Junichi Watahiki

Effects of mastication on mandibular growth evaluated by microcomputed tomography.

It is well known that mastication has a significant influence on mandibular growth and development, but the mechanism behind this effect has not yet been clarified. Furthermore, no studies have examined the effects of changes in mastication on the three-dimensional (3D) morphometry of the mandible. The aim of the present study was to investigate the influences of changes in mastication on mandibular growth and morphology. Twenty-five 3-week-old (at the time of weaning) imprinting control region mice were randomly divided into three groups: mice fed a hard diet (HD), mice fed a soft diet (SD), and mice alternately fed hard and soft diets (HSDs) every week for 4 weeks. The morphometry of the mandible was analysed using 3D microcomputed tomography (muCT). Statistical analysis was undertaken using a t-test. muCT analysis showed that the condylar width was significantly greater in the HD group than in the SD group after 1 week. After 4 weeks, mandibular length was significantly longer and ramus height was greater in the HSD group than in the other two groups. Bone volume was significantly less in the SD group than in the other two groups after 4 weeks. These findings suggest that changes in mastication markedly affect mandibular condylar cartilage growth and mandibular morphology. It is considered that dietary education at an early age is important in order to prevent disruption of the development of the mandible.

A New Method for Alveolar Bone Repair Using Extracted Teeth for the Graft Material

BACKGROUND In the clinical field of jawbone formation, the use of autogenous bone as the graft material is the gold standard. However, there are some problems with this technique, such as risk of infection on the donor side, the limited amount of available bone mass, and marked resorption of the grafted bone. We investigated the potential for using teeth as a bone graft material for jawbone formation because the dental pulp contains stem cells, including undifferentiated neural crest-derived cells. METHODS Alveolar bone defects were created in Wistar rats, and the defects were filled with either tooth or iliac bone graft material, or left as controls. The potential for using teeth as a bone graft material for jawbone formation was measured using real-time polymerase chain reaction, microcomputed tomography, and histologic analysis. RESULTS Polymerase chain reaction revealed that the expressions of P75, P0, nestin, and musashi-1 were significantly higher in teeth than in mandibular bone and iliac bone grafts. Hematoxylin and eosin staining and microcomputed tomography showed that at 8 weeks, tooth graft material produced a similar amount of new bone compared to iliac bone graft material. Osteopontin was expressed in both the tooth and iliac bone graft material at 6 and 8 weeks after surgery. Dentin sialoprotein was expressed in the tooth graft material in the new bone at 6 weeks only. CONCLUSION These results indicate that teeth may be an alternative material to autogenous bone for treating alveolar bone defects by grafting.

Gene Expression Profiling of Mouse Condylar Cartilage during Mastication by Means of Laser Microdissection and cDNA Array

Little is known about the mechanisms of mandibular condylar growth. In this study, gene expression in the mandibular condylar cartilage of young post-natal mice was monitored by means of a cDNA microarray, real-time PCR, and laser microdissection before and after the initiation of mastication (newborn, 7 days, 21 days, initiation of mastication, and 35 days). Insulin-like growth factor-1 (IGF-I), transforming-growth-factor-beta-2 (TGFbeta2), and aggrecan mRNAs were clearly expressed at 21 days, while the expression of osteopontin mRNAs was most clear at 35 days. Parathyroid-hormone-related protein (PTHrP), Indian-hedgehog (Ihh), and insulin-like growth factor-2 (IGF-2) mRNAs were clearly expressed during lactation (newborn and 7 days). Heat-shock-protein 84 (HSP-84) and heat-shock-protein 86 (HSP-86) were clearly expressed at 35 days. These results revealed that gene expression changed during mandibular condylar cartilage growth, and that, interestingly, these changes coincided with the initiation of mastication.

Differences in the developmental origins of the periosteum may influence bone healing

BACKGROUND AND OBJECTIVE The jaw bone, unlike most other bones, is derived from neural crest stem cells, so we hypothesized that it may have different characteristics to bones from other parts of the body, especially in the nature of its periosteum. The periosteum exhibits osteogenic potential and has received considerable attention as a grafting material for the repair of bone and joint defects. MATERIAL AND METHODS Gene expression profiles of jaw bone and periosteum were evaluated by DNA microarray and real-time polymerase chain reaction. Furthermore, we perforated an area 2 mm in diameter on mouse frontal and parietal bones. Bone regeneration of these calvarial defects was evaluated using microcomputed tomography and histological analysis. RESULTS The DNA microarray data revealed close homology between the gene expression profiles within the ilium and femur. The gene expression of Wnt-1, SOX10, nestin, and musashi-1 were significantly higher in the jaw bone than in other locations. Microcomputed tomography and histological analysis revealed that the jaw bone had superior bone regenerative abilities than other bones. CONCLUSION Jaw bone periosteum exhibits a unique gene expression profile that is associated with neural crest cells and has a positive influence on bone regeneration when used as a graft material to repair bone defects. A full investigation of the biological and mechanical properties of jaw bone as an alternative graft material for jaw reconstructive surgery is recommended.

Soft-diet feeding after weaning affects behavior in mice: Potential increase in vulnerability to mental disorders.

Mastication is one of the most important oral functions, and the period during which mastication is acquired overlaps with the term of rapid development and maturation of the neural systems. In particular, the acquisition period after weaning is related to the potential onset of mental disorders. However, the roles of mastication during this period for brain development remain largely unknown. Therefore, we used a series of standard behavioral analyses, assessment of hippocampal cell proliferation, and the expression of brain-derived neurotrophic factor (BDNF), TrkB, and Akt1 in the hippocampus and frontal cortex of mice to investigate the effects of post-weaning mastication on brain function. We fed 21-day-old C57BL6/J male mice either a hard or a soft diet for 4weeks and conducted a series of standard behavioral tests from 7weeks of age. Further, histological analysis with bromodeoxyuridine was performed to compare hippocampal cell proliferation at 7 and 14weeks of age. Real-time polymerase chain reaction was performed to compare BDNF, TrkB, and Akt1 expression in the hippocampus and frontal cortex of 14-week-old mice. Compared to mice fed a hard diet (HDM), soft-diet mice (SDM) showed behavioral impairments, including decreased home cage activity, increased open field test activity, and deficits in prepulse inhibition. These results were similar to those observed in mouse models of schizophrenia. However, no effects were observed on anxiety-like behaviors or memory/learning tests. Compared to HDM, SDM showed significantly decreased hippocampal cell proliferation and hippocampal BDNF and Akt1 gene expression at 14weeks of age. A soft diet after weaning may have resulted in histological and molecular changes in the hippocampus and influenced outcomes of behavioral tests related to mental disorders. Our findings suggest that soft-diet feeding after weaning may affect both physical and mental development of mice, and may increase vulnerability to mental disorders.

Identification of differentially expressed genes in mandibular condylar and tibial growth cartilages using laser microdissection and fluorescent differential display: Chondromodulin-I (ChM-1) and tenomodulin (TeM) are differentially expressed in mandibular condylar and other growth cartilages

Mandibular condylar cartilage can be distinguished from articular and growth cartilages of long bones based on several differences in morphology, physiology, and function between these structures. However, there is almost no information available on the types of genes that contribute to these differences. In this study, genes that were differentially expressed in mandibular condylar and growth cartilages in 1-week-old rats were investigated using fluorescent differential display (FDD) and laser microdissection (LMD). A number of genes were identified by FDD including chondromodulin-1 (ChM-1), which is known to be an angiogenesis inhibitor of endochondral ossification. ChM-1 expression was then compared with that of tenomodulin (TeM) in mandibular condylar and tibial cartilages of 1- and 5-week-old rats using real time PCR (RT-PCR), immunohistochemistry, and in situ hybridization. There was negligible detection of ChM-1 mRNA and protein in mandibular condylar cartilages compared to tibial cartilages of 1- and 5-week-old rats. On the other hand, TeM mRNA was more abundant in mandibular condylar cartilage than in tibial. These observations demonstrated that gene expression in mandibular condylar cartilage differed from other types of cartilage such as articular and growth ones.

Localization and osteoblastic differentiation potential of neural crest-derived cells in oral tissues of adult mice

In embryos, neural crest cells emerge from the dorsal region of the fusing neural tube and migrate throughout tissues to differentiate into various types of cells including osteoblasts. In adults, subsets of neural crest-derived cells (NCDCs) reside as stem cells and are considered to be useful cell sources for regenerative medicine strategies. Numerous studies have suggested that stem cells with a neural crest origin persist into adulthood, especially those within the mammalian craniofacial compartment. However, their distribution as well as capacity to differentiate into osteoblasts in adults is not fully understood. To analyze the precise distribution and characteristics of NCDCs in adult oral tissues, we utilized an established line of double transgenic (P0-Cre/CAG-CAT-EGFP) mice in which NCDCs express green fluorescent protein (GFP) throughout their life. GFP-positive cells were scattered like islands throughout tissues of the palate, gingiva, tongue, and buccal mucosa in adult mice, with those isolated from the latter shown to form spheres, typical cell clusters composed of stem cells, under low-adherent conditions. Furthermore, GFP-positive cells had markedly increased alkaline phosphatase (a marker enzyme of osteoblast differentiation) activity and mineralization as shown by alizarin red staining, in the presence of bone morphogenetic protein (BMP)-2. These results suggest that NCDCs reside in various adult oral tissues and possess potential to differentiate into osteoblastic cells. NCDCs in adults may be a useful cell source for bone regeneration strategies.

Soft-diet feeding reduces prepulse inhibition in young mice after weaning

ferent developmental stages. In the present study, a comprehensive battery of behavioral tasks was applied to longitudinally characterize behavioral phenotypes in NRG1 and wild-type mice. All NRG1 heterozygous mice and their wild-type littermate controls were generated from NRG1 heterozygous breeding pairs. A battery of behavioral tasks (including locomotor activity, hole broad, elevated plus maze, object recognition, social preference test, two-bottle sucrose preference, pre-pulse inhibition, hot plate, and trace fear conditioning) was performed sequentially on the same batch of male and female mice in 2 months of age (postnatal day 63–81) and 6 months of age (postnatal day 189–210). Our preliminary data revealed that NRG1 mutant mice displayed an overall normal behavioral profile in these 2 ages. However, NRG1 deficient females exhibited hyperactivity in the open field in the age of 6 months compared with their wild-type controls. These preliminary findings suggest that NRG1 deficiency might have minor impact on basal behaviors. Further experiments are in progress.