| Objective The purpose of this study is to construct an injectable tissue engineering nucleuspulposus composed of Platelet-rich plasma gel(PRG) as scaffold and rabbit bone marrowmesenchymal stem cells (BMSCs) co-transfected byf adenoviral vectors with TGF-β3andBMP-7as seed cells.Methods Obtain the rabbit BMSCs with the method of direct way. Be sure of theBMSCs through the surface mark and induced differentiation into three differentmesoderm cells.Compose the TGF-β3and BMP-7gene fragment and make sure of their gene order.Establish the TGF-β3adenoviral vector(TGFβ3-pDC316-MCMV-EGFP) and BMP-7adenoviral vector(BMP7-pDC316-MCMV-EGFP) with plasmid pDC316-MCMV-EGFP.Determine the TGF-β3and BMP7expression in mRNA level through Realtime PCR.Divide BMSCs into five groups: A. blank control group; B. immunofluorescence controlgroup with GFP; C. singly transfected by TGF-β3adenovirus; D. singly transfected byBMP7adenovirus; E. co-transfected by both TGF-β3and BMP7adenovirus. After14days， measure the TGF-β3and BMP-7protein expression of group A、C、D、E and theexpression of ACAN、Collagen I、Collagen II、Collagen X and SOX9in mRNA levelthrough Western blot and Realtime PCR respcetively.Prepare the PRP by Lendersberg way. Obtain PRG by mixing PRP with activator. Obtaingrowth-factor-rich supernatant through centrifugalization. Then measure the concentrationof TGF-β1and PDGF-AB of it. Activating PRP by mixing with activator after combiningwith BMSCs and BMSCs co-transfected by both TGF-β3and BMP-7adenovirusseparately, and scan electron microscope (SEM) was used to discover the threedimensional structure of the tissue engineering nucleus pulposus and the growth conditionof co-transfected BMSCs at14days after activation.Result The cells got through the all bone marrow adherence method are able todifferentiate into at least three kinds of mesoderm cells: osteoblast, chondroblast andadipocyte. The cells also express cell surface marker of CD29, CD105, CD166accordingto flow cytometry detection. Gene order sequencing demonstrate that the gene fragments are correct. The tite ofadenoviral vector of TGF-β3and BMP-7are1.495×1010pfu/ml and1.185×1010pfu/mlseparately. The amplified DNA by PCR contain TGF-β3and BMP-7gene fragments testedby electrophoresis.14days after culture of Co-transfected BMSCs, the shape of most cells changed obviously.Western blot showed TGF-β3and BMP-7proteins at a higher level than BMSCs. Theexpression level of ACAN, Collagen I, Collagen II and SOX9gene were much higher thancontrol group(P<0.05), and the expression level of Collagen II in co-transfected group washigher compared to solo-transfected groups(P<0.05). The expression of Collagen X inTGF-β3transfected group and co-transfected group were obviously lower than BMP-7transfected group and control group(P<0.05).The concentration of TGF-β1and PDGF-AB in PRG are351.03±11.15ng/ml and267.38±14.2ng/ml separately, which are obviously higher than the concentration of them inrabbit blood. Scanning electron microscope showed that diameter of ventage in PRG wasbetween40μm to100μm, and co-transfected BMSCs were well-distributed in PRG byputting stylodes into the ventage. The cells growed well in PRG.Conclusion It is pertinent to prepare the BMSCs by direct way. Adenoviral vector ofTGF-β3and BMP-7could transfect the BMSCs and express the TGF-β3and BMP-7withpDC316-MCMV-EGFP. BMSCs transfected by TGF-β3and BMP-7get higher expressionof ACAN, Collagen I, SOX9and lower expression of Collagen X, what demonstrated thatboth TGF-β3and BMP-7are able to induce the BMSCs into nucleus pulposus-likecells(NPCs). PRG preparated through Lenderberg method had a proper structure forco-transfected BMSCs growth. |
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