Proliferation Of Osteoblastic MC3T3-E1 Cells Research Articles

Effects of geranylgeranoic acid in bone: induction of osteoblast differentiation and inhibition of osteoclast formation.

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3-E1 cells, but also up-regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1alpha,25(OH)2D3- or prostaglandin E2 (PGE2)-induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor kappaB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence-accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.

Preparation, solubility, and cytocompatibility of zinc-releasing calcium phosphate ceramics

Zinc is an essential trace element with stimulatory effects on bone formation. Therefore, zinc was doped into β-tricalcium phosphate to develop zinc-releasing biomaterials to promote bone formation. The zinc-doped β-tricalcium phosphate, β-tricalcium phosphate, and hydroxyapatite powders were mixed at a (Ca+Zn)/P molar ratio of 1.60, followed by sintering into a dense body at 1100°C for 1 h. The sintered body was a composite ceramic consisting of zinc-doped β-tricalcium phosphate and hydroxyapatite phases. The composite ceramic contained zinc oxide when the zinc content was higher than 1.20 wt %. The composite ceramic released zinc under pseudophysiological conditions. However, the release of calcium and phosphate decreased with an increase in zinc content in a range higher than 0.12 wt % owing to a decrease in solubility of the zinc-doped β-tricalcium phosphate phase. Proliferation of osteoblastic MC3T3-E1 cells was significantly increased on the composite ceramic with a zinc content from 0.6 to 1.20 wt %, compared with those without zinc. When the zinc content was higher than 1.20 wt %, release of zinc from the zinc oxide caused cytotoxicity. Therefore, the zinc content of the composite ceramic must be <1.20 wt %. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 50, 178–183, 2000.

Bovine Milk Kininogen Fragment 1·2 Promotes the Proliferation of Osteoblastic MC3T3-E1 Cells

The active component on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified from bovine milk. The growth-promoting activity was measured by [3H]thymidine incorporation on the cell. The purified protein showed a molecular size of 17 kDa on SDS-PAGE. Its amino-terminal amino acid sequence was very similar to the internal sequence of bovine high molecular weight (HMW) kininogen, which comprises fragment 1·2. The promotion of proliferation was specific for osteoblastic MC3T3-E1 cells, not for fibroblast BALB/3T3 cells. In blood coagulation, HMW kininogen is considered to be cleaved by a specific enzyme kallikrein. HMW kininogen then releases two peptides, a biologically active peptide bradykinin and fragment 1·2, but the fate of fragment 1·2 is unknown. This milk-derived protein that comprises to fragment 1·2 showed a growth-promoting activity of osteoblasts. We propose the possibility that milk plays an important role in bone formation by supplying the active agent for osteoblasts as well as supplying calcium.

Preparation, solubility, and cytocompatibility of zinc-releasing calcium phosphate ceramics.

Zinc is an essential trace element with stimulatory effects on bone formation. Therefore, zinc was doped into beta-tricalcium phosphate to develop zinc-releasing biomaterials to promote bone formation. The zinc-doped beta-tricalcium phosphate, beta-tricalcium phosphate, and hydroxyapatite powders were mixed at a (Ca+Zn)/P molar ratio of 1.60, followed by sintering into a dense body at 1100 degrees C for 1 h. The sintered body was a composite ceramic consisting of zinc-doped beta-tricalcium phosphate and hydroxyapatite phases. The composite ceramic contained zinc oxide when the zinc content was higher than 1.20 wt %. The composite ceramic released zinc under pseudophysiological conditions. However, the release of calcium and phosphate decreased with an increase in zinc content in a range higher than 0.12 wt % owing to a decrease in solubility of the zinc-doped beta-tricalcium phosphate phase. Proliferation of osteoblastic MC3T3-E1 cells was significantly increased on the composite ceramic with a zinc content from 0.6 to 1.20 wt %, compared with those without zinc. When the zinc content was higher than 1.20 wt %, release of zinc from the zinc oxide caused cytotoxicity. Therefore, the zinc content of the composite ceramic must be <1.20 wt %.

High Mobility Group-like Protein in Bovine Milk Stimulates the Proliferation of Osteoblastic MC3T3-E1 Cells

The active component in bovine milk on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified. Growth-promoting activity was measured by [3H]thymidine incorporation on the cell. The molecular weight of the purified protein was 10 kDa. The amino-terminal sequence of this 10-kDa protein was identical to bovine high mobility group protein (HMG) 1. This 10-kDa protein is suggested to be a basic protein and to have an HMG box, a consensus sequence motif among the HMG family. From these results, we named this protein HMG-like protein. HMG is a ubiquitous nonhistone component of chromatin and considered to be implicated in DNA replication. We found this protein in milk, and it showed a growth-promoting activity. We propose the possibility that HMG-like protein existed in milk and plays an important role for neonate in bone formation by activating osteoblasts.

Effect of 1.25-Dihydroxyvitamin D3 on the Proliferation of Osteoblastic MC3T3-E1 Cells by Modulating the Release of Local Regulators from Monocytes

Some evidence suggests an important role of mononuclear cells at the bone remodeling sites in the coupling of bone resorption to bone formation. Therefore, we examined effects of human monocytes-conditioned medium (CM) treated with 1.25-dihydroxyvitamin D3 [1,25 (OH) 2D3] on the proliferation of osteoblastic MC3T3-E1 cells. 1,25 (OH) 2D3 (10−11-10−8) directly inhibited (3H) thymidine incorporation(TdR) into MC3T3-El cells in a dose-related fashion. 24,25 (OH) 2D3 and 25 (OH) D3 (10−8 and 10−7 M) had only modest inhibitory effect, compared to that of 1,25 (OH) 2D3. CM, per se, on the other hand, significantly stimulated TdR, whereas CM obtained from monocytes treated with 1,25 (OH) 2D3 (10−10 and 10−8 M) significantly inhibited TdR. Treatment of monocytes with 10−7 M 25 (OH) D3 significantly inhibited CM-induced stimulation of TdR to a lesser degree, compared to that of 10−8 M 1,25 (OH) 2D3 and treatment of monocytes with 10−7 M 24,25 (OH) 2D3 did not affect CM-induced stimulation of TdR. The inhibition of TdR by 1,25 (OH) 2D3-treated CM was significantly blocked by 10−6 M indomethacin, an inhibitor of prostaglandin synthesis. Present data first indicate that 1,25 (OH) 2D3 inhibited osteoblast proliferation not only directly but also indirectly through modulating the release of local factors as to bone remodeling from monocytes, and also suggest that its indirect effect via monocytes was mediated at least in part through prostaglandin synthesis.

Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferation.

We examined the binding characteristics of three kinds of steroid hormones--estrogen, androgen, and glucocorticoid--in cultured MC3T3-E1 mouse osteoblastic cells by whole-cell binding assay. The binding studies revealed the presence of a single class of high-affinity binding sites for [3H]17 beta-estradiol, [3H]mibolerone (a synthetic androgen), and [3H]triamcinolone acetonide (a synthetic glucocorticoid). The numbers of binding sites for these steroid hormones were found to be 4534 +/- 819, 14312 +/- 1884, and 24898 +/- 655 sites/cell; and the Kd values were 8.57 +/- 0.62 x 10(-10) M, 1.12 +/- 0.19 x 10(-9) M, and 6.08 +/- 1.24 x 10(-10) M, respectively. We also examined the effects of steroid hormones on the proliferation of MC3T3-E1 cells. 17 beta-estradiol significantly stimulated the proliferation of the cells (130-150% of control). Dihydrotestosterone also significantly stimulated the proliferation of the cells (115% of control); the effect was, however, much less potent than that of 17 beta-estradiol, although the number of binding sites was approximately three times more than that of 17 beta estradiol. Triamcinolone acetonide and dexamethasone had no effect on cell proliferation. These results suggest that estrogen and androgen act directly on osteoblastic cells through a receptor-mediated mechanism, and that androgen is much less potent than estrogen in stimulating the proliferation of MC3T3-E1 osteoblastic cells.

Effect of elevated extracellular calcium on the proliferation of osteoblastic MC3T3-E1 cells: Its direct and indirect effects via monocytes

There has been evidence that elevated calcium concentration at the resorptive site of the bone directly regulates osteoclast function. In the present study, in order to clarify the role of elevated calcium concentration at the resorptive site in the regulation of osteoblast function, not only direct but also indirect effect via human monocytes of the increase in extracellular calcium (Cae) on the proliferation of osteoblastic MC3T3-E1 cells have been investigated in serum-free condition. The increase in Cae enormously stimulated osteoblast proliferation at the concentration of 3 to 20 mM. When human monocytes were cultured at the elevated Cae concentration, monocyte-conditioned medium-induced stimulation of osteoblast proliferation was significantly amplified. Present data demonstrate that elevated Cae has pronounced stimulatory effect on osteoblast proliferation not only directly but also indirectly via monocytes. Calcium released from bone matrix at the resorptive sites might be linked to the coupling of osteoclast and osteoblast functions.

Effect of 17 β -estradiol on the proliferation of osteoblastic MC3T3-E1 cells via human monocytes

Effects of human monocyte-conditioned medium on the proliferation of osteoblastic MC3T3-E1 cells were investigated in serum-free cultured condition. Monocyte-conditioned medium significantly stimulated osteoblast proliferation at the concentration between 10 and 30 %, compared to that in the absence of monocytes, 17 β -estradiol directly stimulated osteoblast proliferation at the concentrations of 10 −8 and 10 −10M. On the contrary, the conditioned medium prepared by monocytes cultured in the presence of 17 β -estradiol at the concentrations of 10 −8 and 10 −10M significantly inhibited osteoblast proliferation. Present data indicate that in addition to direct effect on osteoblasts. 17 β - estradiol affected osteoblast proliferation presumably through modulating the release of several local regulators of bone turnover from monocytes. The effect on osteoblastic activity via monocytes might be linked to the coupling of osteoclast and osteoblast actions.

Transforming growth factor beta modulates proliferation of osteoblastic cells: relation to its effect on receptor levels for epidermal growth factor.

The effect of transforming growth factor beta (TGF-beta) on cellular proliferation of osteoblastic MC3T3-E1 cells was studied with particular emphasis on its effect on modulation of epidermal growth factor (EGF) receptors. In other cells, TGF-beta has been reported to augment EGF receptors. Exposure of MC3T3-E1 cells to TGF-beta initially increased cell surface EGF receptor levels and decreased the rate of DNA synthesis. The initial elevation of EGF receptor levels was due to increased receptor number per cell, not to changes in binding affinity. On the contrary, prolonged exposure (longer than 40 h) resulted in a decrease in EGF receptor and an increase in the rate of DNA synthesis. Thus, the effects of TGF-beta on these cells appears to be biphasic, reflecting complex mechanisms of action; the early effects of TGF-beta may be consistent with cellular differentiation to the osteoblastic phenotype with decreased cellular proliferation, whereas chronic exposure of these cells to TGF-beta stimulated cellular proliferation and inhibited osteoblastic phenotype expression. It is not likely that stimulation of cellular proliferation was through elevation of EGF receptor levels, because TGF-beta did not enhance the stimulatory effect of EGF on cellular proliferation. Thus, we conclude that TGF-beta possesses a stimulatory effect on the cellular proliferation of osteoblastic MC3T3-E1 cells independent of its modulative effect on EGF receptor level.