Differentiation Of MG-63 Cells Research Articles

In Situ Hydrogel with Immobilized Mn-Porphyrin for Reactive Oxygen Species Scavenging, Oxygen Generation, and Risedronate Delivery in Bone Defect Treatment.

We propose a hydrogel immobilized with manganese porphyrin (MnP), a biomimetic superoxide dismutase (SOD), and catalase (CAT) to modulate reactive oxygen species (ROS) and hypoxia that impede the repair of large bone defects. Our hydrogel synthesis involved thiolated chitosan and polyethylene glycol-maleimide conjugated with MnPs (MnP-PEG-MAL), which enabled in situ gelation via a click reaction. Through optimization, a hydrogel with mechanical properties and catalytic effects favorable for bone repair was selected. Additionally, the hydrogel was incorporated with risedronate to induce synergistic effects of ROS scavenging, O2 generation, and sustained drug release. In vitro studies demonstrated enhanced proliferation and differentiation of MG-63 cells and suppressed proliferation and differentiation of RAW 264.7 cells in ROS-rich environments. In vivo evaluation of a calvarial bone defect model revealed that this multifunctional hydrogel facilitated significant bone regeneration. Therefore, the hydrogel proposed in this study is a promising strategy for addressing complex wound environments and promoting effective bone healing.

Onion (Allium cepa L.) Flavonoid Extract Ameliorates Osteoporosis in Rats Facilitating Osteoblast Proliferation and Differentiation in MG-63 Cells and Inhibiting RANKL-Induced Osteoclastogenesis in RAW 264.7 Cells.

Osteoporosis, a prevalent chronic health issue among the elderly, is a global bone metabolic disease. Flavonoids, natural active compounds widely present in vegetables, fruits, beans, and cereals, have been reported for their anti-osteoporotic properties. Onion is a commonly consumed vegetable rich in flavonoids with diverse pharmacological activities. In this study, the trabecular structure was enhanced and bone mineral density (BMD) exhibited a twofold increase following oral administration of onion flavonoid extract (OFE). The levels of estradiol (E2), calcium (Ca), and phosphorus (P) in serum were significantly increased in ovariectomized (OVX) rats, with effects equal to alendronate sodium (ALN). Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) levels in rat serum were reduced by 35.7% and 36.9%, respectively, compared to the OVX group. In addition, the effects of OFE on bone health were assessed using human osteoblast-like cells MG-63 and osteoclast precursor RAW 264.7 cells in vitro as well. Proliferation and mineralization of MG-63 cells were promoted by OFE treatment, along with increased ALP activity and mRNA expression of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappaB ligand (RANKL). Additionally, RANKL-induced osteoclastogenesis and osteoclast activity were inhibited by OFE treatment through decreased TRAP activity and down-regulation of mRNA expression-related enzymes in RAW 264.7 cells. Overall findings suggest that OFE holds promise as a natural functional component for alleviating osteoporosis.

Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells.

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

Molecular mechanism of a novel root-end filling material containing zirconium oxide on the osteogenic/odontogenic differentiation of human osteosarcoma MG-63 cells.

Although the novel root-end filling material containing zirconium oxide (NRFM-Zr) which is hydroxyapatite-based may promote osteoblast differentiation, the molecular mechanism remains unclear. The aim of this study is to investigate it underlying the osteogenic/odontogenic differentiation of human osteosarcoma MG-63 cells induced by NRFM-Zr, compared with calcium silicate-based mineral trioxide aggregate (MTA), and glass ionomer cement (GIC). Firstly, three different types of root filling materials were co-cultured with MG-63 cells, and their cell toxicity, alkaline phosphatase (ALP) activity, and calcium ion concentration were evaluated. Next, gene expression profiling microarray was employed to analyze the impact of the materials on the gene expression profile of MG-63 cells. The results of cell viability revealed that NRFM-Zr group had no significant difference compared to the negative control group. After 5 and 7days of cultivation, both the NRFM-Zr and MTA groups exhibited significantly higher ALP activity compared to the negative control (p < 0.05). Moreover, the NRFM-Zr group had the highest calcium ion concentration, while the GIC group was the lowest (p < 0.05). Gene expression profiling microarray analysis identified 2915 (NRFM-Zr), 2254 (MTA) and 392 (GIC) differentially expressed genes, respectively. GO functional and KEGG pathway analysis revealed that differentially expressed genes of NRFM-Zr, MTA and GIC participated in 8, 6 and 0 differentiation-related pathways, respectively. Comparing the molecular mechanisms of osteogenic/odontogenic differentiation induced by hydroxyapatite-based NRFM-Zr and calcium silicate-based MTA, it was found that they shared similarities in their molecular mechanisms of promoting osteogenic differentiation. NRFM-Zr primarily promotes differentiation and inhibits cell apoptosis, thereby enhancing osteogenic/odontogenic differentiation of MG-63 cells. Furthermore, the inducing efficacy of NRFM-Zr was found to be superior to MTA.

Morphology-Dependent Immunomodulatory Coating of Hydroxyapatite/PEO for Magnesium-Based Bone Implants.

One of the most critical issues concerning orthopedic implants is the risk of chronic inflammation, which poses a threat to the bone healing process. Osteo-immunomodulation plays a pivotal role in implant technology by influencing proinflammatory and anti-inflammatory responses, ultimately promoting bone healing. This study aims to investigate the morphology-dependent osteo-immunomodulatory properties of a hydroxyapatite (HA)/plasma electrolytic oxidation (PEO)-coated WE43 alloy. In this context, following the PEO process with various operational parameters (duty cycles of 50-40, 50-20, 70-40%, and frequencies of 0.5, 0.8, and 1 kHz), a layer of HA was applied as the top coating using a straightforward hot-dip process. The results revealed the formation of the PEO layer with distinct morphologies and pore sizes, depending on the operational parameters. Specifically, a uniform PEO coating with small pore sizes (5.2-5.3 μm) led to the creation of plate-like HA particles, while a random-like HA structure formed on nonuniform surfaces with large pores (7.0-11.1 μm) of PEO. Moreover, it was observed that the plate-like HA coating exhibited higher adhesion strength than the random one (classified as class 2 vs class 3 based on cross-cut standards). Furthermore, electrochemical impedance spectroscopy (EIS) and polarization studies confirmed a substantial increase in the polarization resistance (680 kΩ) and total impedance (48 559.6 Ω) for the plate-like HA/PEO as compared to the substrate (an increase of 1511-fold and 311-fold, respectively) and the random HA/PEO samples (an increase of 85-fold and 18-fold, respectively). In addition, compared to random HA coatings, there was a significant enhancement in the viability (150% control vs 96% control), proliferation, and differentiation of MG63 cells when exposed to plate-like HA coatings. Moreover, surface morphology and chemistry pronouncedly impacted macrophages' viability, morphology, and phenotype. Notably, plate-like HA coatings resulted in a higher upregulation of BMP-2 and TGF-β than proinflammatory cytokines (IL-6 and M-CSF), indicating a polarization of macrophage type 1 (M1) toward type 2 (M2). In summary, the bilayer HA/PEO coating exhibited remarkable osteo-immunomodulatory activity, making it highly appealing for use in bone implant applications.

3D printed OCP bone scaffold with alginate enhancing osteogenic differentiation in MG-63 cells

3D printed OCP bone scaffold with alginate enhancing osteogenic differentiation in MG-63 cells

3D-printed polycaprolactone scaffolds coated with beta tricalcium phosphate for bone regeneration

3D-printing technology is an important tool for the bone tissue engineering (BTE). The aim of this study was to investigate the interaction of polycaprolactone (PCL) scaffolds and modified mesh PCL coated with beta TCP (PCL/β-TCP) scaffolds with MG-63. This study used the fused deposition modeling (FDM) technique with the 3D printing technique to fabricate the thermoplastic polymer and composite scaffolds. Scaffold structure and coating quality were observed under a scanning electron microscope (SEM). MG-63 cells were injected and attached to the mesh-manufactured PCL scaffolds. The biocompatibility of mesh structured PCL and PCL/β-TCP scaffolds could be examined by measuring the viability of MG-63 cells of MTT assay. Bone cell differentiation was evaluated ALP activity by mineralization assay. The results showed that both mesh PCL scaffolds and PCL/β-TCP scaffolds were non-toxic to the cells. The ALP activities of cells in PCL/β-TCP scaffolds groups were significant differences and better than PCL groups in all groups at all experimental dates. The mineralization process was time-dependent, and significantly higher mineralization of osteosarcoma cells was observed on PCL/β-TCP scaffolds at experimental dates. We concluded that both meshes structured PCL and PCL/β-TCP scaffolds could promote the MG-63 cell growth, and PCL/β-TCP was better than the PCL scaffolds for the outcome of MG63 cell differentiation and mineralization.

Comparative Evaluation of the Effect of Two Platelet Concentrates (a-PRF and L-PRF) on the Cellular Activity of Pre-osteoblastic MG-63 Cell Line: An in vitro Study.

Currently, the reconstruction of bone defects with new platelet concentrates is considered a significant challenge in periodontics. This study aimed to evaluate advanced- platelet rich fibrin (A-PRF) and leukocyte- and platelet rich fibrin's (L-PRF) effects on the proliferation and differentiation of MG-63 cells. In this in vitro study, blood samples of five healthy non-smoking volunteers were collected and immediately centrifuged according to the two protocols of Choukroun and Ghanaati, without adding any anticoagulants, to prepare L-PRF and A-PRF. After freezing the clots for one hour, they were crushed and centrifuged once more. After culturing MG-63 cells, the effects of 20%, 10%, 1%, and 0.5% concentrations of A-PRF and L-PRF extracts on cell proliferation and mineralization were evaluated by methyl thiazolyl tetrazolium (MTT) assay and Alizarin Red staining, respectively. Generally, survival and proliferation in the L-PRF group at both time intervals were higher than the A-PRF group and increased with increasing the extract concentration. However, in the A-PRF group, there were no significant differences between the different concentrations, and only the number of cells increased over time. After three days, in the study on mineralization, nodule formation was observed only in the positive control group (osteogenic). In seven days, mineralized nodules were formed in all groups with different concentrations of A-PRF, but not in any of the L-PRF groups. According to the results, L-PRF increased proliferation, and A-PRF exerted a positive effect on the differentiation of MG-63 cells.

Bone marrow mesenchymal stem cell-derived exosomal microRNA-382 promotes osteogenesis in osteoblast via regulation of SLIT2

BackgroundOsteoporosis (OP) is a systemic skeletal disorder with increased bone fragility. Human bone marrow mesenchymal stem cells (hBMSCs) have multi-lineage differentiation ability, which may play important roles in osteoporosis. In this study, we aim to investigate the role of hBMSC-derived miR-382 in osteogenic differentiation.MethodsThe miRNA and mRNA expressions in peripheral blood monocytes between persons with high or low bone mineral density (BMD) were compared. Then we collected the hBMSC-secreted sEV and examined the dominant components. The over-expression of the miR-382 in MG63 cell and its progression of osteogenic differentiation were investigated by qRT-PCR, western blot and alizarin red staining. The interaction between miR-382 and SLIT2 was confirmed by dual-luciferase assay. The role of SLIT2 was also confirmed through up-regulation in MG63 cell, and the osteogenic differentiation-associated gene and protein were tested.ResultsAccording to bioinformatic analysis, a series of differential expressed genes between persons with high or low BMD were compared. After internalization of hBMSC-sEV in MG63 cells, we observed that the ability of osteogenic differentiation was significantly enhanced. Similarly, after up-regulation of miR-382 in MG63 cells, osteogenic differentiation was also promoted. According to the dual-luciferase assay, the targeting function of miR-382 in SLIT2 was demonstrated. Moreover, the benefits of hBMSC-sEV in osteogenesis were abrogated through up-regulation of SLIT2.ConclusionOur study provided evidence that miR-382-contained hBMSC-sEV held great promise in osteogenic differentiation in MG63 cells after internalization by targeting SLIT2, which can be served as molecular targets to develop effective therapy.

Surface characteristics and in vitro biocompatibility of surface-modified titanium foils as a regenerative barrier membrane for guided bone regeneration.

This study evaluated surface characteristics and biocompatibility of surface-modified thin titanium (Ti) foils as a regenerative barrier membrane for future application in guided bone regeneration (GBR) surgery to augment atrophic alveolar bone. Anodic oxidation and post-heat treatment were performed to prepare various Ti foil samples. Then, the in vitro soft and hard tissue compatibility of the samples was evaluated by examining the cell responses using primary human gingival fibroblasts (HGFs) and MG63 human osteoblast-like cells. Investigated Ti foil samples showed marked differences in physicochemical surface properties. Additional 400°C heat treatment applied to the anodized Ti surface led to formation of an anatase titanium dioxide structure and well-organized nanoscale protrusions, and significantly increased surface wettability. Anodization and heat treatment enhanced the growth of HGFs and MG63 cells in Ti foil samples. Additional heat treatment for 10 and 30min further significantly improved the response of HGFs including spreading and proliferation, and upregulated the mRNA expression of cell adhesion- and maturation-related genes as well as the osteoblast differentiation of MG63 cells. Ti foil sample with thin oxide coating obtained by a 30min heat treatment exhibited poor clinical plasticity as a regenerative barrier membrane, which showed complete coating failure in the bending test. Our results indicate that anatase Ti oxide coating of a specific film thickness with nanoscale surface protrusion morphology and hydrophilic characteristics obtained by anodization and post-heat treatment would be an effective approach as a biocompatible Ti regenerative membrane for inducing better regeneration of both gingival tissue and bone.

Generation of graphene oxide and nano-bioglass based scaffold for bone tissue regeneration

Graphene oxide (GO) offers a distinct opportunity in the field of biomedical engineering owing to its exceptionally high mechanical strength, excellent electrical conductivity, high optical transparency, and favorable biocompatibility. In this article, nanocomposite biocompatible GO-based scaffolds (chitosan/gelatin/nanobioglass/GO) Ch-G-NBG-GO were successfully fabricated through freeze drying technique (−40 °C) and evaluated for various physico-chemical and biological properties. The prepared Ch-G-NBG-GO composites have been investigated for their structural, physiochemical, and surface morphology via x-ray diffraction (XRD), high resolution scanning electron microscope, Fourier transform infrared spectroscopy, thermogravimetric analysis (TGA), energy-dispersive x-ray Spectroscopy and, differential scanning colorimetry (DSC) respectively. The morphological analysis showed the porous interconnected network of scaffold formed. Average pore size for the Ch-G-NBG-GO scaffolds were in between 90 and 120 μm, which was very close to the control scaffolds. XRD data revealed the successful incorporation of NBG and GO and distribution across the scaffolds. Porosity of the fabricated scaffolds were in the range between 75.3% and 77.3% which was very close to the control scaffold with 79% porosity. The studies also reveal that after GO incorporation, the weight loss reduced (0.11 ± 0.02–0.095 ± 0.03), scaffolds were firmly stable at room temperature even after a long duration of 28 d. The crystallinity added to the scaffolds due to addition of GO nanoparticles improved the mechanical strength of these scaffolds. The compressive modulus changed from (5.7 to 8.51) MPa after GO addition. Swelling ratio changed drastically especially in case of Ch-NBG-90%GO (4.9 ± 0.04–4 ± 0.01). DSC and TGA data revealed the thermal stability of GO incorporated scaffolds due to the proper interaction between GO/NBG with chitosan-gelatin blend. The scaffold’s potential for bone tissue engineering was evaluated by testing its cytocompatibility for MG-63 cell line. It revealed suitable cell attachment and proliferation of cells compared to the Ch-G-NBG scaffold. MTT assay showed that Ch-G-NBG-GO scaffold below 90% GO concentration possess best biocompatibility. But in case of Ch-G-NBG-90%GO scaffold, the cell proliferation was reduced when compared to control scaffolds. Alkaline phosphatase activity suggested improved osteogenic differentiation of MG-63 cells over GO based scaffolds and this was due to the osteogenic potential of NBG and GO present in the scaffolds. Based on these results, the nano-biocomposite scaffold appears to have the potential for utilization in bone tissue restoration, replacement and regeneration.

Testosterone Enanthate: An In Vitro Study of the Effects Triggered in MG-63 Cells.

The aim of this study was to investigate the effects of the androgenic hormone testosterone enanthate (TE) on human MG-63 cells. MG-63 were cultured for 24 h in the presence of TE at increasing concentrations to assess its lethal dose. Therefore, the suitable concentration for a prolonged use of TE in vitro was assessed by viability assay over 9 days. Finally, MG-63 were exposed to TE for 14 days and assayed for differentiation by qPCR and Alizarin Red S staining. TE in the amount of 100 µM resulted as the maximum dose tolerated by MG-63 cells after 24 h. However, a prolonged exposure in culture TE in the amount of 100 µM showed a cytostatic effect on cell proliferation. On the contrary, TE 10 µM was tolerated by the cells and did not boost cell proliferation, but did enhance new bone formation, as revealed by COL1A1, ALPL, BGLAP, and IBSP gene expression after 3, 7, and 14 days, and calcium deposition by Alizarin Red S staining after 14 days. Based on the current study, 10 µM is the critical dose of TE that should be used in vitro to support bone differentiation of MG-63 cells.

Multifunctional polyethylene imine hybrids decorated by silica bioactive glass with enhanced mechanical properties, antibacterial, and osteogenesis for bone repair

Inorganic/organic hybrids and bioactive glasses demonstrate promising potential as bone substitute biomaterials. A sol-gel hybrid consisting of silica bioactive glass and biodegradable polymer can combine the high bioactivity of a glass with the toughness of a polymer. In this study, multifunctional hybrids with a combination of organic-inorganic hybrid structure class II consisting of polyethyleneimine (PEI) generation 4 (G4) and bioactive glass with enhanced mechanical properties, mineralization, antibacterial, and osteogenesis activities were synthesized by the sol-gel method. Glycidoxypropyl) trimethoxysilane (GPTMS) with different concentrations was used as a covalent bonding agent between PEI polymer and bioactive glass. The effect of GPTMS content was assessed in the presence and absence of calcium in the hybrid structures in terms of morphology, wettability, mechanical properties, antibacterial activity, cell viability, and in vitro osteogenic differentiation properties. By increasing the amount of GPTMS, the compressive strength increased from 1.95 MPa to 2.34 MPa, which was comparable to human trabecular bone. All the hybrids presented antibacterial activity against Staphylococcus aureus, forming an inhibition zone of 13–16 mm. An increase in cell viability of 82.22% in PSCaG90 was obtained after 1 day of MG-63 cell culture. Alkaline phosphatase expression and mineralization of MG-63 cells increased in the PSCaG90 hybrid in the absence of an osteogenic medium compared to PSG60 and PSG90. The PSCaG90 hybrid indicated considerable in vitro osteogenic capacity in the absence of a differentiation medium, expressing high levels of bone-specific proteins including collagen I (COL1A1), Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN), compared to calcium-free hybrids. Overall, our results suggest that the presence of calcium in the PSCaG90 leads to a significant increase in osteogenic differentiation of MG-63 cells even in the absence of differentiation medium, which suggests these hybrid structures with multifunctional properties as promising candidates for bone repair.

Significant transcriptomic changes are associated with the inhibitory effects of 5-aza-2-deoxycytidine during adipogenic differentiation of MG-63 cells.

Our previous study revealed that the treatment of 5-aza-2-deoxycytidine (5-aza) inhibited while treatment of suberoylanilide hydroxamic acid (SAHA) enhanced the adipogenic differentiation of MG-63 cells. In this study, we examined the transcriptomic profiles of the derived adipocyte-like cells from MG-63 cells in the presence of 5-aza (Treatment 1) and SAHA (Treatment 2). Genome wide expression analysis showed high within sample variability for the adipocytes derived with 5-aza versus vehicle. Additionally, the expression profile of 5-aza derived cells was separated from the other sample groups. Differential analysis on the pairwise comparison of 5-aza versus control and SAHA versus 5-aza identified 1290 and 1086 differentially expressed (DE) genes, respectively. Furthermore, some overlap was observed between the up and down-regulated DE genes of 5-aza versus control and SAHA versus control (jaccard score 0.3) as well as between the differentially regulated genes of 5-aza versus control and 5-aza versus SAHA (jaccard score 0.29). A total of 73 transcription factors (TFs) were differentially expressed across all the pair wise comparisons with some overlap between the under and over expressed TFs of 5-aza versus control and 5-aza versus SAHA (jaccard score 0.29). Unsupervised clustering of TFs showed that the samples within the group are consistent in expression and the samples cluster in accordance with the group.Several GO terms related to enhanced adipogenesis such as neutral lipid biosynthetic process, lipid metabolic processes, cellular amide metabolic processes and cellular carbohydrate metabolic processes were enriched in the down regulated genes of 5-aza derived adipocytes versus control, indicating 5-aza inhibit the adipogenic differentiation of MG-63 cells. GSEA analysis on selected gene sets of MAPK and PI3K signaling pathway in MSigDB identified the pathways were up-regulated in 5-aza versus control. This study revealed that inhibition of MG-63 adipogenesis due to 5-aza treatment is associated with large transcriptomics changes and further research is needed to unravel the roles of these genes in the adipogenesis.

Osteogenic effects of exosomes derived from human chorion membrane extracts

ObjectiveHuman chorion membrane extracts (CME) are known to exhibit osteogenic effects when used for treating human osteoblast-like cells (MG63 cells), but the active compound in CME remains unknown. The aim of this study was to identify the presence of exosomes in CME and to determine the osteogenic effect of CME exosomes on MG63 cells.MethodsExosomes were isolated from human placenta CME using the ExoQuick-TC solution and were characterized. The activity and deposition of alkaline phosphatase (ALP) on MG63 cells cultured with or without exosomes in osteogenic induction medium (OIM) were determined. Human amniotic membrane extracts (AME) were used as controls as they had not affected the osteogenic differentiation of MG63 cells in our previous study.ResultsTransmission electron microscopy (TEM) revealed that exosomes isolated from CME and AME (CME-Exo and AME-Exo, respectively) had a cup-shaped structure. NanoSight™ particle tracking analysis (NTA) confirmed that the size of these exosomes was 100–150 nm. In vitro osteogenic experiments demonstrated that the exosomes from CME, but not those from AME, presented increased alkaline phosphatase (ALP) activity and resulted in the mineralization of MG63 cells in a dose-dependent manner.ConclusionExosomes were identified in CME and AME from the human placenta. Further, the exosomes from CME were found to be capable of promoting osteogenic differentiation, suggesting that exosomes are a key component of CME that stimulate the osteogenesis of human osteoblast-like cells. CME exosomes can be developed as promising therapeutic candidates for bone regeneration.

Microstructure, degradation properties and cytocompatibility of micro-arc oxidation coatings on the microwave sintered Ti-15Mg metal-metal composite

The micro-arc oxidation (MAO) coatings were fabricated on the surface of the microwave sintered Ti-15Mg metal-metal composite (MMC). The surface morphologies, phase composition, degradation properties and cytocompatibility of the MAO treated Ti-15Mg MMC were investigated. The XRD and XPS results show that the MAO coatings are mainly composed of anatase and rutile TiO2 crystal phases and amorphous MgO and SiO2 with some –OH groups. There are some differences between MAO pore structure in Ti region and Mg region, but both of the pore sizes increase and the number of pores decreases with increasing the applied voltages. The degradation rate of Ti-15Mg MMC is reduced by the MAO coatings, and with increasing the applied voltages, the degradation rates of the MAO treated samples decrease first, reaching the minimum value at 300 V, and then increase. After immersed in SBF solution for 250 h, the calcium-deficient apatite containing CO32− is detected on the surface of the MAO treated and untreated Ti-15Mg MMC. The results of cell viability and alkaline phosphatase activity show that the MAO treated Ti-15Mg MMCs are more conducive to the proliferation and differentiation of MG-63 cells.

Diosgenin inhibits Wnt/β-catenin pathway to regulate the proliferation and differentiation of MG-63 cells.

Dioscorea opposita Thunb has the effect of anti-osteoporosis, but whether its active ingredient diosgenin (DIO) has an anti-osteoporosis effect is unknown. The purpose of this study is to investigate the effect of DIO on the proliferation and differentiation of MG-63 cells. MG-63 cells were treated with different concentrations of DIO (0.001, 0.01, 0.1 and 1μM) or 20mM Wnt/β-catenin signaling agonist-LiCl, and then their cell cycle and viability were analyzed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), respectively. To investigate osteoblast differentiation, alizarin red staining and ultraviolet spectrophotometer were used to determine the number of calcified nodules and the activity of alkaline phosphatase (ALP), respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were used to detect the expressions of proliferation-related, osteogenic-related and Wnt/β-catenin signal pathway-related factors. After the cells were treated with low-concentration (0.001 or 0.01μM) DIO, cell viability was significantly increased and the proportion of cells in S phase was increased. In addition, low-concentration DIO could significantly increase the expression of Ki67, proliferating cell nuclear antigen (PCNA), osteopontin (OPN), and osteocalcin (BGP), promote osteoblast differentiation, and suppress the expression of β-catenin, Runx2 and cyclinD1. However, high concentrations of DIO showed the opposite effect. Low-concentration DIO obviously reversed the effect of LiCl on decreasing the number of calcified nodules and inhibiting the expression of OPN and BGP in cells. Low-concentration DIO might promote the proliferation and differentiation of MG-63 cell by inhibiting the Wnt/β-catenin signal pathway.

Simvastatin-Loaded Nanomicelles Enhance the Osteogenic Effect of Simvastatin

Objectives. The present study intended to further verify that simvastatin-loaded nanomicelles (SVNs) enhanced the role of simvastatin (SV) in promoting osteoblast differentiation in vitro and to evaluate the effect of SVNs on bone defect repair in vivo. Methods. SVNs were synthesized by dialysis. MG63 cells were subjected to intervention with 0.25 μmol/l of SVNs and SV. A 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit and flow cytometry were used to determine cell proliferation activity, cell cycle distribution, and apoptosis. The osteoblastic differentiation of MG 63 cells was evaluated by measuring alkaline phosphatase (ALP) activity, ALP staining, and the expression levels of the osterix (Osx) and osteocalcin (OC) proteins. In addition, 0.5 mg of SVNs or SV was applied to the skull defect area of rabbits. Micro-CT, hematoxylin and eosin (HE) staining, and Masson’s trichrome staining were used for qualitative and quantitative evaluation of new bone in three dimensions and two dimensions. Results. The SVNs had a mean diameter of 38.97 nm. The encapsulation and drug-loading efficiencies were 54.57 ± 3.15 % and 10.91 ± 0.63 % , respectively. In vitro, SVNs and SV can inhibit the proliferation activity and promote osteogenic differentiation of MG63 cells by arresting MG63 cells at the G0/G1 phase without increasing the apoptosis rate. In vivo quantitative results showed that the bone mineral density (BMD), bone volume (BV)/total volume (TV) ratio, and trabecular number (Tb.N) in the gelatin sponge with SVNs (SVNs-GS) group and gelatin sponge with SV (SV-GS) group were 362.1%, 292.0%; 181.3%, 158.0%; and 215.2%, 181.8% of those in the blank control (BC) group, respectively. Histological results identified the new bone tissue in each group as irregular fibrous bone, and the arrangement of trabecular bone was disordered. There were significantly more osteoblasts and new capillaries around the trabecular bone in the SVNs-GS group and SV-GS group than in both the BC and drug-free nanomicelle (DFNs) groups. Both in vitro and in vivo, SVNs exhibited greater osteogenic efficacy than SV. Conclusion. SVNs significantly improved the osteogenic efficacy of SV.

Regulation of human β-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human β-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

Hydroxyapatite Nanoparticles Fortified Xanthan Gum-Chitosan Based Polyelectrolyte Complex Scaffolds for Supporting the Osteo-Friendly Environment.

Nanoparticle-reinforced polymer-based scaffolding matrices as artificial bone-implant materials are potential suitors for bone regenerative medicine as they simulate the native bone. In the present work, a series of bioinspired, osteoconductive tricomposite scaffolds made up of nano-hydroxyapatite (NHA) embedded xanthan gum-chitosan (XAN-CHI) polyelectrolyte complex (PEC) are explored for their bone-regeneration potential. The Fourier transform infrared spectroscopy studies confirmed complex formation between XAN and CHI and showed strong interactions between the NHA and PEC matrix. The X-ray diffraction studies indicated regulation of the nanocomposite (NC) scaffold crystallinity by the physical cues of the PEC matrix. Further results exhibited that the XAN-CHI/NHA5 scaffold, with a 50/50 (polymer/NHA) ratio, has optimized porous structure, appropriate compressive properties, and sufficient swelling ability with slower degradation rates, which are far better than those of CHI/NHA and other XAN-CHI/NHA NC scaffolds. The simulated body fluid studies showed XAN-CHI/NHA5 generated apatite-like surface structures of a Ca/P ratio ∼1.66. Also, the in vitro cell-material interaction studies with MG-63 cells revealed that relative to the CHI/NHA NC scaffold, the cellular viability, attachment, and proliferation were better on XAN-CHI/NHA scaffold surfaces, with XAN-CHI/NHA5 specimens exhibiting an effective increment in cell spreading capacity compared to XAN-CHI/NHA4 and XAN-CHI/NHA6 specimens. The presence of an osteo-friendly environment is also indicated by enhanced alkaline phosphatase expression and protein adsorption ability. The higher expression of extracellular matrix proteins, such as osteocalcin and osteopontin, finally validated the induction of differentiation of MG-63 cells by tricomposite scaffolds. In summary, this study demonstrates that the formation of PEC between XAN and CHI and incorporation of NHA in XAN-CHI PEC developed tricomposite scaffolds with robust potential for use in bone regeneration applications.