Sarah Peters
Qualitative Research Methods in Mental Health
University of Manchester
h3backgroundh3 chondrogenesis occurs result mesenchymal stem cells mscs condensation chondroprogenitor cell differentiation following chondrogenesis chondrocytes remain resting cells form articular cartilage major challenge osteoarthitic oa cartilage reparation stem cellbased approaches understanding multistep process h3objectivesh3 analyse extracellular protein expression profile human bone marrow mscs hbmscs osteoarthritic patients control donors undergoing chondrogenesis order compare mechanisms involved cartilage extracellular matrix ecm remodeling occurs chondrogenic differentiation process h3methodsh3 hbmscs isolated 3 oa patients 3 healthy controls grown different isotope variants lysine arginine arg6 lys4 control population arg10 lys8 oa population 46 weeks achievement full protein labeling labeled populations subjected differentiation 3d cultures micromasses supplemented chondrogenic inducers 14 days proteins conditioned media two cell populations combined separated 1dsdspage subjected ingel trypsin digestion using automatic digestor resulting peptide mixtures analyzed nanolc coupled online ltqorbitrap xl mass spectrometer quantified using maxquant software perseus tool h3resultsh3 realtime pcr assays showed relevant difference gene expression collagen type ii normal donors compared oa patients moreover chondrocyte phenotype confirmed cases proteglycan immunostainings aggrecan chondroitin6sulfate 14 days chondrogenesis using proteomic approach compared extracellular protein profiles oa normal hbmsc time differentiation among 531 proteins 56 significantly altered levels 35 proteins displayed consistently higher levels oa samples compared normal donors many proteins cartilage specific proteoglycans hyaluronan proteoglycan link protein 1 aggrecan core protein lumican well proteins wellknown role pathogenesis oa like comp mmp3 hand 21 proteins exhibited significantly reduced abundance oa patients compared controls interestingly detected several proteins belong tenascin protein family like tenascinx accelerates collagen fibril formation also found wisp2 decreased day 14 suggesting lower activity wnt signaling pathway oa cells h3conclusionsh3 identification quantification secreted proteins enhance knowledge extracellular regulation chondrogenesis allow identification extracellular markers process moreover lower expression oa patients like tenascinx wisp2 suggest putative usefulness molecular monitorization chondrogenesis cell therapybased approaches cartilage repair h3disclosure interesth3 none declared h3doih3 101136annrheumdis2014eular4997