Antibiofilm Properties and Demineralization Suppression in Early Enamel Lesions Using Dental Coating Materials.

This study aimed to investigate the effects of dental coating materials on Streptococcus mutans biofilm formation. The test materials were PRG Barrier Coat (PRG), BioCoat Ca (BioC), and FluorDental Jelly (FluorJ). Bovine enamel specimens were demineralized to mimic early enamel lesions. The biofilm was developed on a specimen treated with one of the materials by using a modified Robbins device flow-cell system. Scanning electron and fluorescence confocal laser scanning microscopy, viable and total cell counts, and gene expression assessments of the antibiofilm were performed. Ion incorporation was analyzed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. All materials allowed biofilm formation but reduced its volume. FluorJ was the only material that inhibited biofilm accumulation and had a bactericidal effect, revealing 0.66 log CFU in viable cells and 1.23 log copy reduction in total cells compared with the untreated group after 24 h of incubation. The ions released from PRG varied depending on the element. BioC contributed to enamel remineralization by supplying calcium ions while blocking the acid produced from the biofilm. In summary, the dental coating materials physically prevented acid attacks from the biofilm while providing ions to the enamel to improve its mechanical properties.

Wound-healing Processes After Pulpotomy in the Pulp Tissue of Type 1 Diabetes Mellitus Model Rats.

INTRODUCTION: Patients with type 1 diabetes mellitus (DM1) tend to have delayed wound healing, even in the pulp tissue. We hypothesized that hyperglycemia affects odontoblast-like cell (OLC) differentiation and is involved in macrophage polarization. Accordingly, we evaluated dental pulp stem cell differentiation and macrophage phenotypes after pulpotomy. METHODS: After modifying DM1 rat models by streptozotocin, 8-week-old rats' upper left first molars were pulpotomized with mineral trioxide aggregate. Meanwhile, the control group was administered saline. Immunohistochemical localization of nestin, osteopontin, α-smooth muscles (α-SMAs), and CD68 (pan-macrophage marker) was conducted 7 days after pulpotomy. The OLC differentiation stage was determined using double immunofluorescence of nestin and α-SMA. Double immunofluorescence of CD68 and iNOS was counted as M1 macrophages and CD68 and CD206 as M2 macrophages. Proliferating cell nuclear antigen and Thy-1 (CD90) were evaluated by immunofluorescence. RESULTS: In DM1 rats, the reparative dentin bridge was not complete; however, the osteopontin-positive area did not differ significantly from that in controls. Proliferating cell nuclear antigen, indicative of cell proliferation, increased in positive cells in DM1 rats compared with controls. Double-positive cells for α-SMA and nestin indicated many immature OLCs in DM1. CD90 was positive only in controls. CD68-positive cells, especially M1 macrophages, were increased in DM1 rats, allowing the inflammatory stage to continue 7 days after pulpotomy. CONCLUSIONS: The condition of DM1 model rats can interfere at various stages of the wound healing process, altering OLC differentiation and macrophage polarization. These findings highlight the importance of normal blood glucose concentrations during pulp wound healing.

Anti-inflammatory activity of 2-methoxy-4-vinylphenol involves inhibition of lipopolysaccharide-induced inducible nitric oxidase synthase by heme oxygenase-1.

BACKGROUND: 2-Methoxy-4-vinylphenol (2M4VP) is a natural anti-inflammatory compound derived from red wine, but its underlying mechanism remains unclear. Heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, inhibits NO gene expression, while nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor involved in HO-1 production, binds to the antioxidant response element (ARE) in the nucleus and promotes HO-1 transcription. Based on the hypothesis that the inhibitory effect of 2M4VP on NO production is mediated by HO-1, we examined the possible mechanism of the anti-inflammatory activity of 2M4VP in this study. MATERIALS AND METHODS: The anti-inflammatory activity of 2M4VP was analyzed by Griess method, ELISA, qPCR, and Western blotting using LPS-treated macrophage lineage RAW264.7 cells. The impact of 2M4VP on the Nrf2/ARE pathway was also analyzed using immunocytochemistry and an ARE luciferase reporter using HEK293 cells. RESULTS: The results showed that 2M4VP reduced the production of LPS-induced NO and inducible nitric oxidase synthase (iNOS). In addition, 2M4VP increased the expression of HO-1, while pretreatment with the Nrf2 inhibitor ML385 downregulated HO-1 expression. 2M4VP induced Kelch-like ECH-associated protein 1 (Keap1) degradation. Furthermore, it promoted Nrf2 nuclear translocation and increased luciferase activity by binding to the ARE. CONCLUSIONS: 2M4VP induces Keap1 degradation and promotes Nrf2 nuclear translocation. Activation of Nrf2/ARE pathway enhances HO-1 expression and leads to iNOS inhibition for anti-inflammatory function.

Efficacy of Combining an Extraoral High-Volume Evacuator with Preprocedural Mouth Rinsing in Reducing Aerosol Contamination Produced by Ultrasonic Scaling.

The coronavirus disease pandemic has afforded dental professionals an opportunity to reconsider infection control during treatment. We investigated the efficacy of combining extraoral high-volume evacuators (eHVEs) with preprocedural mouth rinsing in reducing aerosol contamination by ultrasonic scalers. A double-masked, two-group, crossover randomized clinical trial was conducted over eight weeks. A total of 10 healthy subjects were divided into two groups; they received 0.5% povidone-iodine (PI), essential oil (EO), or water as preprocedural rinse. Aerosols produced during ultrasonic scaling were collected from the chest area (PC), dentist's mask, dentist's chest area (DC), bracket table, and assistant's area. Bacterial contamination was assessed using colony counting and adenosine triphosphate assays. With the eHVE 10 cm away from the mouth, bacterial contamination by aerosols was negligible. With the eHVE 20 cm away, more dental aerosols containing bacteria were detected at the DC and PC. Mouth rinsing decreased viable bacterial count by 31-38% (PI) and 22-33% (EO), compared with no rinsing. The eHVE prevents bacterial contamination when close to the patient's mouth. Preprocedural mouth rinsing can reduce bacterial contamination where the eHVE is positioned away from the mouth, depending on the procedure. Combining an eHVE with preprocedural mouth rinsing can reduce bacterial contamination in dental offices.

Periodontal Pathogens Inhabit Root Caries Lesions Extending beyond the Gingival Margin: A Next-Generation Sequencing Analysis.

We performed a comprehensive microbiome analysis of root caries lesions using 22 teeth extracted from patients with severe periodontitis. The carious lesions were mechanically collected and cryo-pulverized following tooth extraction. Differences in the microbiome were compared between independent lesions at the supragingival site (SG) and lesions extending beyond the gingival margin (GCB). DNA was extracted and the microbiome was characterized on the basis of the V3-V4 hypervariable region of the 16S rRNA gene using paired-end sequencing on an Illumina MiSeq device. The microbiota in root caries lesions showed compositionally distinct microbiota depending on the location. The most abundant OTUs in the SG group were Streptococcus (26.0%), Actinomyces (10.6%), and Prevotella (7.6%). GCB presented Prevotella (11.1%) as the most abundant genus, followed by Fusobacterium (9.6%) and Actinomyces (8.7%). The SG group showed a lack of uniformity in microbiota compared with the GCB group. The bacterial profiles of GCB varied considerably among patients, including periodontal pathogens such as Porphyromonas, Selenomonas, Filifactor, Peptococcus, and Tannerella. Periodontal pathogens inhabit root caries lesions that extend beyond the gingival margin. This study provides a new perspective for elucidating the microbial etiology of root caries.

A Repeated State of Acidification Enhances the Anticariogenic Biofilm Activity of Glass Ionomer Cement Containing Fluoro-Zinc-Silicate Fillers.

This study aimed to evaluate the anticariogenic biofilm activity of a novel zinc-containing glass ionomer cement, Caredyne Restore (CR), using a flow-cell system that reproduces Stephan responses. Streptococcus mutans biofilms were cultured on either CR or hydroxyapatite (HA) discs mounted on a modified Robbins device. The media were allowed to flow at a speed of 2 mL/min for 24 h while exposed to an acidic buffer twice for 30 min to mimic dietary uptake. Acid exposure enhanced biofilm inhibition in the CR group, which showed 2.6 log CFU/mm2 in viable cells and a 2 log copies/mL reduction in total cells compared to the untreated group after 24 h of incubation, suggesting enhanced anticariogenic activity due to the release of fluoride and zinc ions. However, there was no difference in the number of viable and total cells between the two experimental groups after 24 h of incubation in the absence of an acidic environment. The anticariogenic biofilm activity of CR occurs in acidic oral environments, for example in the transient pH drop following dietary uptake. CR restorations are recommended in patients at high risk of caries due to hyposalivation, difficulty brushing, and frequent sugar intake.

Effect of a novel glass ionomer cement containing fluoro-zinc-silicate fillers on biofilm formation and dentin ion incorporation.

OBJECTIVES: This study is aimed at evaluating the effect of a new glass ionomer cement (GIC) containing fluoro-zinc-silicate fillers on biofilm formation and ion incorporation. MATERIALS AND METHODS: Streptococcus mutans biofilms were developed on two GIC materials: Caredyne Restore (CD) and Fuji VII (FJ); and hydroxyapatite (HA) for 24 h at 37 °C using a flow cell system. The morphological structure and bacterial viability were analyzed using a confocal laser scanning microscopy. Bacterial adhesion during the initial 2 h was also assessed by viable cell counting. To study the ion incorporation, restored cavities prepared on the root surfaces of human incisors were subjected to the elemental mapping of the zinc and fluoride ions in the GIC-dentin interface using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. RESULTS: Morphological observations revealed that biofilm formation in the CD group was remarkably inhibited compared with the HA and FJ groups, exhibiting sparse, thinner biofilm clusters. The microorganisms adhering to the CD group were significantly inhibited, revealing 2.9 ± 0.4 for CD, 4.9 ± 0.2 for FJ, and 5.4 ± 0.4 log colony-forming units (CFU) for HA. The CD zinc ion incorporation depth was 72.2 ± 8.0 µm. The fluoride penetration of CD was three times deeper than that of FJ; this difference was statistically significant (p < 0.05). CONCLUSIONS: Enhanced by the incorporation of zinc and fluoride ions, the new GIC inhibited biofilm formation by interfering with bacterial adhesion. CLINICAL RELEVANCE: A novel GIC comprised of fluoro-zinc-silicate fillers may improve clinical outcomes, such as root caries and minimally invasive dentistry.

A multi-factorial analysis of bone morphology and fracture strength of rat femur in response to ovariectomy.

BACKGROUND: Postmenopausal osteoporosis develops due to a deficiency of estrogen that causes a decrease in bone mass and changes in the macro- and micro-architectural structure of the bone, leading to the loss of mechanical strength and an increased risk of fracture. Although the assessment of bone mineral density (BMD) has been widely used as a gold standard for diagnostic screening of bone fracture risks, it accounts for only a part of the variation in bone fragility; thus, it is necessary to consider other determinants of bone strength. Therefore, we aimed to comprehensively evaluate the architectural changes of the bone that influence bone fracture strength, together with the different sensitivities of cortical and trabecular bone in response to ovariectomy (OVX). METHODS: Bone morphology parameters were separately analyzed both in cortical and in trabecular bones, at distal-metaphysis, and mid-diaphysis of OVX rat femurs. Three-point bending test was performed at mid-diaphysis of the femurs. Correlation of OVX-induced changes of morphological parameters with breaking force was analyzed using Pearson's correlation coefficient. RESULTS: OVX resulted in a decline in the bone volume of distal-metaphysis trabecular bone, but an increase in distal-metaphysis and mid-diaphysis cortical bone volume. Tissue mineral density (TMD) remained unchanged in both the trabecular and cortical bone of the distal metaphysis but decreased in cortical bone of the mid-diaphysis. The OVX significantly increased the breaking force at mid-diaphysis of the femurs. CONCLUSIONS: OVX decreased the trabecular bone volume of the distal-metaphysis and increased the cortical bone volume of the distal-metaphysis and mid-diaphysis. Despite the reduction in TMD and increased cortical porosity, bone fracture strength increased in the mid-diaphysis after OVX. These results indicate that analyzing a single factor, i.e., BMD, is not sufficient to predict the absolute fracture risk of the bone, as OVX-induced bone response vary, depending on the bone type and location. Our results strongly support the necessity of analyzing bone micro-architecture and site specificity to clarify the true etiology of osteoporosis in a clinical setting.

Extracellular matrix with defective collagen cross-linking affects the differentiation of bone cells.

Fibrillar type I collagen, the predominant organic component in bone, is stabilized by lysyl oxidase (LOX)-initiated covalent intermolecular cross-linking, an important determinant of bone quality. However, the impact of collagen cross-linking on the activity of bone cells and subsequent tissue remodeling is not well understood. In this study, we investigated the effect of collagen cross-linking on bone cellular activities employing a loss-of-function approach, using a potent LOX inhibitor, ß-aminopropionitrile (BAPN). Osteoblastic cells (MC3T3-E1) were cultured for 2 weeks in the presence of 0-2 mM BAPN to obtain low cross-linked collagen matrices. The addition of BAPN to the cultures diminished collagen cross-links in a dose-dependent manner and, at 1 mM level, none of the major cross-links were detected without affecting collagen production. After the removal of cellular components from these cultures, MC3T3-E1, osteoclasts (RAW264.7), or mouse primary bone marrow-derived stromal cells (BMSCs) were seeded. MC3T3-E1 cells grown on low cross-link matrices showed increased alkaline phosphatase (ALP) activity. The number of multinucleate tartrate-resistant acid phosphatase (TRAP)-positive cells increased in RAW264.7 cells. Initial adhesion, proliferation, and ALP activity of BMSCs also increased. In the animal experiments, 4-week-old C57BL/6 mice were fed with BAPN-containing diet for 8 weeks. At this point, biochemical analysis of bone demonstrated that collagen cross-links decreased without affecting collagen content. Then, the diet was changed to a control diet to minimize the direct effect of BAPN. At 2 and 4 weeks after the change, histological samples were prepared. Histological examination of femur samples at 4 weeks showed a significant increase in the number of bone surface osteoblasts, while the bone volume and surface osteoclast numbers were not significantly affected. These results clearly demonstrated that the extent of collagen cross-linking of bone matrix affected the differentiation of bone cells, underscoring the importance of collagen cross-linking in the regulation of cell behaviors and tissue remodeling in bone. Characterization of collagen cross-linking in bone may be beneficial to obtain insight into not only bone mechanical property, but also bone cellular activities.

An ENU-induced splice site mutation of mouse Col1a1 causing recessive osteogenesis imperfecta and revealing a novel splicing rescue.

GU-AG consensus sequences are used for intron recognition in the majority of cases of pre-mRNA splicing in eukaryotes. Mutations at splice junctions often cause exon skipping, short deletions, or insertions in the mature mRNA, underlying one common molecular mechanism of genetic diseases. Using N-ethyl-N-nitrosourea, a novel recessive mutation named seal was produced, associated with fragile bones and susceptibility to fractures (spine and limbs). A single nucleotide transversion (T → A) at the second position of intron 36 of the Col1a1 gene, encoding the type I collagen, α1 chain, was responsible for the phenotype. Col1a1 seal mRNA expression occurred at greatly reduced levels compared to the wild-type transcript, resulting in reduced and aberrant collagen fibers in tibiae of seal homozygous mice. Unexpectedly, splicing of Col1a1 seal mRNA followed the normal pattern despite the presence of the donor splice site mutation, likely due to the action of a putative intronic splicing enhancer present in intron 25, which appeared to function redundantly with the splice donor site of intron 36. Seal mice represent a model of human osteogenesis imperfecta, and reveal a previously unknown mechanism for splicing "rescue."

Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly Accelerate the Healing of Calvarial Bone Defects.

Considering the increased interest in cell-based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti-apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3-E1 osteoblastic cells from H2 O2 -induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27-overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity, and fewer apoptotic cells in comparison with control cells. After 4-week of transplantation, both control cell- and Hsp27 overexpressed cell-transplanted groups showed significantly higher new bone formation in comparison with cell-free gel-transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4-week but did not differentiate into bone-forming osteoblasts. In conclusion, cell-containing collagen gel accelerated calvarial bone defect healing in comparison with cell-free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation-based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. J. Cell. Physiol. 231: 1974-1982, 2016. © 2016 Wiley Periodicals, Inc.

Mechanical Loading Stimulates Expression of Collagen Cross-Linking Associated Enzymes in Periodontal Ligament.

Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post-translationally modified by a series of specific enzymes. Among the collagen-modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross-linking and lysyl hydroxylases (LHs) to regulate the cross-linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen-modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intra-peritoneal injection of a LOX inhibitor, ß-aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL-derived cells. The mechanical loading also elevated formation of collagen α-chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2-positive cells was observed only at the bone-side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up-regulating collagen-modifying enzymes and subsequent collagen cross-linking which are important for PDL tissue maintenance. J. Cell. Physiol. 231: 926-933, 2016. © 2015 Wiley Periodicals, Inc.