Human Osteoblast-like Osteosarcoma Cells Research Articles

The Effect of Microcosm Biofilm Decontamination on Surface Topography, Chemistry, and Biocompatibility Dynamics of Implant Titanium Surfaces.

Since the inception of dental implants, a steadily increasing prevalence of peri-implantitis has been documented. Irrespective of the treatment protocol applied for the management of peri-implantitis, this biofilm-associated pathology, continues to be a clinical challenge yielding unpredictable and variable levels of resolution, and in some cases resulting in implant loss. This paper investigated the effect of microcosm biofilm in vitro decontamination on surface topography, wettability, chemistry, and biocompatibility, following decontamination protocols applied to previously infected implant titanium (Ti) surfaces, both micro-rough -Sandblasted, Large-grit, Acid-etched (SLA)-and smooth surfaces -Machined (M). Microcosm biofilms were grown on SLA and M Ti discs. These were treated with TiBrushes (TiB), combination of TiB and photodynamic therapy (PDT), combination of TiB and 0.2%CHX/1%NaClO, plus or minus Ultraviolet-C (UV-C) radiation. Surface topography was evaluated by Scanning Electron Microscopy (SEM) and Laser Surface Profilometry. Surface function was analysed through wettability analysis. Surface chemistry evaluation of the discs was performed under SEM/Energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). Biocompatibility was tested with the cytocompatibility assay using human osteoblast-like osteosarcoma cell line (MG-63) cells. Elemental analysis of the discs disclosed chemical surface alterations resulting from the different treatment modalities. Titanium, carbon, oxygen, sodium, aluminium, silver, were identified by EDX as the main components of all the discs. Based on the data drawn from this study, we have shown that following the decontamination of Ti surfaces the biomaterial surface chemistry and topography was altered. The type of treatment and Ti surface had a significant effect on cytocompatibility (p = 0.0001). Although, no treatment modality hindered the titanium surface biocompatibility, parameters such as the use of chemical agents and micro-rough surfaces had a higher cytotoxic effect in MG-63 cells. The use of smooth surfaces, and photofunctionalisation of the TiO2 layer had a beneficial effect on cytocompatibility following decontamination.

Bisphenol A inhibits osteogenic activity and causes bone resorption via the activation of retinoic acid-related orphan receptor α

Bisphenol A (BPA) has deleterious effects on bone metabolism; however, its underlying mechanism has not yet been comprehensively understood. Here, we investigated whether RORα plays an important role in BPA-induced bone resorption both in vitro and in vivo. We found that BPA (0.1–1 μM) inhibited osteogenic activity (including ALP activity and mineralization), decreased the expression levels of osteoblast markers (such as RUNX2, OSX, and ALP) in human MG-63 osteoblast-like osteosarcoma cells, and inhibited spontaneous vertebral formation in zebrafish larvae. Additionally, BPA diminished β-glycerophosphate-induced osteoblast differentiation and vertebral formation, while simultaneously downregulating the expression levels of RUNX2a, OSX, and ALP. Furthermore, molecular docking data showed that a hydroxyl group of BPA dominantly binds to the H3 (ALA70) and/or H5 (ARG107) of RORα-ligand binding domain with hydrogen bonding (ALA330 and/or ARG367 in the full length of RORα, respectively), which another hydroxyl group of BPA fits into H3, H6, and H7 elements with non-covalent interactions, resulting in the activation of RORα. However, an RORα inverse agonist potently inhibited BPA-induced anti-osteogenic activity and vertebral formation in zebrafish larvae, concomitant with inhibition of osteogenic gene expression. Overall, our findings reveal that BPA inhibits osteoblast differentiation and bone formation by activating RORα. These results suggest that BPA exposure (0.1–1 μM) can cause various bone-resorptive diseases, such as osteoporosis.

Decarboxylated osteocalcin, a possible drug for type 2 diabetes, triggers glucose uptake in MG63 cells.

BACKGROUNDUncarboxylated osteocalcin (GluOC) has been reported to improve glucose metabolism, prevent type 2 diabetes, and decrease the severity of obesity in mice with type 2 diabetes. GluOC can increase glucose uptake in a variety of cells. Glucose metabolism is the main source of energy for osteoblast proliferation and differentiation. We hypothesized that decarboxylated osteocalcin (dcOC), a kind of GluOC, can increase glucose uptake in MG63 cells (osteoblast-like osteosarcoma cells) and influence their proliferation and differentiation.AIMTo investigate the effects of dcOC on glucose uptake in human osteoblast-like osteosarcoma cells and the possible signaling pathways involved.METHODSMG63 cells (human osteoblast-like osteosarcoma cells) were treated with dcOC (0, 0.3, 3, 10, or 30 ng/mL) for 1 and 72 h, and glucose uptake was measured by flow cytometry. The effect of dcOC on cell proliferation was measured with a CCK-8 assay, and alkaline phosphatase (ALP) enzyme activity was measured. PI3K was inhibited with LY294002, and hypoxia-inducible factor 1 alpha (HIF-1α) was silenced with siRNA. Then, GPRC6A (G protein-coupled receptor family C group 6 subtype A), total Akt, phosphorylated Akt, HIF-1α, and glucose transporter 1 (GLUT1) levels were measured by Western blot to elucidate the possible pathways by which dcOC modulates glucose uptake.RESULTSThe glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after short-term (1 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). Glucose uptake of MG63 cells was significantly increased compared with that of the paired control cells after long-term (72 h) treatment with dcOC at different concentrations (0.3, 3, and 10 ng/mL groups, P < 0.01; 30 ng/mL group, P < 0.05). DcOC triggered Akt phosphorylation in a dose-dependent manner, and the most effective stimulatory concentration of dcOC for short-term (1 h) was 3 ng/mL (P < 0.01). LY294002 abolished the dcOC-mediated (1 h) promotion of Akt phosphorylation and glucose uptake without affecting GLUT1 protein expression. Long-term dcOC stimulation triggered Akt phosphorylation and increased the protein levels of HIF-1α, GLUT1, and Runx2 in a dose-dependent manner. Inhibition of HIF-1α with siRNA abolished the dcOC-mediated glucose uptake and substantially decreased GLUT1 protein expression. DcOC intervention promoted cell proliferation in a time- and dose-dependent manner as determined by the CCK-8 assay. Treatment with both 3 ng/mL and 10 ng/mL dcOC affected the ALP activity in MG63 cells after 72 h (P < 0.01).CONCLUSIONShort- and long-term dcOC treatment can increase glucose uptake and affect proliferation and ALP activity in MG63 cells. This effect may occur through the PI3K/Akt, HIF-1α, and GLUT1 signaling factors.

The influence of laser frequency and groove distance on cell adhesion, cell viability, and antibacterial characteristics of Ti-6Al-4V dental implants treated by modern fiber engraving laser

ObjectiveMicro-nano scale surface modification of Ti-6Al-4V was investigated through the fascinated modern fiber engraving laser method. The process was performed at a high laser speed of 2000mm/s, under different laser frequencies (20–160kHz) and groove distances (0.5–50μm). MethodsTopographic evaluations such as Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FE-SEM) were used to identify the quality and regularity of patterns. The proliferation of human osteoblast-like osteosarcoma cells (MG63) was analyzed by MTT assay for up to 72h. Also, the plate counting method was used to quantify the viability potential of the modified surface against Escherichia coli bacteria. ResultsThe cellular viability of the sample modified at the laser frequency of 20kHz and grooving distance of 50μm increased up to 35 and 10% compared to the non-treated and control samples, respectively. In the case of the surface modification at lower grooving distances range between 0.5–50μm, the maximum laser frequency (160kHz) applied leads to lower pulse’s energies and less bacterial adhesion. Otherwise, at groove distances more than 50μm, the minimum laser frequency (20kHz) applied reduces the laser pulse overlaps, increases the cell adhesion and antibacterial properties. SignificanceSurface modification by the fiber engraving laser process significantly enhances the cell adhesion on the surface. As a result of such roughness and cell adhesion enhancement, the surface toxicity feature diminished, and its antibacterial properties improved.

Bioactivity in SBF versus trace element effects: The isolated role of Mg2+ and Zn2+ in osteoblast behavior.

The bioactivity assay originally proposed by Kokubo is one of the most commonly used tests to indirectly evaluate the biocompatibility of bioactive glasses. However, extensive evidence has shown that trace elements present in biomaterials may stimulate cellular behavior in different ways even when no apatite formation is observed, i.e., in biomaterials with low or no bioactivity. To further elucidate this topic, we designed three different SiO2-rich bioglass compositions in which CaO was partially replaced by ZnO and MgO, two oxides known to affect bioactivity as well as osteoblastic behavior. The physicochemical changes induced by the presence of oxides and their effects on biological behavior, as well as the adhesion, proliferation and differentiation of human osteoblast-like osteosarcoma cells (MG-63), were followed by a bioactivity assay in simulated body fluid (SBF). The insertion of ZnO or MgO decreased the glass transition (Tg) and crystallization (Tc) temperatures as a function of the increase in nonbonding oxygens, which was directly reflected in the higher solubility. The release of Mg2+ ions from the MgO-containing samples inhibited the bioactivity in SBF, inducing high cell adhesion and proliferation and moderate ALP activity. The release of Zn2+ also inhibited the bioactivity in SBF but, in contrast to the release of Mg2+, induced low cell adhesion and proliferation and high ALP activity compared to the control.

Antibacterial Copper-Doped Calcium Phosphate Glasses for Bone Tissue Regeneration.

Calcium phosphate glasses are a promising new generation of biomaterials that can simultaneously induce tissue regeneration and controlled release of therapeutic molecules. In this work, novel calcium phosphate glasses containing 0, 2, 4, and 6 mol % Cu2+ were synthesized via room temperature precipitation reaction in aqueous solution. The effect of Cu2+ addition on the glass properties and structure was investigated using thermal analysis, 31P solid-state MAS NMR, Raman spectroscopy, and X-ray diffraction. All glasses crystallize at temperature >500 °C and are mainly formed by Q1 groups. The release of P, Ca, and Cu in solution over time was monitored via inductively coupled plasma-optical emission spectroscopy. It was found that with increasing Cu content, the amount of P and Ca released decreases whereas the amount of Cu released increases. The effect of Cu2+ release on the antibacterial activity against S. aureus, a bacterial strain commonly found in postsurgery infections, has been investigated. The addition of copper has been shown to infer the glasses antibacterial properties. As expected, the antibacterial activity of the glasses increases with increasing Cu2+ content. Cytocompatibility was assessed by seeding human osteoblast-like osteosarcoma cells Saos-2 (HTB85) on the glass particles. A significant increase in cell number was observed in all the glasses investigated. The copper-doped calcium phosphate glasses have proven to be multifunctional, as they combine bone regenerative properties with antibacterial activity. Therefore, they have great potential as antibacterial bioresorbable materials for hard tissue regeneration.

Zinc and strontium based phosphate glass beads: a novel material for bone tissue engineering

Degradable phosphate-based glasses that contain strontium, zinc and calcium were investigated to examine their function as an osteoconductive material. Glass beads of the general formula of (P2O5)–(Na2O)–(TiO2)–(CaO)–(SrO) or (ZnO) were prepared by a melt quench technique followed by milling and spheroidisation. After performing x-ray diffraction on all the samples for glass structure evaluation, glass bead size distribution was initially measured by a scanning electron microscope (SEM). Then, some of these samples were immersed in deionised water to evaluate both the surface changes and measure the ion release rate, whereas other samples of glass beads were incubated in culture media to determine pH changes. Furthermore, human osteoblast-like osteosarcoma cells MG63 and human mesenchymal stem cells were seeded on the glass beads to determine their cytocompatibility via applying CCK assay, ALP assay and Ca assay. SEM images and fluorescence images of confocal microscopy were performed for the cellular studies. While mass degradation and ion release results displayed a significant increase with zinc and strontium incorporation within time, pH results showed an initial increase in pH followed by a decrease. Cellular studies emphasised that all formulations enhanced cellular proliferation. Phosphate glass beads with zinc content 5 mol% and strontium content of 17.5 mol%, (ZnO5) and (SrO17.5) respectively displayed more promising results although they were insignificantly different from that of control (p > 0.05). This may suggest their applicability in hard tissue engineering.

Response of MG63 Osteoblast Cells to Surface Modification of Ti-6Al-4V Implant Alloy by Laser Interference Lithography

The response of human osteoblast-like osteosarcoma cells (MG63) to surface modification of Ti-6Al-4V implant alloy was investigated by Laser Interference Lithography (LIL). In this work, laser interference lithography was employed to fabricate the microstructures of grooves, dots and dimples onto the surfaces of Ti-6Al-4V samples. Two and three beam LIL systems were developed to carry out the experiments. The laser treatment resulted in the increases of the roughness and the contact angle of water on the implant alloy surfaces. The proliferation of osteoblasts was analyzed by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay for the time periods of 4 hours, 2 days, 3 days, and 6 days. The MTT test results demonstrated that the laser treatment surfaces had a positive impact on the proliferation of osteoblast cells after 24 hours. The alloy surface morphology and the morphological changes of MG63 cells cultured on the laser textured Ti-6Al-4V surface were observed by Scanning Electron Microscope (SEM). The SEM results indicated that the osteoblast cells were aligned on grooved surfaces and they were prolonged with the structures. Enzymatic detachment results showed that the 20 µm grooved structures provided the better cell adhesion to the textured Ti-6Al-4V surfaces.

Effect of minocycline hydrochloride ointment on cell attachment and proliferation on titanium disks

To investigate the effect of minocycline hydrochloride ointment on cell attachment and proliferation on titanium disks. Commercially pure (grade 4) machined titanium discs with three different kinds of surfaces (smooth, acid-etched and sandblasted combined with acid-etched) were treated with minocycline ointment for 1 week, and then cleaned in ultrasonic cleanser for 10 minutes. Surface properties were examined by scanning electron microscope (SEM) and roughness tester before and after the treatment. Surface roughness was compared by paired t test. MG-63 (human osteoblast-like osteosarcoma cell) cells were seeded on these three kinds of discs with or without minocycline treatment, and methl thiazolyl tetrazolium (MTT) was performed to investigate the attachment in the 1st day and proliferation in the 4th and 7th day. Data were analyzed by double factor analysis of variance. Surface roughness before and after minocycline application was as follows, Smooth: (0.093 ± 0.025) µm, (0.086 ± 0.026) µm; Acid-etched: (1.100 ± 0.095) µm, (1.009 ± 0.196) µm; Sandblasted combined with acid-etched: (2.837 ± 0.283) µm, (2.968 ± 0.206) µm. No significant changes in roughness were found before and after minocycline application (P values were 0.118, 0.436 and 0.692). SEM examination revealed as similar surface configuration after minocycline application as before, except for some remnant of the minocycline ointment in acid-etched and sandblasted combined acid-etched groups. In MTT test, the growth of MG-63 cells in the 1 st, 4th day and 7th day was not different between groups with and without minocycline application (P values were 0.450, 0.848 and 0.835), and among three groups of different surface (P values were 0.184, 0.579 and 0.331). Minocycline hydrochloride ointment did not affect the surface configuration, surface roughness or the properties for cell attachment and proliferation of titanium discs.

Chemokine gene activation in human bone marrow‐derived osteoblasts following exposure to particulate wear debris

Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-kappaB activation. Inhibition studies with N-acetyl-L-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-kappaB activity and IL-8 chemokine expression involves oxidant signaling and IkappaBalpha-proteasomal degradation. Activation of the NF-kappaB transcription factor, as well as the IL-8 gene, are redox-regulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.

Mechanosensitivity of human osteosarcoma cells and phospholipase C β2 expression

Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 μstrains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC β1, β3, γ1, γ2, and δ1 in all cells, but PLC β2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC β2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC β1 in U-2/OS cells by 35% and PLC β2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC β2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

Expression of Type 2 Iodothyronine Deiodinase in Human Osteoblast Is Stimulated by Thyrotropin

Thyroid hormones play important roles in bone growth, development, and turnover. To exert its biological activity, T(4) needs to be converted to T(3) by iodothyronine deiodinase. In human thyroid gland as well as rat brown adipose tissue, type 2 iodothyronine deiodinase (D2) expression is regulated by a TSH receptor-cAMP-mediated mechanism. TSH receptor knockout mice demonstrated the direct effects of TSH on bone via TSH receptors found on osteoblast and osteoclast precursors. In the present study we investigated the possible expression and function of iodothyronine deiodinase and TSH receptors in human osteoblast-like osteosarcoma (SaOS-2) cells and normal human osteoblast (NHOst) cells. Iodothyronine deiodinase activity was detected in SaOS-2 cells and NHOst cells, and all of the characteristics of deiodinating activity were compatible with those of D2. Northern analysis demonstrated D2 mRNA expression in SaOS-2 cells and NHOst cells. D2 mRNA levels as well as D2 activities were rapidly increased by dibutyryl cAMP or forskolin in SaOS-2 cells and NHOst cells. TSH receptor mRNA was demonstrated in SaOS-2 cells and NHOst cells, and D2 mRNA and D2 activity were stimulated by TSH in both cells. In addition, all T(3) receptor isoforms were detected by RT-PCR in SaOS-2 cells and NHOst cells. The present results indicate the expression of functional TSH receptors and D2 in human osteoblasts and suggest previously unrecognized roles of TSH receptors and local T(3) production by D2 in the pathophysiology of human osteoblasts.

Effects of estriol on proliferative activity and expression of insulin-like growth factor-I (IGF-I) and IGF-I receptor mRNA in cultured human osteoblast-like osteosarcoma cells

The present study was undertaken to elucidate whether estriol (E3) affects the proliferative activity and the expression of insulin-like growth factor-I (IGF-I) mRNA and IGF-I receptor (IGF-IR) mRNA in cultured human osteoblast-like osteosarcoma cells (HOS TE85). In this study, the effects of E3 on cultured HOS TE85 cells were compared with those of 17β-estradiol (E2). HOS TE85 cells were subcultured in phenol red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 72 h and then stepped down to serum-free conditions for an additional 72 h in the absence or presence of E3 (3.52 × 10−9, 3.52 × 10−8, 3.52 × 10−7 mol/l) or E2 (3.67 × 10−8 mol/l). Treatment with either E3 (3.52 × 10−8, 3.52 × 10−7 mol/l) or E2 (3.67 × 10−8 mol/l) resulted in an increase in the number of cultured HOS TE85 cells and their uptake of bromodeoxyuridine. Northern blot hybridization with a IGF-I cDNA probe revealed that RNA prepared from cultured HOS TE85 cells contained IGF-I mRNA transcripts of 1.8, 4.4 and 7.5 kb. Treatment with either E3 (3.52 × 10−9, 3.52 × 10−8, 3.52 × 10−7 mol/l) or E2 (3.67 × 10−8 mol/l) resulted in increased expression of the three mRNA transcripts relative tot hose in untreated control cultures. Semi-quantitative, reverse transcription polymerase chain reaction analysis showed that the 440-bp IGF-IR mRNA transcript was present in HOS TE85 cells and that treatment with either E3 or E2 did not affect the IGF-IR mRNA expression in these cells. These results demonstrate that E3 (3.52 × 10−9, 3.52 × 10−8, 3.52 × 10−7 mol/l) exerts profound effects on the proliferative potential of cultured HOS TE85 cells, compatible with that of E2 (3.67 × 10−8 mol/l), through the induction of IGF-I mRNA expression without affecting IGF-IR mRNA expression in these cells.

Vitamin D compounds exert anti-apoptotic effects in human osteosarcoma cells in vitro

Several studies have demonstrated that vitamin D regulates growth and differentiation in bone cells in vitro. In addition, in vivo studies have shown that vitamin D stimulates bone formation, increases the number of osteoblast precursor cells and prevents bone mineral loss. These observations indicate that vitamin D may have anabolic effects on bone, and thus therapeutic potential in the treatment of osteoporosis. However, little is known about the effects of vitamin D on apoptosis in bone cells and about the contribution of this process to the effect of vitamin D on bone mineral loss. To investigate this aspect in more detail, we studied the effect of 1α,25(OH) 2D 3 and a series of analogues on apoptosis in human osteosarcoma cells. No significant induction of apoptosis was observed with any of the compounds after a 5 day treatment period. In contrast, some of the analogues showed a tendency to protect the cells from undergoing apoptosis. This anti-apoptotic effect of vitamin D was further confirmed by the ability of 1α,25(OH) 2D 3 to suppress camptothecin- and staurosporin-induced DNA fragmentation in the cells. In cultures treated simultaneously with 1α,25(OH) 2D 3 in combination with camptothecin or staurosporin, the level of DNA fragmentation was markedly reduced compared with cultures treated with camptothecin or staurosporin alone. On the basis of the present results, it is therefore concluded that vitamin D displays anti-apoptotic effects in human osteoblast-like osteosarcoma cells in vitro. This observation suggests that besides regulating growth and differentiation, vitamin D exerts its anabolic effects on bone by protecting osteoblastic cells from undergoing apoptosis.

Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells.

Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood. This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells). EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14. EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.

C5a modulation of interleukin-1 beta-induced interleukin-6 production by human osteoblast-like cells.

Periodontal bone resorption is controlled by osteoblast products, including interleukin (IL)-6, which are stimulated by other cytokines and complement components in the pro-inflammatory milieu. This study demonstrated that human osteoblast-like osteosarcoma cells (MG-63) responded to human recombinant (hr) C5a by releasing significant amounts of the bone-resorbing cytokine IL-6. C5a-induced release of IL-6 was enhanced 330% when cells were exposed to IL-1beta prior to C5a challenge at optimal concentrations (1.0 microg/ml C5a, 0.1 ng/ml IL-1beta). Cells simultaneously challenged with these concentrations of C5a and IL-1beta produced a 700% increase in IL-6 release relative to cells challenged with IL-1beta alone. Incubation of IL-1beta-treated cells with anti-human C5a receptor (C5aR) Ab resulted in a 78% suppression of the C5a-induced release of IL-6, but C5aR neutralization did not affect C5a/IL-1beta co-stimulation of IL-6. In addition, neither IL-1beta nor C5a significantly altered the other's cell-surface receptor relative to binding affinity or density. These results indicate that while MG-63 cells express functional C5aRs, the synergistic effect of C5a and IL-1beta on osteoblast IL-6 production is probably controlled by post-receptor signaling events. C5a agonists and antagonist used to alter critical C5a concentrations may present a new point of therapeutic intervention for the treatment of inflammatory bone resorption such as is found in periodontitis.

Fluid shear stress increases interleukin-11 expression in human osteoblast-like cells: its role in osteoclast induction.

It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. The conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate-resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP-positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme-linked immunosorbent assay showed interleukin-11 (IL-11) protein to increase 7-fold in the 24 h-FSS CM. A Northern analysis showed that IL-11 mRNA increased 4-fold in the SaOS-2 cells after 6 h-FSS; however, no IL-6 mRNA expression was detected. Furthermore, the anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. The IL-11 mRNA up-regulation in SaOS-2 cells by the 6 h-FSS was not inhibited by the anti-human transforming growth factor-beta1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7-fold in the 1 h-FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL-11 (mediated by prostaglandins) and thus stimulates bone remodeling.

Implant Surface Characteristics Modulate Differentiation Behavior of Cells in the Osteoblastic Lineage

This paper reviews the role of surface roughness in the osteogenic response to implant materials. Cells in the osteoblast lineage respond to roughness in cell-maturation-specific ways, exhibiting surface-dependent morphologies and growth characteristics. MG63 cells, a human osteoblast-like osteosarcoma cell line, respond to increasing surface roughness with decreased proliferation and increased osteoblastic differentiation. Alkaline phosphatase activity and osteocalcin production are increased. Local factor production is also affected; production of both TGF-beta 1 and PGE2 is increased. On rougher surfaces, MG63 cells exhibit enhanced responsiveness to 1,25-(OH)2D3. Prostaglandins mediate the effects of surface roughness, since indomethacin prevents the increased expression of differentiation markers in these cells.

Expression of thyrotropin receptor on clonal osteoblast-like rat osteosarcoma cells.

The expression of thyrotropin receptor (TSH-R) on various cells derived from bone, including osteoblast-like rat osteosarcoma cells (UMR106 cells), was investigated. TSH receptor mRNA was detected in UMR106 cells by Northern blot analysis. 125I-labeled TSH binding analysis revealed specific high- and low-affinity binding sites (association constants of 5.6 x 10(9) M(-1) and 3.0 x 10(7) M(-1), respectively) on UMR106 cells. Recombinant TSH, but not recombinant human chorionic gonadotropin, increased cyclic adenosine monophosphate (cAMP) production in a concentration-dependent manner in these cells. Furthermore, immunoglobulin Gs from patients with Graves' disease induced cAMP response in UMR106 cells, and the cAMP response index in this cell line correlated with thyroid-stimulating antibody (TSAb) activity detected by Chinese hamster ovary (CHO)-K1 cells transfected with rat TSH-R. We have also demonstrated that recombinant TSH increased cAMP production in human osteoblast-like osteosarcoma (MG63) cells and mouse primary osteoblastic cells. These results suggest that osteoblasts possess functional TSH-R and that abnormal bone metabolism in Graves' disease may be partly explained by the interaction of TSAb with TSH-R in osteoblasts in some patients.

17Beta-hydroxysteroid dehydrogenases in human bone cells.

Interconversion of estrogens by osteoblasts may play a role in regulating bone mass. As a first step toward exploring this possibility, we investigated the expression and activity of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in cultured human osteoblasts (HOB) and osteoblast-like osteosarcoma cells (MG63, TE85, and SaOS-2). Significant 17beta-HSD activity was detected in cell-free extracts of all bone cells with oxidation of estradiol to estrone predominating over reduction. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that the mRNA for 17beta-HSD I was detectable only in MG63 cells, albeit at low levels, while 17beta-HSD II was present in MG63, TE85, and HOB, but not SaOS-2, and 17beta-HSD III was absent from each bone cell type. 17Beta-HSD IV was the only isoform present in all bone cells analyzed. Further analysis of the expression of 17beta-HSD IV in these bone cells by immunoblotting revealed both the full-length 83 kDa protein and the proteolytic 38 kDa form. The kinetic parameters for estradiol oxidation by purified recombinant 17beta-HSD IV (Km = 49.7 microM, Vmax = 79.4 nmol/minute/mg of protein) and its HSD-domain (Km = 79.4 microM, Vmax = 476 nmol/minute/mg of protein) were significantly higher than previously reported, but consistent with the values obtained with crude cell-free extracts of SaOS-2 cells (Km = 98.8 microM, Vmax = 0.07 nmol/minute/mg of protein) which contain only 17beta-HSD IV based on RT-PCR. These studies show that bone cells have the capacity to interconvert circulating estrogens and suggest that bone cell 17beta-HSDs serve primarily to attenuate the continuing actions of estradiol through conversion to its less potent form, estrone, under certain conditions.