Effect Of Self-assembly Peptide Scaffold Loaded With Recombinant Plasmid TGFβ3 On Chondrogenic Differentiation Of Precartilaginous Stem Cells

Objective:To fabricate seed cells for tissue engineering by constructing recombinant humantransforming growth factorβ3(hTGF-β3) and transfecting it into rats' precartilagious stemcells (PSCs).To study the effect of hTGF-β3 transfection into precartilaginous stem cells (PSCs) onproliferation and differentiation into cartilage chondrocytes.Preparing gene-activated matrix (GAM) for cartilage tissue engineering bysynthesizing self-assembling peptide scaffold loaded with TGF-β3 plasmidTo evaluate the effect of different kinds of GAM on PSCs proliferation andchondrogenic differentiation.Methods:Gene engineering technique was introduced to constructed eukaryotic plasmidpcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by immunologicalmicrobeads. Then PSCs were cotransfected with plasmid hTGF-β3 and pcDNA3.1(+)-EGFP by liner polyethyleneimine (PEI). 48 hrs later the transient expression of EGFPwas observed under fluorescence microscope, and the mRNA of hTGF-β3 was detected byRT-PCR.After isolated and purified by immunological microbeads, rats' PSCs were transfectedwith pcDNA3.1(+)-hTGF-β3. MTT, FCM were performed to investigate the effects of proliferation and DNA synthesis, biosynthesis of hTGF-β3 and cartilage associated genesand proteins were examined by qRT-PCR, immunohistology and western blotThe KLD-12 peptides sequences were synthesized using solid phase chemistry andthen purified. 0.5% (w/v) polypeptides solution was self-assembled into gel by PBS andplasmid encoding TGF-β3 was loaded by non-covalent bonds.GAM and PSCs were prepared according to the method before. Group A:TGF-β3-PSCs+KLD12, Group B: PSCs+KLD12-TGF-β3, Group C: PSCs +KLD12;Group D:KLD12. MTT and Hoechst33258 were performed to investigate the effects ofproliferation and DNA content. Biosynthesis of cartilage associated genes (COMP,Aggrecan, ColⅡ, and ColⅩ) were examined by qRT-PCR, sGAG content was detected byAlcian Blue method.Results:PCR product sequencing results verified that it was identical to GenBank. Agaroseelectrophoresis indicated all bands digested by enzyme were in the right rangecorresponding with our expectation. Green fluorescence was observed from PSCs whichwere successfully transfected 48hrs after transfection, and the mRNA and protein ofhTGF-β3 was detected too.hTGF-β3 was stable expressed in PSCs successfmlly. After transfection, PSCs' DNAsynthesis level and proliferation rate were significantly increased. Quantitative real-timePCR and immunological investigation suggested that chondrocyte specific genes andproteins expression were up-regmlated, and so did the deposition of chondrocyte typicalextracellmlar matrix: proteoglycan and typeⅡcollagenKLD-12 sequences were correctly synthesized. Its relative molecular mass was 1466.9.The content of DNA which was released by the plasmid incorporated scaffold reached itspeak in 3d, and maintained at a high level during the next 2 weeks.Proliferation rate of Group A was significant higher than the other three groups.Proliferation difference between Group B and C were not significant until culture time＞10d(p＜0.05). Expression of cartilage associated genes in Group A and B was up-regulated,compared to Group C. The difference of sGAG and DNA content in all the groups was ofstatistical significance. Conclusion(1) It was concluded that the recombinant plasmid was correctly constructed, whichprovided an approach of transgenic therapy for epiphseal injury or other osteochondrodisease repair.(2) Gene enhanced PSCs stabled expressed hTGF-β3 to enhance proliferation and toinduce PSCs differentiation into chondrocytes, which provided an approach for cartilagetissue engineering.(3) Gene activated self-assembling peptide scaffold was corrected synthesized,which can induce seed cells directional proliferation and differentiation, and promotecartilage repair.(4) GAM can be used as vehicle of theoretical genes and seed cells for cartilagetissue engineering. It is still a long way to go as in its infancy.