The aim of this study was to examine the bone regenerative process from fibular periosteum in rats. Twenty male Wistar rats were divided into two groups : a periosteum preservation (PP) group (n=15) and a periosteum removal (PR) group. In the PP group, the fibula was totally removed, but the periosteum and blood supply were preserved. In the PR group(n=5), the fibula was totally removed, including the periosteum. Radiological and histological findings were evaluated after operation. In the PP group, the increase in regenerative bone volume was highest at 1 week. At 2 weeks, the bone volume decreased transiently, but then continued to increase gradually until 4 weeks. There was little change after 4 weeks. The regenerative bone mineral density continued to increase gradually from 5 days until 8 weeks. In the PR group, there was no evidence of regenerative bone. These results suggest that the periosteum has osteogenic capacity and the peak of bone regeneration from the periosteum occurs around 4 to 6 weeks.

View full abstract

A DNA/protamine complex powder, prepared from the reaction between DNA and protamine sulfate solutions, was mixed with water to make a paste. The ALP activity of MC 3T3 E1 cultured on a thin film of the complex paste was higher than that cultured on a plastic well. It was found that DNA/protamine complex induced new bone formation from the results of micro-computed tomography (μ-CT) images and conventional histological sections with hematoxylin and eosin staining in rat cranial defect tests. μ-CT image analyses showed that newly formed bone areas in defects for DNA/protamine complex were significantly higher than those for sham operation (controls) two and three months after surgery. Although the area of red-appearing osteoids reduced with increasing times after surgery, stimulated new bone formation areas were observed around newly formed bone in histological sections stained with Villanuva osteochrome bone stain. Therefore, DNA/protamine complex paste with a biodegradable property will be a useful injectable biomaterial for the repair of bone defects.

View full abstract

To develop an effective method of bone augmentation for dental implants, particularly for patients with a scarce amount of bone, we introduced a new honeycomb-like β-tricalcium phosphate (H-β-TCP) as a scaffold, whose unique geometrical properties induce bone formation along with vasculature. A total of six beagles from 6 to 7 years old were used for this study. Autologous bone marrow-derived mesenchymal cells (BMC) cultured with H-β-TCP were implanted around the dental implants, and healing was compared with conventional bone augmentation using autogenous bone. The 2-wall peri-implant defects were filled with the scaffold material pre-coated with BMP-2 and cultured with BMCs. Judging from the surface contact area between the bone and the implant, at 12 weeks the highest bone formation was found in the experimental group using the BMP-2 coated H-β-TCP with BMCs. This was significantly higher than the control H-β-TCP with BMP-2-coating but without BMC. It was noted that within the tunnels of the implanted H-β-TCP, one or two blood vessels with a diameter of 50-100 micro meter were generated in most cases after 2 weeks. These results suggested that the geometry of the honeycomb like β-TCP with, gave advantages in terms of initial induction of blood vessel formation which is essential for bone augmentation.

View full abstract

Biofilm formation in the oral cavity is postulated to cause oral diseases such as dental caries, periodontal diseases, and peri-implantitis etc. Recently, an artificial mouth system (AMS) was developed to study oral biofilm formation on dental materials in vitro by simulating the human oral environment. The purpose of this study was to monitor the biofilm formation on titanium (Ti) and hydroxyapatite (HA) using AMS and to evaluate the biofilm morphology by scanning electron microscopy (SEM). As bacterial strains, Streptococcus mutans (S.mutans) and Streptococcus sobrinus (S. sobrinus) were employed. The number of bacterial cells on each specimen was evaluated after 5 and 18 hours assays. The number of S. mutans cells after 18 hours was significantly higher than after 5 hours (p<0.05). No significant differences existed between Ti and HA specimens for both S. mutans and S. sobrinus cells in each assay period. The 18 hours assay specimens provided a significantly higher amount of water-insoluble glucan (WIG) by S. mutans than 5 hours specimens (p<0.05). There were no significant differences between Ti and HA in the amount of WIG in each assay period. On SEM observation, biofilm formation of S. mutants and S. sobrinus was recognized on the HA specimen surface and no distinct differences were recognized. Higher magnification observation revealed the formation of pores with a diameter of approximately 5µm. This was due to the pH decrease during biofilm formation. Ti surface could not be influenced by the decrease of pH. This was confirmed by acid etching of HA and Ti. Pores were also formed by acid etching on enamel but not on Ti. It is suggested that the formation of such pores is related with caries formation. In conclusion, biofilm formation on HA and Ti could be monitored using AMS.

View full abstract

In the present study, oral squamous cell carcinoma (OSCC) was induced in a mouse model with 20 μg/ml 4-Nitroquinoline-1-oxide (4NQO) solution in drinking water. 120 six-week-old male Balb/C mice were randomly divided into an experimental group (n=110) and a control group (n=10). They were sacrificed after 16 to 48 weeks of exposure to allow for histopathological and immuhistochemical examinations. Gross changes could be observed, including white changes, leukoplakia, erythroplakia, ulceration and papillary tumor appearance on the mucosa of the tongue dorsum of the experimental group mice during the carcinogenesis period. At the same time, no visible and histopathological changes in tongue epithelium were observed in the control group. Survivin expression was positive in dysplasia and OSCC groups but not in normal mucosa, and correlated positively with PCNA expression. Also, survivin protein staining showed statistical significance in the dysplasia group (p<0.05) but not in the OSCC group (p<0.05). Furthermore, PCNA Labelling Index (PI) in survivin positive specimens were found significantly higher than it in survivin negative specimens (p<0.01). These results showed that survivin might be closely related to cell proliferation, differentiation and carcinogenesis. It also showed that survivin might promote unrestricted multiplication and dedifferentiation of cells, making the tumor taking a malignant behavior through promoting cell mitosis, cell apoptosis, and enhancing cell proliferative activity. Therefore, the detection of expression of survivin and PCNA is helpful for early diagnosis of OSCC.

View full abstract

Salivary glands are important exocrine glands for oral health and initial digestion. Salivary gland dysfunction resulting from irreversible glandular damage usually leads to poor life quality in patients. Recent investigations showed that bone marrow-derived cells (BMDCs) could be grafted into non-hematopoietic cells in multiple tissues. In this study, BMDCs from green fluorescent protein (GFP) mice were transplanted into irradiated C57BL/6 mice to assess the ability of BMDCs to differentiate into parenchymal cells of salivary glands by immunohistochemistry and double fluorescent staining. The data revealed that a population of GFP positive cells showed the characteristics of acinar cells, ductal cells and myoepithelial cells in parotid and submandibular glands, and that some of BMDCs presented amylase expression. These results showed that BMDCs have the ability to differentiate into parenchymal cells of salivary glands with certain glandular cell functions. Such plasticity of BMDCs can be the first step for salivary gland regeneration by stem cells and tissue engineering therapy.

View full abstract

The effect of diabetes mellitus on osseointegration capacity has not been addressed using appropriate animal models. The aim of this study was to investigate the proliferation and differentiation of osteoblastic cells using a type 2 diabetes rat model and 3 different titanium surfaces. We used Goto-Kakizaki rats, a Wistar substrain which develops type 2 diabetes mellitus, with Wistar/ST rats as controls. Titanium disks of grade 2 commercially pure titanium were prepared with surfaces that were machined, sulfuric acid etched, and hydrofluoric acid etched, and rat bone marrow-derived osteoblastic cells were cultured on the 3 different types of titanium surface. In comparison with the controls, the osteoblastic cells of the diabetes rat were inferior in differentiation. The difference with the controls occurred in a relatively small hydrofluoric acid etched group on one of the 3 different surfaces. These results suggest that type 2 diabetes mellitus impairs the differentiation of osteoblastic cells in diabetic rats.

View full abstract

Bone remodeling is achieved through the resorptive activity of osteoclasts and the synthetic activity of osteoblast and it continues throughout life. Cement lines are formed by osteoblasts and are recognized between old and new bone. However, the detailed structure of cement lines has still not been clarified. The present study researched the ultrastructure of cement lines in a ferret femur using light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). On LM, many resorption tunnels were recognized in cortical bones. Some osteoclasts in Howship’s lacunae were observed at the top of the resorption tunnel (cutting cone), and osteoblasts forming some layers were recognized behind osteoclasts (filling cone). The layer of osteoid was observed under osteoblasts, and it was slightly eosinophilic on the decalcified specimen. A very thin layer of cement lines was recognized between osteoid and old bone, and it was slightly basophilic on hematoxylin and eosin (HE). On the non-decalcified sections stained with toluidine blue (TB), cement lines showed anochromasia in the non-etched specimen, but stained with TB in the etched one. On TEM, cement lines were recognized between osteoid and old bone, and they were revealed as extracellular matrices of an afibrillar layer, with a thickness of about 2µm. On BSE analysis, cement lines of osteon were observed as 1- 2µm white lines. Conclusively, the present study indicated that cement lines are extracellular matrices of hypercalcified afibrillar layer having 2µm thickness.

View full abstract

It has been reported that statins increase osteogenic effects by inhibiting the cholesterol synthesis pathway. However, statins are unsuitable for local administration by themselves due to their rapid release in vivo. In order to develop the optimal concentration of fluvastatin-gelatin complexes for use as new osteogenic drugs, the purpose of this study was to investigate the coupled state of fluvastatin with gelatin in vitro. The coupling capacity of fluvastatin with gelatin was determined by measuring the remnant fraction of fluvastatin by ultrafiltration. Fluvastatin was dissolved at various concentrations (100, 200, 300, 400 and 500 µM) into a 3-mg/ml gelatin solution. The complexes (500 µl) of fluvastatin solution and gelatin solution were filtered using a centrifugal ultrafilter at 15000 g in a swing-bucket rotor. The complexes were divided into an ultrafiltrated fraction containing fluvastatins freely released from the complex and a remnant fraction containing fluvastatin coupled with gelatin. The absorbance of the fluvastatin in the ultrafiltrated fraction was measured at 303 nm with an ultraviolet–visible transmission spectrophotometer. The fluvastatin-coupling capacity was then calculated based on a standard curve representing absorbance value vs. fluvastatin concentration. In addition, the osteogenic activity of fluvastatin coupled with gelatin was measured through the HMG-CoA reductase inhibitory activity in the cholesterol synthesis pathway. The results showed that a molecule of gelatin has coupling ability for a specific amount of fluvastatin (291 µM fluvastatin/ 3 mg gelatin). The fluvastatin-gelatin complex had the same osteogenic activity as separately used fluvastatin through the inhibition of HMG-CoA reductase.

View full abstract

The palatine rugae are specialized structures located on the fused hard palate; however, the number, arrangement and direction of the palatine rugae differ among species. Since crocodilians were evolutionally the first animals to show fusion of the palate, the authors focused on the structure of the crocodilian palate. The surface structure of the palate in Alligator mississippiensis at postnatal 60 days was examined by stereomicroscopy and scanning electron microscopy. In addition, histological observation was made by hematoxylin-eosin and Azan staining. Moreover, the urea silver nitrate method was used for nerve staining. On the palate of Alligator mississippiensis, many papilla-like, small conic-shaped projections of about 450-800µm in width and about 500µm in height were observed, although there were no palatine rugae. These projections were distributed over the whole palate, and there was no difference in density by region (anterior, middle, and posterior portion of the hard palate). These projections consisted of keratinized stratified squamous epithelium and connective tissue. In particular, many collagen fibers were observed in connective tissue. The bundles of nerve fibers ran from the connective tissue toward the oral epithelium. Collagen fibers were sparse in the area with the fascicle of nerve fibers under the epithelium of these projections. It is generally known that many nerve fibers and nerve endings are located in the connective tissue of the palatine rugae. Since there are many nerve fibers in the connective tissue of these projections, it is suggested that these projections may act as sensitive organs such as the palatine rugae.

View full abstract

The aim of this study was to examine the effects of orally administered simvastatin on the bone healing process in stroke-prone spontaneously hypertensive rat. Twenty-week-old female stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY) as a control were used. Bone defects were created in the femur of each rat. From the first day following surgery, half of each of the WKY and SHRSP rats were orally administered simvastatin (10 mg/kg/day). The WKY and SHRSP rats were each divided into experimental groups: 1) WKY simvastatin administration group (WKY-S), 2) WKY control group (WKY-C), 3) SHRSP simvastatin administration group (SHR-S), and 4) SHRSP control group (SHR-C). Experimental periods were set at 1, 2 and 4 weeks after surgery. After each experimental period, the animals were sacrificed. Radiographic analysis and histologic and histomorphometric examinations and immunohistochemical staining for BMP-2 were performed on the harvested samples. The data were statistically analyzed. The radiological analysis and histomorphometry parameters showed that the amount of newly formed bone in the trabecular bone area and cortical bone area increased significantly in the WKY-S group compared to that in the WKY-C group at 2 weeks. In addition, a positive immune reaction with BMP-2 was seen in osteoblasts located on the surface of newly formed bone and the outer periosteum around the marginal area of existing bone in WKY-S. In contrast, in both SHR-S and SHR-C 4 weeks after surgery, no differences in bone formation in the trabecular bone area and cortical bone area were recognized as being based on simvastatin administration. In addition, in the expression of BMP-2, there was no significant difference between the presence and absence of simvastatin administration. The oral administration of simvastatin under our experimental conditions had a facilitating effect on bone formation in WKY rats, whereas no effect was recognized in SHRSP rats.

View full abstract

β-TCP is a biodegradable bone regenerative material which has been noted to possess several limitations due to its granular property. Similarly, CM-chitin is a sponge-like material known to decompose in the body. Therefore, in order to improve the use of β-TCP, the combined form of β-TCP/CM-chitin was developed and its bone regenerative properties were tested. This study consisted of four test groups: The empty control group, the hydroxyapatite group, the β-TCP granule group, and the β-TCP/CM-chitin group. These materials were implanted in the mandible of canines and bone samples were harvested at 2 weeks, 1 month, and 3 months after implantation. Histological sections were prepared with hematoxylin-eosin staining. Lastly, degrees of new bone formation in the bone defects were measured by image analysis software. Although the empty control group had a smaller quantity of new bone compared with other groups at 1 month, the empty control group became similar to the latter two groups at 3months. The HA group was similar to the β-TCP group and the β-TCP/CM-chitin group at 1 month, however the HA group was less than the other groups after 3 months. Finally, the quantity of new bone in the β-TCP group and the β-TCP/CM-chitin group increased faster and more sufficiently than in the other groups at all stages. No statistically significant differences were recognized in each group at each stage. Although smaller amounts of β-TCP granules were used in the β-TCP/CM-chitin group, this group showed the same amount of bone formation as the β-TCP group, thus suggesting that CM-chitin does not inhibit bone growth. Furthermore, due to CM-chitin’s sponge-like structure, the β-TCP/CM-chitin group was easier to handle than the β-TCP granules alone. These observations suggest that β-TCP/CM-chitin is a useful bone substitute and may have applications to oral surgery.

View full abstract

Lymphoid enhancer-binding factor 1 (LEF1) is a transcription factor in the Wnt/β-catenin signaling pathway. LEF1 may control odontoblast differentiation. Specificity Protein 6/Epiprofin (SP6) is a transcription factor that mediates the signaling pathway between bone morphogenetic protein (BMP) and the Wnt/β-catenin. LEF1 is decreased by the overexpression of SP6. In contrast, lef1 gene expression is increased in the dental epithelium and mesenchyme of SP6-deficient mice. Thus, LEF1 and SP6 act in an antagonistic manner, being involved in tooth formation. However, this has not been investigated in detail in vivo. In the present study, the expressions of Dickkopf-related protein-1 (DKK-1) (an antagonist to the Wnt signaling pathway), SP6, and phospho-Smad1/5/8 (p-Smad1/5/8) (activated R-Smad in the BMP signaling pathway) were immunohistochemically investigated using serial sections prepared from rat first molar tooth germs in the bell stage on embryonic day 19 and days 10 and 15 after birth, in order to investigate the roles of these factors in differentiation into ameloblasts and odontoblasts. As a result, p-Smad1/5/8 was strongly expressed in preodontoblasts and odontoblasts and also in the inner enamel epithelium and ameloblasts in the secretory stage, suggesting activation of the BMP signaling pathway through p-Smad1/5/8 and involvement of the BMP signaling pathway in differentiation into odontoblasts and ameloblasts and their secretory functions. LEF1 appears in preodontoblasts and preameloblasts and disappears with differentiation, suggesting that it suppresses the differentiation into these cells. In contrast, SP6 and DKK1 expressions are enhanced with odontoblast differentiation. Therefore, the downregulation of the Wnt signaling pathway through SP6 and DKK1 is involved in the disappearance of LEF1 in odontoblasts. SP6 expression was enhanced with ameloblast differentiation, suggesting that the activated BMP signaling pathway suppressed the expression of LEF1 through SP6, thus being involved in ameloblast differentiation.

View full abstract

The posterior area of the mammalian tongue includes two sets of minor salivary glands, namely von Ebner’s glands and Weber’s glands. There have been morphological studies of the minor salivary glands, including reports on the posterior lingual glands of humans, monkeys and rats. However, there has been no report on the minor salivary glands of opossums. The present study was a histological investigation into the morphology of the posterior lingual glands in the gray short-tailed opossum. Lingual tissues were obtained from three opossums. All specimens were fixed in 10% neutral formalin solution, and paraffin sections were made by usual methods. They were stained with hematoxylin and eosin, PAS-alcian blue pH 2.5 and mucicarmine. Weber’s glands were located from the filiform papillae to the back of the circumvallate papilla, and they consisted of seromucous secretory cells showing mucous-rich mixed glands. These mucous cells were stained with alcian blue and/or PAS. The von Ebner’s glands were located from the fungiform papillae to the around of the circumvallate papillae, and they were typically PAS positive serous glands. Conclusively, the present study demonstrated characteristic features of the posterior lingual (Weber’s and von Ebner’s) glands of the gray short-tailed opossum, and it revealed histological data both in accordance with and different from that for the posterior lingual glands of other mammalians.

View full abstract

Because dentists often see patients who are taking bisphosphonate (BIS) drugs, it is important for dentists to be aware of bisphosphonate-related osteonecrosis of the jaw (BRONJ). However, many aspects of the pathogenesis of BRONJ have not yet been clarified. The present study examined the healing process of tooth extraction sockets in ovariectomized rats to elucidate the pathogenesis of BRONJ, particularly the impact of BIS administration in new bone formation. Nine-week old female Wistar rats (6 weeks old during surgical removal of the ovaries) were divided into control (saline treated) and BIS-treated groups (6 in each condition). Alendronate was used for BIS treatment. The maxillary second molar was extracted. Specimens were decalcified with EDTA and embedded in paraffin for serial section. New bone formation and osteoclast counts were determined in H-E and tartrate-resistant acid phosphatase stain, respectively. Bone mineral density was also measured using micro-CT in non-decalcified samples. Contact microradiography (CMR) was taken in polished specimens. Granulation tissue was observed in extraction sockets of both groups at 1 week, and new bone formation was observed at 4 weeks. In the BIS-treated group, the number of multinucleated osteoclasts away from the bone surface increased. New bone formation after tooth extraction increased over time, but it was clearly less in the BIS-treated group compared to the control group. Results showed that BIS inhibited the formation of new bone at extraction socket during the early stage, causing low-density bone trabeculae and the spread of abnormal osteoclasts leading to BRONJ.

View full abstract

This study determines the relationship between best correct visual acuity (BCVA) and phacoemulsification to investigate thefeatures of macular edema associated with cataract phacoemulsification via spectral-domain optical coherence tomography (SD-OCT). Sixty-five patients (65 eyes) with macular edema associated with phacoemulsification were included in this study. Those patients without macular edema before phacoemulsification were observed with the use of Cirrus SD-OCT. The characteristics of edema and exudation were determined according to phacoemulsification with C-scan Cirrus-OCT to measure BCVA and choroidal/retinal thickness in the macula foveal. The choroidal thicknesses in the macula foveal via Cirrus SD-OCT for 1 week, 1 month, and 3 months after phacoemulsification were, 371.2±23.7 μm, 434.7±18.9 μm, and 299.7±18.9 μm, respectively. The retinal thicknesses in the macula foveal were 314.8±19.5 μm, 356.8±15.4 μm, and 259.8±15.4 μm. BCVA ranged from 0.1-0.4 to 0.6-1.2. Serous macula edema and incomplete posterior vitreous detachment were observed. Cystoid spaces in the parafoveal region were seen at the inner nuclear layer, outer plexiform layer (OPR), and outer nuclear layer, and a discontinuous or weak inner segment/outer segment line was often seen in cystoid macular edema (CME). The micromorphological result of Cirrus SD-OCT was either a discontinuous or a weak OPR layer and edema choroida. SD-OCT can cause micromorphological changes in choroidal/retinal features and thicknesses in the macula foveal after cataract phacoemulsification. The results of CME are related to BCVA.

View full abstract

The present study assessed the value of SurgiCase-CMF software in orthognathic surgery for patients with asymmetry. The study consisted of 20 patients with facial asymmetry who visited the Department of Orthodontics in the School of Stomatology, China Medical University, between April 1, 2011, and June 1, 2012. The patients were randomly divided into two groups: an experimental and a control group. Twelve patients in the experimental group underwent 3D-CT scanning before and 6 months after operation. The CT data were stored in DICOM format and imported into SurgiCase-CMF software. Virtual surgery was performed in order to correct asymmetry deformities until a satisfactory outcome was obtained using the software. The reposition distances of bone segments were recorded. The rapid prototypes made by CAD/CAM technique were used to perform operation by surgeons according to virtual surgery design. Actual operations were then performed. The 8 patients in the control group underwent panoramic filmphotography, but did not experience virtual surgery using the SurgiCase-CMF software. The actual operations planning were designed according to clinical examination results. The follow-up period was 6 months. All the patients in the experimental group were satisfied with their facial appearance, while 4 patients in the control group complained about their asymmetrical appearance on follow-up. In the experimental group, SurgiCase-CMF software was also used to indicate important landmark points, lines and angles in order to compare the postoperative symmetry of both sides. Paired t-tests were used, and there was no significant difference between the parameters from the left and right sides, which indicated that the patient with presurgical asymmetry deformities obtained osseous relative symmetry 6 months after operation. We came to the conclusion, therefore, that SurgiCase-CMF software can provide not only effective assistance in orthognathic surgery planning design but also postoperative evaluation of patient operations.

View full abstract

The purpose of this study was to investigate the relationship between the bone mineral density and the contents of osteocalcin calcium and phosphorus in serum with temporomandibular joint osteoarthrosis (TMJOA). The mineral densities of lumbar and neck of femur research groups, including the case group and the control group, were compared. The contents of osteocalcin calcium and phosphorus in serum were also measured and evaluated. The results showed that the mean bone mineral density of the case group (TMJOA group) was lower than that of the control group (healthy control). However, a statistical difference was not reflected. The mineral density of the neck of femur of the case group is lower than that of the controlled group obviously, suggesting that mineral density of the femur was related with TMJOA. It was presumed that the bone mineral density of the femur neck might have relationship with TMJOA. The level of osteocalcin in serum of the case group was significantly lower than that of the control group. Thus it is presumed that osteocalcin had a relationship with TMJOA. The results showed that the capability of bone transformation and formation in TMJOA were all decreased. No relationship was found between calcium phosphorus in serum and TMJOA.

View full abstract

Although studies on the formation of apical periodontitis have somehow been carried out but detailed cellular dynamics remain unclear. We recently established an experiment that could easily be performed using ddY mice. First, under general anesthesia using isoflurane inhalation, the coronal portion of the maxillary first molar was penetrated using a round bur and drill with water irrigation causing pulp exposure until the root apex. Micro computed tomography (R_mCT) was taken over time during observation. Four weeks later, R_mCT confirmed the presence of a radiolucent image at the apex of the tooth, which was then removed for histological examination. The results showed that granulation tissue with fibrosis had gradually formed at the periphery of the abscess. The present method confirmed the effectiveness of the experimental mode to exmine the formation of chronic inflammatory lesions at the root apex.

View full abstract