ERRα regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

The orphan nuclear receptor estrogen-related receptor-α (ERRα) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRα in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRα deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERRα deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERRα in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERRα deficient MSCs and enhanced upon ERRα overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERRα. Under adipogenic conditions, ERRα deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERRα in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERRα may play different roles in bone under different physiological conditions.     

Effects of osteogenic protein-1 (OP-1, BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specific

Bone morphogenetic proteins (BMPs) are a group of cytokines that are characterized by their ability to stimulate osteoblast differentiation and bone formation. However, the influence of BMPs on osteoblastic cells at different stages of differentiation is not known. Since bone matrix proteins are differentially regulated during bone formation we have studied the effects of recombinant human osteogenic protein-1 (rhOP-1; BMP-7) on the expression of these proteins by fetal rat calvarial cells (FRCCs) at discrete stages of osteoblast differentiation. Continuous administration of rhOP-1 to FRCCs, beginning at confluence (day 7), produced a dose-dependent increase in the number, size and mineralization of bone-like nodules formed in the presence of vitamin C and β-glycerophosphate. Within 9 h of administration, rhOP-1 stimulated a 3-fold increase in OPN mRNA which was reflected in a comparable increase in the low phosphorylated, 55 kDa form of osteopontin. In contrast, changes in type I collagen, alkaline phosphatase and bone sialoprotein mRNAs followed the differentiation of preosteoblastic cells, and were increased 2-, 4- and 5-fold, respectively, after 8 days (day 15). When administered at intermediate stages of osteoblast differentiation (days 12, 15 and 18) BSP remained refractory to rhOP-1 whereas the ALP was increased almost 2-fold, independent of the constitutive levels of mRNA expression. To determine the effects on osteoblasts, FRCCs were first grown to the bone nodule-forming stage (day 21) before rhOP-1 was administered. Only modest, transient increases in the expression of ALP and OPN mRNAs were evident whereas OC expression was increased more than 3-fold. In contrast, collagen type I and BSP mRNA levels were not changed significantly. These results suggest that rhOP-1 increases bone formation by promoting osteoblastic differentiation, as indicated by the increased number of bone forming colonies and by increasing the number of osteoblastic cells in the colonies, but not by increasing matrix production by individual osteoblasts. It is also evident that the regulation of bone matrix proteins by rhOP-1 is dependent upon the differentiated state of the cell. © 1996 Wiley-Liss, Inc.     

Expression of bone matrix proteins during dexamethasone-induced mineralization of human bone marrow stromal cells

Glucocorticoids have been shown to induce the differentiation of bone marrow stromal osteoprogenitor cells into osteoblasts and the mineralization of the matrix. Since the expression of bone matrix proteins is closely related to the differentiation status of osteoblasts and because matrix proteins may play important roles in the mineralization process, we investigated the effects of dexamethasone (Dex) on the expression of bone matrix proteins in cultured normal human bone marrow stromal cells (HBMSC). Treatment of HBMSC with Dex for 23 days resulted in a significant increase in alkaline phosphatase activity with maximum values attained on day 20 at which time the cell matrix was mineralized. Northern blot analysis revealed an increase in the steady-state mRNA level of alkaline phosphatase over 4 weeks of Dex exposure period. The observed increase in the alkaline phosphatase mRNA was effective at a Dex concentration as low as 10⁻¹⁰ M with maximum values achieved at 10⁻⁸ M. In contrast, Dex decreased the steady-state mRNA levels of both bone sialoprotein (BSP) and osteopontin (OPN) over a 4 week observation period when compared to the corresponding control values. The relative BSP and OPN mRNA levels among the Dex treated cultures, however, showed a steady increase after more than 1 week exposure. The expression of osteocalcin mRNA which was decreased after 1 day Dex exposure was undetectable 4 days later. Neither control nor Dex-treated HBMSC secreted osteocalcin into the conditioned media in the absence of 1,25(OH)₂D₃ during a 25-day observation period. The accumulated data indicate that Dex has profound and varied effects on the expression of matrix proteins produced by human bone marrow stromal cells. With the induced increment in alkaline phosphatase correlating with the mineralization effects of Dex, the observed concomitant decrease in osteopontin and bone sialoprotein mRNA levels and the associated decline of osteocalcin are consistent with the hypothesis that the regulation of the expression of these highly negatively charged proteins is essential in order to maximize the Dex-induced mineralization process conditioned by normal human bone marrow stromal osteoprogenitor cells. © 1996 Wiley-Liss, Inc.     

TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro,preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

Effect of bone sialoprotein on proliferation and osteodifferentiation of human bone marrow-derived mesenchymal stem cells in vitro

We performed this study to investigate the effects of recombinant human bone sialoprotein (BSP) on the proliferation and osteodifferentiation of human BMSCs(hBMSCs). The hBMSC cultures were divided into 4 groups: control group, 10⁻¹⁰ M BSP group (BSP group), osteogenic medium group (10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/L ascorbic acid, OM group) and BSP + OM group (OM plus10⁻¹⁰ M BSP). Compared with the control group, cell growth of the other three groups slowed down, while fluorescence at the G₀/G₁ phase increased. After 28 days, in the OM group and the BSP + OM group, the proportion of STRO-1-positive cells decreased by 22.7% and 38.4% and ALP activity increased by 50% and 71.43%, respectively. CD271 mRNA expression decreased while Cbfa1, osteocalcin and osterix mRNA levels increased in the OM and BSP + OM groups, and the mRNA level change was greater in the BSP + OM group. After 28 days, the number of nodules in the BSP + OM group was 112.5% more than that in the OM group, but nodules did not formed in the control or BSP group. We conclude that BSP is capable of inhibiting hBMSCs proliferation and enhancing their osteogenic differentiation and mineralization in the presence of OM.     

Phenotypic effects of continuous or discontinuous treatment with dexamethasone and/or calcitriol on osteoblasts differentiated from rat bone marrow stromal cells

Osteoblasts are target cells for glucocorticoids and calcitriol, and their phenotype is greatly modified by these hormones. We investigated the effect of continuous or discontinuous hormonal exposure to osteoblasts derived from rat bone marrow stromal cells in long-term subcultures. Stromal cells were grown in primoculture in presence of dexamethasone (dex), but in following subcultures, dex and/or calcitriol were added just after seeding or after a 7-day hormone-free period. Cell proliferation, alkaline phosphatase (ALP) histochemical staining, and enzymatic bioactivity measurement, osteocalcin (OC), ALP and bone sialoprotein (BSP) mRNA expression were used to study the differential effect on osteoblastic phenotype of various conditions of treatment by dex and calcitriol. In primoculture, the osteoblastic differentiation was confirmed by the formation of calcified nodules and by strong expression of ALP, OC, and BSP mRNAs. In subcultures, proliferation of stromal cells was stimulated by dex and inhibited by calcitriol and by both hormones. Cell proliferation was not modified by hormonal lack during 7 days. Continuous hormonal treatment by dex strongly enhanced OC and BSP mRNAs, but apparently did not modified ALP mRNAs expression. Continuous treatment by calcitriol decreased ALP and the dex-induced BSP expression and stimulated the OC mRNAs level, strongly when associated with dex. The population of ALP+ cells and ALP bioactivity were strongly increased by dex, whereas calcitriol or both hormones decreased them. When the subcultures were undergone without hormonal treatment during 7 days, all osteogenic mRNAs strongly decreased even after hormonal recovery. Dex, calcitriol, and both hormones inhibited ALP mRNAs. OC messengers were only weakly detectable with both hormones. ALP+ cell population and ALP bioactivity were decreased after 14 days of hormonal treatment recovery. These results support that continuous presence of glucocorticoids appears as a major key for the permanent expression of the osteoblastic phenotype that is inhibited by calcitriol, in the rat bone marrow.     

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.     

Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: effects of dexamethasone and calcitriol

During bone loss, osteoblast population can be replaced by adipose tissue. This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone-forming and fat-forming cells in the marrow cavity. Thus, osteoblast and adipocyte pathways seem more closely and inversely related. In the present study, we investigated the effects of dexamethasone (dex) and calcitriol [1,25(OH)(2)D(3)] on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures. Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)(2)D(3). Total cell proliferation, osteoblast and adipocyte-cells number, and -mRNA specific markers were used to study the effects of hormonal treatment on stromal cells. Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)(2)D(3). Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone-forming cell number and increased fat cell population. The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number. Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2. Calcitriol decreased the dex-induced BSP expression but stimulated slightly OC and aP2 mRNA. The effects of both hormones was to increase strongly OC and aP2 mRNA. These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)(2)D(3).     

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.     

Ultrastructure of mineralized nodules formed in rat bone marrow stromal cell culture in vitro.

Rat bone marrow stromal cells were cultured in vitro. At days 14-15 of culture, dense clusters of polygonal cells were formed, and they mineralized 2-3 days later. The cells resembling osteoblasts or young osteocytes were histologically observed to be embedded in mineralized or unmineralized extracellular matrices of the nodules. Next, these mineralized nodules were electron-microscopically examined. The osteoblastic cells associated with the nodules had a well-developed rough endoplasmic reticulum, an evident Golgi apparatus and some mitochondria as their intracellular organellae. Some lysosomes and microfilaments were also visible in the cytoplasms. Moreover, some cells protruded cell processes toward the neighboring cells through the extracellular matrix. The extracellular matrix consisted of numerous collagen fibrils which were striated with 60-70 nm axial periodicity and which was similar to bone tissue collagen. A large number of matrix vesicles were scattered among the collagen fibrils in the unmineralized area of the nodules. In contrast, in the mineralized area, numerous matrix vesicles at different stages of maturation and many calcified spherules were observed. That is the mineralization in this culture system was considered to be initiated in association with the matrix vesicles and to progress along the collagen fibrils. From these findings, it was confirmed by the present study that the mineralized nodules formed in this bone marrow stromal cell culture were ultrastructurally similar to bone and that the mineralization also proceeded by going through the normal calcification process. This culture system is considered to be available to study osteogenic differentiation and calcification mechanisms.     

Association of specific proteolytic processing of bone sialoprotein and bone acidic glycoprotein-75 with mineralization within biomineralization foci

Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15-25-microm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45-50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.     

CARM1 deficiency inhibits osteoblastic differentiation of bone marrow mesenchymal stem cells and delays osteogenesis in mice

Bone repair remains a clinical challenge due to low osteogenic capacity. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that mediates arginine methylation and endochondral ossification. However, the roles of CARM1 in osteoblastic differentiation and bone remodeling have not been explored. In our study, heterozygous CARM1-knockout (KO) mice were generated using the CRISPR-Cas9 system and a model of femoral defect was created. At day 7 postsurgery, CARM1-KO mice exhibited obvious bone loss compared with wild type (WT) mice, as evidenced by reduced bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Deletion of CARM1 in mice lowered synthesis and accumulation of collagen at the injury sites. The alkaline phosphatase (ALP) activity and osteogenic-related gene expression were declined in CARM1-KO mice. To further understand the role of CARM1 in osteoblastic differentiation, bone marrow mesenchymal stem cells (BMSCs) were isolated from the tibia and femur of WT or CARM1-KO mice. CARM1 deletion decreased histone arginine methylation and inhibited osteoblastic differentiation and mineralization. The mRNA sequencing of CARM1-KO BMSCs revealed the possible regulatory molecules by CARM1, which could deepen our understanding of CARM1 regulatory mechanisms. These data could be of interest to basic researchers and provide the direction for future research into bone-related disorders.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Comparative effects of estrogen and antiestrogens on differentiation of osteoblasts in mouse bone marrow culture

Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption, but their anabolic effects have been less explored. In this study, bone marrow cultures were used to compare the effect of 17beta-estradiol (E2), and two triphenylethylene derivatives, tamoxifen (TAM), and FC1271a, and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All, except RAL, stimulated cell proliferation during the first 6 days of the culture. However, in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2, TAM, and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM, FC1271a, and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2, TAM, and FC1271a.     

The Time-Dependent Manner of Sinusoidal Electromagnetic Fields on Rat Bone Marrow Mesenchymal Stem Cells Proliferation,Differentiation, and Mineralization

Electromagnetic fields (EMFs) are used clinically to promote fracture healing and slow down osteoporosis without knowledge of optimal parameters and underlying principles. In the present study, we investigate the effects of irritation for different durations with 15 Hz 1 mT sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs) proliferation, differentiation, and mineralization potentials. Our results show that SEMFs irritation promote rat BMSCs proliferation in a time-dependent manner, and the expression of osteogenic gen [Cbfa 1/RUNX2, bone sialoprotein (BSP), osteopontin (OPN)], alkaline phosphatase activity, and calcium deposition were enhanced after SEMFs treatment depending on the time duration of treatment. To determine the role of MEK/ERK signaling pathway, U0126, a MEK/ERK inhibitor was used. It can suppress rat BMSCs’ proliferation with or without SEMF exposure, and partly attenuate the expression of osteogenesis related proteins (RUNX2, BSP, OPN) which were improved by SEMF. This finding suggests that the effects of SEMF on rat BMSCs’ proliferation differentiation and mineralization are time duration dependent and MEK/ERK signaling pathway plays important role.