Apatites were prepared by varying the ligands of calcium complexes. Two lower crystallinity apatites were prepared from calcium-iminodiacetic acid (Ca-IDA) or calcium- aspartic acid (Ca-Asp) complexes and one higher crystallinity apatite from Ca-IDA complex. Cortical bone responses towards their PLGA composites including different apatites, namely, PLGA/IDA-HA(lower), PLGA/IDA-HA(higher) and PLGA/Asp-HA(lower), were evaluated after implantation into the cortical bone of rabbit tibiae.
Micro-CT observation showed no distinct differences among the three PLGA/apatite composites after 12 weeks of implantation. The original drill holes were partly closed and formation of primary woven bone was observed. Residue of decomposed PLGA was still observed inside the bone marrow.
The histological appearances of porous PLGA/apatite composite scaffolds after 12 weeks of implantation showed similar overall cortical bone response to different PLGA/apatite composites. The original drill hole was not completely filled with new bone. For PLGA/IDA-HA(higher) composite, the original bone defect was partly filled with new bone. Residue of degraded PLGA and remaining apatite was recognized. Both PLGA/IDA-HA(lower) and PLGA/Asp-HA(lower) showed residue of degraded PLGA inside the bone marrow. Remnant lower-crystallinity apatite was not clearly recognized.
In conclusion, apatites with different crystallinity were prepared through Ca-IDA and Ca-Asp complexes. The bone responses towards PLGA/apatite composites with different crystallinity were similar, but the degradation behavior of PLGA/apatite composite was different. It is suggested that the degradation of PLGA in PLGA/apatite composite influences new bone formation in addition to the release and dissolution of calcium from apatite.

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Demineralized dentin matrix (DDM) granules with excellent biocompatibility were designed using extracted human teeth by the cooling-pulverizing and supersonic demineralizing technique. Extracted teeth were pulverized together with saline ice at 12,000 rpm of a ZrO₂ blade for 30 s in a ZrO₂ vessel. The pulverized granules having 0.5-2.0 mm in granular size were dissolved by the supersonic treatment at 120 W and 38 kHz or 600 W and 28-100 kHz in 2.0 % HNO₃ solutions to obtain partially or completely DDM granules. Dissolution efficiencies of the pulverized granules increased with increasing the supersonic time. At 600 W, dissolution efficiencies for DDM treated at 28 kHz were higher than those at other frequencies. The Ca/P molar ratios of the granules were 1.60-1.66, suggesting that partial DDM granules were composites of Ca²⁺-deficient hydroxyapatite (HAp) and collagen. At 120 W, as supersonic time increased, asperity on the surfaces of granules became outstanding due to elution of mineral components. At 45 min, DDM granules were completely demineralized and the weight decreased to about one-fifth. Hard asperity on surfaces and micro-crack were observed. Partially or completely DDM granules were implanted into the subcutaneous tissues of the back region of rats. At 4 weeks, completely DDM granules had a little smaller and more irregular shape than partially DDM ones. Infection and exclusion of DDM granules were not recognized, but inflammatory cell invasion was seen through bio-absorption of DDM due to the enzyme-digestion. To improve surface activity of DDM, DDM granules were soaked at 309.5 K and pH 7.40 in a simulated body fluid. At 48 h, HAp nano-crystals were almost homogeneously coated on the surfaces of completely DDM granules, while at 144 h, porous bone-like apatites were found. The HAp nano-crystals coating for DDM granules would form bioactive surface with specific pore structure and chemical nature.

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Periodontal ligament (PDL) cells are composed of several different kinds of cells including mesenchymal stem cells (MSC). It is well known that these MSC play an important role in the regeneration of periodontal ligament and alveolar bone. The heterodimeric bone morphogenetic protein (BMP-2/-7) is a very potent BMP. However, BMP and TGF-β1 signaling pathways that induce and regulate osteogenesis of HPDL cells are not well understood. It was reported that phosphatidylinositol 3-kinase (PI3K), which is activated by insulin-like growth factor-1 (IGF-1) and subsequently promotes the Akt phosphorylation, might play an important role in BMP-2/-7 and TGF-β1-induced osteogenesis. There are numerous signaling pathways located downstream of PI3K/Akt, including the mTOR/p70S6K pathways that was the focus of this study. Here we show that TGF-β1 is a more potent inducer of osteogenesis of HPDL cells than BMP-2/-7. LY294002, a PI3K inhibitor, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), decreased both the Smad 3 phosphorylation and the osteogenic differentiation induced by TGF-β1 treatment. In contrast, the phosphorylation of Smad1/5/8 by BMP-2/-7 treatment was affected by neither LY294002 nor rapamycin treatment and mTOR inhibition failed to reduce BMP-2/-7-induced osteogenesis of HPDL cells. Thus the PI3K - mTOR pathway is required for TGF-β1-induced osteogenesis but is not essential for osteogenesis induced by BMP-2/-7. In conclusion, the PI3K/mTOR/p70S6K pathway plays different roles in the regulation of osteogenic differentiation by TGF-β1 and BMP-2/-7.

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The purpose of this study was to determine the clinico-pathological characteristics of so-called “denture fibroma” and to perform a review of literature. A comparative analysis was carried out to clarify the epidemiological characteristics of the lesion. A total of 142 cases (average age of 65.2 ± 11.1 years), predominantly female, with so-called “denture fibroma” was examined. The residual alveolar ridge in the posterior region (43.7 %) and in the anterior region (43.0 %) was affected with almost the same tendency. However, significant difference in the frequency between the maxilla and mandible was not observed. Macroscopically, majority of the lesions ranged from 1.1 - 2.0 cm (52.5%) and 0.1-1.0 cm3 (59.0%) in diameter. Histopathologically, myxoid degeneration (55.7%) was the most common feature and the presence of minor salivary gland in the connective tissue (26.2%) was observed simultaneously. In the review of literature, the incidence of denture fibroma ranged from 2.0 to 15.0% of the number of cases investigated. The number of cases among denture wearers ranged from 4.9 to 18.0% as reported. Regarding gender, the lesion affects both male and female with equal predominance. In other patients, the lesion was predominantly observed in elderly people with complete denture. Regarding the site of the lesion, the maxilla was reported to be the favored site. When the anterior and posterior regions were compared, many cases of denture fibroma arose in the anterior region overwhelmingly. A denture worn for more than 10 years was reported to be relevant in the development of so-called “denture fibroma”.

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Periodontitis is an inflammatory disease that leads to the progressive degeneration of the alveolar bone that surrounds and supports the teeth. Without effective treatment, periodontitis can cause tooth loss. One method of treating periodontitis is to use Emdogain®, a material derived from the tooth germ of juvenile swine that promotes periodontal tissue regeneration, including the formation of hard tissue as cementum, alveolar bone. The use of Emdogain® is therefore established in the field of periodontal regenerative therapy. However, because of its swine origin, some patients choose not to be treated with Emdogain®. The active component of Emdogain® has been shown to be a peptide whose sequence corresponds to an amelogenin II precursor. As such, this peptide may function as a growth factor to stimulate cell differentiation and tissue regeneration. In this study, we characterized the effects of the synthetic Emdogain®-derived peptide on the proliferation, adhesion, migration and differentiation of periodontal ligament fibroblasts (HPdLFs), which display properties similar to mesenchymal stem cells. Compared to cells not treated with the synthetic Emdogain®-derived peptide, treated cells showed increased proliferation, initial adhesion, and chemotactic activity. The optimum peptide concentration that stimulated these activities was determined to be 100 ng/ml. We next investigated the effects of the peptide at a concentration of 100 ng/ml on osteogenesis and cementogenesis in HPdLF cells by assaying alkaline phosphatase activity, osteocalcin production, and mineralization. Compared with untreated cells, cells incubated with the peptide showed increased alkaline phosphatase activity after 21 days, increased osteocalcin production after 28 days, and increased calcium deposition after 28 days. Taken together, our data suggest that the Emdogain®-derived peptide stimulates periodontal hard tissue regeneration by stimulating the proliferation, adhesion, and migration of mesenchymal stem cells.

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In recent years, many things have progressed and became clear in the study of genes involved in tooth development. However, the genes associated in the formation of periodontal tissues particular those studies involved in the formation of furcation are few. We have carried out a research to examine the gene expression of cementoblasts in the furcation area in porcine tooth germ. Porcine tooth germs were used as samples excised during the formation of mandibular molars and the cementoblasts around the root and at the furcation were prepared. Total RNA was isolated from the samples followed by PCR amplifying specific genes using PCR templates. Further, significant difference with respect to gene expression was determined by semi-quantitative PCR from the electrophoresis image. Also, after the removal of tooth germs, tissues were processed in routine tissue preparation, embedded in paraffin, sectioned into 5 um thickness, stained with H and E and examined under the light microscope.
Amelogenin, enamelin, ameloblastin, MMP-20, Klk-4 expressions were observed uniformly by the cementoblasts at the furcation of the tooth but only amelogenin and ameloblastin were observed by the cementoblasts around the root of the tooth. Also, a typical strong Msx2 homeobox gene by the cementoblasts at the furcation was observed with an expression level of approximately 64 times than the cementoblasts around the root.
The present study clearly showed that specific genes are involved in the formation of furcation. A strong Msx2 gene expression was observed in the tooth germ and even at the furcation suggesting that Msx2 plays an important role in the process of periodontal tissue formation and maturation of furcation.

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Nodal-Lefty signaling is essential for embryonic stem cell pluripotency and growth control of ES cells, which play an important role in tumorigenic phenotype. We recently reported that TGF-β increases Lefty expression in pancreatic cancer cells. However, a previous study has shown that TGF-β is unable to induce Nodal and Lefty expression in ES or induced pluripotent stem (iPS) cells. We established an iPS cell line from mouse oral tissue via retroviral gene transfer of OCT3/4, Sox2, c-Myc and KLF4. The obtained iPS cells expressed undifferentiated markers, such as OCT4, Nanog and SSEA1. We observed induction of the Lefty gene in these iPS cells, as well as in mouse ES cells in ES medium either with or without TGF-β treatment. This is the first report showing Lefty expression in iPS cells. We then investigated the signaling relationship between TGF-β and mitogen-activated protein kinase (MAPK) by using specific kinase inhibitors, and found that p38 has some effect on Lefty expression. Taken together, these results suggest that iPS cells from oral tissue possess a different mechanism for Lefty expression than mouse ES cells. TGF-β with p38 is able to regulate Lefty expression in iPS cells.

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Aging and mechanical loading by occlusal disharmony are critical and etiological factors for the onset of temporomandibular joint disorders (TMDs). Senescence-accelerated mouse prone 3 (SAMP3) is one of the animal models for studying age-related degenerative changes of various tissues and organs. To evaluate the influences of both the aging and/or the mechanical loading induced by occlusal disharmony on the SAMP3 TMJ condyle, histological and immunohistochemical analyses were performed.
Fifty-five, 4-week-old SAMP3 mice were divided into two groups as an experimental and control. In the experimental group, the maxillary molars were trimmed out from the occlusal plane, which also served as mechanical force loading on the condylar surface. Then the condyles were removed and proceeded for histological and immunohistochemical analysis. The subchondral bone ossification was also evaluated by micro-focusing computed tomography (micro-CT) analysis.
The results indicated that induced occlusal disharmony in an aging background promotes TMD, including osteoarthritis (OA) like disease. OA-like TMJ lesions in the SAMP3 mice, accompanied by mandibular condylar cartilage degradation, were characterized by loss of proteoglycans (PGs) and changes of the localization of the extracellular matrix, such as collagen type I, II and MMP13. Changes in the microarchitecture of the condyle were also caused under OA-like lesion. Disrupted Wnt/β-catenin signaling by means of immunohistochemical analysis of β-catenin contributes to this irreversible cartilage tissue damage.
The present study indicates that the experimentally induced occlusal disharmony (under the age-related background) promotes the TMJ OA accompanied by the alteration to components of the extracellular matrix, and Wnt/β-catenin signaling disorder contributes to these irreversible cartilage tissue damage.

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To determine whether the periosteum influences mandibular head regeneration after condylectomy in rats. male Wistar rats, the periosteum of part of the mandibular head was preserved or removed and the mandibular head was excised up to a section of the neck of the mandible. Radiological and histological findings were evaluated 0, 1, 2, 3, 4, 6, and 8 weeks later.
Although bone regeneration was observed during the first postoperative week in both groups, restoration occurred from the bone stump and from a position distant from the bone stump when the periosteum was conserved. When the periosteum was removed, restoration occurred from the bone stump only. Regenerated bone mass was greater with the preserved periosteum.
Bone mass increased until the sixth week after condylectomy in the periosteum preservation group. Our results confirm the importance of the periosteum in mandibular head regeneration after condylectomy in rats.

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Dental pulp plays an important role in tooth vitality. The pulp of deciduous tooth thus shows vigorous metabolism and repeated, vigorous cell regeneration and proliferation. Furthermore, natural, programmed cell death (apoptosis) frequently occurs as part of this cell regeneration and proliferation process. Apoptosis is defined as cell death governed by intracellular signals (active death of the cell triggered by changes in physiological or external conditions), and the birth of new cells occurs simultaneously with cell death. Apoptosis thus functions as a driving force for morphological and other changes in organisms, and is essential for phenomena such as ontogenetic processes or aging. However, In order to clarify the mechanism of apoptosis in the deciduous tooth pulp cells, we used SuperArray analysis and immunostaining to examine both deciduous and permanent tooth pulp cells. Of the 84 major genes involved in apoptosis, seven showed greater expression in the deciduous tooth pulp cells than in the permanent tooth pulp cells. Gene expression of TP53BP2, CRADD, BAK1, BIRC3, CASP8, CASP2 and TP73 in the deciduous tooth pulp were 16.1, 9.0, 8.5, 8.1, 5.9, 5.8 and 3.2 times greater than those in the permanent tooth pulp cells, respectively.

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In recent years, the expression of desmin intermediate filament in muscle and tendon attachment brought about mechanical stress has been reported. Mastication and swallowing exercises can stimulate the growth of the tongue through functional load. However, the expression of intermediate filaments during development has not been investigated. Moreover, the relevance of the surrounding tissues to the development of the tongue is still unknown. Therefore, the expression of desmin in embryonic mouse tongue during development and the expression of vimentin in mesenchymal tissues were investigated by immunohistochemistry and Western Blot. Results revealed that at E11, there was no distinction between the tongue and mandible. Vimentin expression was detected in the entire tissue at E11 and the expression spread to the surrounding tissues over time. Desmin was not detected at E11 however, at E12, desmin expression was observed at the attachment of genioglossus and intrinsic muscle of the tongue which then became more intense and spread throughout the surrounding tissues. The IHC results coincided with Western Blot analysis. The results suggest that desmin expression at the site of intense mechanical loading plays an important function in mastication and swallowing.

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Chronic periodontitis is a major dental disease associated with continuous inflammation and the mediation of cytokines and chemokines such as Interleukin (IL)-8. Periodontitis processes may also be associated with quantitative changes in IL-8 gene expression. We examined the transcriptional activation and mediation of a novel single nucleotide polymorphism (SNP) at position -845 (from the transcription initiation site) of the IL8 promoter. Moreover, we investigated whether the frequencies of this SNP were associated with susceptibility to chronic periodontitis among Japanese. The fragments of cloned IL8 promoter (-866 to +30) containing the novel SNP (-845T and -845C) were ligated to the luciferase gene. A luciferase assay was then conducted using these reporter plasmids in SCCTM cells. The presence and molecular weight of the -845T-specific binding proteins were analyzed by gel mobility shift and UV-crosslinking competition assays, respectively. SNP analysis was performed with DNA from 56 control subjects and 52 patients by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and disease associations were analyzed by a Chi-square test. These results indicate that transcriptional activity of the IL8 promoter containing -845T was approximately 9-fold higher than when the promoter contained -845C. -845T-specific binding proteins (approximately 55 and 60 kDa) were present in SCCTM cells. Genotype frequencies (T/T and T/C) between the control and patient groups were not significantly different (P = 0.586). These results suggest that transcription of the IL8 gene, particularly mediation of -845T/C, appears to be strictly regulated. This SNP was not associated with chronic periodontitis in the Japanese population.

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The BMP-2/BMP-7 heterodimer (BMP-2/-7) is a potent osteogenic inducer. It is well established that BMP-2/-7 activates mitogen activated protein kinases (MAPKs) in some cells. It is also widely accepted that MAPK signaling cascades play a crucial role in the immediate osteogenic response to BMPs. However, the precise mechanism and role of the MAPK signaling network in osteogenesis is still controversial since there are many kinds of BMP receptors (BMPR) whose signal transduction is very complex and whose signals differ from cell to cell. We investigated whether BMP-2/-7 regulates osteogenic gene expression in human periodontal ligament (HPDL) cells, and how MAPKs might be involved in this process, by treatment of HPDL cells with BMP-2/-7 with or without MAPK inhibitors. We found that the Jun-N-terminal kinase (JNK) is essential for the expression of almost all osteogenic marker genes. Inhibition of extracellular signal-regulated kinase (ERK) using inhibitor had no effect on the expression of early osteogenic marker genes, whereas it significantly down-regulated the expression of late osteogenic marker genes such as Bone sialopotein and Osteocalcin. These results indicate that MAPKs play a crucial role in osteogenesis of HPDL cells.

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Intermittent Recombinant Human Parathyroid Hormone (rhPTH 1-34) is an effective treatment for improving bone mass in patients with osteoporosis; however, its effects on bone regeneration are still unclear. The objective of this study was to evaluate the potential toxicity systemic rhPTH, as well as its ability to regenerate critical-sized defects (CZD) in bone. We used 43 female Wistar rats (body weight, 150 ± 50 g). Critical-sized bone defects in rat calvaria received vehicle alone (Control Group, CG) or daily rhPTH (20μg/Kg/day) by subcutaneous injection (Experimental Group, EG). We evaluated bone healing obtained at the 1st, 3rd, and 6th wks post-surgery by biochemical, soft x-ray, histological, and morphometric studies. In the EG, at the 1st and 3rd wks, many areas of focal osteoblast hyperplasia were found on parietal bone. At the 3rd wk, woven and/or lamellar bone, in an organized interconnected trabecular network, showed disrupted mineralization. At the 6th wk, looped bone was found to have formed patterns on parietal bone. New bone formed in the EG showed significant statistical differences (p = 0.023) at the 6th wk. Systemic rhPTH at the dose of 20μg/Kg/day was able to stimulate bone formation on rat CZD. Also, pre-existing and new bone showed non-proliferative forms of bone hyperostosis (increased non-neoplastic bone).

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Electrochemotherapy (ECT) is currently receiving much attention as a method that can enhance the delivery of antitumour drugs into tumour cells by electroporation. In this study, we examined the effect of cisplatin on metastatic lesions of hamster oral fibrosarcoma by administering it in association with low-voltage electrical pulses. Oral fibrosarcoma was transplanted submucosally into the cheek pouches of 80 hamsters. After transplantation, when the diameter of the metastatic lesion in the left submandibular lymph node had reached 100 mm³, the hamsters were divided into four groups: D+E+ group (cisplatin treatment followed by electroporation; D+E- group (cisplatin treatment); D-E+ group (electroporation without cisplatin treatment); and D-E- group (no treatment). The antitumour effect of cisplatin was marked in the D+E+ group and the metastatic lesion disappeared in some of the animals. These results showed that when ECT consisting of cisplatin plus low-voltage electroporation was applied to metastatic tumour lesions, the antitumour effect of the drug on the lesions was enhanced. Therefore, ECT would seem to be a highly-safe new method that may be of use in the treatment of metastatic tumours.

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Biomimetic calcium phosphate ceramics with excellent bio-absorption and osteoinduction were designed by the partial dissolution-precipitation (PDP) treatment. Beta-tricalcium phosphate (β-TCP) ceramics with single phase were transformed into monetite, hydroxyapatite (HAp) and β-TCP phases by the PDP treatment. The PDP-β-TCP ceramics had a specific surface area of 22.5 m²/g and porosities of 60-80 %. Changes in microstructure of the ceramics before and after PDP treatment influenced body fluid permeation, release characteristics of bone morphogenetic protein-2 (BMP-2) and osteoinduction. ¹²⁵I-labeled BMP-2 was loaded on the four different ceramics, such as spongy-derived bulk of HAp (b-HAp), functionally graded HAp (fg-HAp), β-TCP, and PDP-β-TCP. The four kinds of ceramics were implanted into the back region of subcutaneous tissue in rats and the analyses of the release of BMP-2 and BMP-2 dose-response of bone formation were investigated. In the fg-HAp/BMP-2 and PDP-β-TCP/BMP-2 ceramics, the release time corresponding to the half amount (t _1/2) of BMP-2 were prolonged about 14 and 7 times respectively compared to the ceramics before PDP treatment. BMP-2 dose-response of bone formation study showed the areas of bone tissues were significant higher in the fg-HAp/BMP-2 (5.0μg) and PDP-β-TCP/BMP-2 (0.5μg) ceramics compared to the ceramics before PDP treatment. These results indicated the PDP treatment enhanced BMP-2 release and osteoinduction.

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Osteoporosis is a common metabolic bone disease often affecting postmenopausal women, and various medicines are available that attempt to treat and/or prevent this disease. Hachimi-jio-gan is a traditional oriental (Kampo) medicine used clinically, but it’s mode of action is still unknown. In this study, the effects of Hachimi-jio-gan extract on intestinal calcium absorption were evaluated in an osteoporosis model using ovariectomized (OVX) and sham-operated (SHAM) mice by investigating pharmacokinetic parameters in these animals. The ability of Hachimi-jio-gan extract to inhibit lipopolysaccharide-mediated stimulation of osteoclasts in bones using receptor activator of the NF-kB ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was also investigated. The metabolic behavior of calcium did not differ between OVX and SHAM mice as the pharmacokinetics of calcium was equivalent after intravenous administration. The absolute bioavailability of calcium indicated that the extent of intestinal calcium absorption in the OVX (10.3%) and SHAM (10.7%) mice was at similar low levels. Hachimi-jio-gan extract potentially improved the intestinal calcium absorption by 1.96- and 1.86-fold, respectively. Hachimi-jio-gan extract further suppressed the potent stimulation of RANKL-induced osteoclast differentiation in RAW264.7 cells. These results suggest that Hachimi-jio-gan extract may be suitable for treatment and prevention of osteoporosis through its ability to increase intestinal calcium absorption and suppress osteoclast differentiation.

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A convenient silver staining method generally used in marking proteins in polyacrylamide gel was applied to stain undecalcified tooth sections for clear observation of fine structures in tooth tissues. Ground sections of teeth were directly soaked and stained with a solution prepared according to the staining protocol. Although modification of the staining procedure might be necessary to establish favorable staining condition in tooth sections, observation of fine structures in tooth sections became easy with the staining technique.

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The purpose of the present study was to examine whether the chitosan/ nanometer hydroxyapatite / collagen composite (chitosan/nHAC) incorporating BMP-7 mimetic peptide could accelerate bone regeneration. The chitosan/nHAC composite was prepared and the BMP-7 mimetic peptide introduced into the composite by vacuum adsorption. The released peptide content from the composite was detected using high performance liquid chromatography at different set times. 5 mm diameter cranial bone defects were created on both sides of the parietal bone in 24 adult Sprague-Dawley rats, which were randomized into two treatment groups; one receiving the chitosan/nHAC composite loaded with 1 mg BMP-7 mimetic peptide and the other one receiving the unload composite. Bone healing was evaluated with radiographic and histological analysis. The results showed that significantly improved and effective bone regeneration was achieved with the composite loaded with 1 mg BMP-7 mimetic peptide compared to the unloaded composite. In conclusion, the BMP-7 mimetic peptide in combination with the chitosan/nHAC composite could successfully accelerate the healing of rat’s cranial bone defects. The chitosan/nHAC/BMP-7 mimetic peptide composite is an ideal bone substitute material.

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