Formation of subsurface dentin lesions using a polymicrobial biofilm model.

PURPOSE: To simulate an oral demineralization environment by multiple species of bacteria by inducing subsurface dentin lesions with a polymicrobial biofilm model. METHODS: Polymicrobial biofilms consisting of multiple species of bacteria were generated from stimulated saliva using a high-throughput active attachment model. Biofilms were grown on dentin specimens in McBain medium containing 0, 0.2 or 2.5 ppm F and on glass without fluoride for 192 hours. The medium was refreshed twice daily, after 10 and 14 hours, until 72 hours, followed by treatment for 5 minutes with 400 ppm fluoride. Specimens were recovered after 192 hours. The number of colony forming units (CFU) was measured, and integrated mineral loss (IML) was determined by transversal microradiography. RESULTS: Mineral profiles in specimens grown with 0.2F and 2.5F revealed surface layers and initial lesions distinct from those grown with 0F. IML was significantly lower with 0.2F and 2.5F than with 0F (P < 0.05), although CFUs were similar. CFUs of biofilms grown on dentin in medium containing 0F were 10-fold higher than on glass (P < 0.05). Subsurface lesions on dentin formed consistently, with their growth progression inhibited by application of fluoride. To our knowledge, this is the first report describing the induction of subsurface dentin lesions by a polymicrobial biofilm model, and this model may be useful for studies of demineralization supporting in situ and in vivo models.

Effect of the coating material on root dentin remineralization in vitro.

PURPOSE: A fluoride-releasing coating material containing surface pre-reacted glass-ionomer (S-PRG) filler has become commercially available. However, there has been no detailed investigation of its remineralization effects at various tooth surface regions. The remineralization effects of S-PRG filler-containing coating material at different sites of demineralized dentin surfaces in vitro were evaluated. METHODS: Baseline lesions were prepared on bovine root dentin surfaces by immersion in demineralization buffer and divided into four groups: (B)--baseline lesion; (P)--S-PRG filler-containing material; (V)--S-PRG filler-free coating material as negative control; and (X)--resin-modified glass- ionomer as positive control. Material was applied to half the lesion surface, then P, V and X were remineralized in a gel system. Mineral profiles, integrated mineral loss (IML) and lesion depth (LD) at four regions, i.e. 1--exposed dentin surface adjacent to the material; 2--at a distance from the material; 3--beneath the material near to the edge; and 4--at a distance from the edge, were analyzed by transversal microradiography. Data were analyzed using ANOVA and Games-Howell test with α = 0.05. RESULTS: B showed typical artificial demineralized lesion. The IMLs of V, P and X at regions 1 and 2, and P and X at region 3 were significantly lower than that of B, however, those of V at region 3 and the other three groups at region 4 were not significantly different from that of B. At region 1, P and X showed significantly lower IMLs than V. At region 2, the IML of X showed significantly lower IML than V. There was no significant difference between P and X. The LD values of V, P and X at all regions were not significantly different from that of B. Fluoride, strontium and silicate ions released from the S-PRG filler would provide a favorable environment for remineralization of the demineralized dentin in P.

ADAMTSL6ß protein rescues fibrillin-1 microfibril disorder in a Marfan syndrome mouse model through the promotion of fibrillin-1 assembly.

Marfan syndrome (MFS) is a systemic disorder of the connective tissues caused by insufficient fibrillin-1 microfibril formation and can cause cardiac complications, emphysema, ocular lens dislocation, and severe periodontal disease. ADAMTSL6ß (A disintegrin-like metalloprotease domain with thrombospondin type I motifs-like 6ß) is a microfibril-associated extracellular matrix protein expressed in various connective tissues that has been implicated in fibrillin-1 microfibril assembly. We here report that ADAMTSL6ß plays an essential role in the development and regeneration of connective tissues. ADAMTSL6ß expression rescues microfibril disorder after periodontal ligament injury in an MFS mouse model through the promotion of fibrillin-1 microfibril assembly. In addition, improved fibrillin-1 assembly in MFS mice following the administration of ADAMTSL6ß attenuates the overactivation of TGF-ß signals associated with the increased release of active TGF-ß from disrupted fibrillin-1 microfibrils within periodontal ligaments. Our current data thus demonstrate the essential contribution of ADAMTSL6ß to fibrillin-1 microfibril formation. These findings also suggest a new therapeutic strategy for the treatment of MFS through ADAMTSL6ß-mediated fibrillin-1 microfibril assembly.

Anti-demineralization effect of a novel fluoride-releasing varnish on dentin.

PURPOSE: To investigate the laboratory anti-demineralization effect of a novel fluoride-releasing varnish containing surface reaction-type prereacted glass-ionomer (S-PRG) filler. METHODS: Paired specimens were cut from bovine root dentin. One of each pair was used for the S-PRG group, and the other served as a control (n = 6). A 1 x 3 mm test surface was made on each specimen with the fluoride-releasing varnish. The novel fluoride-releasing varnish is categorized as a two-bottle-type self-etch adhesive. These liquids were mixed, applied on the test surface, and light-cured for 10 seconds. As a control, an S-PRG filler-free varnish was applied in the same manner. Each specimen was immersed in 8% methylcellulose gel demineralization system (1.5 mM Ca, 0.9 mM PO4, 0.1 M acetic acid, pH 5.0) for 7 days at 3 degrees C. The mineral profiles and integrated mineral loss (IML) of the lesions were obtained by transversal microradiography and analytical software. RESULTS: The S-PRG group exhibited significantly thicker surface layer than the control group. Furthermore, the S-PRG group showed significantly lower IML (3,459 vol% xmicropm) than the control group (4,687 vol% xmicropm) ( P < 0.05, Welch's two-sample t-test). The novel fluoride-releasing varnish increased acid resistance of root dentin in the vicinity of the coated surface.

The caries-reducing benefit of fluoride-release from dental restorative materials continues after fluoride-release has ended.

OBJECTIVE: This study tested the hypothesis that the benefit of fluoride-releasing restorative materials continues even after their reserve of fluoride has been depleted. MATERIALS AND METHODS: Pits in perspex blocks simulating cavities were filled with either a fluoride-releasing or a non-fluoride-releasing restorative material and a dentin single-section was placed 1 mm from the edge of the filled pit. These combinations were exposed to an acid gel system. Each demineralized dentin section was separated from the adjacent material and immersed in fresh demineralizing solutions. Transversal microradiographs were taken following the two experimental periods. This study defined ΔΔZ as the increase of integrated mineral loss (ΔZ) during the second acid attack. RESULTS: The first acid attack substantially demineralized the near-surface region (depth < 40 µm) in all samples. The second acid attack, however, did not cause further demineralization in this near-surface region. Instead, it demineralized dentin deeper than 40-60 µm. The ΔΔZ of the material that did not release fluoride was significantly greater than that of fluoride-releasing materials. Negative correlations were found between ΔΔZ and the mineral volume% of the near-surface region and lesion body of the initial lesions. These results indicate that the dentin mineral in the near-surface region is chemically altered to become acid-resistant fluorapatite. In addition, lesion progression during the second period of demineralization, which was fluoride-free, may have been affected for the materials that have high mineral content of the surface layer and lesion body. CONCLUSIONS: It is concluded that dentin surrounding fluoride-releasing materials is protected against demineralization even after the fluoride release has diminished.

Effect of denture base-resin with prereacted glass-ionomer filler on dentin demineralization.

The demineralization of dentin was studied when placed adjacent to one of four experimental denture base-resins. These experimental resins contained polymethylmethacrylate (PMMA) and 0, 5, 10, 20 or 30 wt% surface reaction-type prereacted glass-ionomer (S-PRG) filler, respectively. A dentin thin-section was sandwiched between these resins and subjected to demineralization for 1 wk. Microradiographic analyses showed that the mineral vol% of the surface was increased, and lesion formation was inhibited, in a dose-response relationship with the amount of S-PRG filler. Moreover, the mineral loss decreased, by up to 60%, with an increasing amount of filler. These results indicate that denture base-resins containing S-PRG filler possess a substantial inhibition capability against demineralization, and this may thereby assist in preventing root caries of abutment teeth.

Fluoride release and recharge characteristics of denture base resins containing surface pre-reacted glass-ionomer filler.

The flexural strength, flexural modulus, and the amount of fluoride released from four experimental denture base resins containing 5, 10, 20 and 30 wt% surface pre-reacted glass-ionomer (S-PRG) filler added to the powder were evaluated. The mean flexural strength of the experimental resins, except the 30 wt%, and the flexural modulus of all the resins, complied with ISO 1567 requirements. In the 20 wt% resin, the amount of fluoride released in the initial phase was 1.88 microg/cm2/day, after which the level decreased. After recharging in a 9000 ppm fluoride solution for eight hours, the level of released fluoride increased markedly to 40.21 microg/cm2/16 hrs. Our results show that fluoride levels increased as a function of the S-PRG filler content. After the almost completely discharged resins were recharged, similar fluoride release occurred again. These results suggest that denture base resins containing S-PRG filler have great recharge and release capabilities which may assist in preventing root caries of abutment teeth.

Bonding strength of autopolymerizing resin to nylon denture base polymer.

This study aimed to investigate the shear bond strength of an autopolymerizing resin to a nylon denture base polymer (Lucitone FRS: LT) subjected to different surface treatments, and the results thereof compared with a heat-polymerizing resin and a polycarbonate polymer. Specimens were divided into five groups according to the surface treatment method: polishing (#600), sandblasting, adhesive primer application (resin primer), sandblasting + adhesive primer application, and tribochemical coating (Rocatec system). Following which, specimens were subjected to a shear bond strength test and Si concentrations were measured using an electron probe microanalyzer (EPMA). On shear bond strength, that of LT with tribochemical coating was significantly higher than the other groups. On EPMA results, the surface of LT with tribochemical coating was found to be covered with a silica film. Therefore, findings in this study indicated that silica-coating by Rocatec system was effective in improving the bond strength of nylon denture base polymer to autopolymerizing repair resin.

Synthesis of bone formation deriving biosilanes.

Six silane coupling agents having amide group (biosilanes) were synthesized with the aim to construct the material surface that allows cells to be compatible with it without their destruction. These agents were expected to make a soft landing to cytoplasm through the hydrogen bonding between their amide groups and cells. Evaluations of cell affinity using glass substrates modified with the synthesized biosilanes revealed that many cells remain on the modified glass plate. In addition, the implantation into the body of immunodeficient mouse of a composite material composed of porous hydroxyapatite and osteoblast showed the formation of a bone-like structure.

Three-body-wear resistance of the experimental composites containing filler treated with hydrophobic silane coupling agents.

This paper evaluated the wear resistance of resin composite materials with fillers which were modified with a novel hydrophobic silane coupling agent. The novel silane coupling agent containing hydrophobic phenyl group 3-(3-methoxy-4-methacryloyloxyphenyl)propyltrimethoxysilane (p-MBS) was synthesized. The experimental light-cure hybrid composites containing 85wt% of filler modified with this silane were formulated. Twelve specimens were prepared for the three-body-wear test with the ACTA machine and the collected data were analyzed statistically using a one-way ANOVA and Tukey's multiple comparison test as the post hoc test. The wear of the composites containing fillers treated with p-MBS was significantly lower compared with the composite materials containing fillers pretreated with 3-methacryloyloxypropyltrimethoxysilane or the commercially composites (AP-X and ELS extra low shrinkage) after a wear test for 200,000 cycles (p<0.05). It is suggested that the resin composites containing fillers modified with the novel hydrophobic silane has high wear resistant, because of the coupling layers treated with this silane had an excellent affinity with the base resin and formed a highly hydrophobic layer on the filler surface.

Acid resistance induced by a new orthodontic bonding system in vitro.

The effect of fluoride-containing orthodontic resin bonding systems on acid resistance of adjacent enamel was evaluated using transversal microradiography. Four fluoride-containing orthodontic resin bonding systems: BeautyOrtho Bond (BO), BeautyOrtho Bond+Salivatect (BOS), Kurasper F (KP), Transbond XT (TB) and a resin-modified glass-ionomer (Fuji Ortho LC (FO)) were used. Superbond Orthomite (SB) was used as a non-fluoride material. Rectangular bovine enamel specimens (10X6 mm) were prepared. After curing the materials, nail varnish was applied to the enamel surfaces, leaving a gap of 1 mm from the cured material's periphery. The specimens were demineralized with 8% Methocel MC gel and 0.1 M lactic acid. BO, BOS, and FO revealed shallow lesions and distinct surface layers. The mineral losses of BO, BOS, and FO were significantly lower than those of TB, KP, and SB (p<0.05). In conclusion, the new system induced superior acid resistance in enamel surrounding orthodontic brackets.

Influence of simplified silica coating method on the bonding strength of resin cement to dental alloy.

This study evaluated the effectiveness of a simplified silica coating method (CoJet System) on the bonding strength of resin cements to dental alloy. Bonding strength of the specimens treated with metal primer after alumina sandblasting was compared with those treated with silica coating and silane coupling agent after alumina sandblasting. Furthermore, the influence of silane coupling agent on bonding strength was compared between one-liquid and two-liquid silane coupling agents. Measurement of shear bond strength before and after thermal cycling revealed that the group treated with silica coating in one step without alumina sandblasting yielded high bonding strength. As for the influence of silane coupling agent, treatment with two-liquid silane coupling agent achieved higher mean shear bond strength than with one-liquid silane coupling agent. Findings in this study indicated that silicatization by means of this simplified silica coating method was effective in improving the bonding strength to dental alloy.

Transcriptome database KK-Periome for periodontal ligament development: expression profiles of the extracellular matrix genes.

Specialized connective tissues such as tendon/ligament develop through a series of events that require temporal and spatial expression of numerous genes in mesenchymal progenitors. However, the genes required for tendon/ligament development have not been identified yet. To solve this problem, we made a cDNA library from periodontal ligament and sequenced 11,520 cDNA clones, as a model for investigating tendon/ligament development. The resulting sequence data was assembled to 617 expressed sequence tag (EST) clusters, and an EST database for human periodontal ligament (PDL) was constructed (designated as the KK-Periome database). In the KK-Periome database, the top 13 EST clusters were related to extracellular matrix (ECM) genes. The temporal and spatial expression patterns of these genes during mouse PDL development were examined by in situ hybridization. Among these genes, F-spondin was expressed specifically in dental follicle (DF) cells during tooth germ development, whereas tenascin-N was strongly expressed in the terminally differentiated PDL. This characteristic expression profile was confirmed by in vivo differentiation assay of human PDL (hPDL) cells in the mouse transplant. Thus, the KK-Periome database was proven to be a useful resource for PDL-derived ESTs (transcriptome), and in fact, initial evidence indicated that F-spondin and tenascin-N might serve as markers for DF and PDL, respectively.

Expression of bone extracellular matrix proteins on osteoblast cells in the presence of mineral trioxide.

The biocompatibility of periapical tissue with mineral trioxide aggregate (MTA) affects its ability to repair and regenerate itself. Here we report the cytotoxicity of MTA and how it affects the expression of bone extracellular matrix protein in MC3T3-E1 osteoblast cells. We quantified the cytotoxicity of MTA, amalgam, and Dycal (Dentsply/Caulk, Milford, DE) on MC3T3-E1 cells by measuring the ability of cells to cleave a tetrazolium salt to produce formazan dye during a period of 24, 48, or 96 hours. We used reverse-transcriptase polymerase chain reaction with primer sets for type I collagen, osteocalcin, and bone sialoprotein to measure the gene-expression response of MC3T3-E1 cells treated with MTA. MTA, amalgam, and Dycal were less toxic after 48 hours. MC3T3-E1 cell growth with MTA and Dycal was greater than nonstimulated controls. MTA caused an upregulation of type I collagen and osteocalcin messenger RNA expression after 24 hours. These results showed that, in the presence of MTA, cells grow faster and produce more mineralized matrix gene expression in osteoblasts.

Antibacterial Action of a Condensed Tannin Extracted from Astringent Persimmon as a Component of Food Addictive Pancil PS-M on Oral Polymicrobial Biofilms.

The purpose of this study was to evaluate the antibacterial activity against polymicrobial (PM) biofilms of a condensed tannin extracted from astringent persimmon (PS-M), which is contained in refreshing beverages commercially available in Japan. Salivary PM biofilms were formed anaerobically on glass coverslips for 24 and 72 h and were treated for 5 min with sterilized deionized water (DW), 0.05 and 0.2 wt% chlorhexidine digluconate (CHX), and 0.5-4.0 wt% PS-M solution. The colony forming units (CFU/mL) were determined and morphological changes of the biofilms were observed by scanning electron microscopy (SEM). The CFUs were lower in all PS-M and CHX groups compared to the DW group. PS-M exerted a dose-dependent effect. PS-M (1.53 × 10(7)) at a dose of 4.0 wt% had the same effect as 0.2 wt% CHX (2.03 × 10(7)), regardless of the culture period. SEM revealed the biofilm structures were considerably destroyed in the 4.0 wt% PS-M and 0.2 wt% CHX. These findings indicate that the antibacterial effects of PS-M, a naturally derived substance, are comparable to those of CHX. PS-M may keep the oral cavity clean and prevent dental caries and periodontal disease related to dental plaque, as well as systemic disease such as aspiration pneumonitis.

Effects of resin-based temporary filling materials against dentin demineralization.

This study investigated the in vitro anti-demineralization effects of resin-based temporary filling materials containing surface prereacted glass-ionomer (S-PRG) filler on dentin. Bovine root dentin specimens with a 3×3 mm experimental surface were divided into four treatment groups: DuraSeal (DU) as a control, S-PRG filler-free temporary material (S0), material containing 10% (S10) and 20% (S20) S-PRG filler. Each material was applied to 3×2 mm of the experimental surface, and the specimens were immersed in 8% methylcellulose gel demineralization system for one week at 37˚C. Mineral profiles and integrated mineral loss (IML) of lesions induced on the surface (3×1 mm) adjacent to the materials were computed by transversal microradiography. S10 and S20 yielded thick surface layers and shallow lesion bodies, with significantly lower IML than DU and S0 (p<0.05, Tukey's test). These findings indicate that temporary filling resin-based materials containing over 10% of S-PRG filler content have anti-demineralization effects on adjacent dentin.

Immortalization of cementoblast progenitor cells with Bmi-1 and TERT.

UNLABELLED: A cementoblast progenitor cell line designated BCPb8 was successfully isolated from dental follicle cells immortalized with Bmi-1 and hTERT. BCPb8 showed the potential to differentiate into cementoblasts on implantation into immunodeficient mice. BCPb8 was confirmed to be the first established cementoblast progenitor cell line and will provide a useful model for investigating cementogenesis. INTRODUCTION: The dental follicle is the mesenchymal tissue surrounding the developing tooth germ. During tooth root development, progenitor cells present in the dental follicle are believed to play a central role in the formation of periodontal components (cementum, periodontal ligament, and alveolar bone). However, little more is known about the biology of these progenitors. Previously, we observed that cultured bovine dental follicle cells (BDFCs) contained putative cementoblast progenitors. To further analyze the biology of these cells, we attempted to isolate cementoblast progenitors from immortalized BDFC through expression of the polycomb group protein, Bmi-1, and human telomerase reverse transcriptase (hTERT). MATERIALS AND METHODS: BDFCs were transduced with replication-deficient retroviruses carrying human Bmi-1(LXSN-Bmi-1), and hTERT (LXSH-hTERT) for immortalization. Single cell clones were established from immortalized BDFC, and differentiation into cementoblasts was assessed by implantation into immunodeficient mice. RESULTS AND CONCLUSION: BDFCs expressing Bmi-1 and hTERT showed an extended life span-90 population doublings more than normal BDFCs-and still contained cells with the potential to differentiate into cementoblasts on implantation into immunodeficient mice. From these cells, we established a clonal cell line, designated BCPb8, which formed cementum-like tissue that was reactive to the anti-cementum-specific monoclonal antibody 3G9 and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin, and type I collagen on implantation. Thus, by using Bmi-1 and hTERT, we succeeded in immortalizing cementoblast progenitor cells from BDFC without affecting differentiation potential. The BCPb8 cell line is the first immortalized clonal cell line of cementoblast progenitors and could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for patients with periodontitis.

Formation of inhibition layers with a newly developed fluoride-releasing all-in-one adhesive.

This study evaluated the capability of a novel fluoride-releasing, all-in-one adhesive system on forming inhibition layer (radio-opaque layer) as compared with other adhesive systems. Dentin surface was treated with Imperva bond (IB), FL-BOND (FB), Reactmer bond (RE), or FL-BOND S-1 (FS) (which is a novel system). Untreated specimens were categorized as nonbonding group (NB). After storing for 10 days in de-ionized water, the specimens were cut into halves perpendicularly to the pulp chamber and immersed in a buffered demineralizing solution for four days. Longitudinal sections were cut and microradiographed. The width of inhibition layers adjacent to the adhesive surface--at a depth of 50 microm under the demineralization surface--was analyzed. Microradiography revealed distinct inhibition layers adjacent to the experimental surfaces of FB, RE, and FS. No inhibition layers were observed in NB and IB. In particular, the width of the inhibition layer of FS (12.5 microm) was significantly greater than those of FB and RE. These results indicated that a newly developed all-in-one adhesive system, FS, may have a superior ability of forming inhibition layers adjacent to cavity walls, and that it may also protect dentin against further demineralization in case of secondary marginal caries.

Inhibitory effects of incadronate on the progression of rat experimental periodontitis by porphyromonas gingivalis infection.

BACKGROUND: Incadronate (YM175, disodium cycloheptylaminomethylenediphosphonate monohydrate), a bisphosphonate, has been suggested to prevent the bone resorption associated with periodontitis by inhibiting osteoclast activity. The purpose of this study was to investigate the effect of incadronate in preventing periodontal destruction in rats with Porphyromonas gingivalis-induced periodontitis. METHODS: Periodontitis was induced in 35 Wister rats by inoculating P. gingivalis into the oral cavity and feeding the rats a soft diet for 4 weeks. Incadronate or placebo was administered to the oral cavity of the rats 2 days per week for 2, 4, or 8 weeks. RESULTS: P. gingivalis infection resulted in destruction of the periodontal ligament, reduced bone density, and caused inflammatory cell migration. Radiographic, morphometric, and histological results showed that incadronate had the ability to increase the bone mineral density (quantum level score; cortex 518.9 [placebo 612.8]; sponge 579.8 [placebo 672.0]) and to prevent periodontal ligament destruction (width 0.16 mm [placebo 0.20 mm]; area 0.36 mm2 [placebo 0.54 mm2]) after 8 weeks' administration. Furthermore, the polymorphonuclear leukocyte (PMN) infiltration in gingival tissue was significantly decreased. CONCLUSION: These results showed that incadronate inhibits bone resorption and PMN migration in P. gingivalis-induced periodontitis.

Fracture toughness of resin-modified glass ionomer restorative materials: effect of powder/liquid ratio and powder particle size reduction on fracture toughness.

OBJECTIVES: The objectives of the current investigation were two-fold: (1) to examine the effect of different powder/liquid (P/L) ratios on the fracture toughness of commercial resin-modified glass ionomer cement and conventional glass ionomer cement, and (2) to evaluate the effect of powder size reduction on the fracture toughness of experimental resin-modified glass ionomers in order to improve their physical properties. METHODS: The P/L ratios of the glass ionomer and resin-modified glass ionomers were varied from the manufacturer's recommended ratio to 2.0 and 1.0 by weight. The powder particle sizes for the experimental resin-modified glass ionomers tested were 2, 5, 10 and 25 micro m in diameter. Fracture toughness was determined on ring-shaped specimens with a fatigued pre-crack. RESULTS: The fracture toughness of the resin-modified glass ionomers was significantly higher (p<0.005) than that of the glass ionomer and was not greatly influenced by the P/L ratio. For the experimental resin-modified glass ionomers, it was observed that fracture toughness gradually decreased as the powder particle size became finer. SIGNIFICANCE: The resin components in the liquid play an important role in the improvement of the physical properties of the resin-modified glass ionomer. A reduction in the powder particle size of up to 10 micro m, which resulted in a smoother surface, can maintain high fracture toughness. The high fracture toughness values of the resin-modified glass ionomer may be one of the factors contributing to a favorable clinical outcome in high stress-bearing areas.