Fetal Rat Calvaria Research Articles

Effects of helodermin on fetal rat bone metabolism in vitro

Helodermin belongs to the VIP family of polypeptides. Recent in vivo data suggest that helodermin-like peptides might be involved in the regulation of calcium metabolism. We show that helodermin specifically binds to a secretin-type receptor in osteoblast-like cells from fetal rat calvaria and increases the basal and PTH-stimulated cAMP concentration of these cells. In organ cultures of fetal rat calvaria, helodermin strongly inhibits bone matrix apposition and augments PTH-induced bone resorption. Helodermin-like peptides may thus be capable of enhancing the direct effects of PTH on bone metabolism.

Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone

Osteoprogenitor cells present in single-cell suspensions prepared from fetal rat calvaria (RC) form discrete mineralized three-dimensional bone nodules when cultured long-term in the presence of ascorbic acid and beta-glycerophosphate. These cells (CFU-O) constitute less than 1% of the total cell population under standard culture conditions and their number is increased in the presence of dexamethasone. Using the formation of the bone nodule as a marker for CFU-O, we have now analyzed the proliferation and differentiation capacity of these CFU-O by redistribution and continuous subculture experiments in the presence and absence of dexamethasone. Cell redistribution experiments showed no increase in nodule number after one population doubling with either treatment. After 5.4 population doublings of the entire RC population, nodule number increased up to 2.0-fold in control cultures and 4.5-fold in cultures containing 10 nM dexamethasone. Continuous subculture experiments in which cultures were split 1:3 every 3 day for up to seven subcultures showed that nodule number decreased in parallel with the split ratio in the absence of dexamethasone, while with dexamethasone nodule number was elevated above the number present in primary cultures for 1 or 2 subcultures after which nodule number decreased with the split ratio. Bone nodules were present for up to 18 population doublings. Measurements of nodule area by automated image analysis showed that dexamethasone increased nodule size and that nodule size decreased from primary to 1st to 2nd subculture with or without dexamethasone. The data suggest that dexamethasone selectively stimulates the proliferation of osteoprogenitor cells and that these progenitor cells have a limited capacity for generating daughter cells capable of expressing the bone phenotype.

Stimulation of Bone Matrix Appositionin Vitroby Local Growth Factors: A Comparison between Insulin-like Growth Factor I, Platelet-Derived Growth Factor, and Transforming Growth Factor β*

Many recent in vitro studies have shown effects of insulin-like growth factor I (IGF I), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) on the proliferation and differential functions of bone-forming osteoblasts; however, the question whether these factors might ultimately lead to a net increase or decrease in bone formation has been difficult to assess. In this study, we have used an autoradiographic method based on the incorporation of [3H]proline into freshly synthesized bone matrix to determine the overall effects of these factors on bone matrix apposition in 21-day-old fetal rat calvariae. IGF I, PDGF, and TGF beta increased bone matrix apposition in a dose-dependent manner up to 2-fold within 48 h. In addition, they partially or completely reversed the inhibition of bone matrix apposition observed with PTH. Exogenously added TGF beta was significantly more potent than equimolar concentrations of PDGF or IGF I in stimulating bone formation. Matrix apposition was greatest when IGF I, PDGF, and TGF beta were added simultaneously to the culture medium, indicating that these factors can enhance each other in stimulating bone formation. In conclusion, our results provide direct evidence that IGF I, PDGF, and TGF beta are capable of stimulating bone formation in vitro.

Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.

Effect of cadmium on the bone formation in cultured fetal rat calvaria

To clarify the toxic effect of Cadmium (Cd) on bone, alkaline phosphatase (ALP) activity, which is an osteoblastic marker produced during bone formation, and DNA synthesis were examined in cultures of 21‐day old fetal rat calvaria. Both ALP activity and DNA synthesis were decreased by Cd. Probably Cd affected some stage of mineralization or cell differentiation in bone formation of calvaria. Zn coexisting in the culture medium prevented the inhibitory effect of Cd on ALP activity and DNA synthesis.

Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones

We have compared the effects of of various synthetic amino-terminal forms of human parathyroid hormone-related peptide (PTHrP) of malignancy with synthetic parathyroid hormone (PTH) on the resorptive responses of fetal rat long bones in organ culture. PTH and PTHrP increased 45Ca release at concentrations of 0.1-25 nM. PTHrP (1-40) and bovine PTH (1-34) were more potent than human PTH (1-34) and PTHrP (1-34). However, the slopes of the dose-response curves and the maximal resorptive effects were similar. There was a marked decrease in the potency of amino-terminal PTHrP peptides as the length was decreased. PTHrP (1-29) and PTHrP (1-25) were inactive at 120 nM. Further comparison of bPTH (1-34) and PTHrP (1-34) showed that both could induce bone resorption after a brief (6 hours) exposure and that the response to PTHrP (1-34) was qualitatively similar to that of bPTH (1-34) with respect to enhancement by ACTH and inhibition by calcitonin and glucocorticoids. Hydroxyurea and indomethacin did not block the resorptive response to either agonist. Cyclic AMP production in response to PTHrP (1-34) and (1-40) was similar to that for bPTH (1-34) in ROS 17/2.8 cells. The cyclic AMP (cAMP) response was much smaller in fetal rat long bones and calvariae, and bPTH was more potent than PTHrP. These studies confirm that PTHrP is quantitatively similar in its effects on bone resorption to PTH and are consistent with the two agents acting on the same receptor.

Differential Effects of Continuous and Transient Treatment with Parathyroid Hormone Related Peptide (PTHrp) on Bone Collagen Synthesis*

Parathyroid hormone-related peptide (PTHrp), a polypeptide synthesized by tumors associated with hypercalcemia and known to cause bone resorption, was examined for its effects on bone formation in cultures of 21-day fetal rat calvariae. Continuous treatment with PTHrp for 24-72 h stimulated DNA synthesis, but inhibited [3H] proline incorporation into collagen by about 50%. In contrast, transient exposure to PTHrp at 0.1-1.0 nM for 24 h followed by removal of the factor for 48 h caused an increase in [3H]proline incorporation into collagen and noncollagen protein by 2- and 1.6-fold, respectively. The stimulatory effect was seen in the periosteum-free bone, and was decreased, but not prevented by hydroxyurea. PTHrp at 1-10 nM for 24 h increased medium insulin-like growth factor (IGF) I levels by 2.5-4.4-fold, and the effect was sustained 48 h after the removal of the agent. An IGF I neutralizing antibody prevented the stimulatory effect of PTHrp on bone collagen synthesis. PTH had the same stimulatory effects as those of PTHrp on bone collagen synthesis and IGF I concentrations, although slightly lower doses were needed to observe the enhancement of [3H]proline incorporation into collagen. It is concluded that continuous treatment with PTHrp inhibits, whereas transient treatment stimulates, collagen synthesis; the stimulatory effect appears mediated by an enhancement in the local production of IGF I.

Cortisol Enhances the Anabolic Effects of Insulin-Like Growth Factor I on Collagen Synthesis and Procollagen Messenger Ribonucleic Acid Levels in Cultured 21-Day Fetal Rat Calvariae*

We examined the ability of cortisol to modulate the stimulatory effects of recombinant human insulin-like growth factor-I (IGF-I) on collagen synthesis, procollagen messenger RNA (mRNA) levels and DNA synthesis in 21-day fetal rat calvariae maintained in serum-free organ culture for 24-96 h. Collagen synthesis was quantitated by measuring the incorporation of [3H]proline into collagenase-digestible protein (CDP) and alpha-1(I) procollagen mRNA transcripts were assessed by Northern blot analysis. Cell replication was quantitated by measuring the incorporation of [3H]thymidine into bone. As described previously, 100 nM cortisol had a biphasic effect on CDP labeling, increasing CDP after 24 h and decreasing CDP after 48, 72, and 96 h of culture. IGF-I alone increased CDP labeling by 1.6-fold after 24 h and by 2-fold after 48 or 72 h of culture, and cortisol potentiated this anabolic effect. In the presence of 100 nM cortisol, IGF-I increased CDP labeling by 2.6-fold after 24 h, by 5-fold after 48 h, and by 8-fold after 72 h of culture. A higher concentration of cortisol (1000 nM) also potentiated the IGF-I response on CDP labeling after 96 h of culture. In the presence of 100 nM cortisol, concentrations of IGF-I lower than 10 nM consistently increased CDP labeling and the percent collagen synthesized whereas these concentrations were not always effective without cortisol. PTH, which like cortisol decreased basal CDP labeling, did not enhance the stimulatory effects of IGF-I. Cortisol also enhance the stimulatory effects of IGF-I on alpha-1(I) procollagen mRNA levels indicating that the potentiation of CDP labeling occurs via a pretranslational mechanism. IGF-I had little effect on the incorporation of [3H]thymidine into bone except in the presence of cortisol. Nevertheless, the ability of cortisol to potentiate the stimulatory effect of IGF-I on CDP labeling was independent of cell replication since the enhancement persisted in the presence of aphidicolin, a DNA synthesis inhibitor. Our findings show that physiological concentrations of cortisol can modulate the responsiveness of cells within cultured fetal rat calvariae to the anabolic effects of exogenous IGF-I.

Location of osteoclast precursors in fetal rat calvaria cultured on collagen gels

Although osteoclasts are derived from hematopoietic cells, the exact identity of their precursors and the mechanism for their recruitment onto bone surfaces remain unclear. We wished to study their differentiation in the fetal rat calvaria and to locate its source of osteoclast precursor cells. Osteoclasts were detected by neutral red staining or cytochemical reaction for acid phosphatase of intact bone (cell number and area measured by computerized image analysis) or in cryostat sections of bone (enzyme activity measured by quantitative cytochemistry). Histology of semithin sections of fixed bones was also examined. The 19 day calvariae contained few mature osteoclasts. After 48 h culture on gels of type 1 collagen (1.5 mg/ml) supplemented with 5 mM calcium beta-glycerophosphate, 10 mM proline, and 2 micrograms/ml ascorbic acid, numerous large osteoclasts were seen on their endocranial surfaces. In contrast, cell morphology and enzyme activity deteriorated in bones cultured in liquid medium. The cells that formed in vitro rapidly responded to calcitonin by contraction. Stripping of endocranial membranes from the calvariae prevented osteoclast formation in culture, but these cells were seen when "stripped" bones had been cocultured with their membranes for 48 h or with intact 16 day calvariae (well before the onset of osteogenesis). Few osteoclasts were found when an 0.22 micron filter was inserted between the stripped calvaria and the endocranial membranes. We conclude that the endocranial membranes, which contain the meningeal blood vessels, are a major source of osteoclast precursors and that these cells are present in calvarial tissue even before the onset of osteogenesis.

Effect of heparin on bone formation in cultured fetal rat calvaria

To assess the effects of heparin on bone formation we measured [3H]proline incorporation into collagenase-digestible (CDP) and noncollagen protein (NCP), [3H]thymidine (TdR) incorporation into DNA, and DNA content in 21-day-old fetal rat calvaria cultured in BGJ medium with bovine serum albumin for 24-96 hours. Heparin at 5-125 micrograms/ml decreased TdR incorporation by 26-51% at 24 and 96 hours. At 96 hours, heparin 5, 25, and 125 micrograms/ml decreased [3H]proline incorporation into CDP by 41, 48, and 32%, respectively, with no significant change in NCP. To evaluate the possible role of PGE2 in these inhibitory responses, media PGE2 concentration was measured and the effects of heparin on CDP labeling and DNA synthesis were tested in the presence of indomethacin, piroxicam, and flurbiprofen to inhibit endogenous prostaglandin E2 (PGE2) production and in the presence of a high concentration (10(-7) M) of exogenous PGE2. Heparin did not alter PGE2 production at 24 hours but at 48 hours there was a significant reduction. At 96 hours, indomethacin (10(-6) M) inhibited [3H]-proline incorporation into CDP by 38% but had no effect on the labeling of NCP. Heparin had no further significant inhibitory effect in the presence of indomethacin. Piroxicam and flurbiprofen did not alter DNA content and had a smaller inhibitory effect than indomethacin on the labeling of CDP. Moreover, addition of heparin produced a further inhibition of CDP and DNA content and finally, heparin decreased CDP labeling by 71% in the presence of PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Biphasic Effects of Prostaglandin E2on Bone Formation in Cultured Fetal Rat Calvariae: Interaction with Cortisol*

Previous studies have shown that prostaglandin E2 (PGE2) has both inhibitory and stimulatory effects on the incorporation of proline into collagenase-digestible protein (CDP) in cultured fetal rat calvaria. The present studies were undertaken to analyze further these biphasic effects of PGE2. PGE2 increased [3H]thymidine incorporation at 24 h, and this effect was enhanced in the presence of cortisol (10(-8) and 10(-7) M). An inhibitory effect on CDP labeling was observed at 96 h with PGE2 (10(-6) M) in the absence or presence of indomethacin (10(-6) M), but not in the presence of cortisol (10(-8) or 10(-7) M). When the central osteoblast-rich bone and periosteum were analyzed separately, the inhibitory effect of PGE2, with or without indomethacin, was confined to the central bone. Addition of aphidicolin (30 microM), an inhibitor of cell replication, did not prevent the inhibitory effect of PGE2 on CDP labeling. Analysis of labeled collagen by polyacrylamide gel electrophoresis showed a decrease in labeling of type I collagen in central bone. Moreover, mRNA for alpha 1(I)procollagen was decreased, as measured by dot blot hybridization and Northern blot analysis. Cortisol (10(-8)-10(-6) M) decreased the labeling of CDP as well as noncollagen protein (NCP) at 96 h. In the presence of cortisol, PGE2 (10(-8)-10(-5) M) consistently stimulated labeling of CDP and NCP, with a greater increase in CDP, resulting in an increase in the percentage of collagen synthesized. In the presence of low concentrations of cortisol (10(-8) or 3 x 10(-8) M), PGE2 (10(-7) M) increased CDP labeling by 260-480%, and the absolute value was 145-160% of that in control cultures without any hormone addition. The stimulatory effect was seen in both central bone and periosteum, although absolute values for CDP and percentage of collagen synthesized were higher in central bone. PGE2 (10(-7) M) had similar effects on CDP at 24 and 96 h in the presence of cortisol, and the stimulation at 10(-7) M was the same in the presence and absence of aphidicolin, suggesting that it was not dependent on cell replication. Cortisol decreased labeling of type I collagen, determined by polyacrylamide gel electrophoresis, and alpha 1(I)procollagen mRNA levels, determined by both Northern and dot blot analysis. PGE2 reversed these effects, increasing both radiolabeled collagen type I chains and alpha 1(I)procollagen mRNA levels. These results indicate that PGE2 can regulate bone collagen synthesis at a pretranslational site.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal of Bone and Mineral Research

The number of discrete, three-dimensional bone nodules formed in vitro from a class of osteoprogenitor cells present in fetal rat calvaria cell populations (RC cells) is linearly related to the number of cells plated, implying that this system functions as a colony assay for the expression of osteoprogenitor cells. To determine the effect of fluoride on the expression of these cells, primary RC cells were grown for periods of up to 21 days in alpha-MEM (minimal essential medium) containing 5-15% heat-inactivated fetal bovine serum (FBS), 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and NaF at concentrations from 10 microM to 5 mM. The continuous presence of NaF resulted in an increase in the number of bone nodules with maximal response occurring at 500 microM (p less than 0.001). A similar response at 500 microM NaF was observed also with regard to alkaline phosphatase activity. NaF levels up to 500 microM did not affect the growth of the mixed RC cell population, however, higher concentrations (1 mM) significantly reduced cell numbers (p less than 0.001) suggestive of cytotoxicity. Plating efficiency tests for colony formation in the presence of 0.5 to 2 mM NaF showed that the decreases in nodule formation observed at concentrations above 500 microM correlated with cytotoxicity. NaC1 at 1 mM had no effect on nodule formation, alkaline phosphatase activity, or cell growth. The results show that NaF stimulates osteoprogenitor cell number in vitro and that the maximal effect occurs at concentrations close to toxic levels.

Differential Regulation of Plasminogen Activator and Plasminogen Activator Inhibitor by Osteotropic Factors in Primary Cultures of Mature Osteoblasts and Osteoblast Precursors*

Plasminogen activators (PA) and plasminogen activator inhibitors (PAI) have been implicated in the process of extracellular matrix degradation. To study their role in bone matrix turnover, we examined the activity and regulation of PA and PAI in cultures of periosteal osteoblast-like precursor cells and mature osteoblast-like cells from fetal rat calvariae. Both cell populations released PA activity of the tissue type and a 50K PAI species into the culture medium. However, mature osteoblasts had a strikingly lower PA activity and higher PAI activity than periosteal precursor cells, indicating that osteoblast differentiation is associated with a marked decrease in the PA/PAI ratio. PTH and prostaglandin E2 transiently increased PA activity and decreased PAI activity. In contrast, transforming growth factor-beta decreased PA activity and increased PAI activity. Differential effects of these factors on PA and PAI activity may be involved in the regulation of extracellular matrix deposition by osteoblasts.

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [ 3H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.5-fold with an ED 50 of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED 50 of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.

Direct modulatory effect of hexasodium N,N,N′,N′-ethylenediamine-tetramethylenephosphonate on bone cell function in vitro

The phosphonate hexasodium N,N,N′,N′-ethylenediamine-tetramethylenephosphonate (EDITEMP · Na 6) reduced alkaline phosphatase (Alp) activity and cAMP response to parathyroid hormone (PTH) in primary cultures of foetal rat calvaria cells in a dose-dependent manner, while not affecting culture DNA content. EDITEMP · Na 6 also inhibited the mineralization of three-dimensional bone nodules formed in vitro, but not the number of nodules formed. Bone cell culture DNA content was also reduced by EDITEMP · Na 6 but at concentrations in excess of those needed to modulate osteoblastic cell function. Withdrawal of EDITEMP · Na 6 led to slow but complete recovery of Alp activity. At EDITEMP · Na 6 concentrations of 25 μM and higher, recovery of Alp activity appeared to be independent of protein and/or DNA synthesis. Cell culture acid phosphatase (Acp) activity was not affected by EDITEMP · Na 6. The results indicate that EDITEMP · Na 6 has a direct inhibitory effect on (mature) osteoblastic cell function. In the presence of bone tissue this inhibition also occurred, although not at a relatively low dose level.

Comparison of the Effects of Synthetic Human Parathyroid Hormone (PTH)-(l–34)-Related Peptide of Malignancy and Bovine PTH-(l–34) on Bone Formation and Resorption in Organ Culture*

We have compared the effects of synthetic amino-terminal human PTH-(1-34)-related peptide (PTHrP) of malignancy with those of synthetic bovine PTH-(1-34) in cultures of half-calvariae from 21-day-old fetal rats and of parietal bones from 7-day neonatal mice. Incorporation of [3H] proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), and percent collagen synthesis (PCS) were measured in both systems. Incorporation of [3H]thymidine and cAMP production were measured in fetal rat calvariae. Production of prostaglandin E2 and I2 and bone resorption, as assessed by release of previously incorporated 45Ca, were measured in mouse parietal bones. The effects of PTHrP and PTH were qualitatively similar. At 96 h CDP in rat calvariae was decreased by PTH at a concentration as low as 0.01 nM, while similar effects were seen with PTHrP at 0.1 nM. Effects on NCP were small, so PCS was reduced. At 24 h [3H]thymidine was not altered, but CDP and PCS were decreased by both PTH and PTHrP. cAMP production was increased in fetal rat calvariae at 30 min. Both PTH and PTHrP increased 45Ca release at low concentrations and prostaglandin production at high concentrations in mouse parietal bones. While PTH was about 10-fold more potent than PTHrP, there was no qualitative difference in the responses. These studies further suggest that PTHrP affects bone through the PTH receptor.

Thymosin β4is Expressed in ROS 17/2.8 Osteosarcoma Cells in a Regulated Manner

The differential expression of mRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis of the cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype-dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.

Forskolin has biphasic effects on osteoprogenitor cell differentiation in vitro

Cells isolated from fetal rat calvaria (RC) and maintained in vitro in medium containing ascorbic acid and B-glycerophosphate form three-dimensional, mineralized nodules having the histological, immunohistological, and ultrastructural characteristics of woven bone. We have studied the effects of forskolin (FSK), a diterpene that activates adenylate cyclase, in this system. While 10(-7)-10(-5) M FSK significantly stimulated cAMP levels in RC cells, lower concentrations did not. cAMP levels with 10(-5) M FSK reached a maximum by 30 min at 37 degrees C and returned to basal level in 2-3 hr. Changes in cAMP levels correlated with changes in cellular shape: cells treated with 10(-5) M FSK assumed a stellate morphology, lost microfilament bundles, and reduced their substrate adhesiveness, while cells treated with 10(-9) M were not affected. Exponential growth and saturation densities of FSK-treated cultures were similar to untreated cultures, indicating that FSK was neither toxic nor stimulatory to the population. The effect on bone nodule formation of FSK present continuously depended on concentration: 10(-5) M FSK significantly inhibited the number of nodules formed, while 10(-9) M FSK significantly stimulated bone nodule formation. Single short treatments with either 10(-5) M or 10(-9) M FSK had no effect on nodule formation, but repeated short duration treatments (1 hr every 2 days for 21 days) gave results similar to continuous exposure. These results indicate that intermittent elevations in intracellular cAMP have an inhibitory effect on bone formation. In addition, our work indicates that low concentrations of FSK stimulate differentiation of osteoprogenitor cells possibly through a non-cAMP-dependent process.

In vitro effects of bone- and platelet-derived transforming growth factor-? on the growth of Walker 256 carcinosarcoma cells

Conditioned media from fetal rat calvarial cultures has previously been shown to stimulate the growth of the bone-metastasizing Walker 256 carcinosarcoma cell line. In the current investigation we looked at the possibility that transforming growth factor-beta (TGF-beta), present in conditioned media, and positively correlated with resorption in vitro, may be responsible for the enhanced proliferation of Walker cells cultured in these conditioned media. Purified platelet-derived TGF-beta produced a dose-dependent growth response in Walker cells with an ED50 equal to 0.05 ng/ml. Bone-derived TGF-beta activity in conditioned media, measured by NRK fibroblast colony formation, correlated well with percentage resorption in bone cultures, and growth activity in Walker cell culture. In addition to this, the growth response normally seen with conditioned media cultures of Walker cells was significantly inhibited by the addition of anti-TGF-beta 1 neutralizing antibody. We conclude that TGF-beta is an important growth stimulatory component from fetal rat calvaria.

Effects of ethanol and acetaldehyde on collagen synthesis, prostaglandin release and resorption of fetal rat bone in organ culture

We tested the effect of ethanol and its metabolite, acetaldehyde, on bone formation as measured by [ 3H]proline incorporation into collagenase digestible protein (CDP) and noncollagen protein (NCP), and on DNA synthesis as measured by [ 3H]thymidine (TdR) incorporation in fetal rat calvaria. We also determined the effects of ethanol and acetaldehye on prostaglandin E 2 (PGE 2) release from calvaria and on bone resorption as measured by 45Ca release from fetal rat long bones. Bones were cultured in multiwell plastic dishes (open system) or in stoppered Erlenmeyer flasks (closed system) for 24 to 96 h. In the open system, 1% ethanol (v/v; 172 mM) resulted in a 31% decrease in TdR incorporation at 24 h with no effect on CDP and NCP. At 0.1% (17.2 mM), ethanol increased TdR by 22%, CDP by 73% and NCP by 67% at 24 h, but these effects were not sustained at 96 h. At 24 h, 1% and 0.3% ethanol decreased PGE 2 release by 88% and 75% respectively. This effect was sustained for 96 h only at the higher concentration. In the closed system, 0.1% ethanol increased TdR incorporation by 38% at 24 h. However, there was no effect on the labeling of CDP or NCP. Because its boiling point is 21 °C, acetaldehyde could only be tested in the closed system. Acetaldehyde markedly inhibited bone metabolism. At 24 h, 0.003% (0.54 mM) to 0.01% (1.79 mM) acetaldehyde caused a dose-related inhibition of TdR incorporation from 23 to 45%. At 0.01% and 0.03% acetaldehyde inhibited proline incorporation into CDP by 48% and 94% and NCP by 40% and 74% respectively. In the resorption system, acetaldehyde at 0.01% inhibited PTH stimulated bone resorption, while ethanol (0.1 to 1.0%) had no effect. We conclude that ethanol has little effect on bone formation or resorption; however, the metabolite acetaldehyde is a potent inhibitor of bone formation and resorption. These results may be of significance in the development of osteoporosis associated with chronic alcohol use.