Masao Ozaki

Osteogenic potential for replacing cells in rat cranial defects implanted with a DNA/protamine complex paste.

Osteoinductive scaffolds are required for bone tissue engineering. The aim of the present study was to assess the osteoinductive capacity of deoxyribonucleic acid (DNA)/protamine complexes in a rat model of critical-size calvarial defects. In addition, we investigated whether cultured mesenchymal-like cells (DP-cells) outgrown from DNA/protamine complex engrafted defects could differentiate to become osteogenic cells in vitro. DNA/protamine complexes were prepared by reactions between DNA and protamine sulfate solutions with stirring. Critical-sized (8mm) calvarial defects were created in the central parietal bones of adult rats. Defects were either left empty or treated with DNA/protamine complex scaffolds. Subsequently, micro-computed tomography (micro-CT), histological, and immunohistochemical analyses were performed. Micro-CT and histological assays showed that DNA/protamine complex engrafted defects had enhanced bone regeneration. DP-cells were expanded from explants of DNA/protamine complex engrafted defects using an explant outgrowth culture system. Osteogenesis-related factors were assessed in DP-cells after treatment with an osteoblast-inducing reagent (OIR). After 3months, nearly complete healing was observed for DNA/protamine complex engrafted calvarial defects. Increased alkaline phosphatase (ALP) activity and Alizarin red staining were found for cultured DP-cells. These cells had high expression levels of osteogenic genes, including those for RUNX-2, ALP, osteopontin, and osteocalcin. These results indicated that DNA/protamine complexes could facilitate bone regeneration in calvarial defects. Moreover, in vitro osteogenic induction experiments showed that DP-cells outgrown from DNA/protamine engrafted defects had an osteogenic potential. Based on these results, we suggest that DNA/protamine complexes may recruit osteocompetent cells in these defects, where they differentiate to osteogenic cells.

Immunohistochemical expression of fibrillin‐1 and fibrillin‐2 during tooth development

BACKGROUND AND OBJECTIVE Oxytalan fibers are categorized as a microfibril assembly without elastin deposition, and are unique components in the periodontal ligament (PDL). However, little is known about their formation during PDL development. To clarify the mechanisms of oxytalan fiber formation in developing PDL, we performed immunohistochemical analysis to detect the direct expression of fibrillin-1 and fibrillin-2, which are major components of microfibrils. MATERIAL AND METHODS Frozen sections of lower molars from mice at several stages of growth were prepared without chemical fixation and decalcification using the film transfer method. Immunostaining was performed with anti-fibrillin-1 and -2, and anticytokeratin antibodies. RESULTS Fibrillin-1 was not expressed in the dental follicle during the crown forming stage. At postneonatal day 9, fibrillin-1 expression started with meshwork appearance between the epithelial cells from Hertwigs epithelial root sheath at the root dentin surface. Fibirillin-2 was detected much earlier than fibrillin-1 expression. Fibrillin-2 was expressed with a liner appearance, running parallel to the root axis in PDL, and was partially co-expressed with cytokeratin 14 expression in Hertwigs epithelial root sheath. Furthermore, we detected both fibrillin-1 and fibrillin-2 expression in human PDL. Fibrillin-1 was detected in fibers with a vertically oriented root axis in PDL. Fibrillin-2 was widely expressed in PDL, including around the epithelial cell rests of Malassez. Fibrillin-1 and fibrillin-2 were clearly co-expressed in thick fiber structures in human PDL. CONCLUSION Our results suggest that both fibrillin-1 and fibrillin-2 expression is required to form thick oxytalan fibers in PDL. Based on the expression patterns for fibrillin-1 and fibrillin-2, they have different functions during tooth root and PDL development. Early expression of fibrillin-2 may regulate dental epithelial cell behavior during root and PDL development.

The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Hertwig’s epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-β

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwigs epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-β regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-β was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-β1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-β1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-βsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.

Analysis of dental compensation in cases with mandibular lateral deviation using three-dimensional measurement of gnathostatics models

values were calculated for canine (deciduous canine) cusp, first premolar buccal cusp, second premolar buccal cusp, and first molar mesiobuccal cusp. Moreover, the width was measured between the mandibular and the maxillary palate in the buccal segment and the first molar mesiobuccal cusp, and the improvement and exacerbation groups were compared. Subsequently, the following results were obtained. (1) In the improvement group, the dental arch width increased when the first molars or second premolars made mesial movements after the loss of the deciduous molars. Moreover, in the improvement group, canines, first premolars, second premolars, and first molars for grade 6 children, compared to the exacerbation group, moved significantly to the buccal side. With regard to canines and first premolars, significant distal movement was observed. (2) At grade 6, the arch width was significantly wider compared to children in the exacerbation group. Especially, the width between the first molars was significantly wider in the improvement group in grade 3 than in the exacerbation group. (3) No significant differences were observed between the improvement and exacerbation groups in a leeway space amount, timing of the loss of the deciduous canines and the first primary molars, as well as the eruption timing of the canines, the first premolars, and the second premolars. From the findings, we conclude it is apparent that the crowding of the mandibular incisal region shows improvement. The reasons for this improvement are twofold: (1) an increase in the distal movement of the canines and first premolars; (2) an increase in the dentition width of the buccal segment and first molar in the exchange timing.

Management of the malposition tooth that was caused by dentigerous cyst associated with impacted supernumerary tooth by using an obturator

Abstract This report presents a case of eruption guidance for an upper right central incisor with a dentigerous cyst associated with an impacted supernumerary tooth in a 5-year-9-month-old boy. A swelling was seen in the region of an upper right central incisor. Radiographic examinations showed that a well-defined cystic lesion, containing an impacted supernumerary tooth crown in it, was located between the root of the primary upper right central incisor and a permanent upper central incisor germ. Under general anesthesia, the dentigerous cyst was removed at extraction of the upper impacted supernumerary tooth. And then, a socket was kept open wound. A histopathological examination confirmed the diagnosis of a dentigerous cyst associated with a supernumerary tooth. By using an obturator, we guided eruption of an upper right central incisor with malposition that was caused by the dentigerous cyst, and observed the way of the tooth eruption for two years and seven months. The obturator was inserted in the socket directly for three months until it was closed. After that, the obturator was used as only a removable space maintainer. Eight months after the operation, the obturator was removed because the upper left central incisor began to erupt. Two years and seven months after the operation, both upper central and lateral incisors had erupted completely. In this case, the obturator was used for expectation of natural eruption of permanent incisors, space maintenance and aesthetic recovery. The permanent incisors had been able to be guided to almost correct position without traction.

Measurement of bone conduction characteristics for transmitted vibration sounds of tooth drilling

Abstract Purpose In this study, we measured the vibration caused during dental treatment by an air turbine handpiece and a micro motor handpiece. We then analyzed the obtained data to develop a method for noise cancellation of such vibration during the treatment of tooth decay. We herein describe the audio characteristics of bone conduction sound made by teeth during dental treatment. We measure vibration sounds of an air turbine handpiece and a micro motor handpiece transmitted from teeth to the middle and/or inner ear and obtain its bone conduction characteristics for reducing uncomfortable dental treatment sounds using active noise control technique in the future. Methods First, we measure the frequency characteristics of both acoustic sounds and vibration sounds of two dental handpieces in a special treatment room. Second, we measure the frequency characteristics of bone conduction from teeth to the middle and/or inter ear by actuating a tooth with pure tones of several frequencies in an anechoic chamber. Results The basic rotational frequency of an air turbine head tip with no-load was about 5,500 Hz. A decline of the rotational frequency was confirmed at the turbine head when the bar came in contact with the tooth, and it was proven that the tooth received the effect of the vibration, when the head made contact with the tooth. The results showed that the micro motor handpiece speed varied from a low of 140 to 210 Hz to a medium speed of 280 Hz and a high speed of 700 to 770 Hz. These results were higher than manufacturers specifications of micro motor handpiece. The results of the bone conduction amplitude-frequency characteristics were the best audible amplitude-frequency was near 2,000 Hz in the upper and lower left central incisors.

Assessing anxiety in pre-school dental patients with a doll placement test

The prospect of dental treatment causes many young patients to be anxious and/or afraid. Because it is helpful to know in advance how much anxiety a child is experiencing, we have devised a test in which the child places dolls in a miniature dental office. We used this model to study 48 children, aged three to five years, who visited our paediatric dental office. We prepared a doll-house-sized model of a dental office with a dental chair, a dentist, and dental hygienist. We asked children to place two dolls, one a self-doll and the other a parent/guardian-doll at a location of their choosing in the model clinic. The positions children chose to place the dolls helped us to gauge the anxiety they were experiencing as they anticipated dental treatment. The majority of children we tested were not anxious, and they placed the self-doll in the dental chair. In contrast, we observed that many children who did not place the self-doll on the dental chair were uncooperative during subsequent dental treatment. Thus, our doll-placement test is a valuable predictor of the anxiety of children who will receive dental treatment. Although how children placed the parent/guardian-doll also provided useful information, we gained the most valuable information from whether or not the child put the self-doll on the dental chair.