Etsuo Kasahara

Root canal system of the mandibular incisor

To better assess the efficiency of the mechanical preparation of root canals, 1085 transparent specimens of extracted mandibular incisors were examined for canal configuration, thickness and curvature of the root canals, condition of any accessory canals, and location of the apical foramen. Greater than 85% of the root canals possessed a single canal (Type I). Of specimens in which furcation was observed, only 3% possessed two separate canals (Type III and IV). Fewer than 30% of the specimens showed accessory canals that were mechanically impossible to clean. The majority of the lateral branches were small, greater than 80% of the specimens were smaller than a #15 reamer, and none of the branches were larger than a #30 reamer. Although apical foramina located distal to the apex were observed in about 50% of the specimens, 83.6% of all apical foramina were within 0.5 mm of the apex, and 99.5% were within 1.0 mm. Data on the thickness of the root and main canal in the apical portion and curvature of the root canal suggest that for adequate apical preparation, a #40 reamer must be able to reach the apical constriction.

Root Canal System of the Maxillary Central Incisor

To better assess the efficacy of mechanical preparation of root canals, transparent specimens of 510 extracted maxillary central incisors were investigated for thickness and curvature of the root canal, condition of any accessory canals, and location of the apical foramen. Over 60% of the specimens showed accessory canals that are impossible to clean mechanically. Most lateral branches were small, 80% were the size of a #10 reamer or less, and only 3% were thicker than a #40 reamer. Apical foramina located away from the apex were observed in 45% of the teeth, although nearly 80% of all foramina were within approximately 0.5 mm of the apex, and 95% were within approximately 1.0 mm. Data on the thickness and curvature of the main canal showed that normally it is adequately prepared when reached with a #60 reamer to the apical constriction and supplemented by flare preparation.

Hard Tissue Formation in Subcutaneously Transplanted Rat Dental Pulp

While dental pulp appears to be able to form mineralized matrices that do not always resemble dentin, the precise characteristics of the hard tissue and the mechanism of its induction remain unknown. Therefore, we evaluated hard tissue induced by transplantation of pulp into subcutaneous tissue. Seven days after transplantation, initial hard tissue was formed at the inner periphery of the pulp. After 14 days, this hard tissue expanded inwardly. Mineralized matrix was immunopositive for osteocalcin, osteopontin, and bone sialoprotein, but negative for dentin sialoprotein. Transplantation of GFP-labeled pulp into wild-type rats showed these formative cells to have been derived from the transplant. TEM observation revealed apatite crystals within necrotic cells and matrix vesicles at the initial stage of calcification. These results indicate that pulp cells possess the ability to form a bone- or cementum-like matrix. Calcification of the matrix may occur in necrotic cells and matrix vesicles, followed by collagenous calcification.

Immunohistochemical Localization of α-Smooth Muscle Actin During Rat Molar Tooth Development

The dental follicle contains mesenchymal cells that differentiate into osteoblasts, cementoblasts, and fibroblasts. However, the characteristics of these mesenchymal cells are still unknown. α-Smooth muscle actin (α-SMA) is known to localize in stem cells and precursor cells of various tissues. In the present study, to characterize the undifferentiated cells in the dental follicle, immunohistochemical localization of α-SMA was examined during rat molar tooth development. Rat mandibles were collected at embryonic days (E) 15-20 and postnatal days (P) 7-28. Immunohistochemical stainings for α-SMA, periostin, Runt-related transcription factor-2 (Runx2), tissue nonspecific alkaline phosphatase (TNAP), and bone sialoprotein (BSP) were carried out using paraffin-embedded sections. α-SMA localization was hardly detected in the bud and cap stages. At the early bell stage, α-SMA-positive cells were visible in the dental follicle around the cervical loop. At the late bell to early root formation stage (P14), these cells were detected throughout the dental follicle, but they were confined to the apical root area at P28. Double immunostaining for α-SMA and periostin demonstrated that α-SMA-positive cells localized to the outer side of periostin-positive area. Runx2-positive cells were visible in the α-SMA-positive region. TNAP-positive cells in the dental follicle localized nearer to alveolar bone than Runx2-positive cells. BSP was detected in osteoblasts as well as in alveolar bone matrix. These results demonstrate that α-SMA-positive cells localize on the alveolar bone side of the dental follicle and may play a role in alveolar bone formation.

Immunohistochemical expression of heat shock protein27 in the mouse dental pulp after immediate teeth separation

AimAfter immediate teeth separation, expression of HSP27 in the mouse dental pulp was examined. Immunohistochemistry was performed to examine the incidence of HSP27 expression.Materials and methodsA total of 36 8-week-old ddY mice were used as experimental subjects and a wedge was inserted in between maxillary right molars. The wedge was removed 30 min or 3 h after insertion. Animals were immediately sacrificed after the removal of wedge or until 1 week later and serial sections from paraffin-embedded tissues were prepared. Immunohistochemistry was carried out to examine the expression of HSP27. The untreated side served as the control.ResultsIn the control group, the endothelial cells and some pulp fibroblasts weakly expressed HSP27 suggesting that the expression is due to mechanical stress brought about by physiological masticatory force and pressure from the tongue. In both 30 min and 3 h experimental groups, HSP27 expression was highest at 24 h after wedge removal and the expression remained the same or started to decrease thereafter. The expression decreased at the same level as that of the control group 1 week after wedge removal.ConclusionHSP27 may serve as an indicator of stimulus strong enough to show its expression.

Immunohistochemical Study of Osteodentin in the Unerupted Rat Incisor

To characterize osteodentin in the pre-functional rat incisor, we performed histological and histochemical evaluation of the anterior apex of the incisor in 3-day-old rats. The unerupted incisor was composed of osteodentin, with numerous cells present in the anterior apex. The osteodentin was immunopositive for osteocalcin, bone sialoprotein, and dentin sialoprotein, with an immunolocalization pattern similar to that of dentin. Back-scattered electron microanalysis (BSE) and electron probe microanalysis (EPMA) showed that osteodentin was not uniformly calcified. These results indicate that osteodentin in the rat incisor possesses dentin-like characteristics, and may be fragile in structure.

Immunohistochemical expression of hard tissue related factors in the mouse dental pulp after immediate teeth separation

We examined change of Runx2 and ALP expression in mouse tooth pulp which exposed to teeth separation experiment by immunohistochemistry as a model for conservative dentistry treatment. 8-week-old 36 male ddY mice were used and wedge was inserted between upper 1st and 2nd molars. The wedge was removed 30 minutes as well as 3 hours after the insertion and the samples were prepared extending up to 1 week of time period for regular histopathological and immunohistochemical examinations for ALP and Runx2 expression. The opposite sides without wedge insertion were taken as controls. In the control group pulp, weak expressions of Runx2 and ALP in the vessel endothelial cells as well as the pulp cells were revealed, suggesting the appearance of these genes upon mechanical stress induced by mastication and tongue pressure etc. On the other hand in the experiment group, Runx2 expression increased both in 30-minute and 3-hour teeth separation group. The expression became maximum at 24 hours. Then it gradually decreased and became similar level with the control group at 1-week after the wedge insertion. Similarly ALP expression increased after the wedge insertion and was maximum at 24 hours and then gradually decreased to the levels similar with the control group. These results suggest that when immunohistochemical expression of Runx2 as well as ALP was used as an index, no severe damage occur upon clinical application of wedge insertion.

Potential of Periodontal Ligament Cells to Regenerate Alveolar Bone

Regeneration of periodontal tissues, lost as a result of periodontal disease, is a key objective of periodontal treatment. Although several periodontal regeneration therapies have been devised, the origin of the undifferentiated cells that regenerate periodontal tissues remains unknown. Therefore, in the present study, to clarify the existence of osteoblast progenitor cells in the periodontal ligament, as well as to investigate the mechanism of alveolar bone regeneration without any effects from the original bone, we evaluated osteoblast differentiation induced by transplantation of GFP-transgenic rat molars into the subcutaneous tissues of wild-type rats. Ten days after transplantation, initial alveolar bone was formed apart from the cementum in the bifurcation region. After 20 days, this bone tissue had expanded to almost all of the bifurcation. GFP localization showed that the osteoblasts were derived from the transplant. Alpha-SMA- and BMP4-positive cells were observed near the root surface at 5 days after transplantation. With the progress of alveolar bone regeneration, osteoblasts expressing Runx2 and Osterix appeared in the bone-forming region. These results indicate that periodontal ligament tissue remaining on the root surface after a tooth extraction contains undifferentiated cells that have the ability to regenerate alveolar bone. The process of osteoblast differentiation in this model might be similar to that for normal alveolar bone formation. Thus, periodontal ligament cells might be useful for the regeneration of alveolar bone in tissue engineering applications.

An evaluation of guidelines for the apical enlargement of maxillary premolars

A newly proposed set of experimental guidelines for the final size of apical enlargement was evaluated by instrumenting filling and evaluating a series of extracted maxillary premolars. The guidelines were applied to differing anatomical configurations of this type of tooth, and to teeth in which instrumentation terminated at various distances from the foramen. Comparison was made with the conventional guideline of enlarging to three sizes greater than the first instrument that binds, with instrumentation to 0.5 mm from the foramen. All teeth were vacuum injected with India ink and dried, mechanically enlarged according to the experimental or conventional guidelines, and examined for the presence of apically extruded debris. The teeth were then filled, cleared, and visually inspected for the condition of preparation in the apical portion. For the conventional guideline, preparation was generally judged as poor, and debris was usually forced through the foramen. With the experimental guidelines, adequate preparation was obtained for most canal configurations at 0.5 and 1 mm from the foramen, and at 1 mm the incidence of extruded debris fell sharply. Anatomical conditions possibly contraindicating the experimental guidelines were found and characterized.

Histopathological Safety Evaluation of Newly-Developed MgO Sealer

We aimed to evaluate the subcutaneous tissue reaction to a newly-developed MgO Sealer for root canals. We injected the experimental material and three existing control materials into the dorsal area of 43 male ddY mice. One week and 12 weeks after embedding, the tissue surrounding the embedding sites was removed and histopathological examination was performed. The results demonstrate that the basic histopathological reaction is the formation of fibrous capsules consisting of granulation tissue around the experimental and control embedded materials. Based on our results, we believe that the newly-developed MgO Sealer is as safe as the existing control materials and can be considered for dental use as a root canal sealer.