Differentiation Of Human Bone Cells Research Articles

Topiramate promotes osteogenic differentiation through AMPK-dependent phosphorylation of Smad1/5/9

Topiramate [2,3:4,5-bis-o-(1-methylethylidene) β-D-fructo-pyranose sulfamate; TPM] is one of the most used new-generation antiepileptic drugs. It has been reported to regulate the differentiation of human bone cells. However, the molecular mechanism of TPM in osteoblast differentiation is not fully elucidated. In the present study, we examined the effect of TPM on osteogenic differentiation of C3H10T1/2, MC3T3-E1, primary mouse calvarial cells, and primary bone marrow stem cells (BMSCs). Primary cells were isolated from mice calvaria and bone marrow respectively. Expression of the osteogenic gene was determined by RT-PCR. The osteogenic protein levels were measured by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. Alizarin red s (ARS) staining was performed to measure zebrafish caudal fin regeneration. Treatment of TPM up-regulated the osteogenic genes including distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). In addition, TPM also increased the Dlx5 and Runx2 protein levels, Smad1/5/9 phosphorylation, and alkaline phosphatase (ALP) activity. Furthermore, TPM activated AMPK, and inhibition of AMPK decreased TPM-induced osteogenic differentiation. In the zebrafish model, osteogenic effect of TPM was identified. TPM was increased amputated caudal fin rays of zebrafish. These results demonstrate that TPM enhances osteogenic differentiation via AMPK-mediated Smad1/5/9 phosphorylation.

Biomineralisation with Saos-2 bone cells on TiSiN sputtered Ti alloys

Surface modifications of metallic implants are important in order to protect the underlying metals from the harsh corrosive environment inside the human body and to minimize the losses caused by wear. Recently, researches are carried out in developing bioactive surfaces on metallic implants, which supports the growth and proliferation of cells on to these surfaces. Titanium silicon nitride (TiSiN) hard nanocomposites thin films were fabricated on Ti alloys (Ti-6Al–4V) by pulsed direct current (DC) reactive magnetron sputtering. The films were characterized for its microstructural and electrochemical behavior. The higher charge transfer resistance (Rct) and positive shift in Ecorr value of TiSiN/Ti alloys than the bare Ti-alloys indicates a better corrosion resistance offered by the TiSiN thin films to the underlying substrates. The biological response to TiSiN/Ti alloys and control bare Ti-alloys was measured in vitro using cell-based assays with two main outcomes. Firstly, neither the Ti alloy nor the TiSiN thin film was cytotoxic to cells. Secondly, the TiSiN thin film promoted differentiation of human bone cells above the bare control Ti alloy as measured by alkaline phosphatase and calcium production. TiSiN thin films provide better corrosion resistance and protect the underlying metal from the corrosive environment. The thin film surface is both biocompatible and bioactive as indicated from the cytotoxicity and biomineralization studies.

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro

Both CB(1) and CB(2) cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro. Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing α(v) β(3) or with F-actin rings, or measurement of resorption area. Human osteoclasts express both CB(1) and CB(2) receptors. CB(2) expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB(1) and/or CB(2) antagonists. AND IMPLICATIONS Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB(2) receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

The proliferation and differentiation of human bone cells in response to 25(OH)D and 1,25(OH)2D: Local conversion of 25(OH)D into 1,25(OH)2D?

The proliferation and differentiation of human bone cells in response to 25(OH)D and 1,25(OH)2D: Local conversion of 25(OH)D into 1,25(OH)2D?

Endogenous BMPR-IB signaling is required for early osteoblast differentiation of human bone cells

Osteoblast differentiation is tightly regulated by a number of cytokines and growth factors, including bone morphogenetic proteins (BMP) which stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB and -II). Although the mechanisms which regulate osteoblast differentiation are not fully understood, it is possible that endogenous BMPR signaling could play an important part in this process. To test this hypothesis, we have examined the expression and the functional significance of BMPR during osteoblast differentiation of primary human bone cells. The results showed that although the expression of BMPR-IA and -II transcripts were constantly expressed while the bone cells underwent osteoblast differentiation, the level of BMPR-IB mRNA was transiently, but significantly, up-regulated by threefold on day 3. This increase in BMPR-IB expression was found to be associated with the significant up-regulation of core binding factor alpha 1 (Cbfa1) and alkaline phosphatase (ALP) transcripts as well as the ALP activity, the well-established early markers of osteoblast differentiation. Transfection of bone cells with BMPR-IB small interfering RNA (siRNA) was found to significantly ablate the expression of BMPR-IB which subsequently resulted in reduction of Cbfa1 and ALP mRNA as well as the ALP activity. Moreover, exogenously added BMP-2 failed to rescue osteoblast differentiation of BMPR-IB siRNA-transfected bone cells. In conclusion, the present study has shown that endogenous BMPR-IB signaling is required for early phase of osteoblast differentiation of human bone cells in vitro, suggesting that BMPR-IB could be a therapeutic target for initiating bone healing in vivo.

Correlating cell architecture with osteogenesis: first steps towards live single cell monitoring

Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC) being the primary sites of adhesion. They consist of a large number of clustered transmembrane proteins (integrins). FC integrins connect the cell cytoskeleton with the cell substratum. It has been demonstrated that cell spreading increases osteoblast differentiation in pre-osteoblastic progenitors. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g., bone-specific alkaline phosphatase (bALP)) and late genes (e.g., osteocalcin (OC)). In the present study we have used immunohistochemistry and RT-PCR to determine osteogenic differentiation of human bone cells (HBC). For online monitoring, fluorescently-tagged actin and vinculin were used for transfection of HBCs. Transfection of HBCs with an OC promoter gene construct allowed us to online monitor the gradual process of osteogenesis. We found distinct changes in cell architecture upon osteogenic differentiation thus providing evidence for the connection between cell shape and functional state.

Effects of Soft Laser Irradiation in Human Bone Cells and Periodontal Ligament Cells Derived from Mandible Cultured on Titanium Plates

The objectives of the present study were to evaluate the effects of soft laser irradiation for the proliferation and the differentiation of human bone cells and periodontal cells cultured on titanium plates and plastic dishes both derived from the identical donor.Bone cells derived from mandibular fragments and periodontal ligament (PDL) cells from mandibular first premolars were obtained simultaneously from the identical orthognathic patient. Both cells in the 6th passage were seeded (5×103 cells/well) in 24-well plastic culture dishes with or without titanium plates and incubation continued for 72 hours. During the incubation period, soft laser irradiation was performed none, once, twice, which were served as control, 1×laser, 2×laser, respectively. At the end of incubation period, both cells were used for the assays of DNA synthesis and alkaline phosphatase (ALP) activity. The following results were obtained:1. Bone cells cultured on plastic dishes did not show any significant change of DNA contents and ALP activity by the various time periods of soft laser irradiation. On the other hand, bone cells cultured on titanium plates showed significant increases of DNA contents and ALP activity by the soft laser irradiation, especially by 1×10 minutes laser irradiation group. In this group, the level of DNA contents and ALP activity by titanium-cultured-bone cells were significantly greater than those by plastic-cultured-bone cells.2. In case of PDL cells, the level of DNA contents and ALP activity were constant for any kind of groups with the exception of 2×10 minutes laser group which showed significant DNA increase as compared to control group, suggesting that the existance of titanium plates and/or the irradiation of soft laser were not effective for cultured PDL cells.These results suggest that the proliferation and the differentiation of bone cells cultured on titanium plates can be stimulated by the irradiation of soft laser in vitro.

Evidence That Human Bone Cells in Culture Contain Binding Sites for Osteogenic Protein-1

Recent studies have shown that osteogenic protein (OP)-1 or bone morphogenetic protein (BMP)-7 increases proliferation and differentiation of human bone cells (HBCs) in culture and modulates production of IGF system components. In order to study the mechanism by which OP-1 causes these effects, we sought to test the hypothesis that the effects of OP-1 are mediated at least in part by specific receptors (for OP-1) in HBCs. Binding studies with serum-free cultures of normal HBCs and human osteosarcoma cells showed a maximum binding of 15 - 25% for [ 125I]OP-1; the binding was time- and temperature-dependent in different experiments. Scatchard analysis of [ 125]OP-1 binding to TE85 human osteosarcoma cells showed at least two binding sites, about 30,000 and 60,000 per cell with apparent K d of 2.5 × 10 −10M and 1 × 10 −9M, respectively. [ 125]OP-1 binding to TE85 cells was displaced by unlabeled OP-1 (16-1000 ng/ml) with a 50% displacement at 250 ng/ml. BMP-2 effectively displaced [ 125]OP-1 binding to HBCs while TGF-β1 did not. Affinity cross-linking studies showed that [ 125]OP-1 interacted specifically with three binding sites with apparent M r of 34, 65 and > 205kDa. The findings of this study demonstrate that the effects of OP-1 on HBCs may be mediated in part via BMP-specific receptors.

Osteogenic Protein-1 Stimulates Proliferation and Differentiation of Human Bone Cells in Vitro

The effect of recombinant human osteogenic protein-1 (OP-1) on proliferation and alkaline phosphatase (ALP) activity was studied using human bone cells in culture. TE85, osteosarcoma cells with osteoblastic characteristics and normal bone cells derived from mandible (HBM) were used. OP-1 stimulated 3H-thymidine incorporation in a dose dependent manner in TE85 (5-fold over vehicle control at 3-10 ng/ml) and HBM cells (1.8-fold at 100 ng/ml). In TE85 cells, OP-1 also increased cell number (2.4-fold over vehicle control at 3 ng/ml). OP-1 stimulated ALP activity in TE85 cells (4-fold over vehicle control at 30 ng/ml), but moderately inhibited ALP activity in HBM cells (to 67% of vehicle control at 100 ng/ml). Because 1,25(OH) 2D 3 has been shown to increase ALP activity in many cell types, we also studied if 1,25(OH) 2D 3 modulates the effects of OP-1 on ALP activity. In the presence of 10 −8 M 1,25(OH) 2D 3, a biphasic response occurred in TE85 and HBM cells with stimulation of ALP activity at low dose of OP-1 and inhibition at high dose. Thus, the effect of OP-1 on ALP activity appeared to be modulated by 1,25(OH) 2D 3. Our results suggest that OP-1 could be an important regulator of osteoblast proliferation and differentiation.

Differentiation of normal human bone cells by transforming growth factor-β and 1,25(OH) 2 vitamin D 3

To investigate the role of transforming growth factor-β 1 (TGFβ) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I α 2-procollagen, and osteocalcin. The effect of TGFβ on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGFβ decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGFβ stimulated alkaline phosphatase activity. The addition of 1,25(OH) 2 vitamin D 3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGFβ and 1,25(OH) 2D 3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH) 2D 3 increased osteocalcin mRNA levels, but TGFβ markedly inhibited this stimulation. TGFβ also inhibited production of osteocalcin by the human bone cells. TGFβ appears to modulate differentiation of human bone cells in combination with 1,25(OH) 2D 3 and other factors.

Skeletal growth factor and other growth factors known to be present in bone matrix stimulate proliferation and protein synthesis in human bone cells

The purpose of the study was to investigate the effect of skeletal growth factor/insulinlike growth factor II and other growth factors known to be present in bone matrix on the proliferation and differentiation of human bone cells. Cells were isolated by collagenase digestion from femoral heads obtained during hip replacement operations. Cells were cultured in DMEM medium with 10% calf serum. Third to fifth passage cells were plated in multiwell plates and the medium changed to low serum (0.1%) for 2 days. The medium was changed to serum-free medium prior to addition of growth factors. Cell proliferation was measured by the incorporation of [3H]thymidine into DNA and by the percentage of cells that incorporate bromodeoxyuridine. Protein synthesis was measured by the incorporation of [3H]proline into trichloroacetic acid-precipitable material. Skeletal growth factor/insulinlike growth factor II and insulinlike growth factor I stimulated cell proliferation and protein synthesis in a dose-dependent manner. Alkaline phosphatase-specific activity was not increased by these factors. Transforming growth factor beta 1 did not affect cell proliferation but stimulated protein synthesis and increased the specific activity of alkaline phosphatase. Fibroblast growth factor did not affect any of the cell parameters. These studies suggest that skeletal growth factor/insulinlike growth factor II, insulinlike growth factor I, and transforming growth factor beta 1 may play a role in the local control of the proliferation and differentiation of human osteoblasts.

1,25-Dihydroxyvitamin D3 and human bone-derived cells in vitro: effects on alkaline phosphatase, type I collagen and proliferation.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], but not 24,25-(OH)2D3 stimulates the alkaline phosphatase activity of cultured human bone cell populations. The stimulatory effect of the sterol was dose dependent (10(-10)-10(-7) M), evident by 24 h, and observed over a range of cell densities. Analysis of the radiolabeled collagens synthesised by human bone cell cultures indicated the synthesis of predominantly type I collagen. In the presence of 1,25-(OH)2D3, but not 24,25-(OH)2D3, there was a dose-dependent (10(-11)-10(-9) M) increase in radiolabeled proline incorporation into collagenase-digestible protein and in the amount of collagen synthesized, expressed as a percentage of the total protein synthesis. The effect of 1,25-(OH)2D3 was observed over a range of cell densities and appeared to be specific for the synthesis of type I collagen. The stimulatory effect of 1,25-(OH)2D3 on alkaline phosphatase activity and the increase in proline incorporation into collagenase-digestible protein were accompanied by a dose-dependent (5 X 10(-11) to 5 X 10(-8) M) inhibition of bone cell proliferation. These findings suggest that 1,25-(OH)2D3 is an important modulator of the growth and differentiation of human bone cells in vitro. They are also consistent with the possibility that 1,25-(OH)2D3 has direct effects on bone formation in vivo.