Effect of graphene-oxide-modified osteon-like concentric microgrooved surface on the osteoclastic differentiation of macrophages.

OBJECTIVES: This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO). METHODS: The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05). CONCLUSIONS: Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.

Graphene Oxide-Modified Concentric Microgrooved Titanium Surfaces for the Dual Effects of Osteogenesis and Antiosteoclastogenesis.

Surface modification is an effective method to resolve the biocompatibility, mechanical, and functional issues of various titanium implant materials. Therefore, many researchers have modified the implant surface to promote the osseointegration of the implant and improve the implant survival rate. In this study, we used photolithography to construct concentric microgrooves with widths of 10 µm and depths of 10 µm, to produce an osteon-mimetic concentric microgrooved titanium surface that was further modified with graphene oxide by silanization (GO-CMS). The modified surface had great biocompatibility and promoted the proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) and RAW264.7 macrophages. The concentric microgrooves on the titanium surface guided cell migration, altered actin cytoskeleton, and caused the cells to arrange in concentric circles. The titanium surface of the GO-modified osteon-mimetic concentric microgrooves promoted the osteogenic differentiation of BMSCs and inhibited the osteoclastogenic differentiation of RAW264.7 cells. Subsequently, we constructed an indirect coculture system and found that RAW264.7 cells cultured on a GO-CMS material surface in a BMSC-conditioned medium (BCM) decreased receptor activator of nuclear factor-κB ligand (RANKL) secretion and increased OPG secretion and also that the BCM inhibited osteoclastogenic differentiation. Additionally, the secretion of OSM increased in BMSCs cultured in RAW264.7-conditioned medium (RCM) in the GO-CMS group, which in turn promoted the osteogenic differentiation of BMSCs. In conclusion, the titanium surface of GO-modified osteon-mimetic concentric microgrooves had dual effects of osteogenesis and antiosteoclastogenesis under single and coculture conditions, which is beneficial for implant osseointegration and is a promising method for the future direction of surface modifications of implants.

Biofunctionalization of Microgroove Surfaces with Antibacterial Nanocoatings.

OBJECTIVES: To investigate the physical properties of the modified microgroove (MG) and antibacterial nanocoated surfaces. In addition, the biological interactions of the modified surfaces with human gingival fibroblasts (HGFs) and the antibacterial activity of the surfaces against Porphyromonas gingivalis were studied. METHODS: The titanium nitride (TiN) and silver (Ag) coatings were deposited onto the smooth and MG surfaces using magnetron sputtering. A smooth titanium surface (Ti-S) was used as the control. The physicochemical properties including surface morphology, roughness, and hydrophilicity were characterized using scanning electron microscopy, atomic force microscopy, and an optical contact angle analyzer. The "contact guidance" morphology was assessed using confocal laser scanning microscopy. Cell proliferation was analyzed using the Cell Counting Kit-8 assay. The expression level of the main focal adhesion-related structural protein vinculin was compared using quantitative reverse transcription PCR and Western blotting. The antibacterial activity against P. gingivalis was evaluated using the LIVE/DEAD BacLight™ Bacterial Viability Kit. RESULTS: The Ag and TiN antibacterial nanocoatings were successfully deposited onto the smooth and MG surfaces using magnetron sputtering technology. TiN coating on a grooved surface (TiN-MG) resulted in less nanoroughness and greater surface hydrophilicity than Ag coating on a smooth surface (Ag-S), which was more hydrophobic. Cell proliferation and expression of vinculin were higher on the TiN-MG surface than on the Ag-coated surfaces. Ag-coated surfaces showed the strongest antibacterial activity, followed by TiN-coated surfaces. CONCLUSION: Nano-Ag coating resulted in good antimicrobial activity; however, the biocompatibility was questionable. TiN nanocoating on an MG surface showed antibacterial properties with an optimal biocompatibility and maintained the "contact guidance" effects for HGFs.

Lysophosphatidic acid induces integrin ß6 expression in human oral squamous cell carcinomas cells via LPAR1 coupling to Gαi and downstream SMAD3 and ETS-1 activation.

Integrin ß6 (ITGB6), an epithelial-specific integrin, is upregulated in oral squamous cell carcinomas (OSCC) and is associated with progression and metastasis of OSCC. Lysophosphatidic acid (LPA), an important bioactive phospholipid present in saliva, has also been related to OSCC cell migration and invasiveness. LPA exerts its biological effects through signal transduction pathways that ultimately regulate gene expression. However, it is unclear whether LPA signaling is involved in ITGB6 upregulation in OSCC. Therefore, the aim of the current study was to investigate the role of LPA in the regulation of ITGB6 expression in OSCC cells, and to delineate the molecular signaling pathways involved. Using SAS and HSC-3 OSCC cell lines, we found that LPA increases ITGB6 mRNA expression without affecting mRNA stability, suggesting that LPA acts by regulating ITGB6 gene transcription. In addition, we show that LPA stimulation increases phosphorylation and binding of the transcription factors SMAD3 and ETS-1 to the ITGB6 promoter resulting in ITGB6 active transcription. Finally, we demonstrate that LPA-induced ITGB6 expression is mediated via the LPA receptors 1 (LPAR1) coupling to Gαi. Our findings provide insights into the molecular mechanism underlying ITGB6 overexpression in OSCC and may have important implications for therapeutic purposes.

Pygo2 Regulates Adiposity and Glucose Homeostasis via ß-Catenin-Axin2-GSK3ß Signaling Pathway.

Wnt/ß-catenin signaling plays a key role in regulating adipogenesis through indirectly inhibiting the expression of C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ); however, the detailed molecular mechanism remains poorly understood. Moreover, the factor(s) that determines the Wnt/ß-catenin output level during adipogenesis is also not completely defined. In this study, we showed that Pygo2 exhibited a declined expression pattern during adipocyte differentiation, resulting in an attenuated Wnt/ß-catenin output level. The mechanism study indicated that Pygo2 inhibition led to the downregulation of Axin2, a constitutive Wnt target, in the cytoplasm. Consequently, Axin2-bound GSK3ß was released and translocated into the nucleus to phosphorylate C/EBPß and Snail, resulting in an increase in the DNA binding activity of C/EBPß and decreased protein stability of Snail, which subsequently activated the expression of C/EBPα and PPARγ. Consistent with this, embryonic fibroblasts from Pygo2-/- mice exhibited spontaneous adipocyte differentiation, and adipocyte precursor-specific Pygo2-deficient mice exhibited increased adiposity with decreased energy expenditure. We further showed impaired glucose tolerance and decreased systemic insulin sensitivity in Pygo2-deficient mice. Our study revealed an association between Pygo2 function and obesity or diabetes.

Epigenetic regulation of integrin ß6 transcription induced by TGF-ß1 in human oral squamous cell carcinoma cells.

Overexpression of integrin αvß6 is believed to play an important role in the invasion and metastasis of oral squamous cell carcinoma (OSCC). However, little is known about the molecular mechanisms leading to αvß6 upregulation in OSCC. As the integrin ß6 (ITGB6) is the only partner with αv, the expression of αvß6 is dependent on ITGB6, it is, therefore, pivotal to investigate the mechanisms underlying ITGB6 overexpression in OSCC. We previously reported the cloning and characterization of human ITGB6 gene. In the current study, we further investigated the molecular mechanisms of ITGB6 expression and the upregulation by carcinogenesis related cytokine-transforming growth factor-ß1 (TGF-ß1) in OSCC cells. We first demonstrated that TGF-ß1 can induce ITGB6 mRNA and protein express in a time and concentration dependent manner, and the induced-ITGB6 mRNA was not due to increase the mRNA stability, but regulated at transcriptional level. By using a luciferase reporter assay, site-mutation, RNA interference, and chromatin immunoprecipitation assay, we revealed for the first time that JunB, a member of the activator protein-1 (AP-1) family, is involved in the positive regulation to the ITGB6 transcription induced by TGF-ß1 in OSCC cells. Furthermore, our data also demonstrated that histone acetyltransferase (HAT) CBP mediated histone H3 and H4 hyperacetylation, and RNA Polymerase II recruitment to ITGB6 promoter, facilitated the binding of transcription factor JunB to ITGB6 promoter after TGF-ß1 stimulation. Collectively, these findings demonstrate that JunB and CBP-mediated histone hyperacetylation are responsible for TGF-ß1 induced ITGB6 transcription in OSCC cells, suggesting that epigenetic mechanisms are responsible for the active transcription expression of ITGB6 induced by TGF-ß1 in OSCC cells.