Anatomical study of the zygomaticofacial foramen and zygomatic canals communicating with the zygomaticofacial foramen for zygomatic implant treatment: a cadaver study with micro-computed tomography analysis.

In the present study, anatomical assessment of zygomaticofacial foramina (ZFFs) and zygomatic canals communicating with ZFFs were performed using cadaver micro-computed tomography images. It was suggested that all ZFFs were located above the jugale (Ju)-zygomaxillare (Zm) line, which is the reference line connecting the Ju and Zm, and most were located in the zygomatic body area (ZBA). The anteroposterior position of the ZFF in the ZBA was within a middle to posterior region and was most often located slightly posteriorly in males and closer to the middle of the region in females. The mean distance from the Ju-Zm line to the ZFF in the ZBA was 12.36 mm (standard deviation [SD] 1.52 mm) in males and 11.48 mm (SD 1.61 mm) in females. In zygomatic canals communicating with ZFFs, most zygomatic canals were type I canals, communicating from the zygomaticoorbital foramen and harboring the zygomaticofacial nerve, and the others were type II canals, communicating from the zygomaticotemporal foramen and located near the posterior margin of the frontal process. These results provide useful anatomical information for preventing nerve injury during surgical procedures for zygomatic implant treatment.

Local Irradiation of Mouse Tooth Germ Gives Insight into the Direct Effects of Irradiation on Root Development.

To elucidate the mechanism underlying the failure of root formation after irradiation, we established a method of local irradiation of the molar tooth germ and demonstrated that radiation directly affected dental root development. In the current study, to locally irradiate the lower first molars of 5-day-old C57BL/6J mice, we used lead glass containing a hole as a collimator. We confirmed that our local irradiation method targeted only the tooth germ. The irradiated root was immature in terms of apical growth, and dentin formation was irregular along the outside of the root apices. Moreover, calcified tissue apically surrounded Hertwig's epithelial root sheath, which disappeared abnormally early. This method using a local irradiation experimental model will facilitate research into radiation-induced disorders of dental root formation.

Hmga2 regulation of tooth formation and association with Sox2 and Nanog expression.

Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2-/- mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2-/- mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development.

The effect of the angle of acuteness of additive manufactured models and the direction of printing on the dimensional fidelity: clinical implications.

The influence of the acuteness of the model angle and the direction of printing was investigated on different three-dimensional (3D) printers to understand the fidelity of 3D printing. A Polyjet printer and two fused deposition modeling printers were used in the present study. Computer-aided design (CAD) models were designed in the form of a triangular prism with a height of 15 mm and with varying angles of 60°, 45°, 30°, 20°, 10°, and 5°. From the CAD designs, additive manufacturing (AM) models were printed in three different directions by each of the three printers. To investigate the global fidelity of the 3D printers, the height and surface outlines of the AM model cross sections were measured. The fidelity of the AM models with increasingly acute angles was not accurately reproduced when 3D printed, and the surface outlines of the AM model cross sections were different in each direction of printing for each device. Printing technology and printing direction need to be considered if accurate reproduction of acute angles in fine AM structures in medical models is a necessity.

Isolation and characterization of embryonic ameloblast lineage cells derived from tooth buds of fetal miniature swine.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Anatomical examination of the fibula: digital imaging study for osseointegrated implant installation.

BACKGROUND: Free vascularized fibular flaps are commonly used in jaw reconstruction. CT scan images of the fibula are used in digital planning of jaw reconstruction. In order to fully describe the anatomy of the fibula, an imaging study of the fibula was undertaken. The purpose of the present study was to examine the anatomical structure of the fibula using patient CT images. METHODS: The CT scan images of fibulae of 20 patients were used for the study. The results of the analysis showed that, of the widths, the anterior border of the fibula to the posterior surface was the largest dimension. The shape type analysis showed that the triangular type was most prominent near the head of the fibula, and the irregular type was most prominent towards the lateral malleolus. RESULTS: The results of height and width related to the long axis of implant installation showed that the width of the central section was the largest. With respect to the length of available bone volume, the length near the lateral malleolus was larger than that near the head of the fibula. The results showed that there were significant differences in size between male and female fibulae. CONCLUSION: The present study provides a CT scan based analysis of the anatomy of the fibula. Important information for the optimal site of installation of osseointegrated implants in fibular free flap reconstructions is also provided.

Comparison between virus shedding and fever duration after treating children with pandemic A H1N1/09 and children with A H3N2 with a neuraminidase inhibitor.

BACKGROUND: Shedding of the pandemic virus during an influenza pandemic is thought to persist longer than shedding of influenza viruses during annual influenza seasons, because people have much less immunity against a pandemic influenza. A correlation is thought to exist between the length of virus shedding and the clinical severity of influenza illness. METHODS: We compared the virus isolation rates of children with pandemic A H1N1/09 influenza infection and children with A H3N2 influenza infection after the patients had been treated with one of three neuraminidase inhibitors (NAI) such as peramivir, laninamivir and oseltamivir. The clinical effectiveness of each NAI was assessed on the basis of the duration of the febrile period after the start of treatment. RESULTS: Influenza viruses were isolated from 15 of the 34 patients in the A H3N2 group (mean age 6.2 years) and from 4 of the 25 patients in the A H1N1/09 (mean age 5.6 years) virus group (44.1% versus 16.0%; P<0.05). However, the differences between the duration of fever in the patients in the A H3N2 group and A H1N1/09 group after treatment with the NAIs were not significant. CONCLUSIONS: The virus isolation rates after treatment with each of the NAIs were significantly lower in the A H1N1/09 group, suggesting that the pandemic A H1N1/09 virus was more sensitive to the NAIs than the seasonal A H3N2 virus was. Clinically, there were no significant differences in the effectiveness of the NAIs between the H1N1/09 infected group and H3N2 infected group.

Postnatal mandibular cheek tooth development in the miniature pig based on two-dimensional and three-dimensional X-ray analyses.

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for two-dimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth.

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

The effects of oral xylitol administration on bone density in rat femur.

To examine the effects of oral xylitol administration on rat femur bone density, 36 four-week-old male Wistar rats divided into three groups were fed CE-2 diet (control, n = 12) alone or supplemented with 10% (n = 12) or 20% (n = 12) dietary xylitol for 40 days. Biochemical, morphological, and histological analyses were performed. The 10% and 20% xylitol groups showed higher levels of both serum Ca and alkaline phosphatase activity and lower levels of serum tartrate-resistant acid phosphatase than the control group. Although no significant differences in the three-dimensional bone structure or trabecular bone structure of the femur were observed, both xylitol groups showed significantly higher bone density than the control group. Compared to the control group, the 10% and 20% xylitol groups showed an increase in trabeculae. Thus, oral administration of xylitol appears to affect bone metabolism, leading to increased bone density in rat femur.

Establishment and characterization of the rhabdomyosarcoma cell line designated NUTOS derived from the human tongue sarcoma: Special reference to the susceptibility of anti-cancer drugs.

Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco's modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 microg of streptomycin, 50 U/mL of penicillin and 0.25 microg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, alpha-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.

Novel amelanotic and melanotic cell lines NM78-AM and NM78-MM derived from a human oral malignant melanoma.

Abstract Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs.

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 degrees C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.

Tooth regeneration: implications for the use of bioengineered organs in first-wave organ replacement.

Experiments with animal models have shown that the tooth crown structure can be regenerated using tissue engineering techniques that combine tooth bud cells and biodegradable materials, or by using embryonic tissue and adult stem cells. Moreover, tooth roots and periodontal tissues have been reconstructed by grafting dental stem cells, which leads to the recovery of tooth function, suggesting that tooth regeneration will become possible in humans in the near future. The present article reviews current research on tooth regeneration, discusses a model of tooth replacement that could be used clinically, and proposes a new tooth regeneration approach that overcomes the difficulties associated with the tooth replacement model. Tooth regeneration is an important stepping stone in the establishment of engineered organ transplantation, which is one of the ultimate goals of regenerative therapies.

Distribution of slow muscle fiber of muscle spindle in postnatal rat masseter muscle.

We investigated the properties of the muscle spindle in the masseter muscle at an immunohistochemical level in rats fed for 6 weeks. Slow myosin heavy chain (MyHC) isoforms were measured and intrafusal fibers in the muscle spindle were studied to determine the relationship between the superficial and deep regions of rat masseter muscle after alternated feeding pattern. However, muscle spindles were found in both regions, mainly in the deep region of the posterior superficial region of masseter muscle. The total number of the slow fiber in the intrafusal fiber and number of muscle spindle in the deep region were high from 5 to 8 weeks old in spite of various dimensions of data such as diameter and the compositions of the intrafusal fiber. The relationship of the protein expression of slow MyHC in the two regions at 5 weeks old reversed five weeks later (10 weeks old). This period is an important stage because the mastication system in masseter muscle with muscle spindle may be changed during the alternated feeding pattern of suckling to mastication. The changes may be a marker of the feeding system and of the control by the tension receptor of muscle spindle in this stage of masseter muscle after postnatal development.

Effect of changes in food consistency on NADH-ubiquinone oxidoreductase activity and levels of mRNA for ND1, 51kDa, 75kDa and myosin heavy chain isoforms in two different portions of rat masseter muscle.

We investigated the effect of a change in food consistency on properties of the masseter muscle in 3-week-old rats fed a soft diet for 9 weeks (Group S) and fed a soft diet for 5 weeks followed by a hard diet for 4 weeks (Group S-H). The NADH-O2 oxidoreductase activity, levels of mRNAs transcribed from genes encoding NADH-ubiquinone oxidoreductase (Complex I: ND1, 51kDa, and 75kDa) and myosin heavy chain (MyHC) isoforms and the phenotype of the muscle fibers were measured in the superficial and deep portions of the muscle. In the period from 8 weeks to 12 weeks of age, NADH-O2 oxidoreductase enzyme activity in both the superficial and deep portions of the muscle showed similar patterns in Group S and Group S-H. In contrast, the ND1, 51kDa and 75kDa mRNA levels in the superficial and deep portions of the masseter muscle in the Group S-H were higher than those of Group S in the 12-week-old rats, except for the 51kDa mRNA in the superficial portion of the masseter muscle. MyHC-IIa and MyHC-IId/x mRNA levels in the superficial portion of the masseter muscle were higher in the Group S-H than in the Group S. These observations suggest that short-term feeding stress such as the transition from a soft diet to a hard diet causes changes in oxidative metabolism, in mRNA levels for the Complex I components ND1 and 75kDa, and the mRNA levels for the MyHC isoforms IIa and IId/x in the superficial portion of rat masseter muscle, but no changes in the composition of muscle fiber types.

The internal structure of bony tissue of a human metopic suture by Soft X-ray.

A complete metopic suture was found in the frontal bone of a 79 years old male cadaver. Compact bone matrices were found in the border region of the metopic suture using a Soft X-ray apparatus. The coronal and sagittal sutures were also complete. The appearance of these complete cranial sutures may be due to continuous bone restructuring and resorption in the border region of sutures during brain development of brachy crany.