Rat Calvarial Bone Cells Research Articles

Effects of Different Extremely Low-Frequency Electromagnetic Fields on Osteoblasts

It is well known that the extremely low-frequency electromagnetic field (EMF) can promote the healing of bone fractures, but its mechanism remains poorly understood. The purpose of this study was to examine the response of neonatal rat calvarial bone cells to the rectangular electromagnetic field (REMF), triangular electromagnetic field (TEMF), sinusoidal electromagnetic field (SEMF), and pulsed electromagnetic field (PEMF). The stimulatory effects of EMF were evaluated by the proliferation (methyltetrazolium colorimetric assay), differentiation (alkaline phosphatase (ALP) activity), and mineralization (area of mineralized nodules of the cells). REMF treatment of osteoblasts increased cellular proliferation and decreased ALP activity (p < 0.05). TEMF had an accelerative effect on the cellular mineralized nodules (p < 0.05). SEMF treatment of osteoblasts decreased the cellular proliferation, increased ALP activity, and suppressed mineralized nodules formation (p < 0.05). PEMF promoted the proliferation of osteoblasts, inhibited their differentiation, and increased the mineralized nodules formation (p < 0.05). Moreover, the effects of PEMF on osteoblasts were concerned with the extracellular calcium, P2 receptor on the membrane, and PLC pathway, but the response of osteoblasts on SEMF was only related to PLC pathway. The results suggested that the waveforms of EMF were the crucial parameters to induce the response of osteoblasts.

Biochemical Pathway Mediating the Response of Bone Cells to Capacitive Coupling

Rat calvarial bone cells or mouse MC3T3-E1 bone cells subjected to a capacitively coupled electric field of 20 mV/cm consistently showed significant increases in cellular proliferation as determined by deoxyribonucleic acid content. Verapamil, a membrane calcium channel blocker; W-7, a calmodulin antagonist; indocin, a prostaglandin synthesis inhibitor; or bromophenacyl bromide, a phospholipase A2 inhibitor, each at a concentration that did not interfere with cell proliferation in control cultures, inhibited proliferation in those cultures subjected to the electric field. In contrast, neomycin, an inhibitor of the inositol phosphate cascade, did not inhibit this electrically induced cellular proliferation. Prostaglandin E2 production also was increased significantly with electrical stimulation, and this increase was inhibited by verapamil or indocin but not by neomycin. Thus, the data suggest that the signal transduction mediating the proliferative response of cultured bone cells to a capacitively coupled field involved transmembrane calcium translocation via voltage gated calcium channels, activation of phospholipase A2, and a subsequent increase in prostaglandin E2. Increases in cytosolic calcium and activated calmodulin are implied. The inositol phosphate pathway, unlike its dominant role in signal transduction in mechanically stimulated bone cells, does not appear to play a role in signal transduction in the proliferative response of bone cells to electrical stimulation.

Effect of a low-calcium environment on alkaline phosphatase activity in embryonic rat calvarial bone cells in culture

The effect of a low Ca environment on the alkaline phosphatase (ALP) activity in bone cells was examined. Bone cells were isolated from the calvaria of 18 to 21-day-old fetal rats and cultured in BGJ b or α-minimum essential medium (α-MEM) with calcium concentrations of 1.87 mM (control group), 1.20 mM (middle group) or 0.34 mM (low-Ca group). The control and low-Ca groups grew and reached a confluent state at 10 days of culture and there were no significant differences in the number of cells, total protein, or DNA content between the two groups. The specific ALP activity measured at 10 days of culture of the cells with 0.34, 1.20 and 1.87 mM calcium was 106.3 ± 11.9, 73.3 ± 10.4,and38.8 ± 7.3 unit per mg protein ( n = 9), respectively. When the low-Ca medium was replaced with the control medium during 10 days of culture, the activity decreased to the level of the control group. On the other hand, when control medium was replaced with low-Ca medium, the activity increased to the level of the low-Ca group. After confluence, cell growth in the low-Ca group was poorer than that in the control group with time. The ALP activity markedly increased and the mineralized nodular structures were observed in the control group of α-MEM, but not in the low-Ca group. These findings suggest that stimulation of ALP activity is a compensation mechanism to maintain normal cell functions in a low-Ca condition and that the role of ALP in the low-Ca environment may be different from that on mineralization.

Changes in protein kinase C activity in rat calvarial bone cells cultured in a low calcium environment

Changes in protein kinase C activity in rat calvarial bone cells cultured in a low calcium environment

In Vitro Bone-Cell Response to a Capacitively Coupled Electrical Field

Newborn rat calvarial bone cells were grown to confluence and subjected to a matrix of sine wave 60-kHz capacitively coupled electrical signals of various field strengths, pulse-burst patterns, and duty cycles. Both [3H] thymidine incorporation into DNA and alkaline phosphatase activity were evaluated in field strengths ranging from 0.0001 to 20 mV/cm, with pulse-burst patterns ranging from continuous to 5 milliseconds ON/495 milliseconds OFF, with daily duty cycles ranging from 0.25% to 25%. A significant increase in proliferation occurred in field strengths of 0.1, 1, and 20 mV/cm when the signal was applied continuously for six hours. Significant proliferation also occurred when the 20-mV/cm field was pulsed for six hours at 5 milliseconds ON/495 milliseconds OFF and at 5 milliseconds ON/245 milliseconds OFF. No change in alkaline phosphatase activity occurred in the 20-mV/cm field with any signal. At 1 mV/cm, there was a significant decrease in alkaline phosphatase activity in the continuous signal and in the 5 milliseconds ON/62 milliseconds OFF signal; in the lower fields evaluated, there was an actual decrease in alkaline phosphatase activity with some of the signals. The field strength plays a dominant role in determining the bone-cell's proliferative response, and to a lesser extent the alkaline phosphatase activity response, to a capacitively coupled electric field. The pulse configuration and the duty cycle are also important, but only if the proper field strength is being applied to the cell.

Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.

The interactions of bone cells with their extracellular matrix is of major importance in bone development, repair, and disease. We examined the ability of rat calvarial bone cells to adhere to various matrix proteins and to define the role of integrin cell-substrate adhesion receptors in these interactions. Isolated newborn rat calvarial bone cells prelabeled with 3H-thymidine and plated on plastic wells that had been precoated with serial dilutions of various substrates showed typical dose-response adherence curves to fibronectin, fibrinogen, laminin, vitronectin, and collagen I and IV. Cell adherence to poly-D-lysine, a nonspecific cell adherent, was high at all substrate concentrations > 0.0001 micrograms/ml. A polyclonal anti-rat integrin antibody blocked cell adhesion to all substrates tested except poly-D-lysine. Isolated rat calvarial bone cells were surface labeled with 125I, extracted, and immunoprecipitated with polyclonal antibodies made against the rat integrin complex and peptides derived from the cytoplasmic domains of the alpha 2, alpha 3, and alpha 5 subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced) identified four bands representing a mixture of integrins including the alpha 1 beta 1 laminin/collagen receptor, the alpha 5 beta 1 fibronectin receptor, and the alpha V beta 3 (or possibly alpha V beta 5) vitronectin receptor. These experiments show that bone cells adhere to a wide variety of extracellular matrix proteins via specific integrins. Increased knowledge about the regulation of these receptors and the mechanisms by which they transmit information to the cell will be important for a more complete understanding of bone physiology and pathophysiology.

Changes in protein kinase C activity in rat calvarial bone cells cultured in a low-calcium environment

This enzyme activity was examined in bone cells cultured for 8–10 days; the calcium concentration was 1.87 ± 0.05 ( n = 10) mM in the control medium and 0.34 ± 0.02 ( n = 10) mM in the low-calcium medium. The activity was significantly lower in the low-calcium group than in the control ( p < 0.01). The cytosolic fraction decreased more than the membranous fraction. After restoration to a regular calcium environment, the protein kinase C activity recovered rapidly to near the control value. The extent of recovery was greater in the membranous than in the cystolic fraction. These results suggest that the enzyme was inhibited in bone cells placed in a low-calcium environment, while the sensitivity in the membrane was enhanced.

The inositol phosphate pathway as a mediator in the proliferative response of rat calvarial bone cells to cyclical biaxial mechanical strain.

Isolated newborn rat calvarial bone cells grown in monolayer on polyurethane membranes in specially constructed culture chambers and subjected to a cyclical biaxial mechanical strain of 0.17% at a frequency of 1 Hz for 30 min demonstrated a 16% increase in DNA synthesis during the subsequent 24 h. The metabolites of the inositol phosphate pathway, shown to be an important second messenger in many cell types, were shown to be elevated using high-performance liquid chromatography to separate and quantitate the various inositol polyphosphates. Inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1,3,4,5-tetrakisphosphate reached peak accumulations after 20 s of mechanical strain. Inositol 1,3,4-trisphosphate reached a peak accumulation after 2 min, and inositol 1,2,3,4,5,6 phosphate reached a peak accumulation after 60 min of mechanical strain. Neomycin, an inhibitor of phospholipase C, a membrane-bound enzyme that hydrolyzes phosphatidyl inositol 4,5-bisphosphate to start the inositol phosphate cascade, completely inhibited accumulation of the above inositol phosphates during mechanical straining of the bone cells. Neomycin also completely abolished the increase in DNA synthesis that was seen after a mechanical strain of 0.17%. It is concluded from this study that the inositol phosphate pathway is activated by mechanical strain in bone cells and that this pathway is an important and primary mediator in the transduction of mechanical strain into cellular proliferation in these cells.

Proliferation and macromolecular synthesis by rat calvarial bone cells grown in various oxygen tensions

Perinatal rat calvarial bone cells were isolated by sequential collagenase digestion and grown in oxygen tensions ranging from 1 to 60% O2. Cell proliferation as determined by automated cell counting and DNA content was greatest in the lower oxygen tensions (less than or equal to 9% O2), whereas alkaline phosphatase activity and [35S]sulfate and [14C]proline incorporation were greatest in the higher oxygen tensions (greater than or equal to 13% O2). It is concluded that lower oxygen concentrations favor bone cell proliferation, whereas higher oxygen concentrations favor macromolecular synthesis. These findings, when related to the known pO2 of the fracture callus, suggest the following sequence of events: first, at the time of fracture an ingrowth of osteoprogenitor cells, capillary buds, and primitive mesenchymal cells occurs in the fracture site, a region of low pO2; second, a great increase in cellular proliferation accompanied by an initiation of macromolecular synthesis follows; finally, as the pO2 levels begin to increase, cellular proliferation decelerates, accompanied by an increase in macromolecular synthesis.

The effect of ascorbic acid on the metabolism of rat calvarial bone cells in vitro.

Addition of 50 micrograms/ml sodium ascorbate to confluent cultures of isolated rat calvarium bone cells resulted in a 21% increase in DNA production, a 50-60% increase in incorporation of [14C]proline into collagenous and noncollagenous proteins, and a 200% increase in alkaline phosphatase activity; under identical conditions, [35S]sulfate incorporation into proteoglycans (glycosaminoglycans) was not affected. These results suggest that ascorbate may be important in maintaining or stimulating the osteogenic phenotype of normal bone cells.

Response of cultured bone cells to a capacitively coupled electric field: inhibition of cAMP response to parathyroid hormone.

Fetal rat calvarial bone cells grown in monolayers were exposed to a capacitively coupled electrical field of 2.62 mV/cm for 2.5-30 min. There was a 59% increase in cAMP concentration after electrical stimulation, compared with a 3615% increase after parathyroid hormone (PTH) administration. PTH administration after electrical stimulation resulted in less cAMP synthesis than following either electrical stimulation alone or PTH administration alone. When PTH administration was delayed for 30 min following cessation of the electrical stimulation, there was an inhibition in the cAMP response. This suggests that one possible mechanism of electrically induced osteogenesis is the desensitization of the bone cell to PTH. Since the effect of PTH is to increase bone resorption, desensitizing or blocking the effects of PTH at the bone cell level would result in a net increase in bone formation.

Identification of novel proteins synthesized in bone cells by antibody screening of a cDNA expression library

Novel proteins synthesize predominantly in bone have been identified by antibody screening of bone cell cDNA expression libraries. Two unique cDNAs were identified whose structures do not match any known nucleic acid or protein sequence in the NIH computer bank. The first cDNA clone, BP-I, encoded a mRNA of 2300 bases in size which was expressed at high levels in 17 2.8 rat osteosarcoma cells, rat calvarial bone cells and placenta. A second clone, BP-II, encoded a mRNA of 1500 bases which was expressed at high levels in 17 2.8 osteosarcoma cells and in salivary gland. Expression of both mRNAs in osteosarcoma cells was modulated by the calciotropic hormone, vitamin D. Southern blot analyses indicated that the two cDNAs represented distinct, single copy genes in the rat genome. These novel gene products may serve as potential new markers to study bone turnover in metabolic bone disease.

The effects of calmodulin antagonists on prostaglandin E 2-induced responses in rat calvarial bone cells

Osteoclastic (OC) and osteoblastic (OB) cells were isolated by sequential collagenase digestions of new-born rat calvaria. Prostaglandin E 2(PGE 2) did not alter total calmodulin levels after a 5 or 60 min incubation. The calmodulin antagonists, trifluoperazine (TFP) at 10–50 μM and W-7 (50 μM) inhibited PGE 2-induced increases in calcium uptake by OC cells, but had no effect on control OC or OB calcium levels. W-5 (50 μM), a chlorine-deficient analogue of W-7 with weak anti-calmodulin activity, had no effect. Compound 48 80 (100–500 μg/ml), a highly effective calmodulin antagonist in other systems, had no effect on PGE 2-induced calcium levels or control calcium uptake. There was inhibition of PGE 2-induced increases in cyclic AMP by compound 48 80 (100 μg/ml) in both OC and OB cells but no effect on control levels. TFP at 50 μM inhibited both control and PGE 2-induced increases in cyclic AMP but at 10 μM it lessened only the hormone-induced effect. W-7 (100 μM) inhibited PGE 2-induced increases in OC and OB cyclic AMP but had no effect on control levels; W-5 (50 μM) had no effect on either of these. Dibutyryl cyclic AMP had no effect on control calcium uptake, PGE 2-induced increases or W-7 inhibition of the PGE-2 effect on calcium uptake. The calmodulin antagonists, at doses which had affected only PGE 2-induced increases in calcium uptake and/or cyclic AMP production, had no effect on leucine uptake by OC or OB cells. Lactic-acid production by OC or OB cells only decreased after 50 μM TFP, which suggests a specific, non-metabolic effect related to the other treatments.

Detection of high-affinity glucocorticoid binding in rat bone.

Department of Medicine, Manchester Royal Infirmary, Manchester, M13 9WL (Received 17 October 1977) Recently Feldman, Dziak, Koehler &amp; Stern (1975) have detected high-affinity gluco corticoid receptors in cytosol from isolated foetal rat calvarial bone cells. They estimated the binding constant (Kd) to be 7×10−9 mol/l for [3H]dexame[unk]hasone; however, their estimates were based on a maximum bound: free ratio of only 1%. Their data from cell cultures suggest that such binding may nevertheless be of physiological importance (Chen, Aronow &amp; Feldman, 1977). The very small amount of binding might be due to a low initial concentration of binding sites, to their destruction during or after the trypsin–collagenase digestion, to their high dilution or to a combination of all three factors. We report here a much simpler method whereby we have greatly increased the percentage binding by setting up the incubation in intact calvaria. Calvaría were dissected from foetal rats (19–21 days