Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro

The ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co-culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose-dependent manner. HPL-conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze-thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 10(-5) M indomethacin. Dose-dependent inhibiton of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10(-6) M) or F2 alpha (at 10(-7) M). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.     

Effects of recombinant human interleukin 1 alpha and interleukin 1 beta on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Recombinant human interleukin 1 (rhIL-1)alpha and rhIL-1 beta were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1 alpha and rhIL-1 beta was compared in terms of the units which induced half-maximal [3H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1 alpha and rhIL-1 beta significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-1 alpha and rhIL-1 beta markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1 alpha and rhIL-1 beta increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1 alpha and rhIL-1 beta have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.     

Modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast function and bone metabolism

Recent investigations indicate that the type and amount of polyunsaturated fatty acids (PUFA) influence bone formation in animal models and osteoblastic cell functions in culture. In growing rats, supplementing the diet with omega-3 PUFA results in greater bone formation rates and moderates ex vivo prostaglandin E(2) production in bone organ cultures. A protective effect of omega-3 PUFA on minimizing bone mineral loss in ovariectomized rats has also been reported. The actions of omega-3 fatty acids on bone formation appear to be linked to altering osteoblast functions. Herein we describe experiments with MC3T3-E1 osteoblast-like cells that support findings in vivo where omega-3 PUFA modulated COX-2 protein expression, reduced prostaglandin E(2) production, and increased alkaline phosphatase activity. Other studies indicate that the dietary source of PUFA may affect protein expression of Cbfa1 and nodule formation in fetal rat calvarial cells.     

The role of 5'-methylthioadenosine on rat calvaria cell differentiation.

The role of 5'-methylthioadenosine (MTA), formed during the process of polyamine biosynthesis, on differentiation of osteoprogenitor cells was assessed by its effects on alkaline phosphatase (ALP) activity, bone nodule formation and osteopontin contents of cultured rat calvaria (RC) cells. These three markers were stimulated by exogenous MTA and were depressed by 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, which cleaves MTA to adenine and 5-methylthioribose-1-phosphate. 5-Methylthioribose and 2-keto-4-methylthiobutyrate, metabolites of 5-methylthioribose-1-phosphate, had no effects on ALP activity and bone nodule formation in the presence or absence of DFMTA. On the other hand, adenine enhanced ALP activity, bone nodule formation and osteopontin contents in mineralized nodules and also partially reversed DFMTA-induced inhibition of these three markers. MTA, its metabolites and DFMTA did not affect the growth of RC cells under these culture conditions. These results suggest that adenine formed from MTA is important in the differentiation of RC cells.     

GP130/OSMR is the only LIF/IL-6 family receptor complex to promote osteoblast differentiation of calvaria progenitors

Leukemia inhibitory factor (LIF) and its receptor (LIFR) are "twins" of Oncostatin M (OSM) and OSMR, respectively, likely having arisen through gene duplications. We compared their effects in a bone nodule-forming model of in vitro osteogenesis, rat calvaria (RC) cell cultures. Using a dominant-negative LIF mutant (hLIF-05), we showed that in RC cell cultures mouse OSM (mOSM) activates exclusively glycoprotein 130 (gp130)/OSMR. In treatments starting at early nodule formation stage, LIF, mOSM, IL-11, and IL-6 + sIL-6R inhibit bone nodule formation, that is, osteoprogenitor differentiation. Treatment with mOSM, and no other cytokine of the family, in early cultures (day 1-3 or 1-4) increases bone colony numbers. hLIF-05 also dose dependently stimulates bone nodule formation, confirming the inhibitory action of gp130/LIFR on osteogenesis. In pulse treatments at successive stages of bone nodule formation and maturation, LIF blocks osteocalcin (OCN) expression by differentiated osteoblasts, but has no effect on bone sialoprotein (BSP) expression. Mouse OSM inhibits OCN and BSP expression in preconfluent cultures with no or progressively reduced effects at later stages, reflecting the disruption of early nodules, possibly due to the strong apoptotic action of mOSM in RC cell cultures. In summary, LIFR and OSMR display differential effects on differentiation and phenotypic expression of osteogenic cells, most likely through different signal transduction pathways. In particular, gp130/OSMR is the only receptor complex of the family to stimulate osteoprogenitor differentiation in the RC cell culture model.     

Interleukin 1 stimulates hexose transport in fibroblasts by increasing the expression of glucose transporters

Exposure of quiescent cultures of human gingival fibroblasts (HuGi) and porcine synovicocytes (PSF) to human recombinant interleukin 1 alpha or -beta (IL1 alpha and -beta) enhanced the rate of glycolysis as judged by increased lactate production. The cytokines also increased uptake of [3H]2-deoxyglucose (DG) in a time- and dose-dependent manner. Stimulation of DG uptake was first evident 6-8 h following addition of IL1 and was maximal by 24-30 h. IL1 alpha and -beta were equipotent. Half-maximal stimulation occurred at approximately 1 pM IL1; maximal stimulation (2.5-4.5-fold in HuGi, 3-7-fold in PSF) was obtained with approximately 80 pM IL1. The dose-response curves for lactate production and DG uptake were similar. Increased DG uptake was blocked by specific antisera to IL1 and by inhibitors of protein and RNA synthesis but not by indomethacin, an inhibitor of prostaglandin production. DG uptake was enhanced by IL1 in serum-starved cells in the presence of neutralizing anti-platelet-derived growth factor serum. The effect was therefore not secondary to prostaglandin or platelet-derived growth factor production. No increase in cell cycling was detected in IL1-treated cells under the experimental conditions. Kinetic analysis revealed that the Vmax for DG uptake was increased by IL1 (from 36 to 144 pmol/min/mg of cell protein), whereas the Km was unchanged. HuGi cells were pulse-labeled with [35S]methionine following exposure to IL1. Cell lysates were immunoprecipitated using a specific antiserum raised against human erythrocyte glucose transporter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography of these immunoprecipitates revealed dose- and time-dependent increases in the net rate of glucose transporter synthesis which mirrored the changes in DG uptake.     

Effects of transforming growth factor beta and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells

When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na beta-glycerophosphate, discrete three-dimensional nodules form with the histologic, immunohistochemical, and ultrastructural characteristics of bone (Bellows et al; Calcified Tissue International 38:143-154, 1986; Bhargava et al., Bone, 9:155-163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8-13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF-beta) results in dose-dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2-200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty-eight hour pulses of TGF-beta (0.01-1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF-beta and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF-B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation.     

Effects of interferon alpha on human osteoprogenitor cell growth and differentiation in vitro.

The specific effects of interferon alpha (IFNalpha), on the differentiation pathways of human osteogenic cells are not known. The aim of this study was to investigate possible effects of IFNalpha on osteogenic development by investigating cell differentiation, colony formation (colony forming unit-fibroblastic, CFU-F), cell proliferation, and gene expression, in particular bone morphogenetic protein (BMP) expression, of human bone marrow osteoprogenitor cells. Human bone marrow fibroblasts were cultured with or without the addition of IFNalpha (5-1,000 IU/ml) in the presence and absence of dexamethasone (10 nM) and ascorbate (100 microM), which are agents known to affect osteogenic differentiation. IFNalpha produced a significant dose-dependent inhibition of cell proliferation and alkaline phosphatase specific activity at concentrations as low as 50 IU/ml. IFNalpha (50-1,000 IU/ml) inhibited the stimulation of alkaline phosphatase specific activity induced by ascorbate and dexamethasone. Examination of CFU-F showed dose- and time-dependent inhibitions of colony formation and reductions in both colony size and alkaline phosphatase-positive CFU-F colonies particularly at earlier times. Reactivity with an antibody specific for osteoprogenitors (HOP-26), was reduced in IFNalpha-treated cultures. Northern blot analysis showed a significant dose-dependent up-regulation of BMP-2 mRNA, estrogen receptor alpha mRNA and osteocalcin mRNA expression in ascorbate/dexamethasone cultures. In contrast, IFNalpha significantly inhibited BMP-2 mRNA expression in the absence of ascorbate and dexamethasone. In conclusion, IFNalpha inhibits human osteoprogenitor cell proliferation, CFU- F formation, HOP-26 expression, and alkaline phosphatase specific activity and modulates BMP-2 gene expression. These results suggest a role for IFNalpha in local bone turnover through the specific and direct modulation of osteoprogenitor proliferation and differentiation.     

Purmorphamine enhances osteogenic activity of human osteoblasts derived from bone marrow mesenchymal cells

Purmorphamine is a novel small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells, but there has been no evaluation of its effect on human cells to date. The aim of this study was to investigate the induction of osteogenic activity by purmorphamine in human osteoblasts differentiated from bone marrow mesenchymal cells. Cells were cultured in 24-well plates at a density of 2x10(4)/well in medium containing 1, 2 or 3 microM purmorphamine, or vehicle. At 7, 14 and 21 days, cell proliferation, viability, and alkaline phosphatase (ALP) activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Purmorphamine did not affect cell proliferation or viability, but increased ALP activity and bone-like nodule formation. These results indicate that events related to osteoblast differentiation, including increased ALP activity and bone-like nodule formation, are enhanced by purmorphamine.     

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.     

Comparison of human interleukin-1 beta and its 163-171 peptide in bone resorption and the immune response

Human interleukin-1 beta (IL-1 beta) caused a dose- and time-dependent enhancement of the release of 45Ca from prelabeled mouse calvaria in organ culture. In addition, IL-1 beta dose-dependently stimulated the formation of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha in the calvarial bones. However, IL-1 beta-induced 45Ca release was only partially inhibited by blocking the PGE2 response with indomethacin, suggesting that enhanced PGE2 formation in response to IL-1 beta is not necessary to obtain a bone resorptive effect, but that prostaglandins potentiate the action of IL-1 beta. The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, administered simultaneously with antigen (SRBC) to C3H/HeN male mice, induced a dose-dependent enhancement of specific antibody-producing cells in the spleen (PFC). The degree of PFC stimulation was comparable to that caused by native human IL-1 beta. In mouse bone cultures, neither 45Ca release nor prostanoid formation was stimulated by fragment 163-171. These data indicate that (1) IL-1 beta-induced stimulation of bone resorption is dissociable from IL-1 beta-induced increase of prostanoid biosynthesis and (2) the epitope of the IL-1 beta molecule involved in the immunostimulatory effects may be different from that involved in the stimulatory effects on bone resorption.     

Tumor necrosis factor type alpha (cachectin) stimulates mouse osteoblast-like cells (MC3T3-E1) to produce macrophage-colony stimulating activity and prostaglandin E2

To elucidate the mechanism of tumor necrosis factor alpha (TNF-alpha)-induced bone resorption, the effects of recombinant human TNF-alpha on mouse osteoblast-like cells (MC3T3-E1) were studied. TNF-alpha stimulated MC3T3-E1 cells to produce prostaglandin E2 (PGE2) and macrophage colony stimulating activity (M-CSA) in a dose-dependent manner. TNF decreased alkaline phosphatase (AL-P) activity of MC3T3-E1 cells. These TNF effects were observed at 1 ng/ml (approximately 6 X 10(-11)M). The inhibitory effect on AL-P activity was reversible and the cell growth of MC3T3-E1 cells was only slightly affected by TNF. These findings suggest that both PGE2 and M-CSA stimulated by TNF-alpha are possibly involved in osteoblast-mediated osteoclastic bone resorption, whereas inhibition of AL-P activity may lead to a decrease in bone formation.     

Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: Altered expression of collagenase,cell growth-related,and mineralization-associated genes

Fibroblast growth factors (FGF) are osteoblast mitogens, but their effects on bone formation are not clearly understood. Most in vitro studies examining the effects of FGFs on osteoblasts have been performed only during the initial proliferative stage of osteoblast culture. In these studies, we examined the consequential effect of acidic FGF in cultures of rat fetal diploid osteoblasts that undergo a developmental differentiation program producing a mineralized bone-like matrix. During the initial growth period (days 1–10), addition of acidic FGF (100 μg/ml) to actively proliferating cells increased (P < 0.05) ³H-thymidine uptake (2,515 ± 137, mean ± SEM vs. 5,884 ± 818 cpm/10⁴ cells). During the second stage of maturation (days 10–15), osteoblasts form multilayered nodules of cells and accumulate matrix, followed by mineralization (stage 3, days 16–29). Addition of acidic FGF to the osteoblast cultures from days 7 to 15 completely blocked nodule formation. Furthermore, addition of acidic FGF after nodule formation (days 14–29) inhibited matrix mineralization, which was associated with a marked increase in collagenase gene expression, and resulted in a progressive change in the morphology of the nodules, with only a few remnants of nonmineralized nodules present by day 29. Histochemical and biochemical analyses revealed a decrease in alkaline phosphatase and mineral content, confirming the acidic FGF-induced inhibition of nodule and matrix formation. To identify mechanisms contributing to these changes, we examined expression of cell growth and bone phenotypic markers. Addition of acidic FGF during the proliferative phase (days 7–8) enhanced histone H4, osteopontin, type 1 collagen, and TGF-β mRNA levels, which are coupled to proliferating osteoblasts, and blocked the normal developmental increase in alkaline phosphatase and osteocalcin gene expression and calcium accumulation. Addition of acidic FGF to the cultures during matrix maturation (days 14–15) reactivated H4, osteopontin, type I collagen, and TGF-β gene expression, and decreased alkaline phosphatase and osteocalcin gene expression. In an in vivo experiment, rats were treated with up to 60 μg/kg/day acidic FGF intravenously for 30 days. Proliferation of osteoblasts and deposition of bone occurred in the marrow space of the diaphysis of the femur in a dose-related fashion. The metaphyseal areas were unaffected by treatment. In conclusion, our data suggest that acidic FGF is a potent mitogen for early stage osteoblasts which leads to modifications in the formation of the extracellular matrix; increases in TGF-β and collagenase are functionally implicated in abrogating competency for nodule formation. Persistence of proliferation prevented expression of alkaline phosphatase and osteocalcin, also contributing to the block in the progression of the osteoblast developmental sequence. © 1996 Wiley-Liss, Inc.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression. © 1994 Wiley-Liss, Inc.     

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.     

Pulse application of platelet-derived growth factor enhances formation of a mineralizing matrix while continuous application is inhibitory

Platelet-derived growth factor (PDGF) stimulates chemotaxis and proliferation of osteoblasts, and induces bone formation in vivo. To determine how PDGF might regulate these cells, the effect of PDGF on long-term mineralizing cultures of fetal rat osteoblastic cells was examined. Although PDGF increased cell proliferation in these cultures, continuous treatment with PDGF caused a dose-dependent decrease in mineralized nodule formation. When cells were treated with multiple, brief (1 day) exposures to PDGF at the osteoblast differentiation stage, there was a significant 50% increase in mineralized nodule area. Based on modulation of alkaline phosphatase activity it appears that longer-term exposure to PDGF reduces mineralized nodule formation largely by inhibiting differentiated osteoblast function, while short-term exposure enhances proliferation without inhibiting the differentiated phenotype. Thus, the ultimate affect of PDGF on bone formation is likely to reflect two processes: a positive effect through enhancing cell number or a negative effect by inhibiting differentiated function. The inhibitory effect of PDGF on formation of a mineralized matrix is unlikely to be simply a result of enhanced proliferation of “fibroblastic” cells since cultures treated with PDGF for 3 days and then transferred to new plastic dishes exhibited a 70% increase in mineralized nodule area compared to controls. These results would predict that multiple, brief exposures to PDGF would enhance bone formation in vivo, while prolonged exposure to PDGF, which is likely to occur in chronic inflammation, would inhibit differentiated osteoblast function and limit bone regeneration. J. Cell. Biochem. 69:169–180, 1998. © 1998 Wiley-Liss, Inc.     

Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.