Mineralization Of Osteoblastic Cells Research Articles

Quercetin, a flavonoid, inhibits the proliferation, differentiation, and mineralization of osteoblasts in vitro

It is possible that the flavonoids that are found in many foods might have a protective effect against osteoclastic activity. However, little information is available about the effects of flavonoids on osteoblastogenesis. Therefore, we investigated the effects of quercetin, a flavonoid, on the metabolism of rat calvarial osteoblast-like cells (ROB cells) in culture. The proliferation of cells was markedly inhibited upon exposure of cells to quercetin at 5×10 −6 to 1×10 −5 M. Quercetin 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, quercetin 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, after cells had ceased to proliferate, quercetin reduced the activity of alkaline phosphatase, the level of expression of mRNA for osteocalcin, the rate of deposition of Ca 2+, and the formation of mineralized nodules, all of which are markers of osteoblast differentiation. These findings indicate that quercetin inhibits the proliferation, differentiation, and mineralization of osteoblastic cells.

Localization of zinc after in vitro mineralization in osteoblastic cells.

The present study was designed to investigate the incorporation of zinc (Zn) into cultured UMR-106 osteoblasts in response to mineralization caused by the addition of beta-glycerophosphate. As a result of the induced mineralization, the contents of calcium (Ca), phosphorus (P), and Zn in the monolayer increased, whereas the magnesium (Mg) content did not change. The activity of alkaline phosphatase (ALP) also increased during the process. The zinc distribution in the cell monolayer was studied using Zinquin, a fluorescent zinc ion chelator. The double fluorescent labeling with Zinquin and calcein revealed that zinc was localized both as intracellular vesicles and extracellular clusters, whereas calcium was colocalized with extracellular zinc. These results suggest that zinc is involved in the mineralization process of UMR-106 cells.

Correlation between induction of expression of biglycan and mineralization by C-type natriuretic peptide in osteoblastic cells.

We reported previously that C-type natriuretic peptide (CNP) promotes the differentiation and mineralization of osteoblastic cells [Am. J. Physiol. 270 (Cell Physiol. 39): C1311-C1318, 1996]. However, little information is available about the mechanism of action of CNP in differentiating osteoblastic cells. Using the technique known as differential display-polymerase chain reaction, we attempted to identify the mRNAs whose levels are regulated by CNP in mouse clonal preosteoblastic MC3T3-E1 cells. One species of mRNA whose level was increased by CNP was 99% homologous to the 3'-untranslated region of a mouse gene for biglycan (BGN), a small proteoglycan. BGN is known to be involved in bone formation by osteoblastic cells. Therefore, we investigated the relationship, during the formation of mineralized nodules, between CNP and BGN using calvarial osteoblast-like cells (ROB cells) from newborn rats, that are a good model for studies on bone formation in vitro. Northern blot analysis revealed that transcription of the mRNA for BGN was up-regulated by CNP in ROB cells on days 6 and 8, whereas no effect of CNP was observed on days 3 and 12. Brief treatment with 10(-7) M CNP on days 3 through 9 exclusively enhanced the deposition of calcium, a result that suggests that CNP might regulate the expression of mineralization-related genes and, probably, the gene for BGN during a specific time period.

Endothelins inhibit the mineralization of osteoblastic MC3T3-E1 cells through the A-type endothelin receptor.

We examined the effects of various endothelins on the mineralization of mouse clonal preosteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed mRNAs for endothelin (ET)-1 and the A-type receptor for ET (ETA). A pharmacological study also demonstrated the predominant expression of the ETA receptor. Northern blotting analysis revealed that ETs decreased the expression of mRNA for osteocalcin, which is a marker protein for the maturation of osteoblastic cells. ET-1 also decreased in the deposition of calcium by MC3T3-E1 cells in a dose-dependent manner and it had an inhibitory effect even at 10(-11) M. The rank order of potency of ETs was ET-1 = ET-2 > ET-3. Brief treatment with 10(-7) M ET-1 on days 6-8 alone suppressed mineralization. ET-1 enhanced the rate of production of inositol 1,4, 5-trisphosphate (IP3) in MC3T3-E1 cells, but it had no effect on the rate of production of cAMP. Taken together, our data indicate that ET-1 might inhibit the mineralization of osteoblastic cells via an interaction with the ETA receptor, with generation of IP3 as the intracellular signal.

Phytocomponent p-hydroxycinnamic acid stimulates mineralization in osteoblastic MC3T3-E1 cells

Phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have stimulatory effects on bone calcification and inhibitory effects on bone resorption in rat femoral tissues in vitro. Whether HCA has a stimulatory effect on mineralization in osteoblastic cells is unknown. This study was undertaken to determine the effect of HCA on mineralization in osteoblastic MC3T3-E1 cells in vitro. Cells were cultured for 72 h in a minimum essential medium (alpha-MEM) containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or HCA (10(-7)-10(-5) M) without FBS. Culture with HCA (10(-7)-10(-5) M) did not have a significant effect on cell proliferation and cell death. Deoxyribonucleic acid (DNA) content in osteoblastic cells was significantly increased after culture with HCA (10(-6) or 10(-5) M) for 48 or 72 h. Alkaline phosphatase activity in osteoblastic cells was significantly increased after culture with HCA (10(-7)-10(-5) M) for 24, 48, or 72 h. The results with Alizarin red staining for calcium showed that mineralization was significantly stimulated after culture with HCA (10(-8)-10(-5) M) for 7, 14, or 21 days. This study demonstrates that HCA has stimulatory effects on mineralization in osteoblastic MC3T3-E1 cells.

P-252 - Effect of glucose concentration in media on alkaline phosphatase activity and mineralization in osteoblastic cells.

P-252 - Effect of glucose concentration in media on alkaline phosphatase activity and mineralization in osteoblastic cells.

Inhibition of in vitro mineralization in osteoblastic cells and in mouse tooth germ by phosphatidylinositol-specific phospholipase C

We have examined the effect of phosphatidylinositol-specific phospholipase C (PIPLC) on the in vitro mineralization during cultivation of both osteoblast-like MC3T3-E1 cells and the lower molar tooth germ from mouse embryo. PIPLC not only caused the release of alkaline phosphatase (ALP) from cell membranes into the culture medium but also produced a disturbance of in vitro mineralization in MC3T3-E1 cells and in mouse embryonic tooth germ. These findings strongly suggest that ALP, functioning as an ectoenzyme, is involved in the physiological mineralization process of both bone and teeth.