Rat Calvarial Osteoblastic Cells Research Articles

Pulsed electromagnetic fields inhibit human osteoclast formation and gene expression via osteoblasts

Pulsed electromagnetic fields (PEMFs) can be effective in promoting the healing of delayed union or nonunion fractures. We previously reported that PEMF (Spinal-Stim® by Orthofix, Inc., Lewisville, TX) stimulated proliferation, differentiation and mineralization of rat calvarial osteoblastic cells in culture. In the present work we investigated the effects of PEMF (Physio-Stim® by Orthofix, Inc., Lewisville, TX) on human bone marrow macrophages (hBMMs) differentiated to osteoclasts. PEMF had striking inhibitory effects on formation of osteoclasts from hBMMs from both younger and older women. There were significantly greater changes in gene expression as ascertained by RNAseq from cells from older women. Interestingly, all of the genes identified by RNAseq were upregulated, and all were genes of mesenchymal or osteoblastic cells and included members of the TGF-β signaling pathway and many extracellular matrix proteins, as well as RANKL and osteoprotegerin, indicating the mixed nature of these cultures. From these results, we suggest that PEMF can inhibit osteoclast formation via action on osteoblasts. Thus, PEMF may be very effective for bone mass maintenance in subjects with osteoporosis.

Parathyroid hormone-related protein regulates osteoclast inhibitory lectin expression via multiple signaling pathways in osteoblast-like cells

Osteoclast inhibitory lectin (OCIL) is a recently identified inhibitor of osteoclast formation. A variety of osteotropic factors regulate OCIL expression in osteoblastic cells, however, little information is available to date concerning how this gene is controlled. Using real-time RT-PCR, we examined the regulation of OCIL expression by PTHrp and the signaling pathways used. We demonstrated in rat osteoblast-like UMR-106 cells, rat calvarial primary osteoblastic cells, and murine MC3T3-E1 cells, PTHrp(1-34) increased OCIL expression. In UMR-106 cells, the increase began and reached maximum later than RANKL induction and OPG suppression. cAMP/PKA signaling activators PTH(1-31), forskolin and dibutyryl cAMP (db-cAMP), and calcium ionophore A23187 all increased OCIL levels. In contrast, PKC activator phorbol-12-myristate-13-acetate reduced OCIL expression in short term but induced OCIL mRNA in long term. PKA inhibitor KT5720, mitogen-activated protein kinase (MAPK) cascade inhibitor PD98059, calmodulin antagonist W-7, and Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) inhibitor KN-62 all significantly blunted PTHrp-stimulated OCIL expression. Moreover, PD98059 blocked the stimulation of OCIL by FSK or db-cAMP but not that by A23187. In primarily cultured osteoblasts, the PTHrp induction of OCIL was blocked by KT5720, W-7, and PD98059 as well. The data established that PTHrp(1-34) regulates OCIL expression in vitro through cAMP/PKA, Ca(2+)/CaMK II, and MAPK signaling pathways.

Substance P stimulates late-stage rat osteoblastic bone formation through neurokinin-1 receptors

Substance P (SP) is a widely distributed neuropeptide that works as a neurotransmitter and neuromodulator. Recently, SP receptors, particularly neurokinin-1 receptors (NK(1)-Rs) that have a high affinity for SP, have been observed not only in neuron and immune cells, but also in other peripheral cells, including bone cells. To identify the role of SP in bone formation, we investigated the expression of NK(1)-Rs in osteoblastic cells and the effects of SP on bone formation by rat calvarial osteoblastic cells. Rat calvarial osteoblastic cells were isolated and cultured for 3 weeks in alpha-MEM containing 10% serum, ascorbic acid, dexamethasone, and beta-glycerophosphate. We then investigated NK(1)-R expression, SP effects on osteoblastic bone formation, and osteocalcin mRNA expression in osteoblastic cells. RT-PCR and immunocytochemistry showed that NK(1)-R mRNA was expressed and NK(1)-R was present in 14-day, but not 7-day, cultured calvarial osteoblasts. Bone formation by cultured osteoblastic cells significantly increased after the addition of 10(-8)-10(-6)MSP. During 3 weeks of culture, the addition of SP in the first week did not significantly increase bone formation, whereas adding SP during the first and second week or all 3 weeks significantly increased calvarial osteoblastic bone formation. Furthermore, semi-quantitative RT-PCR indicated that SP stimulated osteocalcin mRNA expression in the osteoblasts at day 14 or day 21, whereas SP did not stimulated the runX2 or type I collagen mRNA expression at day 7 but stimulated them at day 14. These results indicate that SP stimulates bone formation by osteoblastic cells via NK(1)-Rs at late-stage bone formation. These effects were dependent on the expression of NK(1)-R in osteoblastic cells. Our findings suggest that SP secreted from sensory neurons may modulate bone formation after the expression of SP receptors.

Curcumin inhibits the proliferation and mineralization of cultured osteoblasts

The effects of curcumin, which is an important constituent of rhizomes of the plant Curcuma longa Linn, on the metabolism of osteoblasts were examined in cultures of rat calvarial osteoblastic cells (ROB cells). The proliferation of cells was markedly inhibited upon exposure of cells to curcumin at 5 × 10 − 6 to 1 × 10 − 5 M. Curcumin at 1 × 10 − 5 M did not induce apoptosis in ROB cells but arrested cells at the G1 phase of the cell cycle. In addition, curcumin stimulated the expression of mRNA for p21 WAF1/CIP1, which inhibits the activity of cyclin-dependent kinases, and inhibited the phosphorylation of histone H1. Furthermore, curcumin reduced the rate of deposition of calcium and the formation of mineralized nodules. Our results indicate that curcumin might inhibit the proliferation and mineralization of osteoblastic cells through the expression of p21 WAF1/CIP1.

Parathyroid Hormone Uses Multiple Mechanisms to Arrest the Cell Cycle Progression of Osteoblastic Cells from G1 to S Phase

Parathyroid hormone (PTH) plays a major role in bone remodeling and has the ability to increase bone mass if administered daily. In vitro, PTH inhibits the growth of osteoblastic cell lines, arresting them in G(1) phase. Here, we demonstrate that PTH regulates the expression of at least three genes to achieve the following: inducing expression of MAPK phosphatase 1 (MKP-1) and p21(Cip1) and decreasing expression of cyclin D1 at both mRNA and protein levels. The induction of MKP-1 causes the dephosphorylation of extracellular signal-regulated kinase and therefore the decrease in cyclin D1. Overexpression of MKP-1 arrests UMR cells in G(1) phase. The mechanisms involved in PTH regulation of these genes were studied. Most importantly, PTH administration produces similar effects on expression of these genes in rat femoral metaphyseal primary spongiosa. Analyses of p21(Cip1) expression levels in bone indicate that repeated daily PTH injections make the osteoblast more sensitive to successive PTH treatments, and this might be an important feature for the anabolic functions of PTH. In summary, our data suggest that one mechanism for PTH to exert its anabolic effect is to arrest the cell cycle progression of the osteoblast and hence increase its differentiation.

Collagen Type I Prevents Glyoxal-Induced Apoptosis in Osteoblastic Cells Cultured on Titanium Alloy

Advanced glycation end products (AGEs) irreversibly cross-link proteins with sugars and accumulate at a higher age and in diabetes, processes which can interfere with the integration of implants into the tissue. Glyoxal is a highly reactive glycating agent involved in the formation of AGEs and is known to induce apoptosis, as revealed by the upregulation of caspase-3 and fractin (caspase-3 being a key enzyme activated during the late stage of apoptosis and fractin being a caspase-cleaved actin fragment). In this study, we investigated the influence of collagen type I coating on the cytotoxic effect of glyoxal on rat calvarial osteoblastic cells and on human osteosarcoma cells (Saos-2) grown on titanium alloy, Ti6Al4V. Activation of caspase-3 and fractin was measured by counting immunohistochemically stained cells and by flow cytometry with propidium iodide (detection of the apoptosis indicating a sub-G₁ peak). Our results showed an increased number of apoptotic osteoblasts after incubation with glyoxal on Ti6Al4V discs. However, the number of apoptotic cells on collagen-coated titanium was significantly smaller than on uncoated titanium after the same treatment. The present findings demonstrate that osteoblasts treated with glyoxal undergo apoptosis, whereas collagen type I coating of titanium alloys (used for implants) has an antiapoptotic function.

Demonstration of expression of mRNA for particular AMPA and kainate receptor subunits in immature and mature cultured rat calvarial osteoblasts

Reverse transcription polymerase chain reaction revealed expression of mRNA for particular subunits of ionotropic glutamate receptors (iGluR) in primary cultures of rat calvarial osteoblastic cells under immature to mature states. These included GluR3, KA1 and KA2 subunits, in addition to NR1 and NR2D subunits. These results suggest that glutamate may play an unidentified role in mechanisms associated with cellular development through particular subunits of iGluR in rat calvarial osteoblasts.

Group III Metabotropic Glutamate Receptors in Rat Cultured Calvarial Osteoblasts

Reverse transcription polymerase chain reaction revealed expression of mRNAs for particular receptors for the central neurotransmitter l-glutamic acid (Glu) in primary cultures of rat calvarial osteoblastic cells under premature to mature states according to the duration of days in vitro. These included metabotropic Glu receptors (mGluR) such as mGluR4 and mGluR8, in addition to several ionotropic Glu receptor subunits including NR1 and NR2D. Expression of mRNAs was not detected with other mGluR and NR2A-C subunits irrespective of the maturity of cultured cells. The agonist for group III mGluR l-(+)-2-amino-4-phosphonobutyric acid significantly inhibited the forskolin-induced accumulation of cAMP in premature osteoblasts, which occurred in a manner sensitive to prevention by the group III mGluR antagonist (RS)-α-cyclopropyl-4-phosphonophenylglycine. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation and/or differentiation through group III mGluR functionally expressed in rat calvarial osteoblastic cells.

Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: implications for suture fusion and patency.

Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor beta1 [TGF-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5-bromo-2'deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone-associated ECM molecules is potently up-regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture-associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF-beta1 and fibroblast growth factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF-2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than SAG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (*p < 0.001) in FRCs cocultured with SAG suture-derived dural cells as compared with FRCs cocultured alone or with PF suture-derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.

Mechanisms of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells.

Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.

Changes in sphingolipid levels induced by epidermal growth factor in osteoblastic cells. Effects of these metabolites on cytosolic calcium levels

Sphingolipids mediate a number of cellular functions in a variety of cell systems. The role they play in osteoblast signaling is yet unknown. This study investigated the effects of epidermal growth factor (EGF) on the levels of ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P) in rat calvariae osteoblastic cells, and whether these metabolites mediated cytosolic calcium ([Ca2+]I) mobilization in these cells. EGF significantly (P⪡0.05) increased the levels of all three sphingolipids, and the phorbol ester PMA partially inhibited these effects. SPH and S1P markedly increased [Ca2+]Ilevels, with thapsigargin (depletes [Ca2+]Ipools) decreasing the response by 60%. Verapamil (calcium channel blocker) only inhibited ceramide's effects on [Ca2+]I. Furthermore, SPH enhanced the EGF' induced increase in [Ca2+]I. This study demonstrates that ceramide, SPH and S1P mediate [Ca2+]Imobilization in rat calvarial osteoblastic cells, and that EGF induces changes in the levels of these metabolites with PKC playing an important role in the mechanisms regulating these events.

Tissue engineered bone formation using chitosan/tricalcium phosphate sponges.

Chitosan is a biodegradable natural polymer that has been shown to improve wound healing. This study aimed to develop chitosan/tricalcium phosphate (TCP) sponges as tissue engineering scaffolds for bone formation by three-dimensional osteoblast culture. The sponges were prepared by freeze-drying and cross-linking a mixture of chitosan solution with TCP. Fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 56 days. Cell proliferation, alkaline phosphatase (ALPase) activity, and calcium deposition in the cell-sponge constructs were measured at 1, 7, 14, 28, and 56 days. Histologic examination was performed with light microscopy and scanning electron microscopy. Chitosan/TCP sponges supported the proliferation of osteoblastic cells as well as their differentiation as indicated by high ALPase activities and deposition of mineralized matrices by the cells. Light and scanning electron microscopic examination indicated that seeded osteoblastic cells were well attached to sponge matrices and proliferated in a multi-layer fashion. Small bone-like spicules were observed on the sponge matrix at 14 days. Seeded cells appeared to be embedded in the newly formed tissue matrix, which is characteristic of the osteoblast differentiation and their progression into osteocytic cells. The amount of mineralized tissue formed in the sponge at 56 days was significant. These results suggest that the chitosan/TCP sponge is a feasible tool as a scaffolding material to grow osteoblast in a three-dimensional structure for transplantation into a site for bone regeneration.

Phosphatidic acid effects on cytosolic calcium and proliferation in osteoblastic cells

Our previous studies show that epidermal growth factor (EGF) stimulates phospholipase D (PLD)-induced phosphatidic acid (PA) formation in rat calvarial osteoblastic cells. 28 This study investigated the effects of PA on cytosolic calcium ([Ca 2+] i) and proliferation, and the possible involvement of the PLD pathway in EGF effects on [Ca 2+] i and proliferation in rat calvarial osteoblastic cells. PA markedly increased [Ca 2+] i. This response was unaffected by thapsigargin, which depletes [Ca 2+] i pools, blocked by verapamil, a calcium channel blocker, and enhanced by propanolol, an inhibitor of PA-phosphohydrolase. PA also reduced the EGF dependent-[Ca 2+] i increase by 60%, while a PLD inhibitor blocked these effects. Furthermore, PA significantly increased cell proliferation ( P < 0.05) which was inhibited by verapamil and enhanced by H-7 (PKC inhibitor). The PLD inhibitor significantly ( P < 0.05) reduced the EGF-induced increase in proliferation. In summary, PA stimulates rat calvarial osteoblastic cell proliferation and mobilization of [Ca 2+] i using extracellular pools, and EGF's mitogenic effect on these cells requires activation of PLD.

Effects of nitric oxide from exogenous nitric oxide donors on osteoblastic metabolism

We examined the effects of nitric oxide (NO) on the differentiation and mineralization of newborn rat calvarial osteoblastic cells (ROB cells) using exogenous NO donors, sodium nitroprusside, 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)- N-methyl-1-propanamine (NOC-7) and 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18). Sodium nitroprusside and NOC-7 dose-dependently enhanced the rate of production of intracellular cGMP in ROB cells and the rat clonal osteogenic cell line ROB-C26. We used NOC (NOC is the trade name for NO complex manufactured by Dojindo, Kumamoto, Japan) as an NO donor in our experiments because sodium nitroprusside exhibited a marked cytotoxicity. Northern blot analysis revealed that the level of mRNA for osteocalcin, one of the osteoblastic differentiation markers, was enhanced in the ROB cells, which was continuously treated by NOC-18. NOC-18, however, did not affect the level of mRNA for alkaline phosphatase and the activity of alkaline phosphatase. Both the number and the total area of mineralized nodules that are a model of in vitro bone formation were shown to be increased by 10 −5 M NOC-18. Our data suggest that NO might act as a local regulator of the metabolism of osteoblastic cells.

Deceleration by angiotensin II of the differentiation and bone formation of rat calvarial osteoblastic cells.

We examined the effects of angiotensin II (Ang II) on the differentiation of rat calvarial osteoblastic cells and on the formation of bone by these cells. Northern blotting analysis revealed that Ang II inhibited the expression of mRNA for osteocalcin, which is a protein that is specifically expressed during maturation of osteoblastic cells. Ang II decreased the activity of alkaline phosphatase, a marker of osteoblastic differentiation, in the cells, acting via the type 1 (AT1) receptor. We used von Kossa staining to examine the formation of mineralized nodules by osteoblastic cells. Both the number and the total area of mineralized nodules were quantified and shown to be decreased by 10(-7) M Ang II. The accumulation of calcium in cells and the matrix layer was also decreased by Ang II. Binding analysis with subtype-specific antagonists revealed the presence of AT1 receptors for Ang II in this culture system. Ang II caused a marked increase in the rate of production of intracellular cAMP in this system. Our data suggest that Ang II might be intimately involved in osteoblastic metabolism through its interaction with the AT1 receptor.

Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.

Inositol trisphosphate receptor gene expression and hormonal regulation in osteoblast-like cell lines and primary osteoblastic cell cultures.

The inositol trisphosphate receptor (IP3R) is an intracellular calcium channel that mediates the cellular actions of a wide variety of hormones, growth factors, and cytokines. In osteoblastic cell cultures, many bone resorbing hormones increase phosphoinositide turnover, inositol trisphosphate production, mobilization of intracellular calcium, and the secretion of osteoclast recruitment and activating factors. In this study, the effects of 17 beta-estradiol, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), phrobol ester, and serum on IP3R mRNA levels were evaluated in osteogenic-osteosarcoma cells and in primary osteoblastic cultures derived from neonatal rat calvaria. Type-specific reverse transcription polymerase chain reaction (RT-PCR) indicated that all cell types evaluated (G-292, U-2 OS, Saos-2, MC3T3-E1, UMR-106, and calvarial osteoblastic cells) express IP3R mRNA type I; G-292, U-2 OS, MC3T3-E1, and calvarial osteoblastic cells also express type II IP3R mRNA; and UMR-106 and the calvarial osteoblastic cells express type III IP3R mRNA. Northern blot and RT-PCR analyses of human G-292 osteosarcoma cells and rat calvarial osteoblastic cells showed that phorbol ester and serum increase IP3R mRNA levels, whereas 17 beta-estradiol and 1,25(OH)2D3 decrease these levels. In G-292 cells, the effect of 17 beta-estradiol was not due to accelerated IP3R mRNA degradation and required continued protein synthesis. The results show that multiple IP3R types are expressed in osteoblasts and osteoblastic osteosarcoma cells and that this expression is regulated by 17 beta-estradiol and other osteoporotic and antiosteoporotic hormones. These findings indicate that hormonal control of IP3R expression may be relevant in the chronic regulation of osteoblast secretory activity.

Differentiation of osteoblast in vitro is regulated by progesterone.

The regulation of cellular differentiation by progesterone in fetal rat calvarial osteoblasts was investigated. Our results showed that cells cultured in the presence of progesterone had a 7% increase in the alkaline phosphatase activity when compared to untreated cells. The concentration of osteocalcin in the conditioned medium from progesterone treated osteoblasts was 28% higher than that of untreated controls. In addition, administration of progesterone significantly enhanced the number and area of bone nodules. In conclusion, progesterone stimulates the differentiation of fetal rat calvarial osteoblastic cells in vitro.

Simulation of bone resorption-repair coupling in vitro.

In the normal adult human skeleton, new bone formation by osteoblasts restores the contours of bone surfaces following osteoclastic bone resorption, but the evidence for resorption-repair coupling remains circumstantial. To investigate whether sites of prior resorption, more than the surrounding unresorbed surface, attract osteoblasts or stimulate them to proliferate or make new matrix, we developed a simple in vitro system in which resorption-repair coupling occurs. Resorption pits were produced in mammalian dentine or bone slabs by culturing chick bone-derived cells on them for 2-3 days. The chick cells were swept off and the substrata reseeded with rat calvarial osteoblastic cells, which make bone nodules in vitro, for periods of up to 8 weeks. Cell positions and new bone formation were investigated by ordinary light microscopy, fluorescence and reflection confocal laser microscopy, and SEM, in stained and unstained samples. There was no evidence that the osteoblasts were especially attracted to, or influenced by, the sites of resorption in dentine or bone before cell confluence was reached. Bone formation was identified by light microscopy by the accumulation of matrix, staining with alizarin and calcein and by von Kossa's method, and confirmed by scanning electron microscopy (SEM) by using backscattered electron (BSE) and transmitted electron imaging of unembedded samples and BSE imaging of micro-milled embedded material. These new bone patches were located initially in the resorption pits. The model in vitro system may throw new light on the factors that control resorption-repair coupling in the mineralised tissues in vivo.

Second messenger systems stimulated by bradykinin in osteoblastic cells: evidence for B 2 receptors

The effects of bradykinin, analogs and inhibitors on the human osteoblastic osteosarcoma cell lines Saos-2 and G292 and on normal rat calvarial osteoblastic cells were investigated. In all cell types, bradykinin (1 nM-100 microM) caused significant time- and dose-dependent changes in the levels of inositol phosphates. Neomycin inhibited the inositol phosphate response to bradykinin, while indomethacin had no effect. Bradykinin also elicited a dose-dependent increase in free cytosolic calcium concentration. Bradykinin and T-kinin did not affect cyclic AMP levels in these cells. Doses of des-Arg9-bradykinin, a B1 receptor agonist, up to 100 nM did not stimulate the osteoblastic inositol phosphate response. In addition, the bradykinin-stimulated inositol phosphate response was unaffected by des-Arg9-[Leu8]-bradykinin, a B1 receptor antagonist, while it was inhibited by D-Arg-[Hyp3-[beta-(2-thienyl)-Ala]5,8-D-Phe7]-bradykinin, a B2 receptor antagonist. These results suggest that in osteoblastic cells the mechanism of action of bradykinin involves stimulation of the phosphoinositide metabolism and increases in cytosolic calcium levels through activation of B2 receptors.