Application and molecular basis of spheroid culture to the deterioration of periodontal ligament cells due to frequent passage

• Project/Area Number: 19K10133
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 57030:Conservative dentistry-related
• Research Institution: Kyushu Dental College
• Principal Investigator: USUI MICHIHIKO 九州歯科大学, 歯学部, 准教授 (10453630)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: スフェロイド / 歯根膜幹細胞 / 骨分化 / 幹細胞性 / 歯根膜細胞

Outline of Research at the Start

歯根膜細胞の供給源には制限があるため、組織再生に必要な間葉系幹細胞を獲得する際には、単層培養（二次元培養）によって、細胞数を増加させる必要があるが、継代数を重ねるに伴い、増殖能、幹細胞性や多分化能が喪失される。本研究では、スフェロイド培養（三次元培養）を用いて、継代数の増加により生理機能の衰えた歯根膜細胞を回復することができるか否かを検証し、そのメカニズムを解明する。

Outline of Final Research Achievements

In present study, we performed RNA-seq analysis to find genes specifically expressed in hPDLMSC (human periodontal ligament mesenchymal stem cell) spheroids and to analyze their functions. The hPDLMSC spheroid culture group and monolayer culture group were compared by transcriptome analysis. We found that the transcription factor NR4A2 (Nuclear receptor 4A2) was strongly expressed in the spheroid culture group, and knockdown of NR4A2 significantly increased the expression of osteogenesis-related genes, ALP activity, and calcified nodule formation. These results suggest that NR4A2 negatively regulates osteogenesis in hPDLMSC spheroids.

Academic Significance and Societal Importance of the Research Achievements

ヒト歯根膜間葉系幹細胞を用いた細胞治療は、次世代の歯周組織再生治療として期待されている。しかし、再生に適した歯根膜幹細胞の培養条件については不明な点が多い。スフェロイド培養は幹細胞の生理的機能を上昇させることが知られているが、今回我々が見出したNR4A2がヒト歯根膜間葉系幹細胞のスフェロイド培養において、幹細胞性の維持・骨分化抑制に関与している可能性が明らかとなったことは学術的に意義深いと考える。

Research Products

• [Journal Article] Validation of a self-report questionnaire for periodontitis in a Japanese population (2021)
  • Author(s): Iwasaki Masanori、Usui Michihiko、Ariyoshi Wataru、Nakashima Keisuke、Nagai-Yoshioka Yoshie、Inoue Maki、Kobayashi Kaoru、Borgnakke Wenche S.、Taylor George W.、Nishihara Tatsuji
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 1-12
  • DOI: 10.1038/s41598-021-93965-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Mechanism of alveolar bone destruction in periodontitis ? Periodontal bacteria and inflammation (2021)
  • Author(s): Usui Michihiko、Onizuka Satoru、Sato Tsuyoshi、Kokabu Shoichiro、Ariyoshi Wataru、Nakashima Keisuke
  • Journal Title: Japanese Dental Science Review Volume: 57 Pages: 201-208
  • DOI: 10.1016/j.jdsr.2021.09.005
  • NAID: 210000173382
  • Peer Reviewed / Open Access
• [Journal Article] Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis (2021)
  • Author(s): Suga Takenori、Usui Michihiko、Onizuka Satoru、Sano Kotaro、Sato Tsuyoshi、Nakazawa Kohji、Ariyoshi Wataru、Nishihara Tatsuji、Nakashima Keisuke
  • Journal Title: Stem Cells International Volume: 2021 Pages: 1-18
  • DOI: 10.1155/2021/5592804
  • Peer Reviewed / Open Access
• [Journal Article] Sleep duration and severe periodontitis in middle‐aged Japanese workers (2021)
  • Author(s): Iwasaki Masanori、Usui Michihiko、Ariyoshi Wataru、Nakashima Keisuke、Nagai‐Yoshioka Yoshie、Inoue Maki、Kobayashi Kaoru、Nishihara Tatsuji
  • Journal Title: Journal of Clinical Periodontology Volume: 49 Issue: 1 Pages: 59-66
  • DOI: 10.1111/jcpe.13561
  • Peer Reviewed / Open Access
• [Journal Article] Schizophyllum commune β-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria. (2021)
  • Author(s): Chuencheewit Thongsiri, Yoshie Nagai-Yoshioka, Ryota Yamasaki, Yoshiyuki Adachi, Michihiko Usui, Keisuke Nakashima, Tatsuji Nishihara, Wataru Ariyoshi
  • Journal Title: Carbohydrate polymers Volume: 253 Pages: 117285-117285
  • DOI: 10.1016/j.carbpol.2020.117285
  • Peer Reviewed
• [Journal Article] Hematogenous apoptotic mechanism in salivary glands in chronic periodontitis. (2020)
  • Author(s): Shikayama T, Fujita-Yoshigaki J, Sago-Ito M, Nakamura-Kiyama M, Naniwa M, Hitomi S, Ujihara I, Kataoka S, Yada N, Ariyoshi W, Usui M, Nakashima K, Ono K.
  • Journal Title: Arch Oral Biol Volume: 117 Pages: 104775-104775
  • DOI: 10.1016/j.archoralbio.2020.104775
  • Peer Reviewed
• [Journal Article] 3D spheroid culture models for chondrocytes using polyethylene glycol-coated microfabricated chip. (2020)
  • Author(s): Wataru Ariyoshi, Michihiko Usui, Kotaro Sano, Aki Kawano, Toshinori Okinaga, Keisuke Nakashima, Kohji Nakazawa, Tatsuji Nishihara
  • Journal Title: Biomedical research (Tokyo, Japan) Volume: 41 Issue: 4 Pages: 187-197
  • DOI: 10.2220/biomedres.41.187
  • NAID: 130007887499
  • Peer Reviewed / Open Access
• [Journal Article] A Preliminary Study on the Ability of the Trypsin-Like Peptidase Activity Assay Kit to Detect Periodontitis. (2020)
  • Author(s): Iwasaki M, Usui M, Ariyoshi W, Nakashima K, Nagai-Yoshioka Y, Inoue M, Nishihara T.
  • Journal Title: Dent J (Basel) Volume: 8 Issue: 3 Pages: 98-98
  • DOI: 10.3390/dj8030098
  • Peer Reviewed / Open Access
• [Journal Article] Co-cultured spheroids of human periodontal ligament mesenchymal stem cells and vascular endothelial cells enhance periodontal tissue regeneration. (2020)
  • Author(s): Sano K, Usui M, Moritani Y, Nakazawa K, Hanatani T, Kondo H, Nakatomi M, Onizuka S, Iwata T, Sato T, Togari A, Ariyoshi W, Nishihara T, Nakashima K.
  • Journal Title: Regenerative Therapy Volume: 14 Pages: 59-71
  • Peer Reviewed / Open Access
• [Presentation] トランスクリプトーム解析からみえるヒト歯根膜由来間葉系幹細胞スフェロイドの特異的遺伝子発現プロファイルの検討 (2020)
  • Author(s): 菅毅典, 臼井通彦, 鬼塚理, 佐野孝太朗, 豊留-森谷友貴, 花谷智哉, 西原達次, 中島啓介
  • Organizer: 第63回春季日本歯周病学会学術大会