Search

The 5-hydroxytryptamine (5-HT) receptors 5-HT(2A), 5-HT(2B), and 5-HT(2C) belong to a subfamily of serotonin receptors. Amino acid and mRNA sequences of these receptors have been published for several species including man. The 5-HT(2) receptors have been reported to act on nervous, muscle, and endothelial tissues. Here we report the presence of 5-HT(2B) receptor in fetal chicken bone cells. 5-HT(2B) receptor mRNA expression was demonstrated in osteocytes, osteoblasts, and periosteal fibroblasts, a population containing osteoblast precursor cells. Pharmacological studies using several agonists and antagonists showed that occupancy of the 5-HT(2B) receptor stimulates the proliferation of periosteal fibroblasts. Activity of the 5-HT(2A) receptor could however not be excluded. mRNA for both receptors was shown to be equally present in adult mouse osteoblasts. Osteocytes, which showed the highest expression of 5-HT(2B) receptor mRNA in chicken, and to a lesser extent osteoblasts, are considered to be mechanosensor cells involved in the adaptation of bone to its mechanical usage. Nitric oxide is one of the signaling molecules that is released upon mechanical stimulation of osteocytes and osteoblasts. The serotonin analog alpha-methyl-5-HT, which preferentially binds to 5-HT(2) receptors, decreased nitric oxide release by mechanically stimulated mouse osteoblasts. These results demonstrate that serotonin is involved in bone metabolism and its mechanoregulation.

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C2GM was shown to be strongly dependent on osteoblast density. In cocultures of C2GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 x 10(4) cells/cm2), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 x 10(4) cells/cm2). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear.

It has been established, that the osteoclast is derived from the haemopoietic stem cell, but its exact lineage is still controversial. It is sometimes suggested, that osteoclasts and monocytes/macrophages are related cells. It has also been suggested that osteoclast differentiation is regulated by osteoblasts and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In the present paper we addressed the question whether avian monocytes can differentiate into osteoclasts in vitro, using an array of immunocytochemical, enzyme cytochemical and function markers. We have also determined the effects of osteoblasts, osteoblast conditioned medium and 1,25-(OH)2D3 on the expression of osteoclastic features on monocytes during culture. Monocytes developed tartrate resistant acid phosphatase (TRAcP) enzyme activity and antigens for all anti-osteoclast antibodies tested, during culture. However, they did not acquire the ability to resorb dentine and still showed phagocytosis of latex spheres. This indicates that the monocytes developed into cells resembling osteoclasts but lacking their function while retaining the function of macrophages. Osteoblast conditioned medium stimulated TRAcP enzyme activity and proliferation of monocytes in cultures. Addition of osteoblasts or osteoblast conditioned medium to monocyte cultures on dentine in the presence or absence of 1,25-(OH)2D3 did not result in the generation of genuine osteoclasts, nor in pit formation. 1,25-(OH)2D3 appeared to be cytotoxic to the avian monocytes in concentrations considered optimal for mouse osteoclast formation. These results suggest that avian monocytes do not readily differentiate into osteoclasts under in vitro conditions that stimulate osteoclast differentiation from bone marrow derived haemopoietic cells. Furthermore, labelling with anti-osteoclast antibodies and TRAcP as osteoclast-markers should be used only with great caution in the identification of osteoclasts formed in vitro.

We succeeded in the isolation of osteocytes from parietal bones of 16-day-old chick embryos. Isolated osteocytes showed a typical stellate morphology. More than 95% of these cells reacted with the osteocyte-specific antibody OB 7.3. In culture osteocytes formed gap junctions with each other, as could be established by ACAS. Sixteen-millimeter time-lapse microcinematography of the cells also demonstrated the formation of intercellular connections and gap junctions, and portrayed the interaction between osteocytes and osteoclasts: osteocytes seemed to inhibit osteoclast activity. This cinematography also showed the ability of osteocytes to proliferate after they had been disconnected from each other. Thereafter these cells redifferentiated into osteoblasts that became embedded in bone matrix produced by themselves. These findings suggest that osteocytes might be involved in bone formation during remodeling.

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

The present study evaluates the effect of parathyroid hormone (PTH) on intracellular calcium. Intracellular calcium ion concentrations ([Ca2+]i) in fetal rat osteoblasts in primary culture (ROB) and in UMR106-01 osteogenic sarcoma cells were monitored as changes in the ratio (R) of Fura-2 fluorescence intensities in single cells as well as populations of cells. In both single ROB and UMR106-01 cells, addition of 10(-7) M rat PTH1-34 and 3 NIH units/ml human thrombin resulted in heterogeneous responses in R values and therefore [Ca2+]i. PTH-induced calcium responsiveness of ROB was dependent on culture conditions, such that response frequencies were positively correlated with the percentage of fetal calf serum in the culture medium. PTH responsive ROB and UMR106-01 cells had significantly higher resting [Ca2+]i than unresponsive cells. PTH- or thrombin-mediated calcium signalling appeared not to be correlated to alkaline phosphatase activity in single ROB. Low percentages of cells responded to PTH in comparison to thrombin suggesting that an increase in [Ca2+]i is not a common PTH signalling pathway in osteoblasts in primary culture. Our data suggest that activation of this signalling pathway by PTH is culture condition dependent, possibly via a cell-cycle related increase in sensitivity of the pathway.

Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone cells under our experimental conditions. However, our fluorescence measurements of [Ca2+]i in single cells indicate the presence of Ca2+ conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consistent with other studies. We discuss possible roles for GKi, GKCa, and anion channels in acid secretion by OCL and for stretch-activated channels in OCL locomotion.

Primary cultures of embryonic chick osteoblasts consist of a heterogeneous cell population. Patch clamp measurements were done on 1- to 5-day-old osteoblasts, osteocytes, fibroblastlike cells, and cells that could not be classified on morphologic criteria. The measurements showed the omnipresence of depolarization-activated high-conductance channels in cell-attached patches. The whole-cell experiments showed an outward rectifying conductance activating at positive membrane potentials. Channels underlying the latter conductance were found to be K+ conducting in outside-out membrane patches. The activation potential of the outward rectifying K+ conductance shifted to negative membrane potentials upon increasing the intracellular Ca2+ concentration within the range of 10(-8)-10(-3.2) M. The same happened with the activation potential of the K+ channels found in outside-out patches. Finally, inside-out patch experiments directly demonstrated the dependency of the activation potential of K+ channels on Ca2+ ions. Thus the identity and main characteristics of Ca2(+)-activated K+ channels expressed by the various cell types present in chick osteoblast cultures have now been established. Decreased input resistances were found in cells of cultures more than 2 days old. This is consistent with the establishment of electrical coupling between the cells. Functions in which Ca2(+)-activated K+ channels could play a role are discussed.

The fully differentiated osteoblast may be easily recognized in bone tissue. Its cuboidal shape, its position directly opposed to the bone surface and its capacity to produce calcified bone matrix are characteristic. Three other differentiation stages are also reasonably well defined--the preosteoblast, the osteocyte and the lining cell. These differentiation stages are preceded by an unknown number of precursor, progenitor and stem cell stages. Little is known about the regulation of the transitions between the various osteogenic phenotypes and their reversibility or irreversibility. One of the reasons for this is the lack of adequate tools with which to recognize the various differentiation stages. We have developed a number of monoclonal antibodies (in bone) specifically directed against osteocytes, osteoblasts and as yet unidentified cells in the periosteum. The anti-osteocyte monoclonals were used to recognize osteocytes in bone cell cultures and we obtained purified osteocyte populations for metabolic studies. Osteocytes were shown to have binding sites for parathyroid hormone. The antibodies directed against osteoblasts showed that at present our culture conditions are inadequate to allow osteoblast differentiation in vitro.

Two cell populations were isolated from calvaria of chick embryos: PF cells were liberated by collagenase treatment from the periosteum, OB cells from the periosteum-free calvarium. Both populations were cultured in plastic culture dishes. After 6 d of culture, monolayers of each cell type either were scraped off the culture dishes, transplanted on the chorio-allantoic membrane of 7-d-old quail eggs, and cultured there for 6 d, or were used for biochemical experiments. OB transplants proved capable of producing calcified bone matrix, whereas PF transplants formed only fibrous tissue. Biochemically, OB cells showed high cAMP production in the presence of parathyroid hormone (PTH), whereas cAMP production was not stimulated in PF cultures. Lactate production was stimulated by PTH in both populations although somewhat differently. Citrate decarboxylation was high in OB cells and was inhibited by PTH but was low in PF cells, where it was stimulated by the same hormone. The differences in hormonal response between the two cell types made it possible to conclude that PF cultures are relatively free of OB cells. The PF contamination in OB cultures was more difficult to assess. The experiments described in this report show that the OB population contains osteoblasts or osteoblastlike cells which are, under favorable circumstances, capable of bone formation.

Serum-starved chick osteoblast-like cells (OB cells) and periosteal fibroblasts (PF cells) were used to study the proliferative effects of parathyroid hormone (PTH) and prostaglandin E2 (PGE2). Both PTH (10(-11) to 10(-8) M) and PGE2 (10(-9) to 10(-5) M) had a direct, dose-related effect on the de novo synthesis of DNA in OB cells. The PF cells only showed a dose-dependent effect in the presence of PGE2 (10(-9) to 10(-5) M). The hormonally induced proliferation of these cells was shown to be dependent on cell density and stimulation time. An optimal response for both cell types was observed in the cell density range 1.5 to 3.5 micrograms DNA/2 cm2, when stimulated for 18 hours. As cAMP-enhancing substances (N6-dBcAMP, forskolin and IBMX) could mimic the PTH- and PGE2-induced proliferation in OB cells, the increased DNA synthesis was concluded to be mainly caused by enhanced cAMP concentrations.

Patch-clamp measurements were made on osteoblast-like cells isolated from embryonic chick calvaria. Cell-attached-patch measurements revealed two types of high conductance (100-250 pS) channels, which rapidly activated upon 50-100 mV depolarization. One type showed sustained and the other transient activation over a 10-sec period of depolarization. The single-channel conductances of these channel types were about 100 or 250 pS, depending on whether the pipettes were filled with a low K+ (3 mM) or high K+ (143 mM) saline, respectively. The different reversal potentials under these conditions were consistent with at least K+ conduction. Whole-cell measurements revealed the existence of two types of outward rectifying conductances. The first type conducts K+ ions and activates within 20-200 msec (depending on the stimulus) upon depolarizing voltage steps from less than -60 mV to greater than -30 mV. It inactivates almost completely with a time constant of 2-3 sec. Recovery from inactivation is biphasic with an initial rapid phase (1-2 sec) followed by a slow phase (greater than 20 sec). The second whole-cell conductance activates at positive membrane potentials of greater than +50 mV. It also rapidly turns on upon depolarizing voltage steps. Activation may partly disappear at the higher voltages. Its single channels of 140 pS conductance were identified in the whole cell and did conduct K+ ions but were not highly Cl- or Na+ selective. The results show that osteoblasts may express various types of voltage controlled ionic channels. We predict a role for such channels in mineral metabolism of bone tissue and its control by osteoblasts.

Bone cells obtained by digestion of fetal mouse or chicken calvaria were tested for their ability to form or resorb bone in vitro. The isolated cells were precultured for 6 days and subsequently cocultured for 11 days with periosteum-free noninvaded fetal mouse long bone rudiments. Bone formation and resorption during coculture were evaluated by histology and 45Ca release from prelabeled bones. The calvarial origin of cells in cocultures was traced by labeling the cells with 3H-thymidine before coculture, followed by autoradiography. Many osteoblasts and osteoclasts as well as fibroblasts developed from mouse periosteal cells released late in the sequential digestion procedure and previously denoted as "osteoblastlike" (BL). No or few osteoblasts and osteoclasts but many fibroblasts developed from early released cell fractions that have previously been denoted as "osteoclastlike" (CL). Only osteoblasts and fibroblasts but not osteoclasts developed from chicken calvarial cell fractions. The osteoblasts developed primarily from cell fractions from the inner layer of the periosteum, previously denoted as "osteoblastlike" (OB). Cells obtained from the outer layer of the periosteum (PF) gave rise mainly to fibroblasts. These studies show that osteoblast and osteoclast precursor cells are maintained in monolayer cultures of periosteal cell fractions. However, sequential digestion of mouse calvaria does not lead to separation of the two types of bone cells. Rather, osteoclast and osteoblast precursors are released jointly, from the periosteal cell layers closest to the bone surface. In the chicken cell fractions osteoclast precursors are absent after preculture, resulting in a more homogeneous population of osteoblast and fibroblast but not osteoclast precursors.

The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (14C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (3H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one. Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)2D3.

The effects of several prostaglandins on the proliferation of secondary cultures of osteoblast-like cells, as measured by the incorporation of [3H]-thymidine into DNA and total DNA content of the cultures, were studied. PGE2 in the concentration range of 10(-8) to 10(-5) M caused a direct, dose-related stimulation of proliferation, while PGF2 alpha and PGD2 were less effective. PGA2 and 6-keto-PGF1 alpha were inactive in the osteoblasts in concentrations of 10(-7) to 10(-6) M. A similar stimulation profile was observed for the induction of ornithine decarboxylase (ODC, L-ornithine decarboxy-lyase, EC 4.1.1.17): the order of potency of the different prostaglandins in the induction of the ODC activity was PGE2 greater than PGF2 alpha = PGD2; again, PGA2 and 6-keto-PGF1 alpha were without effect in concentrations up to 10(-6) M. These results show that the primary prostaglandins, in order of potency PGE2 greater than PGF2 alpha = PGD2, can have a direct, stimulatory effect on the proliferation of osteoblasts, which is closely related to the induction of ODC activity.

Incubation of osteoblast-like cells with [3H]25-(OH)D3 and varying bovine serum albumin (BSA) concentrations resulted in a dramatic change in the accumulation of 1,25-(OH)2D3 and 24,25-(OH)2D3 in the medium. At 0.1% BSA 1,25-(OH)2D3 formation was transient and 24,25-(OH)2D3 was the main product after 3 h. At 2% BSA accumulation of 1,25-(OH)2D3 was sustained whereas 24,25-(OH)2D3 formation was suppressed. At low BSA levels added [3H]1,25-(OH)2D3 was rapidly metabolized to 1,24,25-(OH)3D3 and more polar metabolites. The effect of increasing BSA concentrations on 25-(OH)D3 metabolism was mimicked by addition of cycloheximide. This indicates that high BSA levels prevent the induction of 24-hydroxylase activity in this system, probably by lowering of the free 25-(OH)D3 concentration. The accumulation of 1,25-(OH)2D3 from 25-(OH)D3 not only depends on the 1 alpha-hydroxylase activity, but also on the further metabolism of 1,25-(OH)2D3 by 24-hydroxylation.

The interactions between two different cell populations within the osteogenic compartment have been examined. The proliferation of periosteal fibroblasts (PF) in the presence or absence of osteoblast-like cells (OB), whose proliferative capacity was inhibited by irradiation, was measured. OB stimulated [3H]thymidine incorporation in PF and parathyroid hormone (PTH) enhanced the stimulation. In the reverse situation, however, PF inhibited OB. Irradiated OB also stimulated 3H-thymidine incorporation in OB, and irradiated PF in PF, but both to a lesser extent. Co-culture experiments showed that direct cell-cell contact was a prerequisite for stimulation of PF. Medium mediated contact between physically separated OB and PF did not stimulate, but rather inhibited PF proliferation. These results demonstrate that OB regulate the proliferation of cells in the PF population and can transmit the proliferation stimulating message of PTH to PF. This implies that in vivo the mature osteoblast may play a pivotal role in the (hormonal) regulation of osteoprogenitor cell proliferation and therefore bone formation.