Bone Organ Cultures Research Articles

17 beta-estradiol increases calcium content in fetal mouse parietal bones cultured in serum-free medium only at physiological concentrations.

Using a bone organ culture system that shows mineralization in vitro, we investigated whether 17 beta-estradiol dose-dependently increases calcium content in cultured calvarial bones in serum-free medium. Fetal mouse parietal bones (3 x 3 mm) were cultured in phenol red-free BGJ medium containing phosphate (3-4 mmol/L), calcium (1-1.25 mmol/L), insulin (6 micrograms/ML), and transferrin (6 micrograms/mL) for 4-5 days. Under these culture conditions, the calcium content of the cultured bones (at dissection 34.0 +/- 4.6 micrograms/bone [mean +/- SD], n = 50) increased by 15-20 micrograms during 4-5 days of culture. 17 beta-Estradiol increased the calcium content significantly at 10(-12) to 10(-11) mol/L, but not at lower (10(113) mol/L) or higher (10(-10) to 10(-9) mol/L) concentrations. 17 alpha-Estradiol had no effect. The stimulatory effect of 17 beta-estradiol was completely inhibited by the antiestrogen agent ICI-182,780. The anabolic effect of 17 beta-estradiol was elicited not only in bones from females but also in those from males. 17 beta-Estradiol had no significant effect on 45Ca release from prelabeled parietal bones. Furthermore, light- and electron-microscopic examinations revealed that bone mineralization proceeded through formation of matrix vesicles, without any metastatic or dystrophic calcification. These in vitro findings suggest that 17 beta-estradiol elicits small, but reproducible, direct effects on calcium content in the parietal bones not only in female but also in male fetal mice at physiological-free E2 concentrations (10(-12)-10(-11) mol/L), which is attainable in serum of normal human subjects. In contrast to in vivo studies, pharmacological doses of 17 beta-estradiol had no anabolic effect on parietal bones. The mechanism of such a biphasic effect of estrogens remains to be elucidated.

Effects of synthetic peptido-leukotrienes on bone resorption in vitro.

Peptido-leukotrienes are short-lived organic molecules known to have potent biological effects as mediators of inflammation, hypersensitivity and respiratory disorders. However, little is known concerning their effects on bone cells. We have shown previously that stromal cells isolated from a human giant cell tumor secrete 5-HETE (5-hydroxyeicosatetraenoic acid) and the peptido-leukotrienes, also known as the cysteinyl leukotrienes LTC4, LTD4, and LTE4. These eicosanoids were shown to stimulate the multinucleated giant cells obtained from these tumors to form resorption lacunae on sperm whale dentine. Here, we show that the peptido-leukotrienes also stimulate isolated avian osteoclast-like cells to form resorption lacunae and to increase their content of tartrate-resistant acid phosphatase. LTD4 increased 45Ca release from murine calvarial bone organ cultures, but not from fetal rat long bone cultures. Isolated avian osteoclast-like cells were chosen to perform receptor binding studies, as this population is the most homogeneous source of osteoclasts available. After the precursors had fused to form multinucleated cells, receptor binding assays were performed. Scatchard analysis of saturation binding data showed a single class of binding sites, with a dissociation constant (Kd) of 0.53 nM and a receptor density of 5,200 receptors per cell. Competition binding studies showed receptor specificity using a specific LTD4 receptor antagonist ZM 198,615. These data show that the peptido-leukotrienes activate highly enriched populations of isolated avian osteoclast-like cells, and also that specific LTD4 receptors are present in this cell population.

Effect of KCA-098 on the function of osteoblast-like cells and the formation of TRAP-positive multinucleated cells in a mouse bone marrow cell population

KCA-098 (3,9-bis(N,N-dimethylcarbamoyloxy)-5H-benzofuro[3,2-c]quinoline-6-one), an analogue of coumestrol (a naturally occurring weak phytoestrogen), dose-dependently increased alkaline phosphatase activity of osteoblastic ROS 17 2.8 cells and freshly-isolated osteoblasts from neonatal mouse calvaria, and reduced cell proliferation. In addition, KCA-098 increased the synthesis of collagenese-digestible protein (CDP) of ROS 17 2.8 cells. On the other hand, KCA-098 had no effect on the basal synthesis of osteocalcin but reduced the 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2D 3)-induced increase in osteocalcin synthesized by ROS 17 2.8 cells. Therefore, KCA-098 had a bidirectional effect on the differentiation of osteoblasts (i.e., stimulating both alkaline phosphatase activity and synthesis of CDP and inhibiting osteocalcin synthesis). However, as KCA-098 stimulated the mineralization of chick embryonic bone in organ culture and recovered the bone density reduced by ovariectomy of rats, it would serve overall to stimulate the differentiation of osteoblasts. On the other hand, KCA-098 inhibited the formation of tartrate-resistant, acid phosphate-positive multinucleated cells (TRAP(+)MNC) induced by 1α,25(OH) 2D 3, parathyroid hormone (PTH), and prostaglandin E 2 (PGE 2) in cultures of mouse bone marrow cells, showing that it inhibits the formation of osteoclastlike cells. Coumestrol and 17β-estradiol had no effect on the proliferation and alkaline phosphatase activity of ROS 17 2.8 cells. They did, however, dose-dependently inhibit osteoclast-like cell formation as well as KCA-098 did, indicating that the main action of coumestrol and 17β-estradiol on bone tissue is the inhibition of bone resorption.

Role of tumor necrosis factor alpha in particulate‐induced bone resorption

The purpose of this study was to determine the role of tumor necrosis factor alpha in bone resorption secondary to mediator release from macrophages exposed to cement particles. The J774 mouse macrophage cell line was exposed to polymethylmethacrylate particles for 24 hours and the resulting conditioned medium was analyzed for prostaglandin E2, tumor necrosis factor alpha, interleukin-1 alpha and beta, and the ability to stimulate release of prostaglandin E2 and 45Ca from radiolabeled mouse calvaria. Macrophage exposure to polymethylmethacrylate particles led to a 9-fold increase in release of tumor necrosis factor alpha (p < 0.01), but did not lead to a significant increase in release of prostaglandin E2, interleukin-1 alpha, or interleukin-1 beta when compared to unexposed cells. Exposure of the macrophages to polymethylmethacrylate particles over a time course from 30 minutes to 96 hours led to an increase in the release of tumor necrosis factor alpha that was initially detected at 30 minutes and was maximum at 48 hours. Incubation of the macrophage-polymethylmethacrylate conditioned medium with rat calvaria significantly increased the release of 45Ca and prostaglandin E2 from the bone. To study the role of release of tumor necrosis factor alpha in bone resorption, the macrophage-polymethylmethacrylate conditioned medium was then preincubated with anti-tumor necrosis factor alpha antibody prior to exposure of the conditioned medium to the calvaria. This preincubation was successful in significantly inhibiting 45Ca release by calvaria (p <0.01) to levels that were not significantly different from the levels of release by unexposed calvaria. Tumor necrosis factor alpha appears to play a critical role in initiating particulate-induced bone resorption. Exposure of macrophages to polymethylmethacrylate particles leads to a significant release of tumor necrosis factor alpha in a time-dependent fashion. This macrophage-polymethylmethacrylate conditioned medium stimulated release of prostaglandin E2 and bone resorption in bone organ culture. The addition of anti-tumor necrosis factor alpha antibody to this in vitro system inhibited the bone resorption stimulated by the macrophage-polymethylmethacrylate conditioned medium and partially suppressed the production of prostaglandin E2. The sequence of events in this model for particulate-induced bone resorption appears to be initiated by the production of tumor necrosis factor alpha by the macrophage, followed by production of prostaglandin E2 by cells in bone, and then by bone resorption.

Regulation of Bone Sialoprotein and Osteopontin mRNA Expression by Dexamethasone and 1,25-dihydroxyvitamin D3in Rat Bone Organ Cultures

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent components of the extracellular matrix of mineralized connective tissues that have been implicated in the formation and remodelling of bone. Although these proteins have similar biochemical properties and are expressed by bone cells during bone formation it has been suggested that they have different functions and that their expression is regulated independently by hormones and cytokines. The precise role of these proteins has, however, yet to be firmly established. Since steroid hormones strongly influence the formation of bone we have analyzed the effects of glucocorticoids and 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3) on the expression of BSP and OPN mRNAs in developing rat bone in vitro using in situ hybridization. In these studies it has been possible to identify the nature and spatial distribution of the cells that respond to these hormones by changes in sialoprotein expression. When cultured in the presence of the synthetic glucocorticoid, dexamethasone (dex), expression of BSP mRNA by hypertrophic cartilage cells in the tibiae and mandible was dramatically increased as were the number of responding cells indicating that glucocorticoids promote differentiation of hypertrophic cartilage cells as well as osteoblasts. Dexamethasone also stimulated a marked (> 5-fold) increase in OPN expression by osteoblasts and cells lining endosteal and periosteal bone surfaces. In contrast to dex, 1,25-(OH)2D3 suppressed BSP expression in osteoblastic cells whereas OPN expression was strongly (> 5-fold) stimulated in all three cultured bone tissues. Histological examination of the tissues showed that cell viability was retained over the culture period. However, in the presence of 1,25-(OH)2D3 considerable resorption of the tissue was evident, with cement and reversal lines being prominent. The increased expression of BSP and OPN by dex is consistent with the stimulation of bone formation by glucocorticoids, whereas the differential effects of 1,25-(OH)2D3 on BSP and OPN may reflect a stimulation of bone remodelling.

Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro

In a previous study of the cellular mechanism underlaying Wolff's law we showed that mechanical stimulation by intermittent hydrostatic compression (IHC) increases bone formation in cultured fetal mouse calvariae compared to non-stimulated cultures. To test whether mechanical stimuli may modulate bone-specific gene expression, we studied the effect of IHC on alkaline phosphatase (AP) expression and enzyme activity as well as collagen and actin mRNA levels in neonatal mouse calvariae and calvarial bone cells. Two cell populations, one resembling osteoprogenitor (OPR) cells and another resembling osteoblasts (OB) were obtained from calvariae by sequential digestion. IHC was applied by intermittently (0.3 Hz) compressing the gas- phase of a closed culture chamber (peak stress 13 kPa, peak stress rate 32.5 kPas −1). In control cultures of calvariae as well as OB and OPR cells, AP activity and AP-, collagen-, and actin-mRNA levels all decreased after one or more days, with the exception of OPR cell collagen expression which increased during culture. IHC treatment upregulated AP, collagen and actin expression and AP activity in calvariae and OB cells, but decreased collagen expression in OPR cells. These results suggest that treatment with IHC promotes the osteoblastic phenotype in bone organ cultures and in osteoblasts. Osteoprogenitor cells seem to react somewhat differently to mechanical stress than osteoblasts. The loss of bone-specific gene expression under control culture conditions, in the absence of mechanical stimuli, suggests that the mechanical environment is important in maintaining the differentiated phenotype of bone cells, and that IHC treatment partially restores this environment in bone cell- and organ cultures.

Intermittent compression stimulates cartilage mineralization.

The effects of intermittent hydrostatic compressive force (ICF; 13 kPa applied at 0.3 Hz frequency), as a substitute for moderate loading in vivo, on ossifying bone organ cultures, were evaluated by means of (histo)-morphometry. In earlier studies, biochemical tests have shown an increased 45Ca intake and an increased alkaline phosphatase activity in bone organ cultures that received ICF, suggesting that ICF promoted matrix mineralization. The purpose of this study was to examine whether an effect of ICF on mineralization can be described by means of histomorphometrical analysis. Fetal mouse metatarsal bone rudiments were cultured for 5 days in serum-free medium, with (experimental) or without (control) ICF. Linear measurements taken during culture demonstrated that the dark zone in the center of the rudiment, representing mineralized hypertrophic cartilage, became significantly longer in the group that received ICF when compared with the control group. This finding was in conformation with the former studies. Histological sections of the rudiments, stained with Goldner's trichrome method were used to study changes at the cellular level and to describe the position and relative amount of mineralizing cartilage matrix (defined as Goldner-positive matrix [GPM]). Histomorphometry demonstrated that ICF treatment significantly increased the length of the hypertrophic cartilaginous zone and enhanced the amount of GPM between the mineralizing hypertrophic chondrocytes. However, the total length of the zone containing GPM was not increased, nor was the future bone collar, consisting of a thin osteoid seam, lengthened by ICF. These data indicate that the cellular processes involved in chrondrocyte hypertrophy were accelerated by ICF, as well as the extracellular processes leading to matrix mineralization. The study supports the earlier conclusion that embryonic bone rudiments are sensitive to mechanical stimulation and that moderate loading promotes their ossification in vitro.

Calvarial and limb bone cells in organ and monolayer culture do not show the same early responses to dynamic mechanical strain

Responses to mechanical strain in calvaria and limb bone organ cultures were compared by measuring cellular glucose 6-phosphate dehydrogenase (G6PD) activity in situ and prostaglandin release. Normal functional strains were recorded in the ulnae (1000 mu epsilon) and calvarium (30 mu epsilon) in vivo in 110 g rats. Organ cultures of ulnae and calvaria from similar animals were loaded to produce dynamic strains (600 cycles, 1 Hz) of 1000 mu epsilon in the ulna, and 100 or 1000 mu epsilon in calvaria. In ulnae, both PGE2 and PGI2 were released and resident osteocytes and osteoblasts showed increased G6PD activity. Neither response was seen in calvaria. However, exogenous PGI2 (10(-5)-10(-9) M) stimulated G6PD activity in osteocytes and osteoblasts in organ cultures of both calvaria and ulnae. In ulnar cells the response was linear, in calvarial cells it was biphasic with maximum activity at 10(-7) M. Osteoblasts derived from ulnae and cultured on plastic plates subjected to dynamic strain (600 cycles, 1 Hz, 4000 mu epsilon) showed increased G6PD activity. There was no such response in similarly treated calvarial-derived cells. Calvarial bone cells differ from those of the ulna in that they do not respond to physiological strains in their locality with increased prostanoid release or G6PD activity either in situ or when seeded onto dynamically strained plastic plates. Cells from both sites in organ culture show increased G6PD activity in response to exogenous PGI2, but their dose:responses differ in shape. These differences may reflect the extent to which functional loading influences bone architecture in these two sites.

The effect of glucocorticoids on osteoblast function. The effect of corticosterone on osteoblast expression of beta 1 integrins.

Prolonged treatment with glucocorticoids is known to produce osteoporosis, which is characterized by a decrease in bone mass. Therefore, we studied the effect of glucocorticoids on the formation of bone and on the expression of beta 1 integrins in a mineralizing organ culture of fetal rat parietal bone. Integrins are a family of integral membrane glycoproteins that mediate the adhesion of cells to extracellular matrix macromolecules and affect the growth and differentiation of cells. In situ hybridization with a 32P-labeled beta 1 integrin cDNA probe was performed on parietal bone, treated with or without 100-nanomolar corticosterone for ninety-six hours, to localize and assess the levels of beta 1 integrin mRNA quantitatively. Corticosterone decreased beta 1 integrin mRNA in the osteoblast layer but not in the periosteum. Northern blot analysis demonstrated a 62 per cent decrease in the levels of beta 1 integrin mRNA in the osteoblast layer of bone that had been stripped of its periosteum. Immunofluorescence microscopy confirmed these results, as they demonstrated a decrease in the levels of beta 1 integrin protein predominantly in the osteoblast layer. This effect was dependent on the concentration of corticosterone. During ninety-six hours of culture, the calcium content and the dry weight of control parietal bone increased 157 per cent and 57 per cent, respectively. However, treatment of these cultures with 100-nanomolar corticosterone inhibited calcification by 24 per cent. The administration of glucocorticoid had no significant effect on the DNA content or dry weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids inhibit the attachment of osteoblasts to bone extracellular matrix proteins and decrease beta 1-integrin levels.

Prolonged glucocorticoid treatment causes osteoporosis in vivo and inhibits bone formation in vitro. We have previously shown that glucocorticoids inhibit calcification and alter osteoblast organization in a mineralizing bone organ culture system. In this study, the effect of glucocorticoids on osteoblast adhesion to bone matrix proteins and integrin expression was examined in primary rat osteoblasts and a transformed rat osteosarcoma-derived cell line ROS 17/2.8. After 24 h of treatment with corticosterone, these cells displayed a concentration-dependent decrease in adhesion to type I collagen and fibronectin. Adhesion was significantly decreased as early as 4 h after glucocorticoid administration. With 100 nM corticosterone treatment for 24 h, inhibition of the adhesion of ROS 17/2.8 cells and primary osteoblasts to fibronectin was 75 +/- 10% and 50 +/- 8%, and inhibition of adhesion to collagen was 31 +/- 10% and 65 +/- 5%, respectively. This effect was specific for osteoblasts, because glucocorticoids did not change the adhesion of fibroblasts. However, glucocorticoids did inhibit the adhesion of all cell types to rat osteonectin. To determine whether the change in osteoblast attachment to collagen and fibronectin was due to an alteration in integrin levels, the plasma membranes of these cells were labeled with [125I]lactoperoxidase, solubilized, and immunoprecipitated with an antibody to beta 1. A 24-h treatment with 100 nM corticosterone caused 80 +/- 2% and 64 +/- 9% decreases in beta 1 levels in primary osteoblasts and ROS 17/2.8 cells, respectively. These results were confirmed with immunofluorescence microscopy, which showed a glucocorticoid-induced decrease in beta 1 staining. Treatment of primary rat osteoblasts and ROS 17/2.8 cells for 72 h with corticosterone also decreased beta 1-integrin messenger RNA levels in a dose-dependent manner. We have demonstrated that the inhibition of integrin expression by glucocorticoids is involved in the decrease in osteoblast adhesion to bone extracellular matrix proteins. These data suggest that integrin modulation may influence osteoblast function and bone formation and, thus, contribute to glucocorticoid-induced osteoporosis.

The anabolic effects of progesterone on fetal rat calvaria in tissue culture.

We examined the effects of progesterone and cortisol on [3H] thymidine incorporation into DNA, total DNA content, and [3H] proline incorporation into collagen using a bone organ culture system. The study consisted of nine experiments conducted at the University of Connecticut after approval by the animal care committee. Fetal rat half calvaria were cultured in the presence of progesterone (10(-5) to 10(-15) mol/L) and/or cortisol (10(-7) mol/L). Statistical analysis was carried out by analysis of variance. There was a biphasic increase in [3H] thymidine incorporation and DNA content of the tissue sample with progesterone treatment, with peaks at both high and low concentrations. Significant increases in [3H] proline incorporation were noted at concentrations of 10(-6) to 10(-10) mol/L of progesterone. Similar responses were observed in the presence of cortisol. Progesterone increased [3H] thymidine incorporation into DNA, total DNA content, and [3H] proline incorporation into collagen proteins in cultured fetal rat calvaria over a wide range of progesterone concentrations in the presence and absence of cortisol.

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins, osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, gamma-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 micrograms/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes.

Integrins and osteoclastic resorption in three bone organ cultures: differential sensitivity to synthetic Arg-Gly-Asp peptides during osteoclast formation.

We investigated possible inhibitory effects of five synthetic Arg-Gly-Asp (RGD)-containing peptides on osteoclastic resorption in three distinct in vitro resorption assays (17-day-old fetal mouse bone organ cultures) that differ in stages of osteoclast differentiation. RGD peptides, which can bind the adhesion receptors called integrins, inhibited osteoclastic resorption (45Ca release) in fetal mouse bone explants in which osteoclast precursors have yet to adhere to the mineralized matrix and develop into mature osteoclasts (metacarpals and coculture system). Treatment of metacarpals with RGD peptides inhibited the formation of multinucleated TRAP+ osteoclasts in the mineralized matrix because their mononuclear TRAP+ osteoclast precursors remained localized in the periosteum. In particular, echistatin, a viper venom protein with known affinity for alpha v beta 3 integrin, and GdRGDSP inhibited osteoclastic resorption dose dependently in these systems (ED50 10(-9) and 10(-4) M, respectively) but did not alter the activity of mature resorbing osteoclasts in radii. In addition, 45Ca release was significantly inhibited by the cyclic peptide GPenGRGDSPCA, which has a relatively higher affinity for the vitronectin than fibronectin receptor(s). In contrast, GRDGdSP, which has a much higher affinity for the fibronectin receptor (than the vitronectin receptors), had no effect on resorption at similar concentrations in any resorption system used. In summary, the data presented in this paper show that peptides with RGD motifs are capable of inhibiting osteoclastic resorption in bone organ cultures. Our studies not only support the hypothesis concerning the importance of alpha v beta 3 in osteoclastic resorption but also suggest an important role of integrin(s) in events preceding the actual resorption of calcified matrix by osteoclasts.

Prostaglandin E 2 Stimulates Preosteoblast Replication: An Autoradiographic Study in Cultured Fetal Rat Calvariae

To determine the mechanism for the anabolic effect of prostaglandins (PGs) on bone, [ 3H]thymidine(TdR) autoradiography was performed to localize the cells that replicate and to determine the fate of these labeled cells over time in cultured 20-day fetal rat calvariae. Treatment of bone organ cultures for 24 or 96 h with cortisol significantly decreased the number of [ 3H]TdR-labeled osteoblasts and periosteal cells. Treatment of bones with 0.1 or 1 μ M PGE 2 alone had no effect on [ 3H]TdR labeling of the osteoblast layer but doubled the number of labeled periosteal cells compared to control bones at 24 h. However, when both cortisol and PGE 2 were added together, the number of labeled osteoblasts and periosteal cells increased. This effect was dependent on the concentration of PGE 2. In bones treated with 0.1 μ M PGE 2 and 0.1 μ M cortisol for 24 h, [ 3H]TdR labeling of periosteal cells was 5.5 times the labeling in bones treated with cortisol alone and osteoblast labeling increased 4.1-fold. Similar results in [ 3H]TdR labeling of osteoblasts and periosteal cells were found with 6-day neonatal mouse calvarin treated with PGE 2 and cortisol. These data demonstrate that PGE 2 in the presence of cortisol increases periosteal cell replication, which leads to an increased number of osteoblasts and increased bone formation.

Thyroid hormones increase insulin-like growth factor mRNA levels in the clonal osteoblastic cell line MC3T3-E1

Thyroid hormones are known to affect skeletal growth and maturation by influencing both bone resorption and bone formation. Their exact mechanism of action, however, is still unknown. Local factors such as prostaglandins, TGF-β or IGF-I were suggested to mediate their effects. Thyroid hormones were reported to stimulate expression of IGF-I mRNA in liver and kidney and to increase IGF-I release from bone organ cultures and osteoblast-like cells. Therefore we studied the effect of thyroid hormones on IGF-I mRNA expression in MC3T3-E1 cells. The cells were grown in culture for 5 to 7 days and treated with triiodothyronine (10 −11 - 10 −6 M) and thyroxin (10 −6 M) for 1–24 h. Cellular mRNA was isolated and subjected to Northern hybridization. The amount of IGF-I mRNA, which is already expressed in this cell line under control conditions, was markedly enhanced by T3 and T4. This effect was found to be dose-dependent with a maximum at 10 −7M and could already be seen after 3 h increasing up to 24 h. Our findings indicate that IGF-I expression in osteoblasts is directly regulated by thyroid hormones. We conclude that IGF-I expression belongs to the phenotypic characteristics of mature osteoblasts, and that thyroid hormones play an important role in differentiation of MC3T3-E1 cells along the osteoblastic lineage.

Effects of medium acidification by alteration of carbon dioxide or bicarbonate concentrations on the resorptive activity of rat osteoclasts.

Little is known about the extracellular conditions or factors that stimulate mature osteoclasts to resorb mineralized tissues. Isolated mammalian osteoclasts are strongly stimulated by protons in HEPES-buffered culture media in the absence of CO2 and HCO3-, but it has been reported that cell-mediated Ca2+ release from bone organ cultures is increased only when media are acidified by reduction of HCO3- concentrations, and not by increasing PCO2 (considered models of metabolic and respiratory acidosis, respectively). We investigated this question using disaggregated rat osteoclasts cultured on dentin slices for 24 h. The number of pits resorbed per osteoclast was stimulated in media acidified by manipulation of either HCO3- or CO2 concentrations. In experiments in which incubator CO2 was varied, resorption was almost abolished in the presence of 2.5% CO2 at pH 7.61 but increased in a stepwise manner up to 1.3 pits per osteoclast when dentin slices were cultured with 10% CO2 at pH 6.97. The depths and widths of pits, measured using a confocal laser reflection microscope, also tended to increase with increasing CO2 and decreasing pH. However, in experiments where pH was lowered by reducing medium HCO3-, pit size decreased, partially offsetting the increased number of pits resorbed per osteoclast. These findings suggest that rat osteoclasts may be more sensitive to stimulation by CO2 acidosis than by HCO3- acidosis, at least in the short term, and may possibly reflect local regulatory processes in bone.

Characterization and autoregulation of latent transforming growth factor beta (TGF beta) complexes in osteoblast-like cell lines. Production of a latent complex lacking the latent TGF beta-binding protein.

We have previously shown that bone organ cultures produce large amounts of latent transforming growth factor beta (TGF beta), which lacks latent TGF beta-binding protein (LTBP). In this study we used the known osteoblast-like cell lines UMR-106, ROS 17/2.8, and MG63 as models to further examine latent TGF beta expression in bone. We found that the osteosarcoma cell line UMR-106 secreted latent TGF beta almost exclusively as a 100-kDa complex lacking LTBP. ROS 17/2.8 cells produced both the 100-kDa complex and also a 290-kDa complex containing the fibroblastic (190 kDa) form of LTBP. MG63 cells (like human foreskin fibroblasts) expressed almost exclusively the 290-kDa complex. To investigate the regulation of latent TGF beta complexes in bone cells we assessed the effects of TGF beta 1 treatment on expression of active and latent TGF beta. TGF beta 1 induced secretion of latent but not active TGF beta in all cell types examined. In human foreskin fibroblast cells, TGF beta 1 and LTBP mRNA were expressed concomitantly. In contrast, in osteosarcoma cell lines autoinduction of TGF beta 1 mRNA was associated with either a delayed increase or no change in LTBP mRNA. In UMR-106 cells LTBP message was virtually undetectable. We postulate that the expression of different latent TGF beta forms by osteoblast-like cells may reflect their maturation states and that different latent TGF beta complexes may have different functions, for example as secretory forms or as matrix storage forms.

Abnormalities of phosphoprotein gene expression in three osteopetrotic rat mutations: elevated mRNA transcripts, protein synthesis, and accumulation in bone of mutant animals.

Osteoclast abnormalities that characterize osteopetrosis, a disorder of bone resorption, may derive from aberrant signals from the osteoblast or the bone matrix. In the present studies, both synthesis and the bone matrix content of the major bone phosphoprotein component, osteopontin, were found to be elevated in three osteopetrotic rat mutations (ia, op, and tl). In whole bone, a twofold increase in the content of the characteristic amino acid O-phosphoserine for osteopontin occurred in op and tl mutant long bone, but a smaller (15%) and more variable increase was observed in ia mutant rat long bone. Extraction of the bone matrix components and partial purification by reverse phase chromatography showed a twofold increase in a phosphoprotein fraction relative to other noncollagenous components. Amino acid analysis and staining characteristics of SDS-PAGE fractionated proteins indicated this to be osteopontin. Organ cultures of calvarial bone from 4 day ia osteopetrotic mutant and normal rats in the presence of 3H-proline showed increased synthesis of this 60 kD protein, which was stimulated by vitamin D. Preparation of total cellular RNA from bone of 2- and 6-week-old mutants and normal rats supported increased synthesis of osteopontin as reflected by hybridization with osteopontin cDNA probe, showing significantly higher levels of mRNA transcripts in ia (3-5 fold), tl (1.4-2 fold), and op (6-25 fold) mutant bone compared to normal littermates. The changes in osteopontin mRNA levels in mutant bone were also examined in relation to other growth and phenotype-expressed genes. The findings of increased accumulation of osteopontin in osteopetrotic bone and increased synthesis by osteoblasts are interesting in light of the previously reported decrease in bone osteocalcin content (Endocrinology, 126:966, 1990), confirmed here by decreased osteocalcin mRNA transcripts. Such aberrations in the composition of skeletal extracellular matrix could be a reflection of or a contributing factor to the osteoclast abnormalities of some of these osteopetrotic disorders.

Evidence that interleukin-1 mediates its effects on bone resorption via the 80 kilodalton interleukin-1 receptor.

Interleukin-1 (IL-1) mediates its effects through two distinct receptors, one of 80 kilodaltons (80 kD) present in athymic lymphocytes and fibroblasts, and one of 60 kD present in cells of the monocyte-macrophage lineage. A novel monocyte cytokine in the IL-1 family which binds to both the 80 and the 60 kD receptors has been purified, cloned, and expressed. As the interleukin-1 receptor antagonist (IL-1ra) has been shown to inhibit bone resorption in organ culture, it is not clear whether these effects are mediated through the 80 or the 60 kD receptor. Recently, neutralizing antibodies (35F5) have been developed to the 80 kD receptor which inhibit IL-1 effects mediated through this receptor. To determine the importance of the 80 kD receptor to IL-1-mediated bone resorption, we used the neutralizing antibodies (35F5) to the 80 kD receptor to determine if they inhibited bone resorption stimulated by IL-1 in bone organ cultures. The 35F5 antibody blocked bone-resorbing activity due to IL-1 completely, and also blocked control or "endogenous" bone-resorbing activity present in murine bone organ cultures incubated in control media. The 35F5 antibody had no effect on bone resorption mediated by tumor necrosis factor (TNF), or parathyroid hormone (PTH). These data suggest that the availability of the 80 kD IL-1 receptor is required for osteoclastic bone resorption mediated by IL-1.

Recombinant human transforming growth factor beta 1 modulates bone remodeling in a mineralizing bone organ culture.

TGF-beta 1 increases cell proliferation and collagen synthesis in osteoblast-like cells and bone organ cultures. However, the effects of TGF-beta 1 on bone resorption remain contradictory. Therefore, the exact role that this growth factor plays in the process of bone remodeling is still not clear. We studied the effects of recombinant human TGF-beta 1 (rhTGF-beta 1) on bone formation and resorption in a mineralizing bone organ culture system. Parietal bones from 20-day-old fetal rat calvariae were cultured up to 7 days in serum-free BGJb medium. They responded to a 1 day pulse or continuous treatment of rhTGF-beta 1 with dose-dependent increases in dry weight, [3H]thymidine ([3H]TdR) incorporation, and collagen synthesis. In contrast, rhTGF-beta 1 reduced the calcium content of the bones. This is not due to increased bone resorption but rather to failure of calcium deposition. The following responses occurred at 1 nM rhTGF-beta 1. Dry weight was increased 25-50% after 6 days in culture. DNA synthesis was increased to a maximum at day 1, reaching twofold of the control level. Adding hydroxyurea at day 0 reduced [3H]TdR incorporation in rhTGF-beta 1 treated bones to 20% of the control and indomethacin abrogated the increase in [3H]TdR stimulated by rhTGF-beta 1 to the control level. Both treatments completely blocked the increase in dry weight induced by rhTGF-beta 1 at day 6. rhTGF-beta 1 stimulated collagen synthesis to reach its maximum at day 2, with a twofold increase in [3H]proline incorporation. Basal alkaline phosphatase activity fell continuously in culture, reaching 35% of day 0 level at day 6. Enzyme activity was not altered by rhTGF-beta 1. Morphologic observations by light and electron microscopy confirmed these findings. In summary, rhTGF-beta 1 altered bone remodeling by increasing organic components and decreasing calcification in a mineralizing bone organ culture system.