Research Projects -
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Bone regenerative therapy using Escherichia coli-derived rhBMP-2 adsorbed in beta-TCP.
Grant number：16K11624 2016.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
NAWACHI Kumiko
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
Bone regeneration using BMP-2 is expected to have the highest therapeutic effect, but there is no BMP-2 product approved in Japan. We succeeded to develop the BMP-2 using E. coli system and it was clarified that the combination of E. coli-BMP-2 and the already approved biodegradable artificial bone, β-tricalcium phosphate (β-TCP), strongly induces bone formation in socket preservation model in dog.
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Search of master regulator of odontoblastogenesis using iPS interference
Grant number：16K15802 2016.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
Ono Mitsuaki
Grant amount：\3380000 （ Direct expense: \2600000 、 Indirect expense：\780000 ）
It is still unknown not only the mechanism but also the master regulator of odontoblastogenesis. Therefore, the final goal of our study is to identify the mater regulator of odontoblastogenesis. We performed the comprehensive analysis from the histological and developmental point of view using RNA-Seq, and could narrow down the several candidate odontoblastogenesis related genes. In the future, we are planing to perform the functional analysis of these candidate genes.
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Chronic muscle pain pathophysiology from the viewpoint of mitochondria dysfunction
Grant number：16K15801 2016.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
MAEKAWA KENJI
Grant amount：\3510000 （ Direct expense: \2700000 、 Indirect expense：\810000 ）
We made an attempt to elucidate the relationship between vitamin D function and muscle pain. This hypothesis was established based on the several previous reports, which describe the positive relationship between taking antilipemic and muscle pain onset, especially seen in hyperlipidemia patients with vitamin D deficiency.
As the results, gene expression levels of vitamin D receptor was significantly higher in trapezius and masseter muscles, which are easily affected by muscle pain than limb muscles, which are not very sensitive to muscle pain. These results suggest the possible relationship between vitamin D function and muscle pain. In addition, expression levels of positive cell number of pain sensitivity marker (c-fos) in central nervous system under noxious stimulus application in masseter muscle tended to be higher in vitamin D deficiency rats than those in rats without vitamin D deficiency. -
Grant number：15H05026 2015.04 - 2019.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B) Grant-in-Aid for Scientific Research (B)
Maekawa Kenji
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
Keratinized gingiva around the tooth and dental implant is critical to maintain a healthy condition of periodontal and peri-implant tissues, however the mechanisms regulating keratinization of gingiva still remain unclear. In this study, we hypothesized that the basement membrane is a critical regulator of keratinization of the oral mucosal epithelium. From our results, we found out that the difference in keratinization of oral mucosa is associated with the composition of the basement membrane. In addition to this, the composition which highly expresses in keratinized gingiva induced human gingival epithelial cells to express keratinized marker in vitro.
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Grant number：15K20439 2015.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
Mino Takuya
Grant amount：\3900000 （ Direct expense: \3000000 、 Indirect expense：\900000 ）
It is well known that peri-implantitis is caused by bacterial infection but the constitution of bacteria causing peri-implantitis is still unclear. We did cluster analysis of NGS to investigate the bacterial flora of peri-implant exudate which was extracted from 6 patients suffering from peri-implantitis. As a result, we found five kinds of bacteria in peri-implantitis site which were not found in periodontitis teeth, and another five kinds of bacteria which were not found in normal implants.
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Identification of master genes for regulating developmental time in regenerated organs
Grant number：15K15707 2015.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
OSHIMA Masamitsu
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
We aimed to study the identification of master genes for regulating developmental time and morphogenesis in regenerated organs. We hypothesized that essential regulatory factors of developmental time and morphogenesis in tooth development could be identified by comparing the deciduous and permanent tooth germs harvested from an identical beagle dog. In this study, we identified the FGF14 and FEZF2 genes as candidates of the regulating factors by the cDNA microarray analysis. Furthermore, we analyzed the effects of these molecules on the murine tooth germ development. We have indicated that FGF14 promoted substantial elongation of the incisor tooth germ by acceleration of ameloblast proliferation. Further study to elucidate the molecular mechanism of FGF14 and FEZF2 on tooth development is necessary for the identification of the regulatory factors of developmental time and morphogenesis in tooth organogenesis.
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Grant number：15K20480 2015.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
OIDA Yasutaka, KUBOKI Takuo, ONO Mitsuaki, Emilio Satoshi Hara
Grant amount：\4030000 （ Direct expense: \3100000 、 Indirect expense：\930000 ）
Osteoarthritis is the most common chronic condition of the joints, affecting approximately ten million Japanese and articular cartilage repair remains a challenging problem. Based on a high-throughput screening and functional analysis, we found that fluocinolone acetonide (FA) strongly potentiated chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF-b3 -treated hBMSCs could completely repair the articular surface.
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Grant number：15K15708 2015.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
Kuboki Takuo
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
Amino acids are essential for life and cell metabolism. We hypothesized that amino acids can regulate the stem cell phenotype and we performed a screening and found tryptophan as stemness regulator of BMSCs. Indeed, migration and colony forming ability of BMSCs was enhanced by tryptophan treatment. In vivo mice bone defect model, Tryptophan accelerated bone healing, and increased bone volume and trabecular number compared to PBS-injected group. In summary, L-tryptophan enhances the stemness and osteoblastic differentiation of BMSCs, and may be used as an essential factor to accelerate bone healing and/or prevent bone loss, such as in the case of ageing and osteoporosis.
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Grant number：26253088 2014.04 - 2018.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
Kuboki Takuo
Grant amount：\41600000 （ Direct expense: \32000000 、 Indirect expense：\9600000 ）
Whole-tooth regeneration therapy has great potential for the replacement of lost teeth．Recently, our group have been reported an in vitro three-dimensional cell manipulation method called the bioengineered organ germ method. However, it is unclear the mechanism of tooth development. Therefore, we performed the comprehensive analysis and could narrow down the several candidate genes. In the future, we are planing to perform the functional analysis of these candidate genes.
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Development of tissue regeneration therapy using host stem cell homing
Grant number：26713053 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A) Grant-in-Aid for Young Scientists (A)
Akiyama Kentaro, KUBOKI TAKUO, ONO MITSUAKI, OSHIMA MASAMITSU
Grant amount：\22490000 （ Direct expense: \17300000 、 Indirect expense：\5190000 ）
In this study, we investigated the mechanism of stem cell accumulation in regeneration site during wound healing process. In the mouse wound healing model, host mesenchymal stem cell accumulation was detected in 1 day after surgery. By using cDNA micro-array analysis, TNFa was detected as one of the stem cell accumulation factor. Thus, we confirmed the effect of TNFa on stem cell function including, cell proliferation, mobilization, immune-modulatory property. While TNFa could inhibit the cell proliferation, cell mobilization was strongly up-regulated. Furthermore, immune-modulatory property was, which was evaluated FASL expression, totally up-regulated. These results indicated that TNFa could stimulate host stem cell function and cause immune tolerance in wound healing site.
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Grant number：26861637 2014.04 - 2017.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
Ono Aya
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
It is still unclear the constitution of bacteria in peri-implantitis. We analyzed the bacterial flora of the exudate around dental implant body suffered from peri-implantitis by NGS. The experimental results were revealed that there are differences between individuals in the bacterial flora, and the peri-implantitis and the periodontal disease were similar bacterial flora and the healthy dental implants and the healthy tooth were also similar in the bacterial flora.
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Therapeutic effect of human dental pulp stem cell on Experimental autoimmune encephalomyelitis.
Grant number：26670837 2014.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
Akiyama Kentaro, KUBOKI TAKUO, ONO MITSUAKI, OSHIMA MASAMITSU
Grant amount：\3250000 （ Direct expense: \2500000 、 Indirect expense：\750000 ）
In this study，we evaluated therapeutic effect of human dental pulp stem cells (DPSCs) against experimental autoimmune encephalomyelitis (EAE) model mice. In comparison with human bone marrow derived mesenchymal stem cells (BMSCs) injection, DPSCs ameliorate clinical symptom including hind leg paralysis and immunological status (up-regulation of regulatory T cells and down-regulation of inflammatory Th 17 cells). However, the accumulation of injected cells into injured nerve system wasn't detected. Thus nerve regeneration mechanism still unclear.
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The identification and functional analysis of attached gingiva related gene and protein
Grant number：25893138 2013.08 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
KUMAZAKI Asuka, KUBOKI Takuo, ONO Mitsuaki, UEDA Junji
Grant amount：\2730000 （ Direct expense: \2100000 、 Indirect expense：\630000 ）
The purpose of this study was to identify the specific gene of attached gingiva. First, to compare the ability of mesenchymal cells derived from attached gingiva and free gingiva, we collected them using out growth method. Mesenchymal cell derived from free gingiva had high ability in cell adhesion, proliferation, and migration, compared with attached gingiva. These data indicated mesenchymal cells derived from attached gingiva and free gingiva were completely different and we hypothesized that the mesenchymal cells were involved in the difference of attached gingiva and free gingiva. Therefore, next we performed the co-culture assay using human squamous cell carcinoma cells and confirmed that cell adhesion and extra cell matrix molecules are important for keratinization of epithelial cells. And from cDNA maicroassay data using mesenchymal tissue and epithelial tissue, several genes were selected as candidate factors of attached gingiva related gene, especially, keratinization.
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The mechanism identification of environmental selective bone induction/suppression of BMP-2
Grant number：25463050 2013.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C) Grant-in-Aid for Scientific Research (C)
Nawachi Kumiko, KUBOKI TAKUO, SONOYAMA WATARU, ONO MITSUAKI, AKIYAMA KENTARO, SHINKAWA SHIGEHIKO
Grant amount：\5070000 （ Direct expense: \3900000 、 Indirect expense：\1170000 ）
Recombinant human BMP-2 (rhBMP-2) has been widely used to treat bone defects or bone-associated diseases in human. However, recently our research group found that rhBMP-2 inhibited bone formation in in the bone marrow space. The purpose of this research was to elucidate the mechanism of environmental selective bone induction/suppression of BMP-2. In the mice model, BMP-2 could not induce the bone formation in the bone marrow rich environment. Moreover, large animal model experiments revealed that rhBMP-2 were suitable for operative procedure in the bone marrow poor environment, such as socket lift and socket preservation.
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Do severe sleep bruxism subjects have the cardiovascular risk due to sleep hypertension?
Grant number：25670819 2013.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
Minakuchi Hajime, KUBOKI Takuo, MAEKAWA Kenji
Grant amount：\3640000 （ Direct expense: \2800000 、 Indirect expense：\840000 ）
Validation of the miniature sleep bruxism (SB) detection/analyzing device has not been evaluated. This study determined the validity of device compared to polysomnogram puls AV recording (PSG). Sensitivity, specificity and accuracy between device and PSG assessment were 1.00，0.88 and 0.93 in case cut-off criteria was set at score 1 to 2. These results suggested that this modified miniature detection/analyzing device would obtain the moderate to high validity in clinical use.
Association between SB and simultaneous comorbid motor activites is unclear. This study aimed to compare the the incidences of body motions between sleep arousal (SA) with SB (SAwSB) and SA without SB (SAw/oSB). Motor activities in SAwSB was significantly higher than that in SAw/oSB (t-test, p<0.01, 95.1+/-6.6 %, 69.2+/-19.5 %). Leg movement and swallowing were significantly frequent among the comorbidities. This suggested that SB can be cocomitantly activated with leg movemnet and swallowing in relation to SA. -
Development of technologies for next-generation regenerative therapies
Grant number：25242041 2013.04 - 2016.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A) Grant-in-Aid for Scientific Research (A)
Tsuji Takashi, KUBOKI Takuo, OSHIMA Masamitsu, EGUSA Hiroshi, TSUBOTA Kazuo, KAGIHARA Yasuhiro, FUJITA Satoshi, KISHIDA Akio, SATO Akio, TAKEDA Akira, TOYOSHIMA Ko-ei
Grant amount：\47190000 （ Direct expense: \36300000 、 Indirect expense：\10890000 ）
In this project, we aimed at the development of fundamental technology for the next generation of organ regenerative medicine. 1) We successfully demonstrated to regenerate of secretory glands including salivary and lacrimal glands by the transplantation of their bioengineered germs, which were reconstituted by using "Organ Germ Methods". 2) To identify the cell seeds for organ regeneration, we demonstrated the possibility for the development of the functional bioengineered 3D integumentary organ system and the realization of organ replacement therapy by using iPS cells. 3) We further achieved the fundamental technology developments of new functional gel materials using the functional sugar chain and self-assembling peptides, for the application to clinical application for organ regenerative medicine. These results indicated that we successfully developed the fundamental technology for clinical application of the wide variety of organ regenerative therapies.
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Development of production technique of tissue derived stem cell using novel reprogramming system.
Grant number：25670818 2013.04 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research Grant-in-Aid for Challenging Exploratory Research
KUBOKI Takuo, ONO Mitsuaki, AKIYAMA Kentaro
Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）
Small non-coding microRNAs (miRNAs) have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. We herein sorted stem-cell-enriched side population (SP) cells from human DPCs and PDLCs, and performed a miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/ progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
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Study of the effect in perioperative oral management
Grant number：24890136 2012.08 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
MINAKUCHI Mami, SOGA Yoshihiko, KUBOKI Takuo
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
The clinical survey of intraoral condition has been performed for the patients who received the surgical therapy of malignant tumors of the gastrointestinal region in order to promote the intraoral management in perioperative periods effectively. And, case report article was published regarding to the relationship between postoperative recovery of the esophageal cancer patient and orally nutrition approaches. Furthermore, 2nd Advanced perioperative dental approach symposium was held at January 26th, 2014. In this meeting, discussions with the nationwide practitioners were performed to exchange the opinion of the newest finding of oral functional management in perioperative periods, develop the novel approach in perioperative oral management and verify these efficacies in order to future applications.
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Human dental pulp stem cell ameliorate Experimental autoimmune encephalomyelitis.
Grant number：24890135 2012.08 - 2014.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up Grant-in-Aid for Research Activity Start-up
AKIYAMA Kentaro, KUBOKI Takuo, ONO Mitsuaki, OSHIMA Masamitsu
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
In this study, we evaluated the therapeutic effect of bone marrow derived mesenchymal stem cells (BMMSCs) or dental pulp stem cells (DPSCs) systemic injection on experimental autoimmune encephalomyelitis (EAE) model mice. In the clinical evaluation, both BMMSCs and DPSCs showed significant therapeutic effect including hind leg paralysis. However, BMMSCs injected group showed, in terms of immunological evaluation, higher up regulation of regulatory T cells (Tregs) in peripheral blood after 40days from EAE induction compare to DPSCs injected group.
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Grant number：24792083 2012.04 - 2015.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B) Grant-in-Aid for Young Scientists (B)
MINO Takuya, KUBOKI Takuo, SONOYAMA Wataru, ONO Mitsuaki, ONO Aya
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
The purpose of this study was to identify the biological markers that are involved in the onset and progression of peri-implantitis. First, we developed a clinical examination protocol by referring to the previous reports and investigated the prospective study for the initial peri-implantitis patient using the developed protocol. The subject of this study was five and, basic information of subjects was shown below, average age: 71.0 ± 4.6 years, average number of implants per patient : 4.8 ± 2.4, average number of implant with peri-implantitis：1.4 ± 0.5) . To identify the biological markers, gingival crevicular fluids around the oral implant and natural tooth were collected by using the paper points. ,The adequate amount of bacterial DNA could be collected for 16S rRNA sequencing using this protocol.