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Background: Focal adhesions (FAs) is the most important process in the first step of osseointegration between preosteoblasts and titanium (Ti). FAs improvement and pre-osteoblasts cell proliferation leads to successful Ti-based dental implants. This study aimed to confirm the applicability of rosmarinic acid (RA) as a functional substance for improving FAs and cell proliferation of MC3T3-E1 preosteoblasts on Ti surfaces during the first stage of osseointegration for successful Ti-based dental implants. Methods: We used MC3T3-E1 preosteoblasts on Ti discs incubated in a medium supplemented with or without 14 μg/ml to decipher the effects of RA on FAs and cell proliferation. FAs and proliferation of MC3T3-E1 cells on Ti discs were assessed via MTT assay. Actin-labeled cells and paxillin contacts were observed and imaged by fluorescent microscopy, and the associated signaling pathways were revealed through western blot analysis. Results: In RA-treated MC3T3-E1 cells on Ti discs, FAs between MC3T3-E1 preosteoblasts and Ti surfaces and the expression of focal adhesion kinase (FAK), phosphorylated FAK and paxillin proteins and filamentous-actin formation increased. RA increased the proliferation of MC3T3-E1 preosteoblasts on the Ti surface as well as the expression of Grab2, Ras, pERK1/2, and ERK1/2. In addition, the expression of secretory leukocyte protease inhibitor and thymosin b4, known as nanomolecules that enhance the interaction between implanted Ti materials and preosteoblasts in the RA-treated MC3T3-E1 preosteoblasts, increased. RA not only increased the FAs of MC3T3-E1 preosteoblasts on the Ti surface through the FAK/Paxillin signaling pathway, but also increased cell proliferation and mitosis through the FAK/Grab2/Ras/ERK1/2 signaling pathway. Conclusion: RA can be applied as an effective functional substrate to improve the FAs and proliferation of MC3T3-E1 preosteoblats on Ti surfaces, which are essential in the first step of osseointegration between implanted Ti and bone tissue for the clinical success of Ti based dental implants.

BACKGROUND: Osteoporosis can be attributed to an imbalance between osteoblastic bone formation and osteoclastic bone resorption. Osteoblasts are the basis of bone formation and are essential for bone growth and maintenance. Disorders of osteoblast function can lead to dysplasia of bone growth and osteoporosis. However, the key genes involved in osteoblast differentiation are still unknown. OBJECTIVE: Bioinformatics was used to identify key genes and upstream miRNAs during osteogenic differentiation of MC3T3-E1 cells and verify their effects on osteogenic differentiation and proliferation of MC3T3-E1 cells. METHODS: The osteogenic induction data of gene expression profile GSE46400 were downloaded from the Gene Expression Omnibus. Differentially expressed genes for osteogenesis differentiation were obtained with limma package. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes were analyzed using DAVID database. The STRING website was used to establish a protein-protein interaction network and CytoHubba plug-in was used to screen the key modules in the network to identify the key genes. MC3T3-E1 cells were cultured and transfected with siRNA and miRNA, separately. The change of prostaglandin-endoperoxide synthase 2 (Ptgs2) and ossification-related gene level was detected by real-time quantitative PCR 72 hours after transfection. The change of PTGS2 protein level was detected by western blot assay 72 hours after transfection. Alkaline phosphatase was quantified 14 days after transfection. Alizarin red staining and quantitative detection were performed 21 days after transfection. RESULTS AND CONCLUSION: (1) 229 differentially expressed genes were screened, of which 114 were up-regulated and 115 were down-regulated. Five genes enriched in the biological process of bone mineralization, including Mmp13, Ibsp, Gpnmb, Ptgs2 and Aspn, were highly expressed. Furthermore, CytoHubba was used to identify that only Ptgs2 was involved in the biological process of bone mineralization, and Ptgs2 was defined as a key gene in the process of osteogenic differentiation. (2) The upstream miRNA of Ptgs2 was predicted by Targetscan, and it was found that mmu-miR-107-3p could regulate Ptgs2 in a targeted way. (3) Knockdown Ptgs2 in MC3T3-e1 cells significantly inhibited cell proliferation and osteogenic differentiation (P < 0.05). After transfection of mmu-miR-107- 3p, cell proliferation and osteogenic differentiation were also inhibited. (4) Dual luciferase reporter gene results showed that mmu-miR-107-3p could directly inhibit the transcription of Ptgs2 (P < 0.05). (5) mmu-miR-107-3p can inhibit the proliferation and osteogenic differentiation of MC3T3-E1 cells by targeting Ptgs2. Ptgs2 may be a key gene in the osteogenic differentiation of MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

In recent years, graphene and its derivatives have attracted much interest in various fields, including biomedical applications. In particular, increasing attention has been paid to the effects of reduced graphene oxide (rGO) on cellular behaviors. On the other hand, dicalcium phosphate (DCP) has been widely used in dental and pharmaceutical fields. In this study, DCP composites coated with rGO (DCP-rGO composites) were prepared at various concentration ratios (DCP to rGO concentration ratios of 5:2.5, 5:5, and 5:10 μg/mL, respectively), and their physicochemical properties were characterized. In addition, the effects of DCP-rGO hybrid composites on MC3T3-E1 preosteoblasts were investigated. It was found that the DCP-rGO composites had an irregular granule-like structure with a diameter in the range order of the micrometer, and were found to be partially covered and interconnected with a network of rGO. The zeta potential analysis showed that although both DCP microparticles and rGO sheets had negative surface charge, the DCP-rGO composites could be successfully formed by the unique structural properties of rGO. In addition, it was demonstrated that the DCP-rGO composites significantly increased alkaline phosphatase activity and extracellular calcium deposition, indicating that the DCP-rGO hybrid composites can accelerate the osteogenic differentiation by the synergistic effects of rGO and DCP. Therefore, in conclusion, it is suggested that the DCP-rGO hybrid composites can be potent factors in accelerating the bone tissue regeneration. [ABSTRACT FROM AUTHOR]