Recently, the development of new dental tissue regeneration therapies, which enable regrowth of enamel, dentin and the tooth itself, have enabled new clinical treatments. Interestingly, it is suggested that dental pulp precursor cells and/or dental pulp stem cells are related to activation and regeneration of dentin. Therefore, there exists the possibility of effective therapies involving stem cell injection instead of the usual dental caries treatment and pulp capping methods. We previously reported the potential of isolated α7 integrin positive (α7⁺) cells, isolated from pure populations of myoblasts in human muscle, to undergo myogenesis and convert to other mesenchymal differentiation programs such as osteoblasts and adipocytes. In the present study, we examined the potential of α7⁺ myoblasts to differentiate into the odontogenic pathway and regenerate dentin. α7⁺ myoblasts were harvested from both human adult and fetal muscle by fluorescence activated cell sorting (FACS). To assess the potential of the α7⁺ myoblasts to differentiate into the odontogenic lineage, aggregates of 10³ cells per 10μl cells were treated with the hanging drop for 2 days. Cell aggregates were then pooled and cultured in suspension with 10^-7mol/l retinoic acid (RA) for 3 days. Cells were plated on gelatin-coated dishes and cultured with bone morphogenetic protein-4 (BMP-4) for 7 days. Odontogenic differentiation was assessed by expression of the odontoblast phenotype markers dentin sialophosphoprotein (DSPP) and dentin sialoprotein (DSP). We also examined modulation of integrin expression and adhesion/motility functions with the extracellular matrix proteins laminin-1, laminin-2 and collagen type I following differentiation into the odontogenic lineage. Following RA and BMP-4 treatment, α7⁺ myoblasts failed to fuse and transdifferentiated along an odontogenic pathway expressing DSPP, DSP and ALP activity. RA and BMP-4 treatment induced α1 integrin and αVβ3 integrin expression, and enhanced adhesion and migration on collagen type I substrates. These observations show that α7⁺ myoblasts are pluripotent stem cells capable of differentiation along odontogenic lineages. Following differentiation to odontoblasts, there is a coordinated switch in the integrin expression profile that potentiates adhesion and motility on extracellular matrix molecules. Our results suggest that α⁺ multipotent muscle stem cells may provide a means to enhance new tissue regeneration therapies of cell injection in dentistry. This study was approved by the UCSF Committee on Human Research and Aichi Gakuin University Ethics Committee.

View full abstract

In recent years, the development of adhesive restorative materials has progressed remarkably with the spread of the concept of minimal intervention (MI) in operative dentistry. Although people had conventionally regarded permeability and adhesive ability as important in the development of adhesive materials, material development has recently been shifting toward functionality which is represented by anti-cariogenicity. However, problems such as loss of fillings and recurrent caries still occur after the restoration. The nanospace into which the bonding agent does not infiltrate in the decalcified dentin collagen etched with acids or self-etching primers is known to exist underneath the hybrid layer. It has been reported that hydrolysis of the remaining decalcified dentin collagen and the bonding agent contacting it occurs over time, eventually causing the adhesion interface between resin and dentin to collapse. To solve this issue, we suggest a blockade of the nanospace, achieved by remineralization of the remaining decalcified dentin collagen using adhesive restorative materials containing resin monomers that possess mineral induction ability, may improve the durability of the adhesion interface between resin and dentin. In this study, we examined in vitro the mineral induction ability of newly developed monomers, compared with that of a model decalcified dentin matrix. We also examined the adhesion ability of 4-META/MMA-TBB resin containing the newly developed monomers to a dentin specimen by means of a micro-tensile bond test. Moreover, we observed the adhesion interface between resin and dentin with a scanning electron microscope (SEM). From these experiments, the following results were obtained: 1. In mineral induction experiments in vitro, the newly developed monomer, CMET, induced hydroxyapatite formation more rapidly than the model decalcified dentin matrix (PV). 2. In the micro-tensile bond test, 4-META/MMA-TBB resin containing the newly developed monomers, CMET and CMEP, at a concentration of 5 and 10% showed a high adhesive ability, equal to that of 4-META/MMA-TBB resin alone as a control. However, bond strength decreased significantly with the increase of concentration. 3. In the SEM observation of the adhesion interface between resin and dentin, a defect with a porous structure and imperfect formation of the resin tag were observed at the adhesion interface in 4-META/MMA-TBB resin containing CMET at concentrations of 30%, 50% and 70% CMET. In conclusion, it was suggested that the newly developed monomer, CMET, promotes remineralization of the remaining decalcified dentin collagen at the adhesion interface. Furthermore, the optimal concentration of CMET to be added to 4-META/MMA-TBB resin may be 10% considering the mechanical properties of the resin.

View full abstract

The purpose of this study was to investigate the toxicity of two root canal materials to immune cells of mice: Canals® (CaN) which is widely used clinically as a sealer, and Vitapex® (VP), which is used for paste root filling materials. Tritium-thymidine uptake into lymphocytes for DNA synthesis derived from male C3H/HeN strain mice was examined on incubation with two root canal filling materials or original exudates into culture medium, and 10- and 100-diluted exudates. After staining with propidium iodide for cell death or annexin V for apoptosis, lymphocytes were analyzed by flow cytometry. The exuded lymphocytes from mice by intraperitoneal injection of exudates of root canal filling materials were analyzed by flow cytometry using FITC-anti Thy1.2 antibody for T-lymphocytes and FITC-anti μ antibody for B-lymphocytes. Statistical analyses were done using Student's t-test, and less than 0.01 was considered to be significant. Tritium-thymidine uptake into lymphocytes decreased significantly by root canal filling materials or exudates. Tritium-thymidine uptake into activated lymphocytes by concanavalin A also decreased by the original exudate or 10-diluted exudate of CaN, and the original exudate of VP only. Propidium iodide-stained lymphocytes incubated with CaN-exudate increased soon and lasted for 48 hours. On the other hand, annexin V-stained lymphocytes incubated with CaN-exudate increased after just 12 hours, and returned to the control level after 24 hours. From the analysis of intraperitoneally exuded lymphocytes by exudate of root canal filling materials, T-lymphocytes tended to increase and B-lymphocytes tended to decrease, compared to the lymphocytes induced by medium only. The results suggested that the root canal filling materials used in this study were toxic to immune cells of mice.

View full abstract

Dissolution of calcium hydroxide may be a key mechanism for the biological properties of mineral trioxide aggregate (MTA). However, the dissolution may also be a factor that affects the physical properties of this material. In this study, we analyzed the distribution of various elements in MTA leached in water, in order to investigate compositional changes that occur during the process of calcium dissolution. MTA (ProRoot® MTA, white) was mixed according to the manufacturer's instructions, placed in cylindrical molds and stored for 24 hours at 37℃ in 100% humidity. The specimens were then suspended in distilled water for 7, 14 and 28 days. Specimens without water immersion served as controls. The distribution of Ca, Si, Bi and Al was analyzed with an electron probe microanalyzer (EPMA) along the cross-section of the specimens. Elemental analysis of the surface was also conducted in selected specimens. Formation of a layer of low Ca concentration (Ca-leached layer) was clearly detected in the periphery of leached MTA specimens. This layer showed a relatively clear boundary and increased in thickness with time. Moreover, precipitates composed mainly of calcium carbonate were formed on the surface of MTA. Si and Al formed a zone of increased concentration within the Ca-leached layer. It was concluded that water exposure of MTA resulted in the formation of a Ca-leached layer, although the process was not a one-way dissolution process but involved surface precipitation of calcium carbonate and Si and Al concentration within the Ca-leached layer.

View full abstract

Since mineral trioxide aggregate (MTA) was developed and reported as an endodontic restoration in the early 1990s, it has been shown that this material offers excellent biocompatibility with the hard tissue on its surface when used for direct pulp capping, furcal repair, repair for root perforation, root-end filling, and apexification, and for inducing tissue regeneration around the filled portion. However, the effects of MTA on the periodontal ligament tissue remain unclear, so this study evaluated the effects of MTA on the attachment, proliferation, and differentiation of human periodontal ligament fibroblasts (HPLF). Two populations of HPLFs derived from two patients were co-cultured with MTA discs (9mm in diameter and 1mm thick). In 24 hours of co-culture, HPLF attached to MTA with both a rough surface and a smooth surface, independently of its surface features. Furthermore, the effects of the variation of cell number seeded on MTA on the proliferation were estimated. When HPLF was seeded at a lower cell density, the proliferation of HPLF was restricted almost completely compared with the control cultured on plastic, while when seeded at a higher cell density, the proliferation rate was similar to the control. HPLFs co-cultured with MTA exhibited induced mineralization after 4 weeks of culture. In 4 days of the culture of HPLF with 5mmol/l CaCl₂, the expression of both osteopontin (OPN) and osteocalcin (OCN) mRNAs was induced, and after 4 weeks of culture, mineralization in both HPLFs was also observed. These results indicated that MTA has cell compatibility for HPLF, and induces the osteoblastic/cementoblastic differentiation of HPLF through calcium released from MTA.

View full abstract

Water delivered by dental unit water systems (DUWS) has been shown to contain high numbers of microorganisms. Biofilms accumulating on the inner surface of the DUWS tubing may be responsible for the high contamination levels of DUWS water. In the United States, the Centers for Disease Control and Prevention has proposed a guideline for DUWS water of ≤500CFU/ml and the American Dental Association has a guideline of ≤200CFU/ml. However, Japan has no evidence-based guidelines to control bacterial numbers in DUWS. We investigated the contamination of DUWS water in seventeen dental units in the Tsurumi University Dental Hospital in 2003, and considered the decontamination material, the material safety data and the application method. Next, we carried out "shock treatment" (a process that chemically removes the biofilm in the DUWS) in 2004. In the shock treatment, a solution was run through the entire DUWS tube at the end of the workday, and was left to sit overnight. At the beginning of the next workday, the lines were purged with ordinary flushing methods. This treatment was carried out on three consecutive nights. The counts from all sampled units ranged from 2.4×10²-6.1×10⁴CFU/ml even for those with DUWS filters or anti-suck-back devices present. Although contamination was reduced after water flushing of most dental units, some still had high contamination levels. Nevertheless, the shock treatment greatly reduced the contamination of the DUWS. However, a cock on the dental unit waterlines entrance and a device to run the solution into the DUWS tube were required, as well as measures to prevent clogging of the tube and water leakage. We also found that it is necessary to continue the periodic treatment, and so we have been regularly performing this treatment.

View full abstract