Abstract
s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S129 (AA)) to investigate the mineralization phase of late chondrogenesis. Monolayers of MC3T3-E1 were cultured for 21D in aMEM + 10%FBS, supplemented with BGP and AA. mRNA expression of typical chondrogenesis markers (Aggrecan (Agg), type II (Col2a1) and X (Col10a1)) collagens and osteogenic markers (Osterix (Osx), Osteocalcin (Ocn) and Osteopontin (Opn)) were assessed by quantitative RT-PCR. We also assessed mRNA expression of Wnts (-4, -5a, -5b, -11) and BMPs (-2, -4, -6, -7). Quantification of Alcian Blue, Safranin O, Sirius red and Alizarin red staining were used to evaluate proteoglycans, collagens and mineralized content respectively. Alkaline phosphatase (ALP) activity allowed to monitor osteogenesis. SDS-PAGE was used to assess the global glycosylation status. Western blot allowed to check canonical (bcatenin) and non-canonical (Calmodulin-Kinase II) Wnt signaling, as well as BMP signaling (Smad and MAPK). Results: During early proliferation (D1-7) and late proliferation/prehypertrophy phases (D7-14) of chondrogenesis, Col2a1 and Agg mRNA strongly increased, but to a lesser extent in COG5-. Proteoglycan staining intensity was lower in COG5-. BMP-4 levels progressively increased and were significantly higher in COG5-. This was consistent with increased phosphorylation of Smad 1/5/8. Wnt-4, -5a and -5b mRNA were evenly increased in both controls and COG5-. BMP-6 and Col10a1 mRNA were increased at D14, but were significantly lower in COG5-. Wnt-11 mRNA increased in controls, but not in COG5-. In mineralization phase (D14 to D21), Col10a1 mRNA and mineralization were strongly increased, but to a lesser extent in COG5-. During osteogenesis, Opn, Osx and Ocn mRNA were significantly higher in controls, alike Alizarin red staining and ALP activity. Protein analysis suggested differences in global glycosylation status, in particular of Wnt ligands. Conclusions: COG5 defect reduces chondrogenesis and osteogenesis. COG5 particularly affected glycosylation andWnt signaling, underlining its critical role in the fate of cells from the articular chondrocyte-bone unit in the course of OA. 232 ROLE OF INTERLEUKIN-10 IN ENDOCHONDRAL BONE FORMATION G. Kim y,z, Y.-K. Jung z, H.-R. Park z, E.-J. Lee z, J.-Y. Choi x, F. Beier {,k, S.-W. Han y,z. yDept. of Rheumatology, Daegu Fatima Hosp., Daegu, Republic of Korea; z Lab. for Arthritis and Bone Biology, Fatima Research Inst., Daegu, Republic of Korea; xDept.s of Biochemistry and Cell Biology, Sch. of Med., WCU program, Kyungpook Natl. Univ., Daegu, Republic of Korea; kDept. of Physiology and Pharmacology, Univ. of Western Ontario, London, ON, Canada; {Children's Hlth.Res. Inst., London, ON,
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