Mouse Osteoblast-like MC3T3-E1 Cells Research Articles

Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

Low frequency pulsed electromagnetic fields exposure alleviate the abnormal subchondral bone remodeling at the early stage of temporomandibular joint osteoarthritis

BackgroundTemporomandibular joint osteoarthritis (TMJOA) is characterized by abnormal subchondral bone remodeling and cartilage degeneration. As a non-invasive biophysical technology, pulsed electromagnetic field (PEMF) treatment has been proven to be efficient in promoting osteogenesis. However, the potential bone protective effect and mechanism of PEMF on abnormal subchondral bone remodeling in TMJOA are unknown.MethodsUnilateral anterior crossbite (UAC) was used to create TMJOA model in rats, and 17β-estradiol (E2) were injected daily to mimic patients with high-physiological levels of estrogen. Mouse osteoblast-like MC3T3-E1 cells treated with recombinant murine IL-1β was used to establish inflammatory environment in vitro. The treatment group were subjected to PEMF (2.0mT, 15 Hz, 2 h/d). Micro-CT scanning, histological staining, real-time PCR and western blotting assays were preformed to observe the changes in the subchondral bone.ResultsAbnormal resorption of subchondral bone induced by UAC, characterized by decreased bone mineral density, increased osteoclast activity and expression of osteoclast-related factors (RANKL) and down-regulated expression of osteogenesis-related factors (OPG, ALP, Runx2 and OCN) at the early stage, could be reversed by PEMF exposure, which was similar to the effect of estrogen. In addition, PEMF exposure and E2 supplement may have a synergistic effect to some extent. Moreover, PEMF exposure could promote the ALP activity and osteogenic mineralization ability of MC3T3-E1 cells. PEMF promoted the expression of factors related to Wnt/β-Catenin signal pathway both in vivo and in vitro.ConclusionsAppropriate PEMF exposure have a protective effect on subchondral bone in TMJOA at early stage, in which canonical Wnt/β-Catenin pathway may be involved. PEMF may be a promising biophysical approach for early intervention of TMJOA in clinic.

2,3-Diphospho-D-glyceric acid inhibits calciprotein particle growth and calcification in MOVAS cells but not in MC3T3-E1 cells

Anionic polyphosphate compounds are well-known to inhibit the formation of hydroxyapatite crystals. Vascular calcification (VC) is associated with an increase in crystalline calciprotein particles (CPPs) in the bloodstream. 2,3-Diphospho-D-glyceric acid (2,3-DPG) is a highly anionic Poly-P compound found in the concave center of erythrocytes where it binds to hemoglobin, thereby reducing oxygen affinity. Since 2.3-DPG exists in plasma, we hypothesized that 2,3-DPG may act as an endogenous inhibitor of VC. In comparison to widely recognized calcification inhibitors such as myo-inositol hexakisphosphate, 2,3-DPG significantly delayed the formation of crystalline CPPs. Additionally, 2,3-DPG inhibited calcification in a mouse vascular smooth muscle cell line (MOVAS) without cytotoxic effects. Taken together with the result that 2,3-DPG did not impact bone-like nodule formation in mouse osteoblast-like MC3T3-E1 cells, these results suggest that 2,3-DPG may inhibit VC selectively.

Uniaxially fixed mechanical boundary condition elicits cellular alignment in collagen matrix with induction of osteogenesis

Osteocytes differentiated from osteoblasts play significant roles as mechanosensors in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae with slender cell processes perpendicular to the trabeculae surface is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, the mechanisms underlying osteocyte network formation remains unclear. Here, we developed a novel in vitro collagen matrix system exerting a uniaxially-fixed mechanical boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured, evoking cellular alignment along the uniaxial boundary condition. Using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension via contraction of actin fibers contributed to the cellular alignment under the influence of isometric matrix condition along the uniaxially-fixed mechanical boundary condition. Furthermore, the cells actively migrated inside the collagen matrix and promoted the expression of osteoblast and osteocyte genes with their orientations aligned along the uniaxially-fixed boundary condition. Collectively, our results suggest that the intracellular tension of osteoblasts under a uniaxially-fixed mechanical boundary condition is one of the factors that determines the osteocyte alignment inside the bone matrix.

Co-electrospun nano-/microfibrous composite scaffolds with structural and chemical gradients for bone tissue engineering

Recent trends in scaffold design for tissue engineering have focused on providing structural, mechanical and chemical cues for guiding cell behaviors. In this study, we presented a structural/compositional gradient nano-/microfibrous mesh by co-electrospinning, using silk fibroin-poly(ε-caprolactone) (SF-PCL) nanofibers and PCL microfibers. The pore size, porosity, and physical property of the gradient meshes were qualified. Cell proliferation of mouse osteoblast-like MC3T3-E1 cells was carried out to estimate the effect of structural and compositional gradients on biocompatibility. Furthermore, the 2-D mesh was rolled up and the compressive property of 3-D cylinder was investigated. The results suggested that the rolled-up gradient cylinder scaffold exhibited higher osteogenic differentiation compared to the pristine nanofibrous cylinder sample. By incorporating Chinese medicine ginsenoside Rg1, sustained release was achieved in composite meshes. Rg1-containing nanofibrous meshes and Rg1 gradient cylinders enhanced the cell proliferation of human umbilical vein endothelial cells (HUVECs). The developed fibrous scaffold may provide structural, compositional, and chemical gradients for bone regeneration. BriefsStructural and chemical gradient fibrous scaffold fabricated by co-electrospinning.

Effects of Rubus coreanus Miquel extracts on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells

Rubus coreanus Miquel (RCM), one a type of edible red raspberry, grows wild in Korea and China and its unripe fruit has been used as a folk medicine for the treatment of impotence and as a diuretic agent. The goal of this study was to investigate the effects of RCM on the proliferation and differentiation of mouse osteoblast-like MC3T3-E1 cells. In this study, RCM extract (1-10 μg/mL) enhanced the viability of osteoblast cell. RCM extract also increased alkaline phosphatase (ALP) activity and osteocalcin secretion of MC3T3-E1 cells (significant at 1-10 μg/mL). Especially, at a concentration of 10 μg/mL ALP activity was increased by approximately 4.3 times in comparison with the control. Alizarin red staining and osteocalcin secretion assay showed that RCM extract stimulates differentiation of osteoblast cells by inducing the activation of mineralization. Moreover, reverse transcriptase polymerase chain reaction and western blotting assay indicated that RCM extract induced the differentiation at mRNA and protein level. These results suggest that RCM extract enhanced the osteoblast function by increasing the proliferation and differentiation of osteoblast like MC3T3-E1 cells.

A bone substitute composed of polymethyl-methacrylate bone cement and Bio-Gene allogeneic bone promotes osteoblast viability, adhesion and differentiation.

Increasing reports on new cement formulations that address the shortcomings of PMMA bone cements and various active components have been introduced to improve the biological activity of PMMA cement. Evaluating the biological properties of PMMA cements reinforced with Bio-Gene allogeneic bone. The MC3T3-E1 mouse osteoblast-like cells were utilized to determine the effects of Bio-Gene + PMMA on osteoblast viability, adhesion and differentiation. The combination of allogeneic bone and PMMA increased the number of adherent live cells compared to both control group and PMMA or Bio-Gene group. Scanning electron microscopy observed that the number of cells adhered to Bio-Gene + PMMA was larger than Bio-Gene and PMMA group. Compared with the control and PMMA or Bio-Gene group, the level of ALP and the number of calcium nodules after osteoinduction was remarkably enhanced in Bio-Gene + PMMA group. Additionally, the combination of Bio-Gene and PMMA induced the protein expression of osteocalcin, osterix and collagen I. The composition of PMMA and allogeneic bone could provide a more beneficial microenvironment for osteoblast proliferation, adhesion and differentiation. PMMA bone cement reinforced with Bio-Gene allogeneic bone may act as a novel bone substitute to improve the biological activity of PMMA cement.

Intracellular Tension of Osteoblasts in Collagen Matrix Under Uniaxially-Fixed Boundary Condition Elicits Osteocyte Alignment with Induction of Osteogenesis

Osteocytes differentiated from osteoblasts play a significant role as mechanosensor in modulating the bone remodeling process. While the well-aligned osteocyte network along the trabeculae is known to facilitate the sensing of mechanical stimuli by cells and the intracellular communication in the bone matrix, understanding these mechanisms in the osteocyte network formation remain poor. Here, we developed a novel in vitro collagen system exerting a uniaxially-fixed boundary condition on which mouse osteoblast-like MC3T3-E1 cells were subcultured for 1 d, evoking cellular alignment along the uniaxial boundary condition. By using a myosin II inhibitor, blebbistatin, we showed that the intracellular tension was strengthened by stabilization of actin filaments along the uniaxially-fixed boundary condition, so that this positive tension-feedback under isometric contraction contributed to the enhanced intracellular tension. After 10 d incubation, cells actively migrated inside the collagen and exerted the promoted expression of osteoblast and osteocyte genes compared with 4 d incubation. Notably, the migrated cells became aligned along the boundary condition after 10 d cultivation, reconstructing the in vivo process of formation of osteocyte network. Collectively, we suggested that the intracellular tension of osteoblasts under uniaxially-fixed boundary condition plays a significant role to determine the osteocyte alignment inside the bone matrix.

Osteogenic cells form mineralized particles, a few μm in size, in a 3D collagen gel culture.

Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with β-glycerophosphate (β-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing β-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.

Combinatorial effects of inorganic ions on adhesion and proliferation of osteoblast-like cells.

Combinatorial effects of three ions, namely silicate (Si), calcium (Ca), and magnesium (Mg) ions, on the adhesion and proliferation of MC3T3-E1 mouse osteoblast-like cells were evaluated. The cells were cultured in single-, dual-, or triple-ion-conditioned culture media with systematically changed ion concentrations. The ranges of Si, Ca, and Mg ion concentrations were set as 10-70, 80-400, and 25-500 ppm, respectively. The numbers of adherent live cells were measured after culturing for 3 h and for 1, 3, and 5 days to examine cell adhesion and proliferation, respectively. Mg ions predominantly enhanced cell adhesion in both the dual-ion (xSi-zMg and yCa-zMg) and triple-ion (xSi-yCa-zMg) systems but had no effect when they acted individually in the single-ion system. Conversely, Si ions predominantly enhanced cell proliferation in most single- and triple-ion-conditioned media. Evaluation of the combinatorial effects of the three ions on cell adhesion and proliferation revealed that the dual- and triple-ion-conditioned media mainly conferred synergistic effects on adhesion but antagonistic effects on proliferation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1042-1051, 2019.

MiRNA-133a-5p Inhibits the Expression of Osteoblast Differentiation-Associated Markers by Targeting the 3' UTR of RUNX2.

Recent studies have recognized the involvement of microRNAs (miRNAs) in the development of osteoporosis, which regulate the balance between osteogenesis and osteoclasis. In this study, we investigated the regulation by miRNA-133a-5p on the osteoblast differentiation-associated markers in the mouse osteoblast-like MC3T3-E1 cells by RUNX2. First, we manipulated the miRNA-133a level in the MC3T3-E1 cells with 20 or 40 nM miR-133a-5p mimics, miR-133a-5p inhibitor, or scramble miRNA. Then, we quantified with real-time polymerase chain reaction (qRT-PCR) the expression of Collagen I, osteocalcin (OCN), and osteopontin (OPN) in the miR-133a-5p-manipulated MC3T3-E1 cells. And the confocal microscopy was also utilized to confirm the regulation by miR-133a-5p on the expression of the three molecules. We also investigated the extracellular matrix (ECM) mineralization and the alkaline phosphatase (ALP) activity in the miR-133a-5p-manipulated MC3T3-E1 cells. In addition, we explored the possible targeting by miR-133a-5p on RUNX2, which was a well-recognized promoter to osteoblast differentiation, with luciferase reporter, qRT-PCR, and Western blotting assay. Results demonstrated that the miRNA-133a-5p mimics markedly reduced, whereas the miRNA-133a-5p inhibitor significantly promoted the expression of Collagen I, OCN, and OPN, the ECM mineralization, and the ALP activity in MC3T3-E1 cells. The alignment analysis demonstrated a high homology between miRNA-133a-5p and the 3' UTR of RUNX2. Moreover, the luciferase reporter assay demonstrated that miRNA-133a-5p targeted the 3' UTR of RUNX2, and inhibited the expression of RUNX2 in both mRNA and protein levels. In conclusion, we identified the inhibition by miRNA-133a-5p to the expression of osteoblast differentiation markers, to the ECM mineralization, and to the ALP activity in MC3T3-E1 cells, by targeting the 3' UTR of RUNX2. Our study suggests that miRNA-133a-5p might be an important target to inhibit osteoblast differentiation in osteoporosis.

Dextrin Promotes Proliferation of Cultured MC3T3-E1 Mouse Osteoblast-like Cells and Their Alkaline Phosphatase Activity: Implications for Potential Application of Dextrin as a Binder of Bone Filling Material

Dextrin Promotes Proliferation of Cultured MC3T3-E1 Mouse Osteoblast-like Cells and Their Alkaline Phosphatase Activity: Implications for Potential Application of Dextrin as a Binder of Bone Filling Material

Effect of nanometer scale surface roughness of titanium for osteoblast function

Surface roughness is an important property for metallic materials used in medical implants or other devices. The present study investigated the effects of surface roughness on cellular function, namely cell attachment, proliferation, and differentiation potential. Titanium (Ti) discs, with a hundred nanometer- or nanometer-scale surface roughness (rough and smooth Ti surface, respectively) were prepared by polishing with silicon carbide paper. MC3T3-E1 mouse osteoblast-like cells were cultured on the discs, and their attachment, spreading area, proliferation, and calcification were analyzed. Cells cultured on rough Ti discs showed reduced attachment, proliferation, and calcification ability suggesting that the surface inhibited osteoblast function. The findings can provide a basis for improving the biocompatibility of medical devices.

High-mobility group box-B1 (HMGB1) mediates the hypoxia-induced mesenchymal transition of osteoblast cells via activating ERK/JNK signaling.

High-mobility group box 1 (HMGB1) is a nuclear protein that involves the binding with DNA and influences chromatin regulation and transcription. HMGB1 activates monocytes and neutrophils, which are involved in inflammation during wounding. In this study, we investigated the promotion of HMGB1 under hypoxia and determined the regulatory role of HMGB1 on the fibrosis of mouse osteoblast-like MC3T3-E1 cells or of human osteoblast MG-63 cells. Results demonstrated that HMGB1 expression was significantly upregulated in MC3T3-E1 or MG-63 cells under hypoxia. We also found that treatment with 10 and 100 ng/mL of HMGB1 significantly promoted the fibrosis-associated markers such as Collagen I, α-SMA, whereas downregulated the E-cadherin, indicating the differentiation of MC3T3-E1 or MG-63 cells into fibroblast cells. Further investigation indicated that the HMGB1 treatment markedly activated the mitogen-activated protein kinases (MAPKs), including extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) phosphorylation, as well as nuclear factor (NF)-κB nuclear translocation. On the other side, using specific inhibitors and shRNAs of protein kinases, we observed that repression of ERK, JNK, p38, and NF-κB all inhibited HMGB1-induced cellular differentiation and migration of MC3T3-E1 cells. In addition, knocking down of advanced glycation end products (RAGE) but not Toll-like receptor (TLR)2 and TLR4 by shRNAs attenuated HMGB1-induced myofibroblast differentiation and migration. In conclusion, our study demonstrated that HMGB1 induced the fibrosis of osteoblasts in vitro via activating the RAGE-MAPK and NF-κB interaction signaling pathways.

Sphingosine-1-phosphate/S1PR2-mediated signaling triggers Smad1/5/8 phosphorylation and thereby induces Runx2 expression in osteoblasts

Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that also plays crucial roles in bone regeneration. Recently, we reported that the S1P receptors S1PR1 and S1PR2 were mainly expressed in osteoblast-like cells, and that the S1P/S1PR1 signaling pathway up-regulated osteoprotegerin and osteoblast differentiation. However, the involvement of S1P/S1PR2 signaling in osteoblast differentiation is not well understood. Here we investigate the role of S1P/S1PR2-mediated signaling in osteoblast differentiation and clarify the underlying signaling mechanisms. We found that an S1P/S1PR2/Gi-independent signaling pathway activated RhoA activity, leading to phosphorylation of Smad1/5/8 in mouse osteoblast-like MC3T3-E1 cells and primary osteoblasts. Furthermore, this signaling pathway promoted nuclear translocation of Smad4, and increased the amount of Smad6/7 protein in the nucleus. S1P also up-regulated runt-related transcription factor 2 (Runx2) expression through S1PR2/RhoA/ROCK/Smad1/5/8 signaling. Moreover, we found that S1P partially triggered S1PR2/RhoA/ROCK pathway leading to bone formation in vivo. These findings suggest that S1P induces RhoA activity, leading to the phosphorylation of Smad1/5/8, thereby promoting Runx2 expression and differentiation in osteoblasts. Our findings describe novel molecular mechanisms in S1P/S1PR2-mediated osteoblast differentiation that could aid future studies of bone regeneration.

Effects of PEMF exposure at different pulses on osteogenesis of MC3T3-E1 cells

ObjectivePulsed electromagnetic fields (PEMFs) were considered to be a factor which may affect osteogenesis of osteoblasts, but the effects were diverse with different PEMF parameters. The aim of the current study is to explore the effects of exposure to PEMFs at different pulse number on osteogenesis of osteoblasts. DesignThe mouse osteoblast-like MC3T3-E1 cells were exposed to 0, 400 or 2800 pulses 400kV/m PEMF and the proliferation, differentiation and mineralization of cells were observed after PEMF exposure by the methods of MTT, biochemical measurement, real-time PCR and Alizarin Red assay. ResultsCompared with 0 pulses groups, the growth curve, alkaline phosphatase (ALP) activity, mRNA level of osteocalcin (OCN) and mineralized nodule formation of MC3T3-E1 cells did not change after 400 pulses PEMF exposure, but decreased after 2800 pulses PEMF exposure. It suggested that under our experimental conditions, only 2800 pulses 400kV/m PEMF exposure can suppress the proliferation, differentiation and mineralization of MC3T3-E1 cells, but 400 pulses 400kV/m PEMF exposure cannot. ConclusionsPulse number is another involved parameter which may influence the effects of PEMF on osteogenesis of osteoblasts.

Influence of cassette design on three-dimensional perfusion culture of artificial bone.

Media perfusion is often required to maintain cell viability within topographically complex 3-dimensional scaffold cultures. Osteoblast-seeded scaffolds for bone regeneration require robust cell proliferation and survival both within the scaffold and over the exterior for optimal osteogenic capacity. Conventional press-fitting cassettes ensure internal fluid flow through the scaffold but may restrict external flow around the scaffold, resulting in a barren (cell-free) external scaffold surface. In this study, we aimed to solve this problem by modifying the cassette structure to enhance external flow in an oscillatory perfusion culture system. Mouse osteoblast-like MC 3T3-E1 cells were seeded in porous ceramic scaffolds and incubated for 3 days either under static culture conditions or in an oscillatory perfusion bioreactor. Scaffolds were held in the bioreactor with either conventional press-fitting cassettes or cassettes with rings to separate the scaffold exterior from the internal cassette wall. The external surfaces of scaffolds maintained under static conditions were well seeded, but cells failed to grow deeply into the core, reflecting poor internal chemotransport. Alternatively, scaffolds cultured by perfusion with press-fitting cassettes had poor cell viability at the cassette-external scaffold surface interface, but cells were widely distributed within the scaffold core. Scaffolds cultured using the modified cassettes with 1 or 2 rings exhibited uniformly distributed living cells throughout the internal pores and over the entire external surface, possibly because of the improved medium flow over the scaffold surface. This modified oscillatory perfusion culture system may facilitate the production of engineered bone with superior osteogenic capacity for grafting.

Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-β estradiol

Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells' response to 17-β estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (α-MEM) cell culture supplemented with 17-β estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 10−8 mol⋅L−1 17-β estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes, of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-β)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to α-MEM supplemented with 17-β estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.

Thermo-sensitive graphene oxide-polymer nanoparticle hybrids: synthesis, characterization, biocompatibility and drug delivery.

We report the covalent interaction mediated assembly of thermo-sensitive polymer nanoparticles (PNPs) on functionalized graphene oxide (GO) nanosheets to create novel GO-PNP hybrids for drug delivery. To this end, thermo-sensitive PNPs with an average diameter of about 50 ± 12 nm were first synthesized with the free radical polymerization reaction, and GO nanosheets were noncovalently modified with a bifunctional linker to provide reactive sites for the binding of PNPs. Finally, GO-PNP hybrids were successfully synthesized by the covalent interaction mediated assembly of PNPs on GO nanosheets. Multi-characterization techniques were utilized to identify the formation of PNPs, the modification of GO nanosheets, and the formation of GO-PNP hybrids. Cell culture experiment with the mouse osteoblast-like MC3T3-E1 cells indicates that the synthesized GO-PNP hybrids have satisfactory biocompatibility. The loading efficiency of drug molecules (Adriamycin, ADR) with GO-PNP (∼87%) is close to that with GO (∼91%), but significantly higher than that with PNPs (∼46%). The release efficiency of GO-PNP hybrids with the highest surface coverage of PNPs (∼85 PNPs per μm2) is about 22%, which is very close to that of PNPs (∼25%) and significantly higher than that of GO (∼11%). Our study indicates that this thermo-sensitive GO-PNP hybrid, when considering the drug loading and release comprehensively, has better performance than both PNPs and GO and thus can be used as a novel nanocarrier for temperature-controllable drug release. The GO-PNP hybrids with and without ADR were applied to kill cancer cells in vitro and the result shows that the GO-PNP hybrid with ADR has an obvious effect on killing cancer cells, and its performance is obviously better than both GO and PNPs. It is expected that this new hybrid material based on GO and PNPs will have great potential for in vivo applications such as to kill target cancer cells by modifying with specific antibodies.

Mechanism of detoxification of the cationic antibacterial monomer 12-methacryloyloxydodecylpyridiniumbromide (MDPB) by N-acetyl cysteine

ObjectivesThe protective effects of N-acetyl cysteine (NAC) against cytotoxicity induced by conventional dental resin monomers have been widely documented. However, its effectiveness to detoxify cationic antibacterial monomers has not yet been elucidated. The aim of the present study was to investigate the possible protective effects of NAC against the cytotoxicity of 12-methacryloyloxydodecylpyridiniumbromide (MDPB) and explore the role of adduct formation in NAC-directed detoxification. MethodsThe influences of NAC on the cytotoxicity of MDPB were studied in mouse osteoblast-like MC3T3-E1 cells using the MTT assay. Ultra-performance liquid chromatography (UPLC) and liquid chromatography–mass spectrometry (LC–MS) analysis were performed to investigate the possible chemical reaction between NAC and MDPB. ResultsWhile only slight reduction in the cytotoxicity of MDPB by NAC was observed immediately after mixing with MDPB, remarkable protection against MDPB-induced cell death was detected when the mixture was tested after 24h of pre-incubation. UPLC and LC–MS analysis revealed that chemical binding of MDPB and NAC occurred under neutral conditions after 24h of pre-incubation. SignificanceOur findings suggest that NAC reduces the toxicity of the cationic antibacterial monomer MDPB, and adduct formation is partially responsible for the detoxification ability of NAC against MDPB-induced cell damage.