Matrix metalloproteinases (MMPs) play many important roles incluging the destruction of extracellular matrix (ECM) and basal membrane, migration of stem cells and endothelial progenitor cells, control of cytokines and their receptors, tissue remodeling, wound healing and angiogenesis. Homeostasis of dental pulp is critical for tooth function. When pulp is damaged or exposed, pulp cells have the potential to differentiate into dentin-forming odontoblasts. However, the mechanism of angiogenesis during pulp wound healing remains unclear. We hypothesized that a member of the matrix metalloproteinases, MMP-3, may play a role in pulp wound healing. To address these issues, we established a rat pulp injury model to investigate the MMP-3 expression during pulp wound healing and examined the effect of MMP-3 in endothelial cells in vitro. Furthermore, we investigated the effect of MMP-3 as a pulp-capping reagent on dentin and pulp regeneration in vivo after amputation. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) analysis revealed that MMP-3 mRNA was unregulated at 24 hours after pulp injury. MMP-3 protein was localized in endothelial cells and/or endothelial progenitor cells in injured pulp in vivo. MMP-3 showed mitogenic, chemotactic and anti-apoptotic activities on human umbilical vein endothelial cells (HUVECs) in vitro. Furthermore, the application of MMP-3 protein on the amputated pulp enhanced angiogenesis and reparative dentin formation in vivo. These results suggest that MMP-3 released from endothelial cells and/or endothelial progenitor cells in injured pulp plays a critical roles in angiogenesis and pulp wound healing.

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Enamel cracks, although generally regarded as a minor symptom in tooth fracture occurring with an external injury, are frequently detected in teeth without any history of injury. These cracks may be recognized as multiple thin lines that run parallel to the tooth axis. Although the cracks might cause several clinical problems such as secondary caries, esthetic disorders, tooth fracture and pulp diseases, they are frequently neglected and their causes and frequencies have not yet been fully studied. Therefore, this clinical survey investigated the frequency of enamel cracks and related symptoms. Eighty patients ranging from teenagers to those in their eighties, all underwent a clinical tooth examination. In each age group, 10 patients were investigated. The enamel cracks were divided into three types (A, B and C) based on differences in the progression of the crack: an enamel crack that was easily confirmed by a simple observation was classified as level C, those confirmed by observation through a magnifying glass were level B, and those confirmed using an activator light and a magnifying glass were level A. Cold air was used to assess the thermal sensitivity of cracked teeth. The results demonstrated that there was a tendency of increased crack incidence with aging, and the incidence was 100% in subjects≧50 years of age. A high incidence was observed in teeth restored with composite resin or a metal inlay restoration in comparison to non-restored teeth in subjects <40 years of age. Level A cracks were the most frequent in young subjects, whereas the frequencies of cracks of levels B and C increased with aging. The frequency of cracked teeth sensitive to cold air was 13.0-16.7% in 20- to 69-year-old subjects, and was not different among various age groups. In conclusion, this study clearly demonstrated age-dependent progression of enamel cracks, suggesting that operative procedures may trigger the formation and/or propagation of enamel cracks. Enamel cracks could be related to the pain response upon cold stimulus, although the frequency of sensitive teeth was not dependent on crack progression.

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Flowable resin composites for posterior direct restoration are important for close filling in complicated cavities, but there have been few reports on the physical properties and changes in physical properties in the oral environment. In this study, we compared the physical properties of universal resin composites with flowable resin composites for posterior direct restoration and investigated the changes in physical properties, bending and compressive strength, in de- and re-mineralization solution as intraoral pH cycle. We used as flowable resin composites, Clearfil Majesty LV (ML), Unifil LoFlo Plus (LP) and Estelite Flow Quick (FQ), and as universal resin composites, Clearfil AP-X (AP), Majesty Posterior (MP), Solare P (SP), Gradia Direct (GD), Beautifil II (BF) and Estelite P Quick (PQ). Cylinder specimens and stick specimens were made, and after immersing them in deionized water, citric acid and remineralization solution for one week, we measured the compressive strength and bending strength by Autograph. We also made cylinder specimens and immersed them in deionized water for one week, and measured the quantity of abrasion. In the compressive strength test, FQ and ML, which are flowable resin composites, showed high strength, followed by PQ and MP, which are universal resin composites, in deionized water. FQ showed the highest strength in citric acid and ML showed a high strength but slightly less than that of FQ, AP, MP and PQ. ML showed the highest strength in remineralization solution, and FQ showed a high strength followed by ML, MP, PQ and AP. A significant difference in compressive strength was found in MP, ML, BF, FQ and PQ after immersion. In the bending strength test, ML showed a high strength followed by MP, PQ, AP in deionized water, followed by BF and FQ. In citric acid, ML and FQ showed high strength followed by MP and PQ, and the results were the same as the citric acid group in remineralization solution. In addition, a significant difference was found in MP, ML, FQ and PQ in bending strength after immersion. In the abrasion test, abrasion was significantly large with SP and LP compared with the other resin composites. In summary, the compressive strength and bending strength of flowable resin composites for posterior direct restoration were almost equal to those of universal resin composites, and no remarkable change of physical properties was seen in compressive strength and bending strength after immersion.

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This study evaluated the improvement of polysiloxane layer at the silica-resin interface, using a mixture of 3-methacryloyloxypropyltrimethoxysilane (3-MPS) and a novel silane coupling agent (MAnF2S2C, n=4, 6, 8) with a hydrophobic fluoroalkyl and a methacryloyl group, such as 3-methacryloyloxypropyl-2-(perfluorobutyl) ethyldichlorosilane (MA4F2S2C). The concentration of each silane solution was adjusted with ethanol to 2 mass%, at various weight ratios of MAnF2S2C (20, 40, 60, or 80mol%) to 3-MPS. The tensile bond strength of the resin composites and the contact angles of the resin monomer mixture (50% Bis-GMA, 50% TEGDMA) to glass plates modified with each silane were measured. MA4F2S2C formed a coupling layer with high bond strength and low contact angle for concentrations of 20-40mol%.

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The use of flowable resins for cementation of composite resin inlays was investigated in order to improve physical or aesthetic problems of inlays cemented with a dual-cured resin cement. The purpose of this study was to estimate the film thickness of flowable resins and the polymerization of flowable resins under the composite resin inlays. Materials tested were 11 flowable resins, a resin for laminate veneer cementation and a dual-cured resin cement. Each material was sandwiched between two glass slabs and a force of 150N was applied to the samples for ten minutes at 4, 23 and 37℃. The film thickness was tested after light-activation. Furthermore, the thickness of the bonding agents was tested after weak or strong compressed air blowing (0.05MPa or 0.10MPa) and light-activation. The polymerization of the flowable resins was tested to measure the surface hardness of the materials that had been light-activated for 40 seconds through the composite resin inlay block of 0 to 5mm in height. The results were as follows: 1. The film thickness of nearly all materials tested decreased as the temperature increased. 2. At 37℃ the sum total of the thickness of each material tested, except one flowable resin, plus that of the corresponding bonding agent with strong compressed air blowing was less than 25μm, which is the standard thickness for luting agent cementation. 3. The surface hardness of nearly all materials tested tended to decrease when the composite resin inlay thickness was approximately 2mm.

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This study evaluated the apical periodontal healing after root-end sealing using Super-Bond C & B®, and root-end filling using Super-EBA™ or ProRoot®MTA, when root canal infection persisted. Apical periodontitis was induced by contaminating root canals with dental plaque in mandibular premolars of 4 beagles. After 1 month, in the SB group (n=15), Super-Bond C & B® was applied to the resected surface following apicoectomy. In the EBA (n=14) and MTA (n=14) groups, a root-end cavity was prepared and filled with Super-EBA™ and ProRoot®MTA. In the control group (n=10), the root-end surface was not filled. The observations of periradicular radiolucency, the histological observation and histometrical analyses were performed at 48 weeks after surgery. The results were as follows. The areas of periradicular radiolucency and histological bone defect areas in the SB, EBA and MTA groups were significantly less than those in the control group. There were no significant differences between the SB, EBA and MTA groups. The percentage of new cementum on the apical dentin surface in the EBA and MTA groups was significantly more than that in the SB and control groups. There were no significant differences between the EBA and MTA groups. The percentage of exposed dentin that was not covered with Super-Bond C & B® or new cementum in the SB group was significantly less than that in the EBA, MTA and control groups. The EBA and MTA groups showed a significantly lower percentage than the control group. The percentage of resorbed dentin in the SB, EBA, and MTA groups was significantly less than that in the control group. There was a significant correlation between the percentage of exposed dentin and the percentage of resorbed dentin in the SB, EBA, and MTA groups. It was suggested that sealing of the resected dentin surface is important for long-term healing. It might be difficult for the new cementum to seal the resected root end surface completely following apicoectomy, and root-end sealing using Super-Bond C & B® has better potential when root canal infection remains.

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We prepared a collagen hydrogel-sponge composite which would serve as a bioactive scaffold for bone engineering. The present study examined the effects of the collagen composite scaffold combined with/without bone morphogenetic protein (BMP) on tissue reaction and osteoinduction after implantation in rat cranial bone defects. In order to create the collagen hydrogel-sponge composite, 1.5% collagen hydrogel cross-linked by ascorbate and copper ion was injected into 4% FC-HAC sponge (6×6×3mm) which was prepared by mixing fibrillar collagen with heat-denatured collagen. A bone defect (4×4×0.5mm) was made in a calvalium of 87 Wistar male rats and the rats were assigned to five experimental groups according to the implantation. The defects were filled with the collagen hydrogel-sponge composite loaded with BMP-2 (BGS group, n=18), the collagen hydrogel-sponge composite (GS group, n=19), the FC-HAC sponge containing BMP-2 (BS group, n=17), the FC-HAC sponge (S group, n=17), or no materials (C group, n=16). Animals were euthanized after 5, 10, and 15 days, and the specimens were analyzed histomorphologically. In the BGS and GS groups, fibroblastic and osteoblastic cells proliferated on the inner side of the collagen hydrogel-sponge composite. Few inflammatory cells were seen in the connective tissue around the remnants of collagen materials. At day 15, newly formed bone area and height in the BGS group were significantly greater than those in any other group (p<0.01). A significantly greater bone induction was measured in the GS group than in the C group (p<0.01). Implanted collagen material in the BGS group significantly disappeared compared to the S group (p<0.05). These results suggested that collagen hydrogel-sponge composite encouraged the proliferation of fibroblastic and osteoblastic cells, and rapid replacement for regenerating tissue and space making, and has a better potential as a scaffold for osteoinduction by combined implantation of BMP.

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The aim of this study was to evaluate the process of healing between the root planed surface and the soft tissue in the healing process after the semilunar coronally positioned flap procedure. Maxillary and mandibular 40 incisors of 8 beagle dogs were used in this study. Prior to the semilunar coronally positioned flap procedure, the gingival recession model was created. A partial-thickness flap of labial gingiva from the canine to the opposite canine was elevated and labial alveolar bone of the subject teeth was removed. Then, the coronal margin of the flap was removed and the flap was sutured at the apically position. Four weeks later, the semilunar coronally positioned flap procedure was performed according to Tarnow's procedure. After root planing, a semilunar incision was made following the curvature of the free gingival margin. A split thickness dissection was made from the initial incision line coronally. This was connected with an intrasulcular incision. The flap was coronally positioned to the CEJ. The tissue was held in place with moist gauze against the tooth for 5 minutes. Clinical observation and histological analysis were performed 0, 1, 2 and 4 weeks after surgery. Successful root coverages were clinically observed at all observation periods. According to histological observation, at week 0 (24 hours), the narrow space between the flap and root surface was filled with clot. Long junctional epithelial attachment had developed within one week, and then did not increase significantly. At week 4, 73.3±29.1% of the root planed surface was adjacent to the epithelium. The length of connective tissue attachment did not show a significant change. Slight root resorption was observed at weeks 1 and 2. New cementum formation was observed on 6.9±11.1% of the root surface. It was concluded that the semilunar coronally positioned flap procedure may produce long junctional epithelium on most of the root planed surface within one week, and limited cementum formation from week 2 to week 4.

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Apexification was performed on an immature tooth with the apex spreading in a blunderbuss-like shape, and standardized root canal models were prepared under the assumption that the apex opening was closed. On each of these models, root canal obturation was carried out by applying 8 different obturation methods: lateral compaction method, Obtura II, Ultrafil, Thermafil, System B, combination of System B and Obtura II, sectional method, and obturation only with ZOE sealer. Based on the images taken from the surface of the root canal wall and on three dimensional CT images taken by a micro focus X-ray CT device, and by calculating the ratio of obturation material in the root canal (obturation ratio), the following conclusions were obtained: among the root canal obturation methods thus applied, the whole of the obturation ratio was the highest in Obtura II, being 98.1%, followed in order by Ultrafil (98.0%), sectional method (97.8%), lateral compaction method (97.7%), combination of System B and Obtura II (97.1%), System B (95.8%), and ZOE sealer obturation (93.3%). It was the lowest when Thermafil was applied, being 87.1%. The obturation ratio of the sliced plane 1mm from the apex was at the highest in Ultrafil being 93.4%, followed in order by System B (90.2%), sectional method (90.0%), Obtura II (88.8%), ZOE sealer obturation (87.9%), combination of System B and Obtura II (86.5%), and lateral compaction method (72.1%). It was the lowest when Thermafil was applied, being 17.8%. In the results of one-way ANOVA, the influence of the type of obturation method on the ratio of obturation was found to be significantly high (p<0.01). When Obtura II, Ultrafil, System B and combination of System B and Obtura II were used, the end of the root canal, which was spread in a blunderbuss-like shape, could be filled with gutta-percha, while obturation was slightly insufficient when the sectional method was applied. When the lateral compaction method and Thermafil were applied, obturation was extremely insufficient. When ZOE sealer obturation was applied, air bubbles of various sizes were widely disperses in the hardened material. In the obturation method where gutta-percha softened by heating or by using a solvent was filled into the root canal, the sealer had to be simultaneously used because gutta-percha tended to peel off from the canal wall. Because air bubbles in the sealer might cause the formation of dead space, precaution had to be taken to prevent the intermingling of air bubbles at the time of mixing or when coating it on the canal wall. These results suggest that, in the root canal with apex spreading in a blunderbuss-like shape and closed by apexification, root canal obturation should be performed by one of the following methods: Obtura II simultaneously using sealer, Ultrafil, System B, or combination of System B and Obtura II.

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In the case of irreversible pulpitis, dental pulp extirpation is necessary for treatment, whereas in reversible pulpitis, the dental pulp is preserved by extraction of the carious tooth. Inflammation in dental pulp tissue is associated with tissue degradation, and matrix metalloproteinases (MMPs) are believed to participate in this destruction. In inflamed pulp, many kinds of inflammatory cytokines such as tumor necrosis factor (TNF)-α, are released from inflammatory tissue. In the present study, we examined whether TNF-α affected the production of MMP-3 in dental pulp fibroblasts and its signaling pathways. TNF-α increased the expression of MMP-3 in a dose-dependent manner in dental pulp fibroblasts. U1026, which are MEK1/2 inhibitors, inhibited the MMP-3 production induced by TNF-α in dental pulp fibroblasts. Moreover, in dental pulp fibroblasts cultured with TNF-α, ERK1/2 was phosphorylated in a time-dependent manner with the maximum phosphorylation at 30min, and U0126 suppressed this phosphorylation of MEK1/2 in TNF-α-stimulated dental pulp fibroblasts. These results suggest that TNF-α may enhance pulp tissue destruction during pulp inflammation in part by regulating MMP-3, and that the MEK1/2-ERK1/2 pathway is involved in MMP-3 production in dental pulp fibroblasts.

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Polymerization shrinkage of dental resin composite generates stresses that are capable of deforming the composite and tooth, debonding the tooth-resin interface and cracking the surrounding tooth structure. Contraction stress is unfavorable for obtaining good prognosis of composite restorations and often induces postoperative discomfort. The objective of this study was to investigate the contraction stresses in bovine enamel generated by adhesive restorations using the indentation fracture method. Six kinds of materials for the adhesive restoration were used in this study: three resin composites (Z100™: 3M ESPE, Herculite XRV: sds kerr and Heliomolar: Ivoclar Vivadent), a conventional glass ionomer cement (Fuji IX GP: GC), a resin-modified glass ionomer cement (Fuji II LC: GC) and a resin composite cement (Panavia®F2.0: Kuraray Medical) for resin composite inlay. Volumetric polymerization shrinkage of the materials except the conventional glass ionomer cement and the resin composite cement was measured using a volume measuring device (AcuVol™, Bisco). The polymerization shrinkage was recorded for 60 minutes from the start of light irradiation for polymerization. Elastic moduli of the six materials were measured using a nano-indentation device (ENT-1100a, Elionix). Contraction stresses generated by the six restorations were calculated. Cracks were introduced with a Vickers indenter near a tapered cylindrical cavity in flat bovine enamel. The ratio of bonded to unbonded surface area (C-factor) was 2. Lengths of the radial indentation cracks were measured before and after the restoration. The contraction stress in bovine enamel was calculated from the crack length and the fracture toughness of the enamel. Following the second measurement of the crack length, interfacial gaps between the enamel and the restorative and crevices in the enamel around the restoration were observed to calculate the ratio of the gap length to the cavity perimeter and to count the number of specimens with an enamel crevice, respectively. The radial cracks were lengthened in all restorations due to the contraction stresses. The calculated contraction stresses were lower than the previously reported values and not significantly different among the restorations, though the polymerization shrinkage and the elastic moduli varied. The gap ratios were more than 90% for the restorations except the resin composite inlay. The resin composite restorations demonstrated more enamel crevices than the other restorations. The contraction stresses generated by polymerization shrinkage propagated the radial cracks, the enamel crevices and the contraction gaps. The formation of enamel crevices and contraction gaps released the contraction stresses in the enamel and at the interface, thus reducing the stresses at the indentation cracks.

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