2004 Fiscal Year Final Research Report Summary
Study of a key factor in chondrogenic differentiation for craniofacial skeletal regeneration
| Project/Area Number |
14207092
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
矯正・小児・社会系歯学
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
YANMAMOTO Teruko Okayama University, Graduate school of Medicine and Dentistry, Professor, 大学院・医歯学総合研究科, 教授 (00127250)
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| Co-Investigator(Kenkyū-buntansha) |
TAKIGAWA Masaharu Okayama University, Graduate School of Medicine and Dentistry, Professor, 大学院・医歯学総合研究科, 教授 (20112063)
YAMASHIRO Takashi Osaka University, Graduate School of Dentistry, Associate professor, 大学院・歯学研究科, 助教授 (70294428)
KURODA Shingo Okayama University, Graduate School of Medicine and Dentistry, Research assistant, 大学院・医歯学総合研究科, 助手 (40332796)
FUKUNAGA Tomohiro Okayama University, Graduate School of Medicine and Dentistry, Research assistant, 大学院・医歯学総合研究科, 助手 (70362994)
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| Project Period (FY) |
2002 – 2004
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| Keywords | laser microdissection / chondrocyte / in situ hybridization / differentiation / regeneration |
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| Research Abstract |
The purpose of the present study was to investigate a key factor in chondrogenic differentiation that play a role in craniofacial skeletal development.
1.We evaluated the expression of CTGF, type I, type II, and type X collagen in chondrocytes of different types of cartilages, including mandibular condylar cartilage, cartilage formed during the healing of mandibular ramus factures, femoral growth plate cartilage, and femoral articular cartilage revealed by in situ hybridization. Among these different types of cartilages we found distinct patterns of CTGF expression. Growth plate cartilage showed localization of CTGF in the hypertrophic chondrocytes that expressed type X collagen with less expression in proliferating chondrocytes that expressed type II collagen, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate
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cartilage were negative for type I collagen and showed sparse expression of CTGF in the proliferating chondrocytes. The spatial distribution of CTGF in the cartilages with type I collagen suggested its role in the early differentiation of those cartilages.
2.Runx1,Runx2, and Sox9 expression was evaluated by in situ hybridization in the growing craniofacial bones of embryonic day (E)12-16 mice. In addition, we evaluated Runx2 expression in the growing face of Runxl knockout mice at E12.5 and Runx1 expression in Runx2 knockout mice at E14.5. Runx1 and Sox9 were expressed in cartilage, and the regions of expression expanded to the neighboring Runx2-expressing osteogenic regions. Expression of both Runx1 and Sox9 was markedly downregulated on ossification. Runx1 and Sox9 expression was absent in actively modeling or remodeling bone tissues in ossified craniofacial bones. Runx2 expression was not affected by gene disruption of Runx1, whereas the expression domains of Runx1 were extended in Runx2^<-/-> mice compared with wildtype mice. These results suggest that Runx1 may play a role in incipient intramembranous bone formation.
3.We investigated that the function of Zac1 in endochondral bone formation. Chondrocytes were isolated from the 17-day embryonic chick sterna. RT-PCR analysis revealed that Zac1 expression was decreased during 3-week culture. To examine the effect of Zac1 on chondrocyte differentiation, we characterized chondrocytes transfected Zac1 gene by chick retrovirus compared to mock transfected group. After retinoic acid(RA) treatment, type IX collagen expression was decreased, and MMP-13 and ADAM-TS5 expression were increased in mock transfected group. In contrast, these changes in Zac1-infected chondrocytes treated with RA were markedly inhibited. These results suggest that Zac1 may play a role in the metabolism of the cartilage.
4.Normal newborn mice mandibular condylar chondrocytes from type I collagen-expressing cells and non-type I collagen-expressing cells were isolated by laser capture microdissection(LCM) and then subjected to microarray analysis. LCM allows isolation of chondrocytes from individual zones of mandibular condylar cartilage for comparative analysis. These techniques will hopefully serve as a guide for the further analysis. Less
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