2014 Fiscal Year Annual Research Report
合成微小環境を用いた軟骨組織のｉｎ ｖｉｔｒｏ構築
| Project/Area Number |
14F04106
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| Research Institution | Okayama University |
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Principal Investigator |
松本 卓也 岡山大学, 医歯(薬)学総合研究科, 教授 (40324793)
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| Co-Investigator(Kenkyū-buntansha) |
HARA Emilio 岡山大学, 医歯(薬)学総合研究科, 外国人特別研究員
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| Project Period (FY) |
2014-04-25 – 2016-03-31
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| Keywords | 軟骨 / 合成微小環境 / DNAメチル化 / 石灰化 |
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| Outline of Annual Research Achievements |
In this study, we are attempting to synthesize a biomimetic microenvironment for fabrication of cartilage in vitro, using materials (e.g., hydrogels) and biological tools, including overexpression of proteins in cells. We hypothesized that chondrogenic differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (hBMSCs) could be regulated by DNA methyltransferases (DNMTs) on specific genes involved in the promotion or suppression of chondrogenesis.
To study the effect of DNMTs on chondrogenesis of hBMSCs, we plan to transfect DNMTs overexpression vectors in the cells, and culture the cells in a 3D micromass culture system.
Since in vitro synthesized cartilage tissue generally undergoes through mineralization, we are also attempting to understand the mechanisms involved in the mineralization process during initial bone formation.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Initially, we analyzed the expression pattern of DNMTS during normal process of chondrogenesis in vitro, and found that DNMT3A and DNMT3B were significantly upregulated. Then, we transfected DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) overexpression vectors in hBMSCs, and induced the cells to differentiate into chondrocytes by culturing the cells in a 3D micromass culture system in chondrogenesis-inducing medium for 21 days. The results showed that overexpression of DNMT3A induced a strong increase in the deposition of cartilage matrix, as detected by toluidine blue staining of histological sections of the micromasses. Similar to DNMT3A, overexpression of DNMT3B also increased the expression of cartilage markers, aggrecan and collagen type II, however, to a less extent.
To analyze the process of mineralization from chondrocytes, we observed the formation of minerals during secondary ossification of mouse femur. We found that mineralization occurs in the initial 7 days after birth, and it progresses relatively quickly until the second week after birth.
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| Strategy for Future Research Activity |
We plan to identify the mechanism of how DNMT3A and DNMT3B regulates chondrogenesis of hBMSCs; more specifically, to identify the sites methylated by DNMT3A and DNMT3B in the promoter or enhancer region of key genes, including SOX9, ACAN, COL2A1.
We also plan to identify the types of mineral formed in the initial process of bone formation, and analyze the maturation process of mineral crystals.
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[Journal Article] The regenerative effects of CCN2 independent modules on chondrocytes in vitro and osteoarthritis models in vivo2014
Author(s)
Tarek Abd El Kader, Satoshi Kubota, Takashi Nishida, Takako Hattori, Eriko Aoyama, Danilo Janune, Emilio Satoshi Hara, Mitsuaki Ono, Yasuhiko Tabata, Takuo Kuboki, Masaharu Takigawa
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Journal Title
Bone
Volume: 59
Pages: 180-188
DOI
Peer Reviewed
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[Journal Article] Efficient bone formation in a swine socket lift model using Escherichia coli-derived recombinant human bone morphogenetic protein-2 adsorbed in β-tricalcium phosphate.2014
Author(s)
Mitsuaki Ono, Wataru Sonoyama, Katushi Yamamoto, Yasutaka Oida, Kentaro Akiyama, Shigehiko Shinkawa, Ryu Nakajima, Hai Than Pham, Emilio Satoshi Hara, Takuo Kuboki
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Journal Title
Cells, Tissues, Organs
Volume: 199
Pages: 249-255
DOI
Peer Reviewed
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