In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria

We investigated the capacity of a clonal osteogenic cell line MC3T3-E1, established from newborn mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the confluent state, to differentiate into osteoblasts and mineralize in vitro. The cells in the growing state showed a fibroblastic morphology and grew to form multiple layers. On day 21, clusters of cells exhibiting typical osteoblastic morphology were found in osmiophilic nodular regions. Such nodules increased in number and size with incubation time and became easily identifiable with the naked eye by day 40-50. In the central part of well-developed nodules, osteocytes were embedded in heavily mineralized bone matrix. Osteoblasts were arranged at the periphery of the bone spicules and were surrounded by lysosome-rich cells and a fibroblastic cell layer. Numerous matrix vesicles were scattered around the osteoblasts and young osteocytes. Matrix vesicles and plasma membranes of osteoblasts, young osteocytes, and lysosome-rich cells showed strong reaction to cytochemical stainings for ALP activity and calcium ions. Minerals were initially localized in the matrix vesicles and then deposited on well-banded collagen fibrils. Deposited minerals consisted exclusively of calcium and phosphorus, and some of the crystals had matured into hydroxyapatite crystals. These results indicate that MC3T3-E1 cells have the capacity to differentiate into osteoblasts and osteocytes and to form calcified bone tissue in vitro.     

Effect of transferrin on alkaline phosphatase activity and collagen synthesis in osteoblastic cells derived from newborn mouse calvaria

The effect of transferrin was tested on osteoblastic cells (clone MC3T3-E1) cultured in serum-free medium containing 1% bovine serum albumin (BSA). Transferrin (Tf) stimulated increases of protein content and protein synthesis, but not of DNA content and cell number, in the cells. This protein also increased alkaline phosphatase activity and collagen synthesis in combination with 1% BSA. Actinomycin D and cycloheximide inhibited alkaline phosphatase activity induced by Tf, suggesting that Tf may enhance de novo synthesis of the enzyme. These results indicate that Tf may be involved in differentiation of osteoblastic cells, but not in their proliferation, in vitro.     

Cartilage-derived morphogenetic proteins enhance the osteogenic protein-1-induced osteoblastic cell differentiation of C2C12 cells

Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) induces differentiation of the pluripotent mesenchymal cell line C2C12 into osteoblastic cells. OP-1 also alters the steady-state levels of messenger RNA (mRNA) encoding for the cartilage-derived morphogenetic proteins (CDMPs) in C2C12 cells. In the present study, the effects of exogenous CDMPs on bone cell differentiation induced by OP-1 in C2C12 cells were examined. Exogenous CDMP-1, -2, and -3 synergistically and dose-dependently enhanced OP-1 action in stimulating alkaline phosphatase (AP) activity and osteocalcin (OC) mRNA expression. AP staining studies revealed that the combination of OP-1 and CDMP enhanced OP-1 action by stimulating those cells that had responded to OP-1 and not by activating additional cells. The combination did not change the mRNA expression of the BMPs and their receptors. CDMP-1 enhanced the suppression of the OP-1-induced expression of the myogeneic differentiation regulator MyoD. CDMP-1 and OP-1 alone stimulated Smad5 protein expression, but the combination of OP-1 and CDMP-1 stimulated synergistically Smad5 protein expression. Thus, one mechanism of the observed synergy involved enhancement of the induced Smad5 protein expression. At the same protein concentration, CDMP-1 is most potent in enhancing OP-1 activity in inducing osteoblastic cell differentiation of C2C12 cells. CDMP-3 is about 80% as potent as CDMP-1, and CDMP-2 is the least potent (about 50% of CDMP-1).     

Co-stimulation with bone morphogenetic protein-9 and FK506 induces remarkable osteoblastic differentiation in rat dedifferentiated fat cells

Dedifferentiated fat (DFAT) cells, which are isolated from mature adipocytes using the ceiling culture method, exhibit similar characteristics to mesenchymal stem cells, and possess adipogenic, osteogenic, chondrogenic, and myogenic potentials. Bone morphogenetic protein (BMP)-2 and -9, members of the transforming growth factor-β superfamily, exhibit the most potent osteogenic activity of this growth factor family. However, the effects of BMP-2 and BMP-9 on the osteogenic differentiation of DFAT remain unknown. Here, we examined the effects of BMP-2 and BMP-9 on osteoblastic differentiation of rat DFAT (rDFAT) cells in the presence or absence of FK506, an immunosuppressive agent. Co-stimulation with BMP-9 and FK506 induced gene expression of runx2, osterix, and bone sialoprotein, and ALP activity compared with BMP-9 alone, BMP-2 alone and BMP-2 + FK506 in rDFAT cells. Furthermore, it caused mineralization of cultures and phosphorylation of smad1/5/8, compared with BMP-9 alone. The ALP activity induced by BMP-9 + FK506 was not influenced by addition of noggin, a BMP antagonist. Our data suggest that the combination of BMP-9 and FK506 potently induces osteoblastic differentiation of rDFAT cells.     

C-type natriuretic peptide enhances osteogenic protein-1-induced osteoblastic cell differentiation via Smad5 phosphorylation

In the present study, we examined the hypothesis that the C-type natriuretic peptide (CNP) enhances osteogenic protein-1 (OP-1) action in stimulating osteoblastic cell differentiation in primary cultures of fetal rat calvaria cell (FRC). CNP enhanced synergistically the OP-1-induced Alkaline Phosphatase (AP) activity and mineralized bone nodule formation in a dose- and time-dependent manner. To examine possible mechanism of the synergy between OP-1 and CNP, the expression levels of key BMP receptors and signaling molecules were examined. Western blot analysis showed that BMPR-IB and -II receptor protein expression was not affected by CNP alone, but was stimulated by OP-1 alone. The combination of OP-1 and CNP did not further increase their protein levels. The Runx2 protein expression level was not altered by CNP alone, but was elevated by OP-1 alone, and was slightly reduced by the combination. The Smad5 protein expression level was slightly decreased by CNP alone, but was stimulated by OP-1 alone, and was not further stimulated by the combination. Smad5 phosphorylation was not stimulated by CNP alone, but was stimulated significantly by OP-1 alone. The combination of OP-1 and CNP further stimulated the OP-1-induced Smad5 phosphorylation. Thus, one mechanism of the observed synergy between OP-1 and CNP involves enhancement of the Smad5 phosphorylation.     

1 alpha, 25-Dihydroxyvitamin D3 and analogues of vitamin D3 induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria

In order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3]. A physiological concentration of 1 alpha, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites--1 alpha, 24-dihydroxyvitamin D3 [1 alpha, 24 (OH)2D3], 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3]--also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1,000 times greater than that of 1 alpha, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.     

Fluorescence activated cell sorting reveals heterogeneous and cell non-autonomous osteoprogenitor differentiation in fetal rat calvaria cell populations

Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone-related protein receptor (PTH1R) as osteoblast markers in dual-color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP(-)PTH1R(-), ALP(+)PTH1R(-), ALP(-)PTH1R(+), and ALP(+)PTH1R(+) populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities ( approximately 150-8,500 cell/cm(2); n = 3). Our results indicate that colony forming unit-osteoblast (CFU-O)/bone nodule-forming cells are found in all fractions, but the frequency of CFU-O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP(-)PTH1R(-), ALP(-)PTH1R(+), ALP(+)PTH1R(-), and ALP(+)PTH1R(+) cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU-O number in the four fractions, with the largest stimulation in the ALP(-) cell populations. However, there was no significant difference in the number or size distribution of CFU-F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non-autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP(-)PTH1R(-) population was capable of forming nodules at the clonal level, at approximately 3- or 12-times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity.     

Thyroid hormone suppresses the differentiation of osteoprogenitor cells to osteoblasts,but enhances functional activities of mature osteoblasts in cultured rat calvaria cells

The effects of thyroid hormone on osteoblastic differentiation and activity were studied in fetal rat calvaria (RC) cells cultured for up to 30 days in medium supplemented with thyroid hormone-depleted serum. In this condition, the cells proliferated and differentiated to form mineralized bone nodules (BN) and expressed osteoblastic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). The continuous presence of triiodothyronine (T3) at 10^-9-10^-8 M in the medium inhibited the osteoblastic differentiation: 34% decrease in ALP activity on day 12 and 60% decrease in BN formation on day 15 at 10^-8 M. T3 at these doses had no effect on the DNA content of RC cells at confluence (day 6). Short-term (48-h) exposure of T3 at 10^-9 M or higher decreased ALP activity when RC cells were differentiating (days 7–11). However, when BN formation by the cells had already reached a plateau (day 28), the activity was increased by treatment with T3 at 10^-7-10^-6 M. OCN production was increased dose dependently by this treatment with T3 (2.1-fold and 1.3-fold of control at 10^-8 M on days 11 and 28, respectively). Similar increases were observed in the levels of OCN mRNA. In addition, increases in phosphorylated OPN in the medium (day 11) and mineralized matrix (day 28) were observed (1.5-fold at 10^-8-10^-6 M), while OPN synthesis and the level of its mRNA were depressed by T3 (60-70% of control at 10^-8 M). These results suggest that T3 regulates osteoblastic differentiation and activity depending on the state of cell differentiation: T3 suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances the functional activity of mature osteoblasts. © 1994 Wiley-Liss, Inc.     

Inhibition of phosphatidylinositol 3-kinase and p70S6 kinase blocks osteogenic protein-1 induction of alkaline phosphatase activity in fetal rat calvaria cells

Published studies reveal that Osteogenic Protein-1 (OP-1) and insulin-like growth factor-I (IGF-I) synergistically stimulate alkaline phosphatase (AP) activity and bone nodule formation in fetal rat calvaria (FRC) cells. In the present study, we examined whether there are interactions between the signal transduction pathways activated by these two growth factors. OP-1 did not significantly affect the levels of IRS-1, IRS-2, the p85alpha subunit of phosphatidylinositol 3-kinase (PI 3-kinase) or the extracellular signal-regulated kinase (ERK)-2, but stimulated ERK-1 protein by twofold. OP-1 also induced phosphorylation of ERK-1 and -2, but not of Akt/protein kinase B (PKB), a protein kinase that is downstream of PI 3-kinase. By comparison, IGF-I increased the levels of the phosphorylated forms of ERK-1 and -2, and Akt/PKB. Inhibition of ERK activation by PD98059 did not significantly alter the stimulation of AP activity by OP-1 or OP-1 in combination with IGF-I. In contrast, inhibition of PI 3-kinase activity by LY294002 blocked the induction of AP activity by OP-1 and OP-1 plus IGF-I. Treatment of cells with rapamycin, an inhibitor of the mammalian target of mTOR, resulted in a 47% and a 53% decrease in the AP activity induced by OP-1 alone and by OP-1 plus IGF-I, respectively. These studies suggest that PI 3-kinase and mTOR contribute to the induction of AP activity by OP-1 and the synergistic effect of OP-1 and IGF-I on AP activity in FRC cells.     

Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation

Osteogenic Protein-1 (OP-1, BMP-7), a member of the bone morphogenetic protein family, stimulates synthesis of biochemical markers characteristic of the osteoblastic and chondrocytic phenotypes and induces new bone formation. Interleukin-6 (IL-6), a cytokine produced by a wide variety of cells, appears to interact with other factors producing different biological effects. In the present study, we showed that OP-1 action in fetal rat calvaria (FRC) cells was enhanced by the combination of IL-6 and the soluble receptor IL-6sR. OP-1 alone induced alkaline phosphatase (AP) activity by 4- to 5-fold above the control. Exogenous IL-6 soluble receptor (IL-6sR) synergistically stimulated the OP-1-induced AP activity and mineralized bone nodule formation by an additional 3-fold. The stimulation was IL-6sR concentration-dependent. The combination of IL-6 and IL-6sR synergistically stimulated OP-1 action by an additional 6- to 7-fold. BMPR-II receptor mRNA expression in FRC cells treated with OP-1 and IL-6 plus IL-6sR was stimulated further, while BMPR-IA, -IB, and ActR-I expressions were not affected. The intracellular signaling molecules Smad2 and Smad5 mRNA expressions were not changed under these conditions. The expression of selected BMP family members (BMP-3, -4, and -6) was altered in FRC cells treated with OP-1 in combination with IL-6 and IL-6sR. The combination of IL-6 and IL-6sR reduced the OP-1-stimulated BMP-3 mRNA levels and enhanced the suppressive effect of OP-1 on BMP-4 and -6 mRNA expressions. In conclusion, the present results demonstrate that exogenous IL-6 and IL-6sR synergistically stimulate OP-1 action in primary cultures of rat osteoblastic cells. One possible mechanism of synergy involves differential regulation of the effects of OP-1 on the expression of the type II BMP receptor and several other BMPs.     

Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells

To clarify the mechanism of the stimulatory effect of statins on bone formation, we investigated the effect of simvastatin, a widely used statin, on osteoblastic and adipocytic differentiation in primary cultured mouse bone marrow stromal cells (BMSCs). Simvastatin treatment enhanced the expression level of mRNA for osteocalcin and protein for osteocalcin and osteopontin, and increased alkaline phosphatase activity significantly (p<0.05). After BMSCs were exposed to an adipocyte differentiation agonist, Oil Red O staining, fluorescence activated cell sorting, and decreased expression level of lipoprotein lipase mRNA showed that treatment with simvastatin significantly inhibits adipocytic differentiation compared to controls that did not receive simvastatin (p<0.05). Lastly, we found that simvastatin induces high expression of BMP(2) in BMSCs. These observations suggested that simvastatin acts on BMSCs to enhance osteoblastic differentiation and inhibits adipocytic differentiation; this effect is at least partially mediated by inducing BMP(2) expression in BMSCs.     

Monocyte chemoattractant protein-1 induces scavenger receptor expression and monocyte differentiation into foam cells

Accumulation of monocytes and the entrapment of oxidized-low-density lipoprotein (ox-LDL) in monocytes are important in the differentiation into "foam" macrophages and the pathogenesis of atherosclerosis. We investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the expression of scavenger receptor (SCR) by using resting monocytes prepared by counterflow centrifugal elutriation. Our results showed that: (1) MCP-1 increased the expression of CD36 SCR by flow cytometric analysis. (2) MCP-1 increased incorporation of 125I-labeled ox-LDL and oil red O staining. (3) MCP-1 and ox-LDL enhanced in vitro transendothelial monocyte migration. (4) These functions were mediated at least in part via extracellular signal-regulated kinase (ERK) pathway. (5) MCP-1 and ox-LDL did not induce monocyte proliferation. Our results imply that MCP-1 is involved in the inflammatory process of atherosclerosis through the induction of SCR expression via the ERK pathway and differentiation of monocytes into foam macrophages, as well as induction of monocyte migration.     

Selective emergence of differentiated chondrocytes during serum-free culture of cells derived from fetal rat calvaria

Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.     

Oxidized LDL induces transcription factor activator protein-1 in rat mesangial cells

It has been shown that oxidized low-density lipoprotein (ox-LDL), through the activation of glomerular cells, stimulates pathobiological processes involved in monocyte infiltration into the mesangium. The underlying molecular mechanisms are not fully understood. The present study showed that ox-LDL strongly induced AP-1 binding activity in rat mesangial cells (RMCs) in a dose- and time-dependent manner, reaching the maximal activation at 250 microg ml(-1) within 24 h. The results from mobility shift assays and Western blotting analysis revealed that this AP-1 binding increase involved c-Jun, but not c-Fos. Moreover, this ox-LDL-increased AP-1 binding was inhibited by several protein kinase (PK) inhibitors: the protein kinase C (PKC) inhibitor Bisindolylmaleimide I, the cAMP-dependent PK (PKA) inhibitor H89, and the tyrosine PK (PTK) inhibitor genistein. Protein phosphorylation represents mitogen-activated protein kinase (MAPK) activity. Therefore, we examined the role of ox-LDL on the activation of mesangial cell JNK/SAPK, the only recognized protein kinase that catalyses phosphorylation of c-Jun. The incubation of mesangial cells with ox-LDL induced phosphorylation of JNK1/SAPK dose dependently, with the maximal response at 150 microg ml(-1). This study demonstrates that multiple kinase activities are involved in the mechanism of ox-LDL-induced AP-1 activation in mesangial cells, and ox-LDL stimulates AP-1 through JNK-c-Jun other than MEK-c-Fos signalling pathway.     

Wnts differentially regulate colony growth and differentiation of chondrogenic rat calvaria cells

The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20-40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved.     

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

 Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell’s osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems. Accepted: 25 August 1998     

Recovering the osteoblastic differentiation potential of mesenchymal stem cells derived from diabetic rats by photobiomodulation therapy

Autologous cell-based therapy for bone regeneration might be impaired by diabetes mellitus (DM) due to the negative effects on mesenchymal stem cells (MSCs) differentiation. Strategies to recover their osteogenic potential could optimize the results. We aimed to evaluate the effect of photobiomodulation (PBM) therapy on osteoblast differentiation of rats with induced DM. Bone marrow MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB, respectively). dOB were treated with PBM therapy every 72 hour (660 nm; 0.14 J; 20 mW; 0.714 W/cm², and 5 J/cm²). Cell morphology, viability, gene and protein expression of osteoblastic markers, alkaline phosphatase (ALP) activity, and the mineralized matrix production of dOB-PBM were compared to dOB. PBM therapy improved viability of dOB, increased the gene and protein expression of bone markers, the ALP activity and the mineralized matrix production. PBM therapy represents an innovative therapeutic approach to optimize the treatment of bone defects in diabetic patients.