Research Projects -
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人工知能による深層学習を応用した運動障害性咀嚼障害の多軸診断支援システムの開発
Grant number：20K10071 2020.04 - 2023.03
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
大野 彩, 窪木 拓男, 森田 瑞樹, 菊谷 武, 百田 龍輔
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
本申請研究では、①咀嚼運動を撮影した動画から評価できる「咀嚼機能評価プロトコール」の診断結果が、十分な信頼性・妥当性を有するかどうかを、臼歯部移送試験や専門医の診断等と比較して評価する。また②そのプロトコールを用いて、患者および正常咀嚼者の動画撮影および診断を行い、教師データを収集する。そして、③教師データを人工知能（AI)深層学習に供し、AIによる運動障害性咀嚼障害診断システムを開発することを目的としている。
本年度は、人を対象とする医学系研究に関する倫理指針に則り、本研究計画について倫理審査委員会の承認を得た。そして、動画を用いた運動性咀嚼機能評価法の信頼性・妥当性の確認のため、まずは健常者にてプロトコールおよび咀嚼解析システムの精度確認を行った。その結果、撮影角度、影や眼鏡等の影響による運動検出精度の低下が明らかとなった。そのため、システムの改良を行い、運動検出精度を向上させることに成功した。 -
下顎骨後方移動術に伴う睡眠呼吸障害の発症リスクおよび施術基準の確立
Grant number：19K10382 2019.04 - 2023.03
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
中村 政裕, 川邉 紀章, 片岡 伴記, 鬼頭 慎司, 森本 泰宏, 水口 一, 窪木 拓男, 宮脇 卓也
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
骨格性下顎前突患者に対し安全に下顎骨後方移動術を施行するためには、術後の上気道の狭窄やそれに伴う睡眠呼吸状態への影響に配慮しなければならない。過去の研究においても、下顎骨後方移動術を行った患者の上気道形態の変化は調べられているが、これらは覚醒時に撮影したセファログラムやコンピューター断層撮影画像を解析した静態評価であった。そのため、本研究は、上気道の動態および三次元解析が可能であるMRI movieおよびvolumetric MRIを用いて下顎骨後方移動術前後の睡眠時における上気道形態の変化を観察し、下顎骨の後方移動量と睡眠時の上気道の容量との関連を解明し、睡眠時の上気道形態変化と睡眠時呼吸状態との関連を調べることで睡眠呼吸障害の発症リスクを明らかにすることを目的としている。
本年度の研究実績として、研究を開始するにあたり、臨床研究実施計画書および研究説明書、同意書等を作成し、岡山大学臨床研究審査専門委員会への申請を行った。臨床研究審査専門委員会の承認が得られたため、患者への同意説明を開始した。 -
オーラルフレイル回避を目指した筋機能訓練の有効性検討と新規個別スキームの開発
Grant number：19K10225 2019.04 - 2023.03
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
水口 一, 三木 春奈, 水口 真実, 窪木 拓男
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
摂食機能の低下の抑制が可能となれば，健常高齢者の要介護状態への転落を遅延できるかもしれなという研究仮設のもと，高齢者の筋機能維持を筋機能訓練の観点から検証することを当初の目的としていた。その一方で，睡眠時の口腔周囲筋の機能亢進が筋機能訓練の一助となりうる可能性にも着目した。その結果，高齢者の就寝時の筋活動量と筋機能との関連についても検討を行うこととなった。
第一に，就寝中の咬筋の筋活動を簡便かつ適正に定量化することを目的に，音声動画撮影を追加したpolysomnography（PSG）検査をもとに，筋電図による睡眠時筋活動検査システムについて検討を行った。しかしながら従来の睡眠時の口腔周囲筋の活動評価は，筋電図をもとに一定の閾値を超えた筋活動の頻度を基準としてきたため，掻痒や体動による筋電図波形の亢進も筋活動の亢進として誤認されてしまう場合があり，その信頼性に問題があることが明らかとなった。すなわち，我々が行った動画記録を伴うポリソムノグラフ（PSG）検査において，咬筋の筋活動が亢進したイベントのうち，80.7±16.0%が体動や掻痒等による非特異的な筋活動やアーチファクトに起因したものであった事が示された。
本研究結果は，筋電計単体による口腔周囲筋の評価には多くの偽陽性イベントが含まれていることを示すものであり，従来の検査方法，評価方法の妥当性を高く評価できないとう結果となった。事実，閉塞性睡眠時無呼吸（OSAS）症状が出現する際には低酸素状態となることから，交感神経活動の亢進，微小／睡眠覚醒，筋活動の亢進が出現し，その結果，筋活動の亢進が認められることが知られている。
そこで，検査が容易な筋電図検査において，真の口腔周囲筋の活動と偽の筋電図の亢進現象（OSAS関連性，嚥下関連性，動作関連性，その他）を識別する手法の確立が急務と考えられた。 -
基底膜構成分子の誘導制御による低侵襲角化歯肉獲得療法の確立
Grant number：19H03841 2019.04 - 2023.03
日本学術振興会 科学研究費助成事業 基盤研究(B) 基盤研究(B)
前川 賢治, 窪木 拓男, 冨田 秀太, ハラ エミリオ・サトシ, 大橋 俊孝, 大野 充昭
Grant amount：\17030000 （ Direct expense: \13100000 、 Indirect expense：\3930000 ）
基底膜直下に存在する角化歯肉由来間葉細胞と非角化歯肉由来間葉細胞の遺伝子発現の相違を明らかにすることで，上皮細胞の角化に関わっている間葉細胞からのシグナル分子を抽出することが可能であると考える。そこで，令和元年度は，レーザーマイクロダイセクション法とRNA-Seq を組み合わせた候補因子の抽出を目的に，以下の実験を実施してきた。
間葉組織は，筋肉や脂肪など様々な組織を含むことから，マクロレベルで口蓋粘膜から間葉組織を採取すると，様々な組織を含んでしまう。基底膜直下の間葉組織の遺伝子発現解析を正確にするには，特異的に組織を採取することが可能なレーザーマイクロダイセクション法を用いる必要がある。そこで，マウスの口蓋粘膜（角化粘膜）と頬粘膜（非角化粘膜）の凍結組織切片を作製し，サンプルの厚み，固定方法，染色方法，レーザーの強度の調整等，様々な条件検討を行い，本組織において最適な条件を見出した。そして，これらのサンプルからRNAを抽出し，RNA-seqが可能な質の高いRNAが回収できていることを，TapStation (アジレント)にて確認した。
また、in vitroにおいて，角化粘膜の間葉組織に高発現している遺伝子をRNA-seq解析から抽出後，さらなる候補因子の絞り込みをin vitroにて行う予定である。そこで，令和元年度は，ヒト口腔扁平上皮癌由来の口腔粘膜上皮細胞であるTR146と，ヒト口腔粘膜細胞由来線維芽細胞を用いた三次元共培養実験モデルを構築すべく，コラーゲンゲルの種類，濃度や細胞の濃度などの条件検討を行い，正常な口腔粘膜組織に類似したin vitro モデル構築に適正な条件の絞り込みを終えた。 -
The clarification of bone formation and absorption mechanism in the bone marrow microenvironment
Grant number：19H03842 2019.04 - 2022.03
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)
Ono Mitsuaki
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
BMP-2 has been widely studied for its potent ability to induce osteoblast differentiation and ectopic bone formation, and is already in clinical use worldwide. On the other hand, we have shown that BMP-2 inhibits bone formation in the bone marrow. However, the mechanism by which BMP-2 suppresses bone formation remains unclear. Therefore, we analyzed the detail of BMP-2 induced bone. Single cell RNA-seq analysis revealed that BMP-2-induced bone formed bone marrow with hematopoietic function. These results suggest that BMP-2 has the ability to regenerate bone marrow as an organ, and may suppress bone formation in the bone marrow in order to reserve space for hematopoiesis.
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Bone marrow microenvironment of MRONJ and application of Escherichia coli derived BMP-2
Grant number：19K10246 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Nawachi Kumiko
Grant amount：\4160000 （ Direct expense: \3200000 、 Indirect expense：\960000 ）
The pathogenesis of medication - related osteonecrosis of the jaws (MRONJ) remains largely unknown. Therefore, it is essential to elucidate the pathogenesis of MRONJ and to develop a treatment for this disease. We hypothesized that BMP-2, which has strong osteogenic potential, might be useful in the treatment of MRONJ. In fact, a mouse MRONJ model was created and rhBMP-2 was administered into the extraction socket. The results showed that bone formation in the extraction socket was suppressed in the non-transplanted group, whereas bone formation was significantly enhanced by rhBMP-2 transplantation. These results suggest that BMP-2 may be a new treatment for MRONJ.
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Examination for the effect of chewing on cognitive function and comprehensive search for biomarkers for early diagnosis of dementia
Grant number：19K10205 2019.04 - 2022.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
Mino Takuya
Grant amount：\4290000 （ Direct expense: \3300000 、 Indirect expense：\990000 ）
Whether chewing stimulation increase BDNF consistency or not has not been revealed on humans. We conducted 60 minutes chewing stimulation experiments with gum base on healthy male adults to investigate BDNF consistency in saliva and plasma using the BDNF ELISA kit. As a result, there were no increase of BDNF consistency in saliva and plasma during 60 minutes chewing stimulation experiments.
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Grant number：18K19646 2018.06 - 2020.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory) Grant-in-Aid for Challenging Research (Exploratory)
Kuboki Takuo
Grant amount：\6240000 （ Direct expense: \4800000 、 Indirect expense：\1440000 ）
Stem cells are required for lifelong homeostasis and regeneration of organs and tissues in mammals. However, aging of stem cells reduces cellular function and results in dysfunctional organs and tissues. Therefore, maintenance of the stemness of stem cells is of crucial importance, although its mechanisms are still unclear. The aim of this study was to identify the master regulator for the maintenance of stemness of bone marrow mesenchymal stem cells (BMSCs). First, we compared the RNA expression patterns between BMSCs derived from young and old mice, as well as between human BMSCs and human dermal fibroblasts using RNA-seq, and found that 30 transcription factors were highly expressed in BMSCs derived from young mice and in human BMSCs. Next, we found that several transcription factors could inhibit the induction to pluripotent stem (iPS) cells using iPS interfering method. The detailed function of these transcription factors in BMSCs are now being investigated.
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Understanding of molecular mechanisms of tooth development using iPS interference and application to tooth regeneration techniques
Grant number：18H02991 2018.04 - 2021.03
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)
Kuboki Takuo
Grant amount：\17550000 （ Direct expense: \13500000 、 Indirect expense：\4050000 ）
Whole-tooth regeneration is ultimate goal in dental field and the goal of this study is to identify the master transcription factors that characterize tooth germ derived epithelial and mesenchymal cells and develop method to generate those cells. First, we analyzed the mouse embryonic tooth germ and human stem cell from the apical papilla (hSCAP) by RNA-Seq and performed iPS interference to identify the potential transcription factors. Finally, candidate transcription factors were overexpressed in human adult dermal fibroblast (hADF), resulting that hADF was induced into the cells similar to tooth germ derived mesenchymal cells.
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The functional analysis of WISP1 gene aimed at the prevention of osteoarthritis
Grant number：18K09682 2018.04 - 2021.03
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)
Maeda Azusa
Grant amount：\4420000 （ Direct expense: \3400000 、 Indirect expense：\1020000 ）
The purpose of this study was to elucidate the relationship between WISP1 gene and osteoarthritis (OA). In this study, we found that severity of OA mouse models in the Wisp1 deficient (Wisp1-KO) mouse was milder compared to wild type mouse. We also found that expression level of genes related to the extracellular matrix degrading proteases in the Wisp1-KO-OA knee joint tissue was lower compared to wildtype, it is suggesting that WISP1 is involved in pathogenic mechanism and progress of OA by promoting the secretion of ECM-degrading proteases.
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Molecular understanding of the accumulation of mesenchymal stem cells in vivo by photo-manipulation techniques and its application to bone-related diseases
Grant number：17H04399 2017.04 - 2021.03
Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
Takarada Takeshi
Grant amount：\16640000 （ Direct expense: \12800000 、 Indirect expense：\3840000 ）
In this study, we generated a tTA-dependent photoactivatable Cre-loxP recombinase knock-in mouse model (TRE-PA-Cre mice) using a CRISPR/Cas9 system. These mice were crossed with ROSA26-tdTomato mice (Cre reporter mouse) to visualize DNA recombination as marked by tdTomato expression. We demonstrated that external noninvasive LED blue light illumination allows efficient DNA recombination in the liver of TRE-PA-Cre:ROSA26-tdTomato mice transfected with tTA expression vectors using hydrodynamic tail vein injection. The TRE-PA-Cre mouse established here promises to be useful for optogenetic genome engineering in a noninvasive, spatiotemporal, and cell-type specific manner in vivo.
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Understanding of periodontal disease and peri-implantitis with stem cell disfunction
Grant number：17H04392 2017.04 - 2020.03
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)
AKIYAMA Kentaro
Grant amount：\16640000 （ Direct expense: \12800000 、 Indirect expense：\3840000 ）
In this study, we investigated how the bone marrow-derived mesenchymal stem cells (MSCs) function is affected by host aging. And then, how the suppressed MSCs function contributes to the development of inflammatory diseases in periodontal tissues such as periodontal disease and peri-implantitis. As a result, in the experimentally induced periodontal disease model mice, increased bone resorption was observed in aged mice. Moreover, the function of MSCs derived from 50-week-old mice showed lower cell proliferation and osteoblast differentiation ability with higher adipocyte differentiation ability. More interestingly, MSCs from aged mice showed decreased immunoregulatory capacity.
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Grant number：17K11750 2017.04 - 2020.03
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)
Kimura-Ono Aya
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
Recently, we successfully regenerated the cortical bone-like bone, which is important to maintain the long-term stability of regenerated bone, using Escherichia coli-derived rhBMP-2 (E-rhBMP-2) adsorbed in PLGA membrane in rat model. The purpose of this study was to evaluate the E-rhBMP-2/PLGA membrane using peri-implantitis canine model. First, we generated the peri-implantitis model using beagle dogs and transplanted the autogenous bone to evaluate this animal model. Interestingly, autogenous bone graft could not recover the bone defect caused by peri-implantitis. Next, we transplanted the β-TCP containing E-rhBMP-2 in bone defects and covered with PLGA membrane containing E-rhBMP-2. Only β-TCP containing E-rhBMP-2 was transplanted as control group. As a result, PLGA membrane containing E-rhBMP-2 induced bone formation compared with control group. In conclusion, the PLGA membrane containing E-rhBMP-2 was efficient in bone formation in a peri-implantitis model in beagle dogs.
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必須アミノ酸トリプトファンによる幹細胞老化制御機構の解明・骨質改善治療への応用
Grant number：17K11751 2017.04 - 2018.03
日本学術振興会 科学研究費助成事業 基盤研究(C) 基盤研究(C)
笈田 育尚, 窪木 拓男, 大野 彩, 宝田 剛志, 大野 充昭
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
口腔インプラント治療は，人工歯根が歯槽骨や顎骨と結合することにより強固な骨支持を得るため，骨量と骨質が重要な因子となる．しかし，日本人は欧米人と比べ歯槽骨が解剖学的に菲薄で，インプラント体埋入のために骨造成が必要な場合も少なくない．また，高齢化の進む日本で増加傾向にある骨粗鬆症患者へ口腔インプラント治療がなされる場合も多く，多くの研究者が骨造成や骨質改善に関する研究を進めてきた．
我々は，これまでの研究から骨髄由来間葉系幹細胞の幹細胞性維持という観点からスクリーニングし，同定したトリプトファンが，骨質改善や骨の創傷治癒を促進することが明らかにした．この結果は，トリプトファンの投与が口腔インプラントの骨結合促進においても有用である可能性を強く示唆するものである．しかし，口腔インプラントの埋入に伴うトリプトファンの投与が，①骨のリモデリングを担う骨芽細胞，破骨細胞や間葉系幹細胞にどのような影響を与えるのか，また，②インプラント体の初期固定や長期予後に有意に働くのか，また，トリプトファンによる幹細胞の活性化が骨粗鬆症をはじめとする老化疾患に対して有効なのか，未だその詳細は明らかでない．
そこで，本研究ではトリプトファンの骨代謝関連細胞に与える効果の検討を行うこととした．トリプトファンと間葉系幹細胞の骨芽細胞分化との関係性は明らかにしてきたが，破骨細胞との関係性は未だ不明である．はじめに，トリプトファンが破骨細胞分化に与える影響を検討した．すなわちトリプトファンを投与したマウスの大腿骨を経時的 (0, 3, 7, 14日)にサンプリングし，組織学的，分子生物学的に検討する計画をした．しかし本研究では研究期間が短く解析，評価するまでには至らなかった．
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Grant number：16H06990 2016.08 - 2018.03
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
Hara Emilio Satoshi, Matsumoto Takuya, Okada Masahiro, Kuboki Takuo, Nagaoka Noriyuki, Hattori Takako
Grant amount：\2860000 （ Direct expense: \2200000 、 Indirect expense：\660000 ）
We investigated the initial stages of endochondral ossification during secondary ossification of mouse femur epiphysis from the Life Science and Material Science viewpoints. We found that the very initial starting point of mineral formation in the epiphysis occurs at post-natal day 6 (P6).
Electron microscopy-based ultrastructural analysis showed that cell-secreted matrix vesicles were absent in the early steps of osteoblast-independent endochondral ossification. Instead, chondrocyte membrane fragments were found in the fibrous matrix surrounding the hypertrophic chondrocytes. EDS analysis and electron diffraction study indicated that cell membrane fragments acted as nuclei for newly formed calcospherites which initially showed an amorphous calcium phosphate phase, and then transformed into apatite crystal phase. These findings would be valuable to develop novel organic-inorganic hybrid materials for manipulation of biomineralization. -
Examine the effect of HMGB1 on bone marrow derived mesenchymal stem cell
Grant number：16H06991 2016.08 - 2018.03
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
Komi Keiko, KUBOKI Takuo, AKIYAMA Kentaro, ONO Mitsuaki, MARUHAMA Kotaro, YOSHIOKA Yuya, KUNITOMO Masayoshi
Grant amount：\2990000 （ Direct expense: \2300000 、 Indirect expense：\690000 ）
In recent years High mobility group box 1 (HMGB 1) is known as a factor promoting tissue regeneration. It is also reported that HMGB1 induces mesenchymal stem cells in the marrow to the damage skin. In this study, we investigated the influence of HMGB1 on bone marrow derived mesenchymal stem cells. In the mouse wound healing model, host mesenchymal stem cell accumulation was detected in 1 day after surgery, and HMGB1 was expressed around the stem cell accumulation site in same day. We examined the effect of HMGB1 on mesenchymal stem cells, but the effect on stemness, stem cell proliferation and migration was not clear. Further studies on multiple differentiation and immunomodulate function that may be affected in the presence of HMGB1 will be necessary in the future.
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Identification of risk factor of CKD and pathogenic bacteria of infectious complications in dialysis
Grant number：16H06989 2016.08 - 2018.03
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
KOYAMA Eri, KUBOKI Takuo, ONO Aya, ONO Mitsuaki, AKIYAMA Kentaro
Grant amount：\2860000 （ Direct expense: \2200000 、 Indirect expense：\660000 ）
In our country, the chronic kidney disease (CKD) patients increase year by year, and it is reported that oral health is related to life prognosis and the onset of complications in CKD patients, and the relationship between oral and kidney is attracting a great deal of attention.
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Therefore, the goal of this study is to identify the bacterial flora in shunt infection and clarify the relationship between oral flora and shunt infection. From the results of 16S rRNA analysis, the ratio of staphylococcus in saliva of several patients was abnormally high and oral flora of saliva was similar to that of infected shunt．And we also found that the oral disease-related bacteria existed in the infected shunt. -
Evaluation of upper airway size using Magnet Resonance Imaging
Grant number：16K11787 2016.04 - 2020.03
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)
KATAOKA Tomoki
Grant amount：\4680000 （ Direct expense: \3600000 、 Indirect expense：\1080000 ）
MRI movie and volumetric MRI, that could evaluate a soft tissue kinetically and three-dimensionally, were used to evaluate upper airway size. While the original aim to make a treatment standard of patients with obstructive sleep apnea syndrome was not accomplished, we established the evaluation method of the upper airway using MRI movie and volumetric MRI, and clarified the usefulness of MRI movie that could evaluate a state of upper airway kinetically. As a result, mandibular body length and anteroposterior position were correlated with upper airway size, although the anteroposterior length and position of maxillary bone, and the vertical position and length of mandible were not correlated.
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Grant number：16K11590 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)
Minakuchi Mami
Grant amount：\4550000 （ Direct expense: \3500000 、 Indirect expense：\1050000 ）
The results of comprehensive analyses of the intestinal flora and metabolites in the elderly requiring long-term care showed that the ratio of bacterial flora X and Y, at the division level, were significantly higher in overweight and underweight individuals, respectively. At the genus level, the bacterial flora Z and A were identified, respectively, as a bacterium positively and negatively correlated with BMI. Moreover, it became clear that some samples of underweight individuals could be distributed in the same cluster.
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Grant number：16H05524 2016.04 - 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)
Ono Mitsuaki
Grant amount：\17160000 （ Direct expense: \13200000 、 Indirect expense：\3960000 ）
Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, in this experiment, we revealed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application.