Human Osteoblast-like MG-63 Cells Research Articles

Fabrication of Dielectrically Sensitive Carbon Nanotube/Polyethylene Nanohybrid Shish-Kebab Structure for Potential Tissue Engineering Applications

Carbon nanotube (CNT)/polymer nanohybrid shish-kebab (NHSK) structure is a unique approach to functionalize CNTs and mimic the hierarchical architecture of native collagen fibrils in the extracellular matrix (ECM), owing to their favorable geometric similarity. This study thus aimed at combining the unique NHSK architecture of CNTs with their favorable dielectric properties to fabricate a collagen-like, dielectrically sensitive and biocompatible material. The NHSK structure was fabricated with CNTs and polyethylene (PE) by means of a solution crystallization technique. Configuration of PE in terms of molecular weight and structure was found to be one of the important factors that determined the morphology of the NHSK. Moreover, the material was exposed to dielectrophoresis (DEP) to guide the deposition of CNT/PE NHSK into the directional pattern. The PE-decorated CNTs exhibited enhanced dielectrophoretic sensitivity and controllable structural alignment, while the neat CNTs did not. Human osteoblast-like MG-63 cells were cultured on the scaffolds to examine their biocompatibility and results indicated that the nanotopography of the kebab crystals on the CNTs facilitated cell adhesion and proliferation while the DEP-patterned NHSK structure further boosted cell attachment and viability, thus suggesting that the CNT/polymer nanohybrid shish kebab structure has potential applications in tissue engineering.

In vitro degradation and cytocompatibility of magnesium alloy coated with Hf/PLLA duplex coating

A novel and bifunctional hafnium/poly (l, l-lactide) duplex coating (Hf/PLLA) was fabricated on magnesium alloy via magnetron sputtering coupled with dip-coating method. Microstructure, composition, in vitro degradation and cytocompatibility of coating were examined systematically. The in vitro electrochemical tests revealed that the Hf/PLLA duplex coating has significantly suppressed the degradation of magnesium alloy with the low corrosion current density of 0.02 μA/cm2 and high corrosion resistance of 324.5 KΩ cm2. Simultaneously, the cytocompatibility of magnesium alloy was further improved, as observed by the superior cell adhesion and proliferation of human osteoblast-like MG63 cells on the duplex coating.

Hydroxyapatite Bioceramics Reinforced with Silica-Coated Graphene

Challenges facing graphene-reinforced composites include insufficient interfacial strengths, filler agglomeration, and their potential cytotoxicity. To tackle these challenges, silica-coated graphene (S-G) is used to toughen a representative bioceramic, hydroxyapatite (HA), and HA-based composites were prepared by park plasma sintering. By adding 2 wt.% silica-coated graphene oxide, a precursor to S-G, the fracture toughness of HA could be increased by 84%. Compared with graphene/HA pellets, the S-G/HA samples showed superior mechanical properties. In addition, the incorporation of S-G could promote the proliferation and differentiation of human osteoblast-like cells MG63, and showed no adverse effect on their viability. Therefore, the multifunctional bioceramic-based materials presented here possess great potential in load-bearing orthopedic implants.

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.

Insulin and osteocalcin: further evidence for a mutual cross-talk

PurposeIn the last few years, bone has been recognized as an endocrine organ that modulates glucose metabolism by secretion of osteocalcin, an osteoblast-specific hormone, that influences fat deposition and blood sugar levels. To date, however, very few in vitro models have been developed to investigate, at the molecular levels, the relationship between glucose, insulin and osteocalcin. This study aims at covering this gap.MethodsWe studied osteogenic differentiation, osteocalcin gene expression, and osteblast-mediated insulin secretion, using cultured MG-63 human osteoblast-like cells that underwent glucotoxicity and insulin resistance. In addition, we investigated whether a correlation existed between hyperglycemia and/or insulin resistance and total osteocalcin serum concentrations in patients.ResultsWhile insulin and low glucose increased osteocalcin gene expression, disruption of insulin signaling in MG-63 osteoblasts and high glucose concentration in cell culture medium decreased osteocalcin gene transcription and reduced osteogenic differentiation. Concomitantly, insulin secretion was significantly impaired in rat INS-1 β-cells treated with conditioned medium from insulin resistant MG-63 cells or cells exposed to high glucose concentrations. Also, chronic hyperglycemia, but not insulin resistance, inversely correlated with circulating osteocalcin levels in patients.ConclusionOur results further support the existence of an endocrine axis between bone, where osteocalcin is produced, and pancreatic β-cells, and add new insights into the molecular details of this relationship. These findings may contribute to the understanding of osteocalcin regulation and its role in metabolism.

Effect of polygonimitin C on bone formation and resorption in human osteoblast-like MG63 cells

Purpose: To investigate the effect of polygonimitin C (PC) on bone formation and resorption in human osteoblast-like MG63 cells.Methods: MG63 cells were treated with PC at doses of 0, 20, 40 or 80 μg/mL for 48 h, with an untreated group as control. The effect of PC on alkaline phosphatase (ALP) activity in MG63 cells was investigated by p-nitrophenyl phosphate disodium hexahydrate assay. Western blot assay was used to evaluate the effect of PC on the expressions of osterix (OSX), bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), fibronectin (FN), type I collagen (COL I), osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) proteins in MG63 cells.Results: ALP relative activity in MG63 cells treated with PC at 20, 40 or 80 μg/mL (123.58, 137.74 or 159.62 %, respectively) was significantly (p < 0.05 or 0.01) higher than that in control group (99.37 %). Expressions of OSX, BMP-2, RUNX-2, OC, FN, COL I and OPG proteins in MG63 cells treated with PC at 20, 40 or 80 μg/mL were significantly (p < 0.01) higher than those in control group. However, there were no statistically significant differences in RANKL protein expression between PC-treated MG63 cells and control group.Conclusion: These results show that PC exerts protective effects against osteoporosis by promoting bone formation and inhibiting bone resorption. Thus, PC may be useful in the development of new antiosteoporosis drugs.Keywords: Polygonimitin C, MG63 cells, Bone formation, Bone resorption, Osteoporosis

Daidzein stimulates osteogenesis facilitating proliferation, differentiation, and antiapoptosis in human osteoblast-like MG-63 cells via estrogen receptor–dependent MEK/ERK and PI3K/Akt activation

Daidzein, a natural soy isoflavone, has a structure similar to estradiol and exhibiting bone-sparing effects against osteoporosis. However, the molecular mechanisms of osteogenesis remain unclear. We hypothesized that daidzein stimulates osteogenesis through estrogen receptor (ER)–dependent signal pathways. To test this hypothesis, we investigated the effects of daidzein compared with 17β-estradiol on proliferation, differentiation, and cisplatin-induced apoptosis in human osteoblast-like MG-63 cells containing 2 ER isoforms. The results showed that daidzein stimulated cell proliferation by altering cell cycle distribution, promoted cell differentiation by increasing the alkaline phosphatase activity and collagen content, and reduced cell apoptosis associated by up-regulating the expression of Bcl-xL. The above actions of daidzein were prevented by cotreatment with the ER antagonist ICI 182780. Using small interfering RNA technology, we further demonstrated that the effects of daidzein on alkaline phosphatase activity, collagen content, and cell apoptosis are mediated by both ERα and ERβ, whereas the effects on cell proliferation are primarily mediated by ERα. However, the effects of 17β-estradiol on osteoblastic proliferation and survival are mediated by both ER isotypes, and the effects on osteoblastic differentiation are primarily mediated by ERα. The use of specific inhibitors indicated that activation of the mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) and phosphoinositide 3-kinase/protein kinase B or PKB (PI3K/Akt) signaling pathway at least partially accounts for these effects of daidzein. Taken together, the results indicate that daidzein stimulates osteogenesis through facilitating proliferation, differentiation, and antiapoptosis in human osteoblast-like MG-63 cells via activation of MEK/ERK and PI3K/Akt in an ER-dependent manner.

Continuous Fabrication and Assembly of Spatial Cell-Laden Fibers for a Tissue-Like Construct via a Photolithographic-Based Microfluidic Chip

Engineering three-dimensional (3D) scaffolds with in vivo like architecture and function has shown great potential for tissue regeneration. Here we developed a facile microfluidic-based strategy for the continuous fabrication of cell-laden microfibers with hierarchically organized architecture. We show that photolithographically fabricated microfluidic devices offer a simple and reliable way to create anatomically inspired complex structures. Furthermore, the use of photo-cross-linkable methacrylated alginate allows modulation of both the mechanical properties and biological activity of the hydrogels for targeted applications. Via this approach, multilayered hollow microfibers were continuously fabricated, which can be easily assembled in situ, using 3D printing, into a larger, tissue-like construct. Importantly, this biomimetic approach promoted the development of phenotypical functions of the target tissue. As a model to engineer a complex tissue construct, osteon-like fiber was biomimetically engineered, and enhanced vasculogenic and osteogenic expression were observed in the encapsulated human umbilical cord vein endothelial cells and osteoblast-like MG63 cells respectively within the osteon fibers. The capability of this approach to create functional building blocks will be advantageous for bottom-up regeneration of complex, large tissue defects and, more broadly, will benefit a variety of applications in tissue engineering and biomedical research.

Estrogen stimulates osteoprotegerin expression via the suppression of miR-145 expression in MG-63 cells.

Osteoprotegerin (OPG) is implicated in the pathogenesis of postmenopausal osteoporosis, and other metabolic bone diseases caused by estrogen deficiency. Previous studies have demonstrated that estrogen may stimulate OPG expression in osteoblast cells at the transcriptional level; however, whether estrogen can regulate OPG expression at a post-transcriptional level remains elusive. The present study aimed to investigate the role of microRNA (miRNA) in estrogen-mediated OPG production in human osteoblast-like MG-63 cells. The results from ELISA, western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) confirmed that estrogen may upregulate OPG expression. Mechanistic studies indicated that estrogen increased the activity of a luciferase reporter harboring the OPG 3′-untranslated region (3′-UTR). Bioinformatics analysis demonstrated that there is a potential targeting site in the OPG 3′-UTR for miRNA (miR)-145, which is associated with osteoblast differentiation. The results of an RT-qPCR suggested that estrogen suppressed miR-145 expression. In addition, dual-luciferase assay, RT-qPCR and western blot analysis indicated that miR-145 directly targets and negatively regulates OPG expression. Furthermore, transfection of cells with miR-145 mimics was able to partially inhibit the induction of OPG expression by estrogen, thus confirming the role of miR-145 in estrogen-mediated OPG induction. Taken together, the results of the present study demonstrated that estrogen may post-transcriptionally regulate OPG expression through suppression of miR-145 expression.

Ethnomedicine based evaluation of osteoprotective properties of Tinospora cordifolia on in vitro and in vivo model systems.

Indian ethnomedicine acclaims the use of Tinospora cordifolia (TC) in the treatment of bone fractures and vat rakta (gout). The objective of the study is to investigate the effects of alcoholic extract of Tinospora cordifolia on bone remodeling (involving osteoblastic and osteoclastic actions) in vitro and protect against ovariectomy-induced bone loss in vivo. Human osteoblast-like cells MG-63 and primary osteoblast cells isolated from rat femur were used as osteoblast models and RAW macrophage cell line 264.7 induced to take up osteoclastic lineage using RANK ligand were used as osteoclast models in the current study. Sirius red staining, quantification of osteocalcin, cytopathological analysis by Hematoxylin/eosin staining and semiquantitative reverse transcription PCR (RT-PCR) was carried out to ascertain the effects of T. cordifolia extract on osteoblast cells. MTT assay was perfomed to understand the influence of T. cordifolia extract on osteoclast cells. Adult female Sprague-Dawley rats were used as in vivo models to study the effect of T. cordifolia on ovariectomy induced bone loss. Radiological (DEXA analysis), Biochemical (markers of bone formation and resorption), histopathological (Hematoxylin/eosin staining) and histomorphometric analysis of the bone was carried out. Treatment with T. cordifolia extract resulted in enhanced collagen deposition, increased levels of osteocalcin, increased expression of osteogenic genes all indicative of favourable osteoblastogenesis. Treatment with T. cordifolia extract did not exert any significant influence on the proliferation of osteoclasts. Pretreatment with T. cordifolia extract at a dose of 50mg/kg body wt/day orally for 21days followed by treatment for 12 weeks post ovariectomy was able to prevent ovariectomy-induced bone loss in vivo. Results of the study support the use of T. cordifolia in Indian ethnomedicine for the treatment of bone diseases and fractures.

Effects of low‐frequency ultrasound treatment of titanium surface roughness on osteoblast phenotype and maturation

Low-frequency ultrasound is widely used in the treatment of chronically infected wounds. To investigate its feasibility as a method for insitu restoration of metal implant surfaces in cases of peri-implantitis, we evaluated how low-frequency ultrasound affected surface properties of and response of human osteoblast-like MG63 cells to titanium (Ti). Three Ti surfaces [hydrophobic/smooth (pretreatment, PT); hydrophobic/rough (sandblasted/acid-etched, SLA); and hydrophilic/rough (SLA processed and stored hydrophilicity, mSLA)] were subjected to 25kHz ultrasound for 10min/cm2 . Substrate roughness, chemical composition, and wettability were analyzed before and after ultrasound application. Osteoblastic maturation of cells on sonicated disks was compared to cells on untreated disks. Ultrasound treatment altered the topography of all surfaces. Contact angles were reduced, and chemical compositions were altered by ultrasound on PT and SLA surfaces. Cell response to sonicated PT was comparable to untreated PT. Alkaline phosphatase was increased on sonicated SLA compared to untreated SLA, whereas DNA, osteocalcin, BMP2, osteoprotegerin, and VEGF-A were unchanged. Cells produced less osteocalcin and BMP2 on sonicated mSLA than on untreated mSLA, but no other parameters were affected. These results show that low-frequency ultrasound altered Ti surface properties. Osteoblasts were sensitive to the changes induced by ultrasound treatment. The data suggest that the effect is to delay differentiation, but it is unclear whether this delay will prevent osseointegration. These results suggest that low-frequency ultrasound may be useful for treating implant surfaces insitu leading to successful re-osseointegration of implants affected by peri-implantitis.

Accelerated cell-surface interlocking on plasma polymer-modified porous ceramics

Excellent osseointegration of permanent implants is crucial for the long lasting success of the implantation. To improve the osseointegrative potential, bio-inert titanium alloy surfaces (Ti6Al4V) are modified by plasma chemical oxidation (PCO®) of the titanium-oxide layer to a non-stoichiometric, amorphous calcium phosphate layer. The native titanium-oxide film measuring only a few nanometers is converted by PCO® to a thick porous calcium phosphate layer of about 10μm. In a second step the PCO surface is combined with a cell adhesive plasma-polymerized allylamine (PPAAm) nano film (5 and 50nm). Independent of the PPAAm coating homogeneity, the human osteoblast-like MG-63 cells show a remarkable increase in cell size and well-developed filopodia. Analyses of the actin cytoskeleton reveal that the cells mold to the pore shape of the PPAAm-covered PCO, thereby establishing a strong attachment to the surface. Interestingly, we could demonstrate that even though our untreated PCO shows excellent hydrophilicity, this alone is not sufficient to facilitate fast cell spreading, but the positive surface charges mediated by PPAAm.This multilayer composite material guarantees enhanced interlocking of the cells with the porous surface.

Effects of a Hot-Water Extract of Allium hookeri Roots on Bone Formation in Human Osteoblast-Like MG-63 Cells In Vitro and in Rats In Vivo.

Allium hookeri is a wild herb found mainly in the Himalayas, growing at altitudes of 1400-4200 m. A.hookeri is widely consumed as a vegetable and herbal medicine in Asia, but its effects on bone health have not been reported previously. This study investigated the effects of a hot-water extract of A.hookeri roots on bone formation. The hot-water extract significantly increased the proliferation of in vitro human osteoblast-like MG-63 cells and the stimulatory effects on osteoblast differentiation were noticeably greater for the hot-water extract than for daidzein (a positive control), as reflected by alkaline phosphatase activity, collagen content, and mineral deposition. Expression of the bone-remodeling marker osteocalcin production and bone microstructural parameters were significantly improved in Sprague-Dawley rats in vivo after oral treatment with the hot-water extract compared with their control (saline-administered) counterparts. The chemical compounds of the hot-water extract were characterized by liquid chromatography-mass spectrometry, and alliin, sinapic acid, and ferulic acid, which exert beneficial effects on bone health, were identified. These findings indicate that A.hookeri can be used as a natural resource for increasing bone formation. This is the first report of the anabolic effects of A.hookeri extracts on bone formation in vitro and in vivo.

Insulin released from titanium discs with insulin coatings-Kinetics and biological activity

Local administration of insulin from a titanium surface has been demonstrated to increase bone formation in non-diabetic rats. The authors hypothesized that insulin was released from the titanium surface and with preserved biological activity after the release. Thus, in the present in vitro study, human recombinant insulin was immobilized onto titanium discs, and the insulin release kinetics was evaluated using Electro-chemiluminescence immunoassay. Neutral Red uptake assay and mineralization assay were used to evaluate the biological effects of the released insulin on human osteoblast-like MG-63 cells. The results confirmed that insulin was released from titanium surfaces during a six-week period. Etching the disc prior to insulin coating, thickening of the insulin coating and incubation of the discs in serum-enriched cell culture medium increased the release. However, longer storage time decreased the release of insulin. Furthermore, the released insulin had retained its biological activity, as demonstrated by the significant increase in cell number and a stimulated mineralization process, upon exposure to released insulin. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1847-1854, 2017.

Cysteine Prevents Menopausal Syndromes in Ovariectomized Mouse.

Cysteine (Cys) is well known to be involved in oxidation-reduction reactions, serving as a source of sulfides in the body. Amino acids are known to improve menopausal symptoms and significantly reduce morbidity. This study aims to find an unrevealed effect of Cys with estrogenic and osteogenic actions. Ovariectomized (OVX) mice were treated with Cys daily for 8 weeks. Estrogen-related and osteoporosis-related factors were analyzed in the vagina, serum, and tibia. Cys was treated in estrogen receptor (ER)-positive human osteoblast-like MG-63 cells and ER-positive human breast cancer Michigan Cancer Foundation-7 (MCF-7) cells. Cysteine administration ameliorated overweightness of the body and vaginal atrophy in the OVX mice. Cysteine increased the levels of alkaline phosphatase (ALP) and 17β-estradiol in the serum of the OVX mice and improved the bone mineral density in the OVX mice. In MG-63 cells, Cys increased the proliferation, ERβ messenger RNA (mRNA) expression, and estrogen response element (ERE) activity. Cysteine increased the ALP activity and the phosphorylation of extracellular signal-regulated kinase. In MCF-7 cells, Cys also increased the proliferation, ERβ mRNA expression, and ERE activity. Taken together, these results demonstrated that Cys has estrogenic and osteogenic activities in OVX mice, MG-63 cells, and MCF-7 cells. The novel insights gained here strongly imply the potential use of Cys as a new agent for postmenopausal women.

A preliminary study for novel use of two Mg alloys (WE43 and Mg3Gd).

In this study, two types of magnesium alloys (WE43 and Mg3Gd) were compared with Heal-All membrane (a biodegradable membrane used in guided bone regeneration) in vitro to determine whether the alloys could be used as biodegradable membranes. Degradation behavior was assessed using immersion testing with simulated body fluid (SBF). Microstructural characteristics before and after immersion were evaluated through scanning electron microscopy, and degradation products were analyzed with energy dispersive spectrometry (EDS). To evaluate the biocompatibility of the three types of materials, we performed cytotoxicity, adhesion, and mineralization tests using human osteoblast-like MG63 cells. Immersion testing results showed no significant difference in degradation rate between WE43 and Mg3Gd alloys. However, both Mg alloys corroded faster than the Heal-All membrane, with pitting corrosion as the main corrosion mode for the alloys. Degradation products mainly included P- and Ca-containing apatites on the surface of WE43 and Mg3Gd, whereas these apatites were rarely detected on the surface of the Heal-All membrane. All three type of materials exhibited good biocompatibility. In the mineralization experiment, the alkaline phosphatase (ALP) activity of 10 % Mg3Gd extract was significantly higher than the extracts of the two other materials and the negative control. This study highlighted the potential of these Mg-REE alloys for uses in bone regeneration and further studies and refinements are obviously required.

Response of Human Osteoblast to n-HA/PEEK--Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite.

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Epigallocatechin-3-gallate Promotes Osteoblastic Activity in Human Osteoblast-like Cells

Purpose: To investigate the effect of epigallocatechin-3-gallate (EGCG) on bone metabolism and osteoblastic activity. Methods: MG-63 human osteoblast-like cells were treated with varied concentrations of EGCG. Alkaline phosphatase (ALP) activity and matrix mineralization assays were carried out on the treated and untreated MG-63 human osteoblast-like cells. Beta-catenin mRNA level was determined by quantitative real-time polymerase chain reaction (q-PCR). Results: The results showed that EGCG treatment significantly increased ALP and mineralization activities at concentrations of 15 and 30 μM, in a dose-dependent manner. Furthermore, EGCG treatment significantly increased beta-catenin mRNA expression by 70.7 ± 11.0 and 126.7 ± 35.1 %, respectively, at EGCG concentration of 15 and 30 μM. In addition, the stimulating effect of EGCG treatment on ALP activity was abolished by co-treatment with ICI 182,780, an antagonist of estrogen receptor (ER). Again, the increase in beta-catenin MRNA, when treated with EGCG, was inhibited by co-treatment with ICI 182,780. Conclusion: EGCG promotes osteoblastic activity in human osteoblast-like cells, by Wnt signaling through estrogen receptor (ER) pathway. Keywords: Epigallocatechin-3-gallate, Osteoporosis, Osteoclast, Proliferation, Estrogen receptor

TNF-α is upregulated in T2DM patients with fracture and promotes the apoptosis of osteoblast cells in vitro in the presence of high glucose

Fracture healing is regulated by proinflammatory mediators such as tumor necrosis factor-α (TNF-α), which poses influence on the balance between bone formation and remodeling. And the diabetes is thought to contribute to the delayed diabetic fracture healing. In the present study, we examined the promotion to proinflammatory cytokines and chemokines in type 2 diabetes mellitus (T2DM) patients with bone fractures, and then evaluated the promotion to TNF-α by the high glucose treatment in human osteoblast-like MG-63 cells and the regulatory role of the promoted TNF-α on the MG-63 cell apoptosis. It was demonstrated that there were significantly-upregulated high-sensitivity C-reactive protein (hsCRP) TNF-α, IL-1β, IL-6, IFN-γ-inducible protein 10 (IP-10) and RANTES in T2DM patients with bone fracture. And the promotion to TNF-α and IL-1β was confirmed in vitro in both mRNA and protein levels in high glucose-treated MG-63 cells. And either TNF-α or high glucose reduced the viability of MG-63 cells, promoted apoptosis and upregulated apoptosis-associated markers, such as released cytochrome c, cleaved caspase 3 and lyzed PARP. Moreover, there was a synergistic effect between TNF-α and high glucose. The viability reduction and the apoptosis induction of MG-63 cells were significantly higher in the group with both TNF-α and high glucose treatments, than in the group with singular TNF-α treatment. In conclusion, our study demonstrated that proinflammatory cytokines and chemokines were promoted in T2DM patients with bone fracture or in osteoblasts by the high glucose stimulation. TNF-α and high glucose synergistically reduced the viability and induced the apoptosis in the osteoblast-like MG-63 cells in vitro. It implies the significant regulatory role of TNF-α in the delayed fracture healing in T2DM.

Effect of various concentrations of Ti in hydrocarbon plasma polymer films on the adhesion, proliferation and differentiation of human osteoblast-like MG-63 cells

Hydrocarbon polymer films (ppCH) enriched with various concentrations of titanium were deposited on microscopic glass slides by magnetron sputtering from a Ti target. The maximum concentration of Ti (about 20at.%) was achieved in a pure argon atmosphere. The concentration of Ti decreased rapidly after n-hexane vapors were introduced into the plasma discharge, and reached zero values at n-hexane flow of 0.66sccm. The decrease in Ti concentration was associated with decreasing oxygen and titanium carbide concentration in the films, decreasing wettability (the water drop contact angle increased from 20° to 91°) and decreasing root-mean-square roughness (from 3.3nm to 1.0nm). The human osteoblast-like MG-63 cells cultured on pure ppCH films and on films with 20at.% of Ti showed relatively high concentrations of ICAM-1, a marker of cell immune activation. Lower concentrations of Ti (mainly 5at.%) improved cell adhesion and osteogenic differentiation, as revealed by higher concentrations of talin, vinculin and osteocalcin. Higher Ti concentrations (15at.%) supported cell growth, as indicated by the highest final cell population densities on day 7 after seeding. Thus, enrichment of ppCH films with appropriate concentrations of Ti makes these films more suitable for potential coatings of bone implants.