Regulation of human gingival fibroblast gene expression on microgrooves: A DNA microarray study / 대한치과보철학회지

PURPOSE: We aimed to investigate the gene expression of human gingival fibroblasts on microgroove surface using DNA microarray. MATERIALS AND METHODS: Microgrooves were applied on grade II titanium discs to have 0/0 µm (NE0, control group), 60/10 µm (E60/10, experimental group) of respective width/depth by photolithography. The entire surface of the microgrooved Ti substrata was further acid etched and used as the two experimental groups in this study. Human gingival fibroblasts were cultured in the experimental group and the control group, and total RNA was extracted. The oligonucleotide microarray was performed to confirm the changes of various gene expression levels between experimental group and control group. Changes of gene expression level were determined at the pathway level by mapping the expression results of DNA chips, using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. RESULTS: Gene expression levels on E60/10 and NE0 were analyzed, there were 123 genes showing significant differences in expression more than 1.5 times on E60/10 microgrooved surface compared to NE0 surface, and 19 genes showing significant differences in expression more than 2 times. The KEGG pathway analysis confirmed the changes in gene expression levels under experimental conditions. Cell signaling, proliferation, and activity among the various gene expression results were identified. CONCLUSION: Microgrooved surfaces induce gene expression changes and related cell signaling. According to the results of this study, microgrooves can be used as the surface of various biomaterials which need to improve cell activity through gene expression changes and activation of cell signaling.

Effect of titanium surface microgrooves and thermal oxidation on in vitro osteoblast responses / 대한치과보철학회지

PURPOSE: We aimed to investigate the effect of combined various microgrooves and thermal oxidation on the titanium (Ti) and to evaluate various in vitro responses of human periodontal ligament cells (PLCs). MATERIALS AND METHODS: Grade II titanium disks were fabricated. Microgrooves were applied on titanium discs to have 0/0 microm, 15/3.5 microm, 30/10 microm, and 60/10 microm of respective width/depth by photolithography. Thermal oxidation was performed on the microgrooves of Ti substrata for 3 h at 700degrees C in air. The experiments were divided into 3 groups: control group (ST), thermal oxidation group (ST/TO), and combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO). Surface characterization was performed by field-emission scanning microscopy. Cell adhesion, osteoblastic differentiation, and mineralization were analyzed using the bromodeoxyurdine (BrdU), Alkaline phosphatase (ALP) activity, and extracellular calcium deposition assays, respectively. Statistical analysis was performed using the oneway analysis of variance and Pearson's bivariate correlation analysis (SPSS Version 17.0). RESULTS: In general, the combined microgrooves and thermal oxidation group (Gr15-TO, Gr30-TO, Gr60-TO) showed significantly higher levels compared with the control (ST) or thermal oxidation (ST-TO) groups in the BrdU expression, ALP activity, and extracellular calcium deposition. Gr60-TO group induced highest levels of cell adhesion and osteoblastic differentiation. CONCLUSION: Within the limitation of this study, we conclude that the Ti surface treatment using combined microgrooves and thermal oxidation is highly effective in inducing the cell adhesion andosteoblastic differentiation. The propose surface is also expected to be effective in inducing rapid and strong osseointegration of Ti oral implants.

Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation

PURPOSE: To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR. RESULTS: The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-microm-wide/10-microm-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen alpha1 mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0. CONCLUSION: The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs.

Effect of etched microgrooves on hydrophilicity of titanium and osteoblast responses: A pilot study

PURPOSE: The aim of this pilot study was to investigate the effect of etched microgrooves on the hydrophilicity of Ti and osteoblast responses. MATERIAL AND METHODS: Microgrooves were applied on Ti to have 15 and 60 micrometer width, and 3.5 and 10 micrometer depth by photolithography, respectively. Further acid etching was applied to create Ti surfaces with etched microgrooves. Both smooth- and acid-etched Ti were used as the controls. The hydrophilicity of Ti was analyzed by determining contact angles. Cell proliferation and osteogenic activity of MC3T3 mouse preosteoblasts were analyzed by bromodeoxyuridine assay and alkaline phosphatase (ALP) activity test, respectively. One-way ANOVA, Pearson's correlation analysis and multiple regression analysis were used for statistics. RESULTS: Etched microgrooves significantly increased the hydrophilicity of Ti compared to the smooth Ti. 60 micrometer-wide etched microgrooves significantly enhanced cell proliferation, whereas the osteogenic activity showed statistically non-significant differences between groups. Result of the osteogenic activity significantly correlated with those of hydrophilicity and cell proliferation. Hydrophilicity was determined to be an influential factor on osteogenic activity. CONCLUSION: This study indicates that increase in hydrophilicity of Ti caused by etched microgrooves acts as an influential factor on osteogenic activity. However, statistically non-significant increase in the ALP activity suggests further investigation.

Micropatterned grooves and acid-etching on titanium substrata alter viability and gene expression of adhered human gingival fibroblasts: A pilot study / 대한치과보철학회지

STATEMENT OF PROBLEM: Prior to determining an optimal width of micropatterned grooves provided on titanium substrata, we have done a pilot study using surface topographies in combined microm and submicrom levels. PURPOSE: The purpose of this study was twofold 1) to assess the proliferation and 2) to analyze the expression of genes encoding the intracellular signaling proteins involved in cell-substratum adhesions and adhesion-dependent G1 phase cell cycle progression of human gingival fibroblasts plated on smooth and microgrooved/acid-etched titanium substrata. MATERIAL AND METHODS: Three groups of titanium discs as NE0 (smooth Ti substrata), E15 (Ti substrata with microgrooves of 15 micrometer of spacing and 3.5 micrometer in depth and with further acidetching), and E30 (Ti substrata with microgrooves of 30 micrometer spacing and 3.5 micrometer in depth and with further acid-etching) served as the human gingival fibroblasts' substrata. Viability and proliferation of fibroblasts were determined using an XTT assay. Gene expressions of fibronectin, alpha5 integrin, CDK4, and p27(kip) were analyzed in RT-PCR. Cell-substratum interactions were analyzed in SEM. RESULTS: From the XTT assay at 24 h incubation, the mean optical density (OD) value of E15 was significantly greater than the values of E30 and NE0. At 48 and 96 h however, the mean OD values of E30 were significantly greater than the values of E15 and NE0. No differences in the expression of PCR transcripts at 96 h incubations were noted between groups, whereas at 48 h, an unexpected increase in the expression of all the transcripts were noted in E15 compared with other two groups. Fibroblasts were observed to orient and adhere inside the microgrooves. CONCLUSION: Micropatterned grooves and acid-etching on Ti substrata alter viability and gene expression of adhered human gingival fibroblasts.