Modulation Of Adhesion, Proliferation And Differentiation Of Mesenchymal Stem Cells Transfected By Transforming Growth Factor β₁ Gene On PLGAmodified With RGD Peptide

Background: Cell adhesion to scaffold and triggering signals that direct cellproliferation and differentiation play a central role in osteal tissue engineering. Howtomodulatethisprocessisakeytopicinresearchnowadays.Objective: Theproliferation,adhesionanddifferentiationofMSCtransfectedbytransforming growth factorβ1 gene on the PLGAscaffold modified by RGD peptidewas investigated to explore the effect of TGF-β1 gene and RGD peptide on cellbiologicalbehavior.Methods: A heterobifunctional cross-linker, Sulfo-LC-SPDP was used toimmobilize GRGDSPC on the surface of PLGA. Recombinant plasmid pcDNA-TGF-β1 was transfected into MSC by liposome Lipofectamine 2000, and then thetranfected MSC was screened with G418 culture medium, at last stable transfectioncell was formed. In this experiment there are four groups:Group Ais simple MSC onPLGA, Group B is transfection MSC on PLGA, Group C is simple MSC on PLGAmodified with RGD peptide, and Group D is transfection MSC on PLGA modifiedwith RGD peptide. The two kinds of MSCs were seeded respectively onto the twokindsofPLGA,incubatedfor1d,2dand3d,thecelldensitywerecalculatedandwereused as index of cell proliferation. MSCs were seeded onto the scaffolds, and incubated for 4h and 12h at standard condition, in each experiment the number ofadhesive cells was counted to evaluate the efficiency of cell adhesion, thenFITC-conjugated phalloidin was used to stain the cytoskeleton of cells incubated for24h. MSCs were cultured on the scaffolds for 7d, 14d and 21d with osteogenicsupplements,andthentheirALPactivitywasmeasured.Results: Cell density in Group B was higher than in Group A, in Group D washigherthaninGroupCforthecultureof1-3d,butnosignificantdifferencewasfoundbetween Group Aand Group C, Group B and Group D. After 4h and 12h culture theefficiencyof cell adhesioninGroupC washigher thanGroupA,GroupD washigherthanGroupB,butnosignificantdifferencewasfoundbetweenGroupAandGroupB,Group C and Group D. After 24h culture the qualityof cell adhesion in Group C andGroup D were better than Group A and Group B, and their cytoskeleton were morerobust. Group C expressed higher ALP activity than Group A, so did Group D thanGroupBafterculturedfor14d,but nosignificant differencewasfoundin7dand14d.AndGroupBexpressed higherALPactivitythan GroupAin7d, 14d,and21d,sodidGroupDthanGroupC.Conclusion: TGF-βgene promoted cell proliferation, but RGD peptide had noeffect in cell proliferation. RGD peptide enhanced cell adhesion to scaffold andheightened the efficiency of adhesion, but TGF-βgene had no effect in cell adhesion.RGD peptide promoted MSC differentiate to osteoblast after cultured for 14d, andTGF-βgenepromotedMSCdifferentiatetoosteoblastinearlystage.