Effects of N-acetylcysteine on TEGDMA- and HEMA-induced suppression of osteogenic differentiation of human osteosarcoma MG63 cells.

Triethyleneglycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are major resinous components of dental restorative materials and dentin bonding adhesives. Resin monomers are known to cause cytotoxicity in mammalian cells via oxidative stress and inhibit differentiation of dental pulp cells and osteoblasts. This study was aimed to investigate whether oxidative stress was involved in the inhibition of TEGDMA- and HEMA-induced differentiation. TEGDMA and HEMA reduced alkaline phosphatase (ALP) activity and the mRNA expression of the osteopontin (OPN) gene in MG63 cells at noncytotoxic concentrations. On the other hand, N-acetylcysteine (NAC) did not affect ALP activity at concentrations below 10 mM. Reduced ALP activity and OPN mRNA expression by TEGDMA were partially recovered via cotreatment with NAC. However, NAC did not exhibit significant effects in HEMA-treated cells. Glutathione (GSH) levels were also down-regulated by both TEGDMA and HEMA. The addition of NAC induced the partial recovery of GSH in cells treated with 0.5 mM TEGDMA. On the other hand, the levels of GSH in HEMA-treated cells were not affected by NAC. These results suggest that oxidative stress is involved in the suppression of differentiation by TEGDMA. Translocation of Nrf2 from the cytoplasm to the nucleus has been known to play a role in the suppression of osteogenic differentiation by oxidative stress. However, Nrf2 did not move into the nucleus in resin monomer-treated MG63 cells, suggesting the contribution of other signaling pathways to the suppressive effects of resin monomers.

In vitro cytocompatibility of N-acetylcysteine-supplemented dentin bonding agents.

INTRODUCTION: Cytotoxic resin components of dentin bonding agents are known to cause oxidative damage and suppress odontogenic differentiation of dental pulp cells. Because antioxidants were found to protect cells from cytotoxicity of resin monomers in previous studies, we investigated the effect of N-acetylcysteine (NAC) on cytotoxicity and anti-differentiation activity of bonding agents. METHODS: Human dental pulp cells were treated with the extracts of dentin bonding agents (Prime & Bond NT, Adper Single Bond, and Dentin Cement), and then cell viability, alkaline phosphatase (ALP) activity, and matrix mineralization were observed. To assess the effects of NAC, NAC was directly added into culture media or mixed with bonding agents before extraction. Release of NAC from bonding agents was also observed by high-performance liquid chromatography. RESULTS: NAC enhanced ALP activity and mRNA expression of dentin sialophosphoprotein gene, whereas extracts of dentin bonding agents inhibited the induction of ALP activity. When the cells were treated with extracts of the bonding agents, the NAC in the culture media reduced the cytotoxicity of the bonding agents. When NAC was incorporated into bonding agents, a protective effect was only seen for Prime & Bond NT containing more than 1% NAC. The disruption of ALP activity and matrix mineralization in pulp cells was partially reversed by NAC only in Prime & Bond NT-treated cells. High-performance liquid chromatography analysis of NAC showed that the amount of NAC effluxed from Prime & Bond NT was not greater than that effluxed from Adper Single Bond. CONCLUSIONS: NAC was useful for reversing cytotoxicity and anti-differentiation effects of Prime & Bond NT on human dental pulp cells.

Enhancement of the ALP activity of C3H10T1/2 cells by the combination of an oxysterol and apatite.

Biomimetic apatite coating has been used to load osteogenic biomolecules onto the surface of titanium implants. Apatite on the surface of biomaterials is thought to function as a reservoir of biomolecules as well as enhancing osteoconductivity. In this study, 20alpha-hydroxycholesterol (20alpha-HC), an osteogenic oxysterol, was used to induce differentiation of a mouse embryo fibroblast cell line (C3H10T1/2) by loading the oxysterol on biomimetically coated apatite of titanium discs. We found that the phosphatase (alkaline phosphatase (ALP)) activity of 20alpha-HC was significantly higher with ascorbic acid than alone, suggesting a need for ascorbic acid as a co-factor. When 20alpha-HC was added into the apatite coating solution, the ALP activity of the C3H10T1/2 cells did not increase on the apatite surface, even in the presence of ascorbic acid. However, ALP activity increased dramatically when 20alpha-HC was loaded by volatilization of EtOH from the apatite coat after dipping discs in 20alpha-HC-dissolved EtOH. Interestingly, ascorbic acid was not needed for this increase in ALP activity, suggesting a synergistic effect of 20alpha-HC and apatite. The concentration of calcium ions, a major component of apatite, affected the osteogenic effect of 20alpha-HC, and the increase in ALP activity was attenuated by L-type calcium channel inhibitors, verapamil and nifedipine. These results demonstrate that calcium ions released from apatite are important in the synergistic effect of 20alpha-HC and apatite.

Distinct differentiation properties of human dental pulp cells on collagen, gelatin, and chitosan scaffolds.

OBJECTIVE: The growth and differentiation properties of human dental pulp cells (HDPC) were investigated on a variety of natural scaffolds, including 2 types of collagen, gelatin, and chitosan. STUDY DESIGN: Cell attachment and growth rates of HDPC on collagen (type I and type III), gelatin, and chitosan were observed. Alkaline phosphatase (ALP) activity, mRNA expression of differentiation-related genes, and mineralization of the HDPC on each scaffold were assessed. RESULTS: Dental pulp cells attached and proliferated rapidly on collagen and gelatin, but chitosan did not properly support cell growth. The cells plated on gelatin exhibited high ALP activity, but not as high as cells plated on collagen. The expression peak of osteocalcin (OCN) mRNA from cells grown on collagen was found earlier and followed by dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1) mRNA expression. In cells grown on gelatin, however, OCN mRNA transcripts appeared at a later period of culture with no increase in DSPP or DMP-1 mRNA. Intensely mineralized extracellular matrix was seen in cells grown on collagen, but gelatin did not allow enough mineralization of cells in differentiation-inducing media. CONCLUSION: Collagen supported proliferation and differentiation of HDPC, and the expression of DSPP and DMP-1 mRNA was reduced on gelatin.

The differentiation-inducing effect of conditioned media obtained from dental pulp cells.

OBJECTIVE: Conditioned media (CM) from human dental pulp cells (HDPC) was investigated for its effects on the proliferation and differentiation of HDPC and MG63 cells. STUDY DESIGN: CM prepared from the primary culture of HDPC was used for the culture of HDPC and MG63. Cell growth, alkaline phosphatase (ALPase) activity, mRNA expression of differentiation-related genes, and mineralization of the HDPC and MG63 cells in the media containing CM were assessed. RESULTS: HDPC CM increased the cell growth of HDPC but decreased MG63 cell growth. ALPase activity and the mineralization of both HDPC and MG63 were enhanced by HDPC CM. The CM also up-regulated the expressions of DSPP, DMP-1, and OCN mRNA in HDPC. Pretreatment of HDPC CM with a neutralizing antibody against TGF-beta completely eliminated the effect of CM on ALPase activity in HDPC. CONCLUSION: HDPC was able to secrete odontogenic differentiation-inducing factors, in which TGF-beta seems to a key element of the CM effects.

Effects of TEGDMA and HEMA on the expression of COX-2 and iNOS in cultured murine macrophage cells.

OBJECTIVE: This study is aimed to investigate the effects of triethyleneglycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) on expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in cultured murine macrophage cell line RAW 264.7. METHODS: For mRNA gene expression analysis of COX-2 and iNOS, RAW 264.7 cells were exposed to TEGDMA and HEMA, and mRNA of each gene was observed by using a reverse transcriptase polymerase chain reaction (RT-PCR) assay. The Western blotting method was applied for detection of COX-2 and iNOS proteins extracted from RAW 264.7 cells treated with the resin monomers. Prostaglandin E2 (PGE(2)) was quantified by immunoassay with commercially available ELISA kits. RESULTS: TEGDMA and HEMA induced the expression of COX-2 mRNA dose-dependently in RAW 264.7 cells. The expression of COX-2 mRNA was up-regulated at 5 h of exposure to both TEGDMA and HEMA, and diminished thereafter. The resin monomers did not affect the expression of iNOS mRNA. Up-regulation of COX-2 protein was confirmed in the cells treated with TEGDMA and HEMA. Production of PGE(2) was also enhanced by TEGDMA. However, HEMA did not affect PGE(2) biosynthesis, although HEMA up-regulated the expression of COX-2 mRNA and protein. SIGNIFICANCE: These findings suggest that TEGDMA and HEMA can be a critical factor of inflammation related to resin-based dental biomaterials, and that COX-2 is involved in the inflammatory reaction of the resin monomers.

Effects of root canal sealers on lipopolysaccharide-induced expression of cyclooxygenase-2 mrna in murine macrophage cells.

Cyclooxygenase-2 (COX-2) is a useful biomarker of the inflammatory potential of biomaterials in vitro. In this study we investigate the effects of soluble extracts from 3 selected root canal sealers (AH26, Sealapex, and N2 Universal) on COX-2 mRNA expression in cultured murine macrophage cells. Root canal sealers and the addition of lipopolysaccharide (LPS) both produced significant increases in COX-2 mRNA expression in RAW 264.7 macrophages. In addition, both Sealapex and N2 Universal produced a synergistic 6- to 8-fold increase in COX-2 mRNA expression, whereas AH26 did not demonstrate synergy with LPS. These results suggest that LPS and certain root canal sealers have a synergistic effect on the inflammatory responses of macrophages. Under the conditions of this in vitro study, the results suggest that one potential mechanism of periapical inflammatory reactions might be the synergistic effects of certain root canal sealers on LPS-induced COX-2 expression by macrophage cells.

Inhibitory effects of root canal sealers on the expression of inducible nitric oxide synthase in lipopolysaccharide-stimulated murine macrophage cells.

Excessive production of nitric oxide (NO) is associated with inflammation. In the present study, we examined the effects of root canal sealers (N2 Universal, Sealapex, and AH26) on NO production and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Root canal sealers decreased NO synthesis in LPS-induced RAW 264.7 macrophages in a dose-dependent manner. RT-PCR and Western blot analysis demonstrated markedly lower levels of iNOS mRNA and protein in LPS-activated macrophage cells treated with root canal sealers compared with untreated cells. From these results, we conclude that root canal sealers do not inhibit NO synthesis by direct inhibition of the enzyme, but rather through inhibition of iNOS mRNA expression (leading to a decrease in iNOS protein expression). Our data, therefore, suggest that root canal sealers may be an effective inhibitor of LPS-induced inflammatory effects in macrophage cells. Further in vitro and in vivo studies are necessary to confirm the effects of root canal sealers on the inflammatory processes.