Cultures Of Fetal Rat Calvaria Cells Research Articles

The EP4-ERK-dependent pathway stimulates osteo-adipogenic progenitor proliferation resulting in increased adipogenesis in fetal rat calvaria cell cultures

We previously reported that fetal rat calvaria (RC) cells are osteo-adipogenic bipotential and that PGE2 receptors EP2 and EP4 are involved in bone nodule formation via both common and distinct MAPK pathways in RC cell cultures. Because PGE2 participates in multiple biological processes including adipogenesis, it is of interest to determine the additional role(s) of PGE2 in RC cells. PGE2 increased the number of adipocyte colonies when RC cells were treated during proliferation but not other development stages. Of four EP agonists tested, the EP4 agonist ONO-AE1-437 (EP4A) was the most effective in promoting adipogenesis. Concomitantly, EP4A increased the number of cells with BrdU labeling and gene expression of CCAAT/enhancer binding protein (C/EBP)δ and c-fos but not peroxisome proliferator-activated receptor γ2 and C/EBPα. Amongst MAPK inhibitors, U0126, an inhibitor of MEK1/2, abrogated the EP4A-dependent effects. Our results suggest that the PGE2–EP4-ERK pathway increases the number of osteo-adipogenic bipotential progenitor cells, with a resultant increase in adipogenesis in RC cell cultures.

Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis

Periodontitis is characterized by periodontal tissue inflammation and alveolar bone loss. The intermittent administration of parathyroid hormone (PTH), a major regulator of bone remodeling, has been demonstrated to stimulate osteoblastic activity. Although the systemic administration of PTH has been reported to protect against periodontitis-associated bone loss, the effect of the topical administration of PTH is unclear. In this study, the effect of intermittent administration of PTH on osteoblastic differentiation was examined in cultured calvaria cells and then the effect of topical and intermittent administration of PTH was determined by measuring the recovery of alveolar bone loss after inducing experimental periodontitis in rats. Alkaline phosphatase activity and bone nodule formation were measured in fetal rat calvaria cells. Experimental periodontitis was induced by placing nylon ligature around rat maxillary molars for 20 d. After ligature removal (day 0), PTH was topically injected into buccal gingiva three times a week for 10 wk. Micro-computed tomography analysis and histological examination were performed on days 35 and 70. Intermittent exposure of PTH in calvaria cells increased alkaline phosphatase activity and bone nodule formation by 1.4- and 2.4-fold, respectively. Ligature procedures induced marked alveolar bone loss around the molars on day 0 and greater bone recovery was observed in the PTH-treated rats on day 70. An increase in osteoid formation on the surface of alveolar bone was detected in the PTH-treated rats. Intermittent treatment with PTH stimulated osteoblastic differentiation in fetal rat calvaria cell cultures, and topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis.

Osteoblast and chondroblast differentiation.

Recognition of discrete commitment and differentiation stages requires characterization of changes in proliferative capacity together with the temporal acquisition or loss of expression of molecular and morphological traits. Both cell lines and primary cultures have been useful for analysis of transitional steps in the chondroblast (CB) and osteoblast (OB) lineages. One striking feature is that OBs and CBs share expression of some molecules, including newer markers such as epsilon BP (galectin-3), while also having unique markers. The fact that hypertrophic chondrocytes appear able to downregulate cartilage markers and upregulate OB markers also points to an interesting lineage relationship that needs to be explored further. Recently, we have focused on the osteoprogenitors that divide and differentiate into mature OBs forming bone nodules in fetal rat calvaria cell cultures. We use cellular, immunocytochemical, and molecular approaches, including PCR on small numbers of cells, to discriminate stages. Nodule formation is characterized by loss of proliferative capacity and sequential increased marker expression, that is, alkaline phosphatase (AP), followed by bone sialoprotein (BSP), and osteocalcin. Upregulation of collagen type I and biphasic expression of osteopontin, with two peaks corresponding to proliferation and differentiation stages, also occurs. A variety of other molecules are also upregulated in the mature OB, including epsilon BP and CD44s. By replica plating and PCR, we have begun to study the expression of the messenger RNAs (mRNAs) for potential regulatory molecules (e.g., PTHrP) and their receptors (e.g., PTHR, FGFR-1, and PDGFR alpha) and have found all to be modulated during the progression from committed osteoprogenitor to mature OB.(ABSTRACT TRUNCATED AT 250 WORDS)

Tri-iodothyronine (T3) and dexamethasone interact to modulate osteoprogenitor cell differentiation in fetal rat calvaria cell cultures

We investigated the role of 3,5,3'-tri-iodothyronine (T3) in regulating differentiation of osteoprogenitor cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985-1992; 1987). In standard culture medium containing 15% fetal bovine serum FBS), low concentrations of T3 (0.001-0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1-100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T3 markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T3 was present prior to confluency. Higher concentrations of T3 (1-100 nM) decreased nodule number whether or not Dex was added. We then cultured cells in medium containing FBS from which T3 and T4 were removed by treatment with AG-1 chi-10 resin. In both + or - Dex conditions, bone nodule formulation was increased 1.5 to 2-fold in T3, T4-depleted medium when compared with cultures maintained in standard culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with both anabolic and catabolic effects on bone tissue. To investigate the effect of LIF on bone formation in the absence of a resorption cycle, we used fetal rat calvaria cell cultures and quantified bone nodule production, which provides a colony assay to analyze the effects of factors on osteoprogenitor differentiation and bone formation. In these cultures, dexamethasone (Dex) stimulates bone nodule formation. In dose-response experiments, LIF inhibited bone nodule formation by cells cultured with (+Dex; ID50 = 250 U/ml) or without (-Dex; ID50 = 30 U/ml) 10(-8) M Dex. Residual nodules were small and poorly mineralized. Continuous exposure to LIF (500 U/ml) up to day 25 did not affect either the growth rate or saturation density of the cultures, but decreased alkaline phosphatase activity and bone nodule production, with greater inhibition in -Dex cultures. Exposure to LIF (500 U/ml) for 3 days early during nodule formation (about day 10) reduced bone nodule numbers to the same extent as continuous treatment in -Dex cultures and significantly, but less markedly, in +Dex cultures; earlier and later pulses had no effect. Northern blot analysis of expression of messenger RNAs of bone related proteins in cultures pulsed (-Dex) at various stages of development showed marked inhibition of alkaline phosphatase, bone sialoprotein, and osteocalcin; slight inhibition of type I collagen; early stimulation of osteopontin; and no effect on Secreted Protein, Acidic and Rich in Cysteine/osteonectin. These results suggest that LIF is an inhibitor of bone nodule formation in these cultures, acting at a stage when late osteoprogenitors and/or early osteoblasts are present, and that Dex may modulate the effects of LIF by shifting effective doses to higher concentrations.

Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphate

Osteoid nodules form but do not mineralize in fetal rat calvaria cell cultures grown in α-minimal essential medium with 10% fetal bovine serum in the absence of Na β-glycerophosphate (β-GP). To study factors involved in the initiation and progression of mineralization, cultures were treated with β-GP and radiolabelled with 0.1–0.2 μCi/ml 45Ca after nodules had formed (17–19 days in medium without β-GP). Concentrations of β-GP from 1 to 14 mM induced a dose-dependent increase in 45Ca uptake. 45Ca uptake was restricted to nodule-containing cultures and did not occur in cultures without nodules. Continuous labelling over 72 h compared with 2 h pulses over the same time period showed that little mineralization occurred over the first 8–12 h and that the rate of mineralization was maximal and constant after 24 h exposure to β-GP. Calcium uptake from medium was slow during the first 12 h of β-GP exposure but increased rapidly thereafter until the medium calcium concentration reached a steady state of between 0.5 and 0.6 mM. Measurement of calcium concentration in the medium after mineralization had been initiated (24 h after β-GP exposure) showed a linear calcium uptake into nodules ( r = 0.990) over a 7 h period at a rate of 9.2 μg calcium/h/culture. Initiation of mineralization was prevented by 100 μM levamisole, but not by 100μM dexamisole. When 100μM levamisole was added 24 h after mineralization had been initiated by the addition of β-GP, the progression of mineralization was unaffected. Similarly, after mineralization had been initiated for 24 h by 10 mM β-GP, mineralization continued independent of the presence of β-GP. The data show that the initiation and progression of mineralization are separate phenomena and that organic phosphate and alkaline phosphatase play a crucial role in the initiation of mineralization but are not required for the continuation of mineralization of bone nodules.

Effects of acidic fibroblast growth factor and epidermal growth factor on subconfluent fetal rat calvaria cell cultures: DNA synthesis and alkaline phosphatase activity

The effects of acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) were examined in subconfluent fetal rat calvaria cell cultures, in the presence of 2% serum. Maximal effect of aFGF and EGF on DNA synthesis measured by [ 3H]thymidine incorporation was observed after 18 h. aFGF stimulated DNA synthesis by 3.5-fold with an ED 50 of 0.75 ng/ml while a 2.3-fold EGF stimulation was recorded with an ED 50 of 0.067 ng/ml. 5-Bromo-2-deoxyuridine staining showed a higher stimulation of proliferation in the scattered cells than in the cell clusters. An 18 h aFGF or EGF treatment decreased alkaline phosphatase (ALP) activity by 40 and 23%, respectively, as compared with control cultures. This inhibition was more pronounced after 48 h in the presence of the effectors but no modification of the ALP electrophoretic mobility was observed. These data suggest that aFGF is a less potent mitogen than EGF and a higher inhibitor of ALP activity in fetal rat calvaria cell culture.