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The control of macrophage polarization is important in bone tissue regeneration such as osseointegration. In this study, a coating method was developed to improve the osseointegration of titanium (Ti) implants by generating an immunomodulatory effect. The surface of the Ti discs was coated with a poly(lactide

Purpose Osteoradionecrosis (ORN) is known to be a refractory disease in the oral and maxillofacial field. The purpose of this study was to examine the effects of pentoxifylline (PTX) and tocopherol (TP) on an ORN animal model focused on bone healing. Materials and methods A total of 48 Sprague–Dawley rats were used: 40 received a single irradiation dose of 35 Gy on the left mandible, and eight were used as the nonirradiated control group. The rats received PTX (T1, C1), TP (T2, C2), a combination of PTX and TP (T3, C3), or normal saline (T4, C4). Three weeks after irradiation, the mandibular posterior teeth were extracted. The rats were sacrificed 4 weeks after extraction. Results In the T3 group, bone volume/tissue volume was 19.62 ± 16.03 (%), bone mineral density was as 0.31 ± 0.16 (g/cm3) in the micro-CT analysis, which were higher than that of other groups (p = 0.025, p = 0.012, respectively). In the histological analysis, bone regeneration was the most prominent in the T3 group. The ratio of empty lacunae was the highest in the T4 group, 68.77 ± 15.47 (%, p = 0.004). Immunohistochemistry showed that the expression of TNF-α was relatively lower in the T3 than in the T4 or T2 groups. The RT-qPCR showed the expression level of PECAM, VEGF-A, and osteocalcin was more than twofold as high as in the T3 group compared to the other groups. Conclusion The combination of PTX and TP appears to promote angiogenesis and osteogenesis in a rat ORN model. Therefore, PTX and TP might be useful in the treatment and prevention of ORN.

Immune reactions affect tissue regeneration and restoration as well as the occurrence and resolution of inflammation around biomaterials. Various kinds of immunomodulating techniques are being developed to acquire optimized immune reactions for local tissue regeneration. The strategies for immunomodulation are different depending on the types of scaffolds, targeted tissue, conditions of hosts, etc. Hydrogels are the most biocompatible biomaterials for tissue engineering, as they can provide highly hydrated environments similar to natural conditions. In accordance with the tremendous increase in the use of 3D printing technologies, various types of hydrogels are being applied as printing materials in the field of tissue engineering. Implanted hydrogels inevitably induce immune reactions, which will influence 3D-printing-induced tissue regeneration eventually. Understanding the immune-properties of hydrogels is undoubtedly advantageous for designing and constructing the biocompatible 3D hydrogels. In this chapter, basic immuno properties of hydrogels such as collagen, gelatin and alginate are described, and the relationship between immune reactions and tissue regenerations is also discussed. Furthermore, several techniques for immunomodulation using the hydrogels to enhance healing and regenerative processes are introduced.

Phosphatidylserine-containing liposomes (PSLs) can mimic the anti-inflammatory effects of apoptotic cells by binding to the phosphatidylserine receptors of macrophages. MGF-E8, a bridge molecule between phosphatidylserine and macrophages, can promote M2 polarization by activating macrophage integrin with its arginine-glycine-aspartic acid (RGD) motif. In this study, to mimic MGF-E8, PSLs presenting RGD peptide (RGD-PSLs) were prepared, and their immunomodulatory effects on macrophages and the bone tissue regeneration of rat calvarial defects were investigated. RGD peptides enhanced the phagocytosis of PSLs by macrophages, especially when the PSLs contained 3% RGD. RGD-PSLs were also more effective than PSLs for the suppression of lipopolysaccharide-induced gene expression of proinflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) as well as CD86 (M1 marker) expression. Furthermore, RGD promoted PSL-induced M2 polarization: 3%-RGD-PSLs significantly enhanced the mRNA expression of Arg-1, FIZZ1, and YM-1, as well as CD206 (M2 marker) expression. In a calvarial defect model, a significant increase in M2 with a decrease in M1 macrophages was observed with 3%-RGD-PSL treatment compared with the effects of PSLs alone. Finally, new bone formation was also accelerated by 3%-RGD-PSLs. Thus, these results suggest that the intensive immunomodulatory effect of RGD-PSLs led to the enhancement of bone tissue regeneration.

Macrophage polarization has become increasingly important for the improvement of the biocompatibility of biomaterials. In this study, we coated Ti discs with phospholipids (phosphatidylserine/phosphatidylcholine [4:1 mole/mole]) by evaporating the solvent under vacuum, and observed the polarization of RAW 264.7 cells cultured on the discs. The coated discs were hydrated before cell culture was added. The shape of the hydrated phospholipids varied with the concentration of loaded phospholipids: a perforated layer (0.1 mM), tubules and spheres (1 mM), and spheres (10 mM). RAW 264.7 cells exhibited different morphologies, depending on the concentration of phospholipids. On the coated discs, the gene expression and protein release of TGF-&beta, 1, VEGF, Arg-1, and TNF-&alpha, were downregulated, especially with 10 mM phospholipids. The stimulation of mRNA expression and the protein release of those genes by IL-4 and LPS were also disturbed on the phospholipid-coated discs. In conclusion, the polarization of RAW 264.7 cells was prevented by hydrated phospholipids on Ti discs.

AIM To evaluate the effects of hydrophilic dental resin monomers, triethylene glycol dimethacrylate (TEGDMA) and hydroxyethyl methacrylate (HEMA), on the polarization of a human monocyte cell line (THP-1). METHODOLOGY THP-1 cells were treated with resin monomers at noncytotoxic concentrations for 48 h and were analysed for CD86 and CD206 expressions using flow cytometry. The cells were stimulated for polarization in the presence of resin monomers (co-treatment) or after treatment with monomers (pre-treatment). CD86 and CD206 mRNA in co-treated cells was evaluated using quantitative real-time polymerase chain reaction. The release of TNF-α and TGF-β by pre-treated and co-treated cells was assessed using enzyme-linked immunosorbent assay. Morphological changes of macrophages during polarization were observed using bright-field microscopy. One-way analysis of variance was used for statistical analysis. RESULTS TEGDMA (1 mmol L-1 ) and HEMA (2 mmol L-1 ) did not induce CD86 and CD206 expressions in THP-1 cells but rather inhibited their expressions in the co-treated cells. The inhibitory effects also appeared at the transcription level. However, the expression of surface markers was not affected by pre-treatment with resin monomers. The release of TNF-α and TGF-β by M1- and M2-stimulated cells, respectively, was suppressed by co-treatment (P

Placement of dental implants initiates inflammatory foreign body response, in which macrophages play a central role and affect the subsequent tissue healing process such as bone formation. The purpose of this study was to fabricate phosphatidylserine (PS)-containing supported lipid bilayers (SLBs) on a titanium surface to regulate the polarization of macrophages, a critical factor that affects following tissue healing and regeneration. The fluorescent recovery after photobleaching images showed that the percentage of PS had a critical influence on the fluidity, and 20% PS had the highest fluidity. Furthermore, more expanded and elongated cells were observed in the SLB-coated groups. transforming growth factor-β1 and vascular endothelial growth factor, the key cytokine markers of M2 macrophage polarization, were increased in the SLB-coated groups, especially in the 20% PS group. Consistently, cells cultured on the SLB-coated titanium exhibited the distribution of CD206+ , which is a M2 macrophage specific maker. The results of this study demonstrated M2 polarization of macrophages on PS-SLB-coated titanium discs, which suggests the application of PS-SLB as an immune-regulating coating material to improve tissue reactions to dental implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2625-2633, 2018.

Inhibiting liposome uptake by macrophages using polyethylene glycol (PEG) surface modifications is a widely used approach for extending the half-life of liposomes circulating in the blood. However, the biological effects of PEGylated liposomes on macrophages have not yet been thoroughly investigated. The purpose of this study was to examine the effects of PEGylated phosphatidylserine-containing liposomes (PEG-PSLs) on the expression of two inflammation-associated cytokines, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), in the murine macrophage-like cell line RAW 264.7. Previous studies have demonstrated that PSLs inhibit TNF-α secretion and enhance TGF-β synthesis in macrophages by mimicking apoptotic cells. We found that PEGylation differentially affected the TNF-α and TGF-β levels. The PSL-mediated inhibitory effect on TNF-α secretion was enhanced by PEGylation, and PEG-PSLs decreased TGF-β levels compared with non-PEGylated PSLs. Fluorescence-activated cell sorting analysis demonstrated that 1% PEGylation disturbed the incorporation of PSLs into macrophages. The interference of uptake is thought to extend the binding interaction between PS to PS receptors for PSL-mediated inhibition of TNF-α expression. Together, these findings indicate that PEG-PSLs can prevent TNF-α secretion without increasing TGF-β levels in macrophages, and they support the potential clinical use of PEG-PSLs as anti-inflammatory agents with a relatively low potential to induce tissue fibrosis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1479-1486, 2017.

Phosphatidylserine (PS) is known to enhance biomineralization due to the ability to accumulate calcium ions. In this study, the effects of PS on odontogenic differentiation and mineralization of human dental pulp cells (HDPCs) were investigated using phosphatidylserine-containing liposomes (PSLs). PSL was slightly cytotoxic at 125 µM in growth medium, and ALP activity was up-regulated in the PSL-treated HDPCs at non-cytotoxic concentrations. Mineralization was also enhanced by PSL, while mRNA expressions of DSPP and OCN genes were slightly attenuated. The mRNA expression of Runx2 was not altered by PSL. It is thus likely that PSL selectively affected odontogenic differentiation processes of HDPC. Finally, the interaction between PSL and HDPC was investigated by staining with annexin V-FITC in PSL-treated HDPC. It was found that PS was gradually incorporated into HDPC cytoplasm for several days. The results of this study suggest that PSL is able to stimulate dentin formation in dental pulps.

Biomedical implants tend to induce fibrous encapsulation which can cause malfunction of devices and local discomfort of patients. The purpose of this study was to reduce foreign body-induced fibrous capsule formation by immunomodulation of macrophages. Polyethylene-glycol-grafted liposomes containing phosphatidylserine (PEG-PSLs) were used to modulate macrophages. Mixed cellulose ester (MCE) membranes coated with a PEG-PSLs-entrapped alginate-gelatin matrix were subcutaneously implanted into rats, and the thickness of the fibrous capsule around each MCE membrane was analyzed after four weeks. PEG-PSLs significantly reduced fibrous capsule thickness, while liposomes containing phosphatidylserine (PSLs) did not affect fibrosis. In in vitro assays, PEG-PSLs suppressed TGF-β1 secretion and multinucleated giant cell (MGC) formation in IL-4-treated RAW 264.7, a murine macrophage cell line. Although PSLs inhibited MGC formation, they exerted no effect on the secretion of TGF- β1, which is known to be an important factor in tissue fibrosis. Therefore, our results suggest that PEG-PSLs reduce fibrous capsule formation by mediating the suppression of TGF-β1 secretion from macrophages.

Macrophages are key players in innate immune responses to foreign substances. They participate in the phagocytosis of biomaterial-derived particles, angiogenesis, recruitment of fibroblasts, and formation of granulation tissues. Most macrophage functions are achieved through the release of various cytokines and chemokines; the release profile of cytokines is dependent on the phenotype of macrophages, namely proinflammatory M1 or antiinflammatory M2. M1 and M2 macrophages coexist during an inflammatory phase, and the M1/M2 ratio is considered to be an important factor for wound-healing or tissue regeneration. This ratio depends on the chemical and physical properties of biomaterials. To obtain a favorable foreign body reaction to biomaterials, the phenotypes of the macrophages can be modulated by cytokines, antibodies, small chemicals, and microRNAs. Geometrical surface fabrication of biomaterials can also be used for modulating the phenotype of macrophages.

A new maltol derivative (2) along with three known maltol derivative (1) and flavonol glycosides (3 and 4) were isolated from the dried flowers of Sophora japonica. Based upon the results of combined spectroscopic methods, the structure of new compound (2) was determined to be maltol-3-O-(4'-O-cis-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-β-glucopyranoside, an isomer of 1. These compounds strongly inhibited the action of sortase A (SrtA) from Streptococcus mutans, a primary etiologic agent of human dental caries. The onset and magnitude of inhibition of the saliva-induced aggregation in S. mutans treated with compound 2 (4×IC50) were comparable to the behavior of untreated srtA-deletion mutant.

Macrophages are key players in innate immune responses to foreign substances. They participate in the phagocytosis of biomaterial-derived particles, angiogenesis, recruitment of fibroblasts, and formation of granulation tissues. Most macrophage functions are achieved through the release of various cytokines and chemokines; the release profile of cytokines is dependent on the phenotype of macrophages, namely proinflammatory M1 or antiinflammatory M2. M1 and M2 macrophages coexist during an inflammatory phase, and the M1/M2 ratio is considered to be an important factor for wound-healing or tissue regeneration. This ratio depends on the chemical and physical properties of biomaterials. To obtain a favorable foreign body reaction to biomaterials, the phenotypes of the macrophages can be modulated by cytokines, antibodies, small chemicals, and microRNAs. Geometrical surface fabrication of biomaterials can also be used for modulating the phenotype of macrophages.

During restoration of damaged teeth in dental clinics, dentin bonding agents are usually overlaid with restorative resin composites. The purpose of this study was to investigate the effects of restorative resin composites on cytotoxicity of dentin bonding agents. Dentin bonding agents were placed on glass discs, pre-cured and uncured resin composite discs. Bonding agents on the glass discs and composite resins discs were light cured and used for agar overlay cytotoxicity testing. Dentin bonding agents on composite resin discs exhibited far less cytotoxicity than that on glass discs. The polymerization of resin composite increased the surface hardness and decreased the cytotoxicity of bonding agents. In conclusion, composite resins in dental restorations are expected to enhance the polymerization of dentin bonding agents and reduce the elution of resin monomers, resulting in the decrease of cytotoxicity.

Inhibiting liposome uptake by macrophages using polyethylene glycol (PEG) surface modifications is a widely used approach for extending the half-life of liposomes circulating in the blood. However, the biological effects of PEGylated liposomes on macrophages have not yet been thoroughly investigated. The purpose of this study was to examine the effects of PEGylated phosphatidylserine-containing liposomes (PEG-PSLs) on the expression of two inflammation-associated cytokines, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), in the murine macrophage-like cell line RAW 264.7. Previous studies have demonstrated that PSLs inhibit TNF-α secretion and enhance TGF-β synthesis in macrophages by mimicking apoptotic cells. We found that PEGylation differentially affected the TNF-α and TGF-β levels. The PSL-mediated inhibitory effect on TNF-α secretion was enhanced by PEGylation, and PEG-PSLs decreased TGF-β levels compared with non-PEGylated PSLs. Fluorescence-activated cell sorting analysis demonstrated that 1% PEGylation disturbed the incorporation of PSLs into macrophages. The interference of uptake is thought to extend the binding interaction between PS to PS receptors for PSL-mediated inhibition of TNF-α expression. Together, these findings indicate that PEG-PSLs can prevent TNF-α secretion without increasing TGF-β levels in macrophages, and they support the potential clinical use of PEG-PSLs as anti-inflammatory agents with a relatively low potential to induce tissue fibrosis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1479-1486, 2017.

M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration. To investigate the role of macrophages in dentin regeneration, this study examined the effects of M1 and M2 macrophages on the odontogenic/osteogenic differentiation of human dental pulp cells (HDPCs) using the conditioned media (CM) of the activated human monocyte cell line THP-1.M1 and M2 macrophages were induced by lipopolysaccharide/interferon-γ and interleukin-4, respectively, and the phenotypes were confirmed by flow cytometry. Macrophage CM was prepared at 2-day intervals for a period of 6 days, which included the first 2 days of activation. The CM obtained on days 4 (M1CM-4 day and M2CM-4 day) and 6 (M1CM-6 day and M2CM-6 day) were tested for their ability to promote the alkaline phosphatase (ALP) activity of HDPCs. M2CM-4 day was also examined for its effects on the messenger RNA expression of dentin sialophosphoprotein and osteocalcin genes and the matrix mineralization of HDPCs. Tumor necrosis factor alpha and transforming growth factor beta 1 (TGF-β1) in M1CM and M2CM, respectively, were quantified by an enzyme-linked immunosorbent assay. To verify the role of TGF-β1, M2CM-4 day was pretreated by a TGF-β blocking antibody and was examined for its effect on the ALP activity of HDPCs.M2CM-4 day and M2CM-6 day enhanced the ALP activity of HDPCs (P .05). Furthermore, M2CM-4 day promoted the messenger RNA expression of the dentin sialophosphoprotein gene and matrix mineralization (P .05), whereas M1CM did not affect ALP activity. The enzyme-linked immunosorbent assay detected large amounts of TGF-β1 in M2CM-4 day and M2CM-6 day. The TGF-β blocking antibody suppressed the ALP-enhancing activity of M2CM-4 day (P .05). Furthermore, the same amount of TGF-β1 as in M2CM-4 day increased ALP activity to a similar level as M2CM-4day-treated HDPCs.The CM of M2 macrophages enhanced the odontogenic/osteogenic differentiation of HDPCs. M1CM did not affect the ALP activity of HDPCs at least in the absence of M1-type inducers. The effects of M2CM on HDPCs were likely caused by TGF-β1. Therefore, M2 macrophages are expected to support dentin regeneration in dental pulps.

N-Acetylcysteine (NAC), a well-known thiol-containing antioxidant, may alleviate the toxicity of poly (methyl methacrylate) (PMMA) bone cement through the inactivation of the monomer components in the cement by its sulfhydryl moiety. This study aimed to reveal the effects of the NAC on the polymerization heat, degree of conversion (DC), mechanical properties, and residual monomer release of PMMA bone cement. The addition of NAC (0, 0.25, 0.50, 0.75, and 1.00 wt%) to PMMA powder significantly reduced the maximum polymerization temperature and steadily delayed the setting time. The DC of all experimental PMMA bone cements reached as high as 91% after 7 days of incubation, as determined by Fourier transform infrared spectroscopy (FTIR). The experimental groups showed steady declines in flexural strength and surface hardness as a fraction of increasing NAC. Meanwhile, the mechanical properties of the experimental groups increased with the duration of time. The amount of methyl methacrylate (MMA) leached into deionized water was analyzed using high performance liquid chromatography (HPLC) and was found to increase with an immersion time up to 24 h, after which no subsequent change was observed. The results showed a linear correlation between the thermal and mechanical properties and the released MMA with the DCs of the experimental groups. The potential usefulness of NAC in developing more biocompatible PMMA bone cement for increased working performance was highlighted in this study. Open image in new window

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.

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.

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 (PGE2) 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 PGE2 was also enhanced by TEGDMA. However, HEMA did not affect PGE2 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.

Objectives Simulating the optical properties of natural tooth would be the final goal for esthetic restorative materials. Filler distribution in resin composites determines the scattering in composite materials, which in turn would influence the color parameters such as lightness, chroma and hue. The objective of this study was to determine the influence of filler size and amount on the color parameters of experimental resin composites. Method Color of 11 experimental resin composites with two different sized fillers (LG: 0.77 μm and SG: 0.50 μm) in 10–70 wt% was measured by a spectrophotometer. Color coordinates (CIE L*, a* and b*), chroma and hue angle were determined. Optical constants including scattering coefficient (S), absorption coefficient (K) and light reflectivity (RI) were calculated. To determine the influence of the amount of filler on the optical parameters, Pearson correlations between the amount of filler (%) and color parameters and optical constants were calculated. Correlations between the optical constants (S, K and RI) and color parameters were calculated (p Results S value increased as the amount of filler increased. RI value generally increased as the amount of filler increased for LG filler group, and increased up to 40% filler for SG filler group. CIE L* value increased as the amount of filler increased in both of LG and SG filler groups. CIE L* value was highly correlated with S and RI values for both filler groups (r = 0.961–0.974). Conclusion Lightness was highly correlated with the amount of filler, S and RI values (r = 0.932–0.974). But the correlation coefficients between the amount of filler and chroma/hue were moderate (r = 0.406–0.827); therefore, pigmentation would be tried to simulate the color of resin composites to those of natural tooth. Optical properties of resin composites could be partly simulated to those of teeth by controlling the filler distribution.

Objective The cytotoxic mechanisms of root canal sealers (Sealapex, AH26, and N2 Universal) were studied in vitro with MC3T3-E1 osteoblastic cells. Study design MC3T3-E1 cells were cotreated with root canal sealers and antioxidants, and concentrations of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured. DNA fragmentation was observed after treatment with the sealers. Results N-Acetylcystein (NAC) prevented N2 Universal– and AH26-induced cytotoxicities. However, ascorbic acid and Trolox did not affect the cytotoxicity of the sealers. N2 Universal and AH26 significantly decreased the GSH pool within a 3-hour treatment period. Unlike GSH levels, the ROS levels were not altered by the sealers. Cytotoxicity of Sealapex was not affected by NAC, and there were no changes of GSH/glutathione disulfide levels in cells treated with Sealapex. Conclusion Cytotoxicities of N2 Universal and AH26 are caused by an intracellular GSH depletion without a burst of ROS. Sealapex may cause cytotoxicity in a way different from N2 Universal and AH26.

UDMA, a dental resin monomer, was analyzed for its capacity to induce oxidative stress and apoptosis. UDMA exhibited a dose-dependent cytotoxicity, and the cytotoxic effect was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant, suggesting involvement of oxidative stress in cell death caused by UDMA. The preventive effects of NAC were also observed in UDMA-induced apoptosis in RPC-C2A pulp like cell line. A fragmented DNA ladder pattern, characteristic of apoptosis, was shown by treatment with UDMA, but NAC blocked the DNA fragmentation. The preventive effects of NAC on apoptosis were also confirmed by Annexin V staining. In conclusion, UDMA likely induces apoptotic cell death via oxidative stress. By applying the same analysis, AH26, a resin-based root canal sealer, was found to cause oxidative stress and apoptosis. NAC lowered cytotoxicity and DNA fragmentation of AH26, suggesting that oxidative stress is involved in apoptotic cell death caused by AH26.

Background Dental resin monomers that are leached from the resin matrix due to incomplete polymerization can affect the viability and various functions of oral tissues and cells. In this study, the effects of triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) on odontogenic differentiation of human dental pulp cells (HDPCs) were examined. To mimic clinical situations, dental pulp cells were treated with resin monomers for 24 h prior to the analysis of alkaline phosphatase (ALP) activity and mRNA expression of genes related to pulp cell differentiation. To elucidate the underlying signaling pathways, regulation of mitogen-activated protein (MAP) kinases by resin monomers was also investigated. Results The ALP activity of HDPCs was reduced by TEGDMA and HEMA at noncytotoxic concentrations. The mRNA expression of dentin sialophosphoprotein (DSPP), osteocalcin (OCN), and osteopontin (OPN) was also downregulated by resin monomers. However, DSPP expression was not affected by hydrogen peroxide (H2O2). Among the MAP kinases examined, ERK activation (ERK phosphorylation) was not affected by either resin monomers or H2O2, whereas JNK was phosphorylated by TEGDMA and HEMA. Phospho-p38 was upregulated by HEMA, while TEGDMA and H2O2 suppressed p38 phosphorylation. Conclusions Exposure to TEGDMA and HEMA for a limited period suppresses differentiation of HDPCs via different signaling pathways.

Objective This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress. Methods A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI). Results All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC 50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N -acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 μM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls. Significance These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.

The objective of this study was to determine the changes in color and translucency of dental porcelain-repairing resin composites compared to dental porcelain after thermocycling. Color and spectral reflectance of three shades (A2, A3, and A3.5) of one brand of dental porcelain and three basic shades (A2, A3, and A3.5) and three combinations (A2/A3, A3/3.5, and A2/A3.5) of three brands of porcelain-repairing resin composites (ABT, FSP, and TCR) were measured, before and after thermocycling for 3000 cycles, relative to the illuminant D65. The specimen was 2 mm in thickness, and 1 mm of each shade was layered to make combined shades. Changes in color (ΔE*ab) and translucency parameter (ΔTP) were calculated. A general linear model by the material (porcelain or resin composite) and shade was used to compare differences (α = 0.05). The range of color changes was 0.68–1.67 in porcelain, 0.56–1.30 in ABT, 2.28–3.10 in FSP, and 0.36–1.15 in TCR. The range of ΔTP was 0.45–0.96 in porcelain, −0.48 to 0.94 in ABT, −1.31 to 0.82 in FSP, and −0.51 to 1.91 in TCR. After thermocycling, changes in color and TP were correlated with the shade of the material, but not with the material. The discrepancy in the changes of color and translucency after thermocycling between porcelain and porcelain-repairing resin composites should be considered when selecting repairing materials. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006

Objectives Assuming that color changes after aging are related to changes in translucency of materials, the purpose of the present study was to determine the correlation between the changes in color and the changes in scattering and absorption properties after accelerated aging with representative dental esthetic restorative materials: glass ionomer, resin-modified glass ionomer, compomer, and resin composite. Methods Color was measured according to the CIELAB color scale in the transmittance and reflectance modes and used to calculate changes in color (ΔE*ab), color coordinates (ΔL*, Δa*, and Δb*), translucency parameter (ΔTP), scattering coefficient (ΔS), absorption coefficient (ΔK), and light reflectivity (ΔRI) after accelerated aging. Simple correlations between each pair of the changes in optical values were calculated, and multiple regression analysis was used to determine the parameters influencing the changes in color and color coordinates (p = 0.05). Results In the resin composite and compomer, ΔS, ΔK, and ΔRI values were approximately zero, whereas ΔS was as high as 8.9 in the glass ionomer. For most comparisons, correlation coefficient (r) was between 0.700 and 0.997. ΔL* was found to have a major influence on color changes, and ΔS, ΔTP, and ΔRI influenced ΔL*. Therefore, changes in scattering and absorption properties, after aging, were closely correlated with changes in color and color coordinates, especially in glass ionomer-based filling materials. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2007

The first step in the assessment of biocompatibility of new biomaterials is in vitro toxicity tests that include cytotoxicity and genotoxicity. In this study, we applied genetically engineered yeast cells to the assessment of cellular oxidative stress, a major cytotoxic effect of metal-based biomaterials, and genotoxicity. For measurement of oxidative stress, a gene of green fluorescence protein (GFP) was fused to a chromosomal copy of SOD1 gene, of which encoded protein is expressed responding to intracellular oxidative stress caused by most metal ions. After exposure to metal ions, the yeast cells exhibited fluorescence dose-dependently even at sub-lethal concentrations. For genotoxicity test, a multicopy plasmid bearing a RAD54 promoter fused to GFP was used as a sensing part of yeast cells. The monomers of dental resin (HEMA, TEGDMA and UDMA) were tested for their mutagenicity. Among the monomers, TEGDMA exhibited the increase of fluorescence. This result indicates that the test with yeast may produce the scores of genotoxicity closer to those obtained with mammalian cells rather than bacteria. In conclusion, this study showed the usefulness of yeast cells for in vitro biocompatibility test of biomaterials.

The objective of this study was to measure the differences in color and color parameters between dental porcelain and porcelain-repairing resin composites. The colors of three shades (A2, A3, A3.5) of one brand of dental porcelain, three original shades (A2, A3, A3.5), and three combinations (A2/A3, A3/3.5, A2/A3.5) of three brands of porcelain-repairing resin composites (ABT, FSP, TCR) were measured. The specimens were 2 mm thick, and 1 mm of each shade was layered to make combined shades. Differences in color (DeltaE(ab) (*)), lightness (DeltaL*), chroma (DeltaC(ab) (*)), and hue (DeltaH(*)) between porcelain and resin composite were calculated. Color difference was calculated as DeltaE(ab) (*) = (DeltaL*(2) + Deltaa*(2) + Deltab*(2))(1/2), chroma difference was calculated as DeltaC(ab) (*) = (Deltaa*(2) + Deltab*(2))(1/2), and hue difference was calculated as DeltaH(ab) (*) = (DeltaE(ab) (*2) - DeltaL*(2) - DeltaC(ab) (*2))(1/2). The influence of porcelain shade, brand of resin composites, and shade of resin composites were analyzed by three-way analyses of variance, and the differential influence of color parameters on color difference was analyzed with multiple regression analysis (alpha = 0.05). Differences in color and color parameters were influenced by the porcelain shade, brand and shade of resin composites. The DeltaE(ab) (*) value was in the range of 2.2-16.9. The DeltaE(ab) (*) value was correlated with DeltaC(ab) (*) (standardized correlation coefficient, beta = - 0.85), DeltaL* (beta = - 0.52), and DeltaH(ab) (*) (beta = 0.08). Between the same shade designated pairs of porcelain and repairing composite, color difference was perceptible. Therefore, studies to improve the color matching between porcelain and repairing resin are recommended.

Using FM4-64 to label endosomes and Abp1p-GFP or Sac6p-GFP to label actin patches, we find that (1) endosomes colocalize with actin patches as they assemble at the bud cortex; (2) endosomes colocalize with actin patches as they undergo linear, retrograde movement from buds toward mother cells; and (3) actin patches interact with and disassemble at FM4-64–labeled internal compartments. We also show that retrograde flow of actin cables mediates retrograde actin patch movement. An Arp2/3 complex mutation decreases the frequency of cortical, nonlinear actin patch movements, but has no effect on the velocity of linear, retrograde actin patch movement. Rather, linear actin patch movement occurs at the same velocity and direction as the movement of actin cables. Moreover, actin patches require actin cables for retrograde movements and colocalize with actin cables as they undergo retrograde movement. Our studies support a mechanism whereby actin cables serve as “conveyor belts” for retrograde movement and delivery of actin patches/endosomes to FM4-64–labeled internal compartments.

The objectives of this study were to determine the wear resistance of dental resin composites, and to evaluate the influence of wear-simulating apparatus on wear. Nine commercial resin composites were studied. Wear was simulated with an oral wear simulator (Proto-Tech), which simultaneously incorporates the wear mechanism of attrition and abrasion in three-body wear mode, and with a pin-on-disk type friction tester (Rhesca, Japan). Composite specimens were subjected to 50,000 cycles of wear against a dental porcelain antagonist. After wear simulation, parameters for the determination of wear amount, such as wear volume, maximum wear depth, mean of maximum wear depth, and average wear depth were measured with the use of a 3D profilometer. For the oral wear simulator, the range of wear volume was 4.3–9.2 × 10−2 mm3, and there were significant differences among the composites (p < 0.05). In other parameters, there were no significant differences among the composites. For the pin-on-disk tester, the range of mean of maximum wear depth was 7.5–26.3 μm, which was significantly different among the composites (p < 0.05). Correlation coefficient (r) between the wear volume from the oral wear simulator and the mean of maximum wear depth from the pin-on-disk tester was 0.52. Conclusively, dental resin composites showed significantly different wear resistance. Wear simulating apparatus influenced the amount of wear, and suitable parameters for the determination of wear amounts should be selected in each apparatus. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 71B: 414–420, 2004

The objective was to evaluate the color changes after curing, polishing, and thermocycling of a nano-filled resin composite. A nano-filled composite was grouped into two shades of enamel (EN) and translucent (TL). One hybrid composite was used as a control (CL). Color of specimens of 10 mm in diameter and 2 mm in thickness was measured on a reflection spectrophotometer with SCE geometry under the D65 illumination over white and black backgrounds. Color before curing and after curing, polishing and thermocycling was measured. Color change (ΔE), translucency parameter (TP), and contrast ratio (CR) were compared. Average ΔE after curing was 4.6 in the EN group and 10.4 in the TL group compare to 2.9 in CL. Polishing caused average ΔE of 3.3–3.6, which was not different by the shade group (p > 0.05). After 2000 cycles of thermocycling, average ΔE was 1.4–1.8, which was not different by the shade group (p > 0.05). TP values increased after curing in the EN group, but decreased in the TL group (p < 0.05). TP values after thermocycling decreased in the EN group but did not change in TL (p = 0.05). TP values of TL shades were higher than those of EN regardless of specimen conditions (p < 0.05). Changes in CR values showed similar trends to those of TP values in translucency. Changes of color and translucency after curing, polishing, and thermocycling varied by the shade group. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 71B: 16–21, 2004

Metal ions are released from dental alloys into the oral environment, which can cause biological responses over short and extended periods. Since most toxic metal ions are capable of inducing oxidative stress on cells through the mitochondrial respiratory chain, mitochondria may contribute to and be a target of metal toxicity. In this study, we investigated the effect of metal ions on growth of the budding yeast, Saccharomyces cerevisiae, and on the morphology and function of yeast mitochondria. Moreover, we tested whether mitochondrial respiratory activity contributes to metal toxicity. Metal ions affected yeast cell growth. The toxicity of metal ions to yeast cells, ranked in decreasing order are as follows: Hg > Ag > Au > Cu, Ni, Co, Zn. This result mostly correlates with the degree of toxicity of those metal ions to growth of human cells. The MIC90 of Hg, Ag and Au ions in synthetic complete media are 0.325, 5 and 320 microM, respectively. None of the toxic metal ions resulted in loss of mitochondrial respiratory activity. However, respiration-deficient rho0 cells appeared to be resistant to Ag ion, but not to Hg and Au ions. Furthermore, at high concentrations, Ag ion caused morphological changes in mitochondria. These studies indicate that yeast may be used as a model system to screen for toxic effect of metals ions from dental alloys, and that oxidation activity in mitochondria may play a role in acute toxicity of silver ion.

Background: Rapid wound healing of oral soft tissue may reduce the opportunity of infection and discomfort of patients. Previous studies have demonstrated that enhancement of angiogenesis is an effective way to accelerate wound repair. In this study, to enhance angiogenesis and healing of palatal wounds, dimethyloxalylglycine (DMOG) was applied to a rat palatal wound model. DMOG is known to inhibit oxygen-dependent degradation of hypoxia inducible factor-1 alpha (HIF-1 alpha), which can lead to up-regulation of angiogenesis markers, favoring wound repair. We also evaluated the effects of DMOG on cell migration and HIF-1 alpha expression of rat palatal (RP) cells. Furthermore, mRNA and protein expression of vascular endothelial growth factor (VEGF) were analyzed in DMOG-treated RP cells. Methods: Primary cultures of rat palatal (RP) cells were obtained from Sprague-Dawley (SD) rats. Effects of DMOG on cell viability and migration of RP cells were evaluated by using a formazan and culture insert, respectively. VEGF mRNA was observed by real-time PCR, and VEGF and HIF-1 alpha proteins were detected by Western blotting. For the animal study, excisional wounds, 3 mm in diameter, were made at the central part of the palate of SD rats. DMOG with hyaluronic acid ointment was topically applied three times during 1 week, and then wound closures were quantitated photographically and histologically. Results: DMOG was cytotoxic to RP cells at concentrations higher than 2 mM and did not affect cell migration at non-cytotoxic concentrations. mRNA and protein expression of VEGF were significantly stimulated by DMOG treatment. The protein level of HIF-1 alpha was also stabilized in RP cells by DMOG. In the animal study, groups treated with 1 mg/ml DMOG showed an increase of rat palatal wound contractures. Conclusions: DMOG enhanced wound healing of rat palatal mucosa, which was likely due to the angiogenic effect of the agent.

Actin cables, bundles of actin filaments that align along the long axis of budding yeast, are crucial for establishment of cell polarity. We fused green fluorescent protein (GFP) to actin binding protein 140 (Abp140p) and visualized actin cable dynamics in living yeast. We detected two populations of actin cables: ( i ) bud-associated cables, which extend from the bud along the mother-bud axis, and ( ii ) randomly oriented cables, which are relatively short. Time-lapse imaging of Abp140p–GFP revealed an apparent increase in the length of bud-associated actin cables. Analysis of movement of Abp140p–GFP fiduciary marks on bud-associated cables and fluorescence loss in photobleaching experiments revealed that this apparent elongation occurs by assembly of new material at the end of the cable within the bud and movement of the opposite end of the cable toward the tip of the mother cell distal to the bud. The rate of extension of the tip of an elongating actin cable is 0.29 ± 0.08 μm/s. Latrunculin A (Lat-A) treatment completely blocked this process. We also observed movement of randomly oriented cables around the cortex of cells at a rate of 0.59 ± 0.14 μm/s. Mild treatment with Lat-A did not affect the velocity of movement of randomly oriented cables. However, Lat-A treatment did increase the number of randomly oriented, motile cables per cell. Our observations suggest that establishment of bud-associated actin cables during the cell cycle is accomplished not by realignment of existing cables but by assembly of new cables within the bud or bud neck, followed by elongation.

Background: Immobilizing bioactive molecules and osteoconductive apatite on titanium implants have investigated direct ossification. In this study, hydroxycholesterol (HC) was immobilized with apatite on titanium through simply adsorption or sandwich-like coating. Three kinds of hydroxycholesterol were chosen to induce ossification: 20α-hydroxycholesterol (20α- HC), 22(S)-hydroxycholesterol (22(S)-HC) and 25-hydroxycholesterol (25-HC).The effects of HC/apatite coating on ossification abilities were evaluated in vitro and in vivo. Results: At 6 d, adsorbed apatite/25-HC and apatite/22(S)-HC coating exhibited some cytotoxicity, while the cell viability of apatite/20α-HC coating was similar as apatite coating. Immobilizing HC with apatite significantly enhanced the ALP activities compared with apatite coating. There was no significant difference in ALP value between adsorbed apatite/HC coating and sandwich-like apatite/HC/apatite coating. When compared with apatite coating, the mineral deposition improved by adsorbed HC with apatite at higher concentration in vivo. Conclusions: When compared with apatite coating, immobilizing HC with apatite coating induced the ossification in vitro and in vivo.

Purpose This study aimed to evaluate the effects of fibronectin and oxysterol immobilized on machined-surface dental implants for the enhancement of cell attachment and osteogenic differentiation, on peri-implant bone healing in the early healing phase using an experimental model in dogs. Methods Five types of dental implants were installed at a healed alveolar ridge in five dogs: a machined-surface implant (MI), apatite-coated MI (AMI), fibronectin-loaded AMI (FAMI), oxysterol-loaded AMI (OAMI), and sand-blasted, large-grit, acid-etched surface implant (SLAI). A randomly selected unilateral ridge was observed for 2 weeks, and the contralateral ridge for a 4-week period. Histologic and histometric analyses were performed for the bone-to-implant contact proportion (BIC) and bone density around the dental implant surface. Results Different bone healing patterns were observed according to the type of implant surface 2 weeks after installation; newly formed bone continuously lined the entire surfaces in specimens of the FAMI and SLAI groups, whereas bony trabecula from adjacent bone tissue appeared with minimal new bone lining onto the surface in the MI, AMI, and OAMI groups. Histometric results revealed a significant reduction in the BIC in MI, AMI, and OAMI compared to SLAI, but FAMI demonstrated a comparable BIC with SLAI. Although both the BIC and bone density increased from a 2- to 4-week healing period, bone density showed no significant difference among any of the experimental and control groups. Conclusions A fibronectin-coated implant surface designed for cell adhesion could increase contact osteogenesis in the early bone healing phase, but an oxysterol-coated implant surface designed for osteoinductivity could not modify early bone healing around implants in normal bone physiology. Graphical Abstract

During the past decade significant advances were made toward understanding the mechanism of mitochondrial inheritance in the yeast Saccharomyces cerevisiae. A combination of genetics, cell-free assays and microscopy has led to the discovery of a great number of components. These fall into three major categories: cytoskeletal elements, mitochondrial membrane components and regulatory proteins. These proteins mediate activities, including movement of mitochondria from mother cells to buds, segregation of mitochondria and mitochondrial DNA, and equal distribution of the organelle between mother cells and buds during yeast cell division.

The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes . Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.