Until now the developmental process of masticatory muscle (derived from the branchial arches) is not well documented. Thus, in order to clarify the developmental process of murine masseter muscle, Hepatocyte Growth Factor (HGF), Insulin-like Growth Factor-1 (IGF-1) and Myosin Heavy Chain (MyHC) were evaluated by histological examination, western blotting and RT-PCR analyses on embryonic days 14 (E14), 16 (E16) and 18 (E18). E14 showed that the mandibular bone development is initiated by the appearance of the ossification centre in the peripheral zone of Meckel cartilage. Whereas, E16 demonstrated presence of developing masseter muscle at the lower part of the mandible composed of immature bone. On the contrary, E18 showed larger masseter muscle and more mature mandibular than E16. Interestingly, both IGF and HGF proteins and genes increased at initial stage of masseter muscle development, and decreased with the time. A significant change in composition of MyHC was detected in E18 compared with E14 and E16. This result indicate that murine masseter muscle develops in accordance with the surrounding tissue, where IGF-1 and HGF could be an important modulator of stem cell-derived myoblast proliferation and myogenic differentiation, and MyHC-2a and 2d are strongly expressed by mature muscle fibres during the developmental process of the masseter muscle.

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Apatites with different level of crystallinity, Ca-deficient hydroxyapatite (CDHA) powder with low crystallinity and the mixture of carbonated hydroxyapatite (CHA) and CDHA powder with higher crystallinity, were prepared through from Ca-EDTA complex. Porous composite scaffolds of poly(lactide-co-glycolide) (PLGA) and apatites were manufactured by solution casting/particles leaching method. The biological properties of two composites, composite scaffold A (PLGA and CDHA with low crystallinity) and composite scaffold B (PLGA and CHA/CDHA mixture with higher crystallinity) were examined. The MC3T3E-1 osteoblast-like cells were cultures towards the composite scaffolds A and B. Morphological observation by scanning electron microscope revealed that cells firmly attached inside the micropores of the composite scaffold A, but that a few cells were present inside the composite scaffold B, although there were no significant difference in the cell viability between composite scaffold A and B. Two porous composite scaffolds A and B were implanted into the cortical bone of tibiae of rabbits. After 4 weeks’ implantation, bone response towards porous composite was histologically evaluated. High-resolution microfocus X-ray computed tomopraphy observation and histological appearances revealed that composite scaffold A provided greater degree of new bone formation than composite scaffold B. The original drill hole was partly filled with new bone in composite scaffold A. In conclusion, PLGA/apatite porous composite scaffold with low level of crystallinity apatite is one of promising materials for bone substitutes or scaffold materials for bone tissue engineering.

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Enhancement of osteoblastic adhesion and migration on a titanium (Ti) surface increases the successful rate of osseointegration in implant therapy by stimulating osteoblastic differentiation. It has been reported that Ti surfaces coated with fibronectin (FN) or GRGDSP peptide derived from FN had improved initial cell attachment and supported osteoblastic cell differentiation. However, the differences in gene expression by osteoblasts grown on FN and GRGDSP coated Ti disks have not been elucidated. Ti disks were coated with FN or GRGDSP peptide, then MC3T3-E1 osteoblastic cells were cultured on those surfaces. After 15 days, the levels of gene expression of the cells were examined. Those levels were mostly similar between the FN and GRGDSP coated disks, though that of discoidin domain receptor-1 (DDR-1) was significantly greater in cells cultured on the FN coated disks. The elevated mRNA level of the DDR-1 gene was then confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR assays. In addition to the ability of the RGD domain to bind with integrins, FN also binds to fibrillin, proteoglycan, and collagen by their specific binding domains, thus the higher level of DDR-1 gene expression seen in our experiments may have been induced by those and not by the RGD domain. For future developing biomaterial as coating for Ti implants using synthetic peptides, binding domains other than the RGD domain in FN may be useful and important.

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Ameloblastoma (AB) is the most common odontogenic epithelium-derived tumor, but has unclear etiology. Various ligand/receptor systems are implicated in ameloblastoma development. In this study, we explored the expression of erythropoietin (EPO), platelet-derived growth factor (PDGF) and their receptors (EPOR, PDGFR) in ABs, and the potential origins of ABs from keratocystic odontogenic tumors (KCOTs) and tooth germs in control groups. Immunostaining was performed using antibodies for EPO, EPOR, PDGF-A and PDGFR-α. Statistical analysis was performed based on immunoreactivity. We found that EPOR expression was significantly higher in ABs than in KCOTs (P<0.05), but was similar to that of tooth germs. EPOR expression was also significantly different between the AB subtypes (P<0.05). Nevertheless, EPO expression was only significantly different between tooth germs and KCOTs (P<0.05). Immunoreactivity for PDGFR-α and PDGF-A was stronger in ABs than in KCOTs and tooth germs. However, significant differences were only found between individual groups or among types or subtypes of ABs (P<0.05 or P<0.01). In addition, the expression of both EPOR and PDGFR-α in the recrudescent ABs was significantly greater than in primary ABs (P<0.01). We conclude that EPO, EPOR, PDGF-A and PDGFR-α are essential for the growth of human teeth as well as for oncogenesis, development, cell differentiation and biological behavior of odontogenic neoplasm.

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The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ, lineage committed progenitor cells, or precursor cells for osteoblasts. In this study, we investigated gene expression in human dental follicle cells (hDFC) during osteogenic differentiation in vitro. hDFC were cultured with mesenchymal stem cell osteogenic induction medium (MSCOIM) or growth medium (MSCGM). The ability of hDFC to differentiate into osteoblasts was examined using the alizarin red S and von Kossa stains, and alkaline phosphatase (ALP) activity. Gene expression profiling was performed with oligonucleotide microarray. Then, mRNA levels were confirmed using real time-PCR. The morphology of hDFC cultured with MSCGM exhibited fibroblast-like spindle shapes and became cuboidal shaped in MSCOIM for 3 days. Calcium deposition was observed in hDFC cultured with MSCOIM by staining with alizarin red S and von Kossa. ALP activities increased in hDFC during osteogenic differentiation. Expression monitoring of 54,675 genes in hDFC cultured for 3 days identified 951 genes with a greater than twofold difference in intensity between hDFC cultured with MSCOIM and MSCGM; 522 genes were up-regulated and 429 genes were down-regulated. Gene expression of IGF-II and IGFBP-2 increased during the hDFC differentiation into osteoblasts. Gene expression profile analysis revealed an interesting feature of hDFC in the initiation phase of osteogenic differentiation. IGF-II and IGFBP-2 also have important roles in hDFC during differentiation. Here we suggest that the dental follicle may serve as a therapeutic cell reservoir for bone regeneration.

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This study used micro-computed tomography (Micro-CT) to evaluate occlusal dental caries non-destructively. Twenty-seven extracted molar teeth were scanned using Micro-CT, and 3-D images were reconstructed from the data. The extent of caries was evaluated from images extracted at various angles, and recorded as 2-D images. To assess the validity of Micro-CT, six teeth were randomly selected and cut approximately 100-μm-thick sections in the same plane as that evaluated using Micro-CT. The specimens were examined histologically and with contact microradiograms (CMR). The fine structure was observed using Micro-CT. The Micro-CT image was equivalent to the histological examination or CMR. In all, 78 measuring points in all of the teeth were analyzed using KaVo DIAGNOdent, a laser-based caries-detection device. Spearman’s rank correlation coefficient between the DIAGNOdent score and the extent of caries was 0.70, and suggested a good correlation between DIAGNOdent and Micro-CT for examining occlusal caries. The sensitivity and specificity were calculated for DIAGNOdent using Micro-CT as the ‘gold standard’. The sensitivity and specificity for enamel caries were 0.74 and 1.00, respectively. The values for dentin caries were 0.64 (sensitivity) and 0.91 (specificity). These results were similar to those of recent studies using histological examination as the gold standard. We concluded that Micro-CT is useful for non-destructive examination in the evaluation of occlusal caries.

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Cementum is divided into acellular cementum and cellular cementum based on the absence and presence of cementocytes in the cementum matrix. Since the distribution of acellular and cellular cementum is affected by physical conditions such as aging and pathological conditions like inflammation and hyper occlusal pressure causing hypercementosis. Moreover, it has been reported in many text books that acellular and cellular cementum distribute irregularly. However, it is generally known that acellular cementum is located from the cervical margin to the cervical half or one-third and cellular cementum is observed from the apical half or two-thirds to the apical portion. In the present study, we have observed labio-lingual grinding sections of extracted human permanent teeth used for student practice (50 incisors and 78 molars) and classified the distribution pattern of acellular and cellular cementum into five types as following: 1) Typical distribution type of the acellular and cellular cementum as explained above, 2) Type with thick acellular cementum at the cervical portion. In this type, thick acellular cementum is observed at the cervical portion, although the acellular and cellular cementum is distributed as same as type 1 except in the cervical portion, 3) Type with thick cellular cementum at the cervical portion. This type showed that thick cellular cementum is distributed at the cervical portion instead of acellular cementum in type 2, 4) Type with acellular cementum from the cervical area to apical portion. This type showed acellular cementum covering from the cervical to apical portion of the tooth root surface, although the cellular cementum was located near the dentino-cement junction, 5) Type with cellular cementum from the cervical to apical portion. Cellular cementum distributed from the cervical to apical portion of the tooth root surface, although the acellular cementum is located inside the surface cellular cementum. In type 5, we found holes of various sizes and shapes in the hypertrophied cementum. Since these holes were large and cement canaliculus surrounding the holes extended toward the holes, it is suggested that the hypertrophied cementum enfolded a part of the periodontal ligament.

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It has been reported that simvastatin (SV), which is a cholesterol synthesis inhibitor and a therapeutic drug for hypercholesterolemia, stimulates bone morphogenetic protein 2 (BMP-2) expression in osteoblasts. The purpose of this study is to evaluate the efficacy of oral administration of SV on the bone healing process in the cortical bone defects of the tibiae of ovariectomized (OVX) rats. Cortical bone defects (1mm diameter) of the tibia were created through the medial cortex and medulla. After 1 day of operation, SV/physiological saline solution (10mg/kg) was orally administrated as experimental group. Only physiological saline solution was orally administrated as sham group. OVX rat was used as a model of osteoporosis. There were no significant differences in body weight, calcium and phosphorus concentration and alkaline phosphatase activity in serum between SV and sham groups during 3 weeks of administration. On the contrary, the serum level of total cholesterol of SV group was significantly lower than sham group. After 3 weeks of administration, the tibia bone was excised. Micro high-resolution microfocus X-ray computed tomography showed the formation of new bone in SV group. Quantitative analysis for bone mineral density (BMD) was performed through three-dimensional images. BMD in SV group was significantly higher than sham group. Histological sections stained with hematoxylin and eosin were observed under light microscope. In the sham group, thin granulation tissue with small amount of new bone and blood clot were observed inside the defects. A few osteoblasts were identified around new bone. On the contrary, the bone defect areas were filled with new bone in SV group Cand a greater number of osteoblasts was observed. New bone was also identified inside bone marrow. Interestingly, in SV group there were fewer amount of osteoclast-like giant cells around newly bone compared with the sham group. Immunostaining for osteocalcin, osteonectin, osteopontin, BMP-2 and BMP-4 was performed to investigate the mechanism of the efficacy of bone healing process by SV administration. SV group showed stronger osteoblastic protein expression than sham group after immunostaining. The positive cell rates of SV group for anti-osteocalcin, anti-osteonectin, anti-BMP-2 and anti-BMP-4 antibodies were significantly greater than sham group. In conclusion, the present results revealed that oral administration of SV might provide a beneficial effect on bone healing process in OVX rats, which could be a promising drug for bone regeneration in osteoporosis therapy.

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