hydroxyproline), the number of bone cells increased during PTH treatment. To simulate repletion, PTH was removed after 3-9 days. Within 6 days of removal of PTH, resorption (osteoclast numbers and 45Ca release) decreased to control levels and bone formation increased to exceed untreated control levels. Autoradiographs of [3H]proline incorporation suggested an increase in the number of active bone forming cells (compared to untreated controls) after removal of PTH. These cellular and metabolic changes were similar to changes which occur during depletion and repletion in vivo. The results support the hypothesis that reversal of the resorptive processes initiated by PTH in organ cultures can occur in the absence of circulating factors. The apparent increase in the components of bone formation that were observed after PTH withdrawal may have resulted from generation of increased numbers of osteoblastic cells during PTH treatment.

Bone Resorption↗

PubMed1988

Bovine skeletal growth factor stimulates protein phosphorylation of chicken bone cells in vitro.

1. Bovine skeletal growth factor (SGF), a potent bone cell mitogen, stimulated protein phosphorylation in cultured chicken calvarial cells. 2. SDS-PAGE followed by autoradiographic analysis of the cellular proteins indicated that [32P] incorporation was enhanced in several proteins in response to 10 ng/ml of SGF (the maximum stimulatory mitogenic dose for these cells). 3. Under conditions favoring tyrosine kinases, SGF stimulated phosphorylation of at least 6 proteins in crude calvarial cell membrane fraction, and caused a time-dependent stimulation of phosphorylation of angiotensin II by crude calvarial cell membrane fractions. 4. Thus, our data demonstrate that SGF stimulates protein phosphorylation in bone cells, and suggest that at least some of the protein phosphorylation involves tyrosine residues.

Angiotensin II↗