Osteoporosis is a skeletal disorder characterized by reduced bone strength and an increased risk of fractures. Bone strength is exacerbated by various factors that influence bone mineral density (BMD) and bone quality with increasing age. We have developed a novel supplement diet for osteoporosis prevention that contains fructo-oligosaccharide, isoflavone, and calcium citrate in addition to calcium phosphate (high mineral diet: HMD), thereby increasing the Ca and P content. The present study aimed to clarify whether rats with osteoporosis fed a HMD showed improved BMD compared with rats fed a normal mineral diet (NMD). The experiment used 20-week-old ovariectomized rats divided into an NMD group (Group 2, n=8) and a HMD group (Group 3, n=8). This study also used 20-week-old sham-ovariectomized rats fed NMD as controls (Group 1, n=8). After 8 weeks and 24 weeks on the diet, this study examined the changes in BMD, bone mineral content (BMC), and 3-dimensional (3D)-map using micro-computed tomography (CT) imaging of the shaft of the femur. In the 3D-map, both Groups 1 and 3 showed high BMD on the inner portion of the cortical bone, while Group 2 showed slightly lower BMD at the same location. Decreased estrogen secretion in Group 2 significantly affected bone metabolism, lowering both BMD and BMC compared to Group 1. However, while Group 3 also showed decreased estrogen secretion, BMD and BMC were higher than in Group 2. These findings indicate that HMD increases both BMD and BMC, and is therefore more effective than NMD for improving BMD and bone quality.

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Periodontal ligament (PDL) cells are comprised of heterogenous cell populations including mesenchymal stem cells and osteogenic progenitor cells. We previously reported that differentiation of human PDL cells into osteoblasts requires phosphoinositide 3-kinase (PI3K) activities. Here, we investigate osteoblast differentiation using TGF-β1, BMP-2/BMP-7, and dexamethasone (Dex) in human PDL cells, focusing on the PI3K/Akt pathway. We found that in TGF-β1-treated cells, Dex increased IGF-1 expression as well as phosphorylated Akt, which is a main target molecule of PI3K. Downregulation of IGF-1 expression and enhanced phosphorylation of Akt were observed in BMPs with Dex-treated cells, even though alkaline phosphatase expression and activities were enhanced. These results indicate that IGF-1 is a key regulator in TGF-β1-induced osteogenesis, but it is not required in osteoblast differentiation initiated by BMPs. Because BMPs require PI3K activation for osteoblast differentiation, and because BMP treatment upregulates Akt phosphorylation, signaling molecules other than IGF-1 may support BMP-induced osteoblast differentiation.

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Biodegradable materials, such as poly (lactic acid) (PLA), have been applied to regenerate bone. However, pure PLA is brittle, so it can be modified by blending ε-caprolactone (PCL). These biodegradable materials have no known functional activity. In the present study, a copolymer of PLA/PCL (PLCL) film was fabricated by dissolving the materials in chloroform. Carboxylic acid groups (COOH) of PLCL were chemically introduced onto the PLCL film surface by 0.5N NaOH hydrolysis. Apatite formation was evaluated on an alkaline hydrolyzed PLCL film (PLCL-COOH) after immersion in Hanks’ balanced salt solution (HBSS, pH7.4) without organic species. Water adsorption in (PBS) PLCL-COOH phosphate buffered saline was also monitored by comparing PLCL. PLCL-COOH provided a greater degree of apatite precipitation than PLCL after 3 and 7-day immersion. Weight loss was significantly different between PLCL-COOH and PLCL films from 3 days until 124 days of immersion in PBS (p<0.05). The contact angle of the PLCL-COOH surface with respect to double distilled water significantly decreased compared with PLCL (p<0.05). Thus, it was predicted that PLCL-COOH would show a better biological response and be applicable in new types of dental materials in dental treatment.

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The aims of this study were to observe the growth of vascular endothelial cells (VECs) and the vascularization of novel porcine acellular dermal matrix (PADM), and to investigate the methods for modifying heterologous dermal substitutes in order to improve their compatibility. Twenty-four Wistar rats were divided into a PADM group and a HADM group, which were transplanted with PADM and HADM, respectively. At the 3rd day, and at the 1st, 2nd, and 4th week after the operation, the dermal substitute was removed, and HE staining and CD34 and factor VIII immunohistochemical staining were performed. The distribution and proliferation of VEC, the vascularization, and histological changes in the dermal substitute were observed. At the 3rd postoperative day, tiny capillaries sprouted in the dermal substitute and began to proliferate. At the 1st postoperative week, the new capillaries extended into the dermal substitute. At the 2nd postoperative week, the new capillaries had entered the dermal substitute and further increased in number, and were more mature than the capillaries in the 1^st postoperative week (P<0.05). At the 4th postoperative week, the capillaries were distributed everywhere in the dermal substitute, along with further matured VECs, but the capillary number was not significantly increased compared with the 2nd postoperative week (P> 0.05). There was a good VEC proliferation in implanted PADM, with immigrated fibroblasts and matured capillaries. The novel PADM showed good tissue compatibility.

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Recycling unnecessary teeth for use as a bone grafting material should be a valuable approach to enhance the bone-forming process in alveolar bone defects and to reduce material costs. However, the process of bone formation using grafting material is not fully understood. The aim of this study was to assess the fate of material including milled teeth by immunohistological observation in a rat incisor extractionmodel. Freeze-milled incisor material was injected into an extraction socket in a rat mandible bone. Four weeks after the operation, the mandible was sampled and frozen sections were prepared by the sticking film method, after which DMP-1 and CD68 were visualized by immunohistochemical staining. The results revealed that milled tooth particles remained around the socket at four weeks post-operation, and showed different reactions depending on the particle size. Small particles (3-10 μm) were phagocytosed by CD68-positive cells, whereas bone regeneration occurred around large (30-60 μm) particles. These results suggest that the injection of crushed teeth could provide an optimum scaffold and growth factors for bone formation in extraction sockets.

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This study investigated the antibacterial activity and cell adhesion of commercially pure titanium (Ti) surfaces modified by zinc plasma immersion ion implantation (Zn-PIII) treatments for dental implant application. An X-ray photoelectron spectrometer, atomic force microscope, and scanning electron microscope were utilized to analyze the chemical composition and surface topography of Zn-PIII Ti specimens. Porphyromonas gingivalis (P. gingivalis), common oral bacteria which often cause complications in combination with dental implantation, were seeded onto the modified Ti surfaces for 48h in order to study the antibacterial effect of the surface. The attachment and proliferation of murine osteoblasts (MC3T3-E1) on the Zn-PIII Ti surface were investigated using acridine orange fluorescence staining and a scanning electron microscope. The results showed that the Zn-PIII treatment influenced the surface topography of the Ti specimens, and that Zn singles on Zn-PIII-treated Ti surfaces increased with increased Zn-implanted time. The numbers of bacteria that adhered to Zn-PIII-treated Ti surfaces were significantly less than for untreated Ti, and gradually decreased with increasing Zn concentration. In addition, the adhesion and proliferation of MC3T3-E1 on Ti were improved by Zn-PIII treatment (p < 0.05). The results suggest that the Zn-PIII modified surface-layer-formed Ti could consequently restrain bacterial adhesion of P. gingivalis, and could enhance the cell proliferation and adhesion to Ti surfaces for dental implant application.

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The objective of this study was to evaluate the usefulness of regenerative bone from periosteum as a new graft material for the repair of bone defects. Regenerative bone at 1, 4, and 8 weeks (RB1, RB4 and RB8) was harvested from the fibular periosteum of donor rats as graft materials. These regenerative bones were transplanted into the mandibular bone defects of recipient rats in the experimental groups (RB1, RB4 and RB8 groups). Normal fibulas (NF), which were harvested from donor rats, were transplanted in the control groups (NF groups). Immediately, 2, and 4 weeks after transplantation, radiological and histological findings were evaluated. Bone healing was observed in all groups. In particular, the height and width ratios in the RB1 groups were significantly higher than those in the NF groups, and RB1 resisted resorption as compared with the other bone grafts. The bone mineral density in the RB1 groups was significantly less than that in the NF groups. These results indicate that regenerative bone from periosteum is a useful reconstructive material. The properties of regenerative bone after transplantation differ depending on the timing of regeneration.

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High-mobility group box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE) are thought to play key roles in the progression of chronic inflammatory diseases. We recently showed that HMGB1 and RAGE might be involved in the progression of human gingival inflammation as novel inflammatory mediators. The molecular mechanisms governing the ability of RAGE to induce ligand-specific responses in gingival inflammation are still unknown. We identified HMGB1/S100A4/NF-κβ pathway, a global regulator of inflammation, as a major RAGE-responsive molecule induced by Porphyromonas gingivalis (P. gingivalis) in rat gingiva. Consistent with the observation that RAGE can be activated by multiple ligands, the elevated expression of RAGE-mediated HMGB1 and S100A4 expression in gingival inflammation. Phosphorylation of NF-κβ and IL-1β immunoreactivity was stronger in P. gingivalis challenged gingiva compared to the control. Quantitative real-time RT-PCR confirmed the increased expression of HMGB1, RAGE, and IL-1β mRNA in P. gingivalis challenged gingiva. Our results indicate that RAGE is a major target gene shared by HMGB1 and S100A4 and the coordinated action of RAGE and NF-κβ leads to the induction of gingival inflammation.

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The present study aimed to investigate cellular behavior on nanoscale features of a titanium surface by controlling the deposition time in NaOH. These effects were then evaluated for osteogenic differentiation of rat bone marrow cells to potentially increase the success rate of titanium implants. Titanium disks were left untreated or soaked in 10 M NaOH for 5 min, and 1h, 3h, 9h and 24 h. Scanning electron and probe microscopy were used to evaluate the nanoscale features. Rat bone marrow cells were seeded on the specimens in osteo-genic differentiation medium. Alkaline phosphatase activity, osteocalcin production, and mineralization were then analyzed. Statistical significance was analyzed using one-way ANOVA followed by the Tukey test. Nanofeatures were detected at 1 h after NaOH treatment and were well established at 9 h. Alkaline phosphatase activities of specimens soaked for 1 h or 3 h were significantly different from specimens soaked for 9 h or 24 h after 14 days of differentiation. Osteocalcin production and calcium deposition between untreated specimens and specimens soaked for 5 min, as well as between specimens soaked for 9 h and 24 h, were significantly different after 21 days. It was found that the nanoscale modification of a titanium implant surface by NaOH treatment affects osteoblastic differentiation of bone marrow cells and enhances mineralization. This study found that modification of titanium surfaces with NaOH could be an effective method of improving their biological properties. Further developments in nanotechnology may help improve osseointegration of titanium implants.

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Oral squamous cell carcinoma (OSCC) is one of the most common types of human cancer in the world. Oral leukoplakia (OLK) is the most commonly diagnosed premalignant lesion in the oral cavity and it is also most associated with the development of OSCC. However, the mechanism of malignant transformation from OLK to OSCC is not completely understood. In this study, we attempted to evaluate expression of Aurora A and the main effectors in the Ras/Raf/MEK/ERK pathway in OSCC and OLK clinical tissues. Protein expression was analyzed by immunohistochemical technology in 25 tissue samples of OLK and 21 OSCC clinical tumor specimens. Upon analyzing the levels of Aurora A, phospho-MEK1/2 and phospho-ERK1/2, a remarked increase was observed in tumors compared to OLK tissues. No statistically significant difference between tumor and OLK tissues was found for pRaf-1 S259 and ERK1/2. These results suggest that the increase in Aurora A, phospho-MEK1/2 and phospho-ERK1/2 expression was closely correlated with malignant transformation of OLK. Furthermore, Aurora A, phospho-MEK1/2 and phospho-ERK1/2 might be coordinated for valuable prognostic indicators of malignant transformation of OLK. In addition, more than 50% pERK1/2 immunopositive cells appears to be a good cut off value to predict the malignant transformation of OLK.

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The aims of this study were to produce rough surfaces on zirconia by laser treatment and to examine how changes in surface topography affect tissues surrounding zirconia implants. Threaded zirconia implants with a diameter of 2 mm and length of 7 mm were used. The experimental implants had surfaces treated with laser (YAG laser) irradiation (R-ZIs). The controls were not treated with laser irradiation (S-ZrIs). Twenty R-ZIs and twenty S-ZrIs were placed in the tibiae of 8-week-old male SD rats. The peri-implant tissues with implant bodies were collected 4 weeks after implant placement. Light microscopic and histomorphological evaluations were performed, and removal torque (RTQ) was measured. The bone-implant contact (BIC) ratio was approximately 1.25 times higher for R-ZrI than for S-ZrI on the side of the cortical bone, indicating a statistically significant difference (p<0.05). There was no statistically significant difference in their BIC ratios on the side of the bone marrow. On the cortical bone side and bone marrow side, there was no statistically significant difference between R-ZrI and S-ZrI in the peri-implant bone area (BA), the area of peri-implant bone within the implant threads. RTQ was approximately 7 times higher for R-ZrIs than for S-ZrIs, indicating a statistically significant difference (p<0.05). In this study, the results of the animal experiment revealed new bone formation in the surroundings of the zirconia implants at 4 weeks after implant placement, indicating achievement of osseointegration. The results suggest that laser-produced rough implant surfaces effectively enhance osseointegration.

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The aims of this study were to create experimental implants by coating rough plastic surfaces with a thin layer of titanium, and to use the experimental implants in an animal experiment to investigate whether differences in the surface characteristics of the implant affect the peri-implant bone reaction during the period of osseointegration. Titanium rods of diameter 1.6 mm and length 7 mm were treated by acid etching (AE) or sandblasting followed by acid etching (SA), and replicas were made from plastic. Experimental implants were created by depositing a thin layer of titanium on the plastic replicas by DC-magnetron sputtering, and the surface characteristics of the experimental implants were evaluated. The experimental implants were placed in the tibias of eight-week-old male SD rats. The rats were sacrificed and the implants harvested at 3, 5, 10, 14, 21 and 28 days after implant placement. The samples were examined by optical microscopy and micro-CT to confirm peri-implant new bone growth. Examination of the experimental implants by SEM imaging showed that the different surface conditions (SA and AE) had been faithfully recreated. TEM observation and XPS analysis confirmed that the coating was titanium. The surface roughness of SA and AE was 2.68±0.536 μm and 0.47±0.069 μm, respectively. With AE, the BMD of peri-implant trabecular bone showed that bone mineralization progressed not on the surface of the implant but at sites a small distance away. At day 28 after placement of the implant, when osseointegration was complete, the BMD value in the region near the implant surface was higher in SA than in AE. Furthermore, the BV/TV value was high at an earlier stage in SA than AE. The results showed that the SA surface was better than the AE surface for achieving osseointegration.

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The aim of this study was to prepare zirconia implants with laser-modified surfaces and to evaluate peri-implant tissue response and osseointegration in an animal study. The experimental zirconia implants received one of the following surface treatments and were placed in the tibiae of SD rats: vertical irradiation with a fiber laser (vertical fiber laser), horizontal irradiation with a fiber laser (horizontal fiber laser), vertical irradiation with a Neodymium-doped yttrium orthovanadate (YVO4) laser (vertical YVO4 laser), and horizontal irradiation with a YVO4 laser (horizontal YVO4 laser). The control implants were smooth surfaced. Tibiae with implant bodies were collected 28 days after implant placement, and removal torque values were measured. Tissue sections were prepared for light microscopy, and the bone-implant contact (BIC) ratio and the peri-implant bone area (BA) were measured. The vertical fiber-laser implants had a mean BIC that was significantly higher than other implants. The mean BIC of the vertical fiber-laser implants was approximately 4.2 folds of the value of the control implants on the cortical bone side and approximately 2.7 folds of the value of the control implants on the bone marrow side. The mean BA was significantly higher in the vertical YVO4-laser implants. The vertical fiber-laser implants had a mean torque removal value that was approximately 2.4 folds of the value of the control implants and approximately double of the value of the vertical or horizontal YVO4-laser implants. The horizontal fiber-laser implants had a mean removal torque value that was approximately double of the value of the control implants and approximately 1.7 folds of the value of the vertical or horizontal YVO4-laser implants (p<0.05). Both types of lasers were useful in implant surface treatment to enhance osseointegration of zirconia implants.

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To establish a large regenerative cartilage without any scaffolds, we cultured chondrocytes on N-Isopropylacrylamide (NIPPAM)-coated dishes with 3 × 3 mm² grids, producing many cell sheets. We then attempted to have the cell sheets form into pellets, which we term cartilage elements, and have the cartilage elements fuse with each other. Finally, we regenerated the large cartilaginous constructs with some stiffness. The NIPPAM coating on culture dishes, which enables to detach cultured chondrocytes only by the decrease in incubation temperature, was useful for preparing many chondrocyte sheets with a homogenous size. The cell sheets could provide effective matrix production and become spherical to form the cartilage elements. For their fusion, those elements were jammed into an agarose mold and were cultured for 3 weeks. The regenerative constructs made by fusing the cartilage elements derived from human or beagle chondrocytes were subcutaneously transplanted into nude mice and the cell-donor beagles, respectively, showing fair cartilage regeneration 2 months after transplantation. They could also prevent severe foreign-body reactions that often occur when using some scaffolds. Thus, by mass production of homogenous cartilage elements using the NIPPAM-coated dishes and their mutual fusion in the agarose mold, mature cartilage was three-dimensionally regenerated without any scaffolds.

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In this study, a kind of Mg-4Zn-3HA alloy was investigated as a biodegradable material. Magnesium alloys can be totally degraded in the body, and their corrosion products are not deleterious to the surrounding tissues. We conducted a comprehensive investigation of the microstructure, mechanical properties, in vitro degradation assessments, and in vitro cytotoxicity evaluations of Mg-4Zn-3HA alloy. Our observations of the microstructure show that the Mg-4Zn-3HA alloy is porous, and its structural characteristic resembling nature bone make this alloy suitable for use in implants. The results of in vitro degradation indicate that the corrosion resistance is improved with the extension of immersion time, and XRD and EDS analysis proves that the corrosion products on the surface of Mg-4Zn-3HA alloy contain hydroxyapatite (HA), Mg (OH)₂ and other magnesium-substituted calcium phosphate, which could reduce the degradation rate. The degradation process of magnesium alloy and the formation of corrosion layer are also discussed in this work. The corrosion products contain HA and Mg (OH)₂, which can promote good biocompatibility. No significant cytotoxicity to MC3T3-E1 cells is detected in the extraction medium, and MC3T3-E1 cells are able to adhere and spread on the corrosion layer of the Mg-4Zn-3HA alloy. Mg-4Zn-3HA alloys have the potential to be used for biomedical applications.

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The aims of the current study were to determine whether side population (SP) cell selection is possible, to show an efficient approach for purifying rat dental pulp (DP) and dental germ (DG) stem cells, and to compare phenotypic differences with bone marrow SP cells. SP cells, isolated from murine adult incisor DP and DG based on exclusion of the DNA dye Hoechst 33342, exhibited potent stem cell activity compared to main population cells. Both bone and dentin are mineralized tissues and are structurally similar. However, in contrast to bone marrow stem cells, DP stem cells do not support establishment of hematopoietic marrow. To investigate whether the phenotype of DP or DG SP cells is distinct from bone marrow SP cells, we used FACS to isolate SP cells from biologically distinct tissues (bone marrow, DP and DG) and characterized expression of several surface marker genes. Direct comparison of surface marker expression on SP cells isolated from different tissues identified surface markers common to dental tissue SP cells as well as those specific to bone marrow SP cells. FACS analysis of specific differentiation markers confirmed the similarity of surface marker expression between bone marrow SP cells and DG SP cells. However, marker expression in DP SP cells was considerably different from that in bone marrow SP cells. Because these tissues consist of multiple types of cells, heterogeneity of these tissues might explain the differences in surface marker profiles. We concluded that isolating DP SP cells with a phenotype that is distinct from that of bone marrow SP cells is possible.

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The aim of this study was to evaluate a novel technique for inducing osteogenesis through original periosteal distraction in a canine mandible model. A periosteal distraction device was rigidly fixed to the mandible in three adult canines (i.e., six sides total). Two sides of 1 dog served as the control. In the experimental group, periosteal distraction was started 4 weeks after placement of the periosteal distraction device. The periosteum was distracted 0.5 mm per day until a height of 3 mm was reached. Distraction continued for 8 weeks. The animals were killed 14 weeks postoperatively. Radiographs were obtained after the devices were fitted and again after distracting the periosteum. Computed tomography (CT) was performed at weeks 1 and 8. Transverse images using OsiriX software were constructed based on the periosteal device to observe the amount of augmentation. Images over time were superimposed on the reference image using Photoshop software. It revealed the changes with time, which were measured. All 6 periosteal distraction devices remained rigidly fixed to the lateral surface of the mandibles. At completion of distraction, all operative sites had healed without evidence of infection or wound dehiscence. The amount of bone augmentation was calculated by comparing CT imagees. The experimental group showed increased width in the area of bone growth and in the bone itself on all six sides. Average increases were 0.7 mm in bone width and 3.31 mm in the area. Bone area and bone width were decreased slightly in the control group. There were significant differences between controls and the experimental group regarding bone width and bone area. We achieved bone augmentation using a custom-made periosteal distraction device. The amount of bone augmentation was calculated by comparing CT images.

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CD81 belongs to a family of cell-surface proteins (tetraspanins) known to be up-regulated in rheumatoid arthritis (RA) synoviocytes. Recently, we showed that small interfering RNA targeting CD81 (CD81 siRNA) has a therapeutic effect on RA. We also showed that CD81 siRNA decreased the expression of TNF-α in SW982 cells, suggesting that TNF-α stimulates the expression of synoviolin via CD81. Here, we show that a monoclonal antibody (MAb) against CD81, which was raised by immunization with recombinant CD81, decreased the proliferation of rat C6 glioma cells, showing that this antibody is functionally active and probably inhibits cell proliferation through the inhibition of CD81 function on the cell surface.

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