In Vitro Experimental Study On PAd-bFGF-GFP Transfected Bone Marrow Mesenchymal Stem Cells And Decellularized Scaffolds To Develop Tissue-engineered Ligaments

ObjectiveNatural healing fails to take effect in severe injury of ligaments.Replacement grafts are usually required. However,Synthetic replacements orautografts and allografts can't achieve satisfactory results. Tissueengineering ligament construct including the establishment of the supportstructure of the ligament,introduction of seed cells to grow and produceextracellular matrix on the stent to carry out alterations to the originalstent and ultimately the formation of self-renewal ligament tissue. Nowadays,with the rapid development of biotechnology, tissue-engineered ligaments(TELs) have provided a new strategy for injured ligaments replacement.Acellular ligament was deprived of cellular components and eliminated off theimmunogenity while retaining the natural shape and property of the ligamentbiomechanical properties.It, as a natural scaffold material, has receivedwidespread attention. Bone marrow mesenchymal stem cells (BMSCs) arecurrently popular seed cells. Experiments show that the basic fibroblastgrowth factor (bFGF) can promote bone marrow mesenchymal stem cell growth andsecretion of ligament-specific extracellular matrix. Introducing growthfactor into cell to improve seed cell capacity and make change in vivomicroenvironment to promote the formation of organs and tissues, as a meanof gene therapy, is the hot topic. In this study, the adenovirus vector wasused to introduce basic fibroblast growth gene into bone marrow mesenchymalstem cells.Then tansfected BMSCs were cultured with acellular ligament tobuild TELs.Methods1. Isolatation and culture of the BMSCs and Ligament Fibroblast (LF).Isolation and culture of the BMSCs and LF was through femoral punctureand Ficoll density gradient centrifugation,tissue culture method separately.Proliferation activity of the two cells was assessed by MTT assay.BMSCs wereconfirmed by multiple lineage differentiation capacity. 2. Construction of the recombinant adenovirus vectors containing the bFGFgenes by Gateway technique.We constructed recombinant adenovector by gateway technique with onlytwo main steps:BP reaction to construct entry Clone and LR reaction toconstruct expression clone.Then expression clone were packaged in293cellsto form the tareget virus.We amplified the recombinant adenovirus and detectedvirus titer. We detect the expression of bFGF through RT-PCR and western-blotafter293cells were transfected.3.To investigate the expression of bFGF in culture medium after BMSCs weretransfected and the effect of bFGF on cell proliferation and production ofextracellular matrix proteins specific to ligaments in cells compared amongpAd-GFP-BMSCs,BMSCs,pAd.bFGF-GFP.BMSCs, LF.A. Transfection efficiency of adenovirus vector to BMSCs was dected byflow-cytometry analysis with different MOI valure.B.The most appropriate MOI was determined by MTT after BMSCs weretransfected with pAd-bFGF-GFP with different MOIC The secrection of bFGF was dected by ELLISA.D The effect of bFGF on cell proliferation was assessed by MTT.Productionof extracellular matrix proteins (collagen Ⅰ, collagen Ⅲ, andvimentin)specific to ligaments in cells was determined by mRNA expression ofRT-PCR compared among BMSCs, LF,BMSCs,pAd-GFP-BMSCs.4. Construction of tissure engineering ligaments by decellularizedligament scaffolds and seeded cellsRabbit patellar tendons were decellularized with1%DCA as perfomed in ourearly experiment.Then decelluarized ligament scaffolds were seeded in vitrowith pAdbFGF-GFP-BMSCs, pAd-GFP-BMSCs, BMSCs and LF to fabricate tissureengineering ligaments. The growth and proliferation of seeded cells on thescaffolds was estimated by MTT. Production of extracellular matrix proteins(collagen Ⅰ, collagen Ⅲ, and vimentin)specific to ligaments in cells wasdetermined by mRNA expression of RT-PCR compared among BMSCs, LF,BMSCs, pAd-GFP-BMSCs. In pAdbFGF-GFP-BMSCs seeded scaffolds histological analysisthrough confocal microscope and HE staining was conducted to observe cellsgrowth and distribution. Biomechanical measure was made to valuae theinfluence of coculture in vitro to decellularized ligaments.Results1.We successfully isolated and obtained the BMSCs and LF. BMSCs and LFwere similar in the general cell morphology such as both of them had spindleshape with whirlpool-like growth pattern.There were significant highproliferation in BMSCs compared to LF after MTT assay.BMSCs candifferentiate into bone cell,fat cell and chondrocyte.2.Recombinant adenovirus plasmid was constructed successfully determinedby electrophoresis after PCR amplification.The result of gene sequencing wascorrect. Recombinant adenovirus titer was4.85×109pfu/ml. bFGF was expressedafter293cells were transfected by pAd-bFGF-GFP vectors dected by real timeRT-PCR and western-blot3.BMSCs were effectively transfected in vitro. Cell proliferation wasgreatest when BMSCs were transfected at MOI200. bFGF protein was detected48h after transfection by ELISA.Expression level reached peak at7d, thendecreased gradually but bFGF was still detectable at14days after transfection.Proliferation of pAd-bFGF-GFP-BMSCs was significantly high than othergroups by MTT assay.Realtime RT-PCR showed that expression of collagen typeⅠ, collagen type Ⅲ, was higher significantly in pAd-bFGF-GFP-BMSCs than theother groups at two week. There was no significant difference in vimentinexpression between pAd-bFGF-GFP-BMSCs and LF.4． Proliferation of pAd-bFGF-GFP-BMSCs was significantly high thanother groups by MTT assay. Realtime RT-PCR showed that expression of collagentype Ⅰ, collagen type Ⅲ, was higher significantly in pAd-bFGF-GFP-BMSCsthan the other groups. There was no significant difference in vimentinexpression between pAd-bFGF-GFP-BMSCs and LF.HE stain and confocalmicroscopy showed that pAdbFGF-GFP-BMSCs infiltrated excellently intodecellularized scaffolds. Biomechanical measure showed that the maximum load of the decellularized ligament scaffolds with and without cell culture hadno significant difference. Stiffness was significantly low in cell culturegroups than decellularized ligaments scaffolds alone.Conclusions1． BMSCs were achieved easily, expanded massively to reach the demandfor tissure engineering ligaments in vitro after culture for2weeks. LF wasobtained successfully though by much more traumatic approach. BMSCs wasdetermined by multiple lineage differentiaon capacity2． pAd-bFGF-GFP vectors with high titer and purity were constructed,which can be used safely in vitro.3BMSCs were effectively transfected by pAd.bFGF-eGFP. Secrection of bFGFwas detected for at least14days. Proliferation of pAd-bFGF-GFP-BMSCs wassignificantly higher than other groups. Expression of matrix prorein wassignificantly higher in pAd-bFGF-GFP-BMSCs group than the other groups.4.Proliferation and infiltration of pAd-bFGF-GFP-BMSCs was excellent indecullarized ligaments. Expression of matrix prorein was highersignificantly in pAd-bFGF-GFP-BMSCs group than the other groups.Biomechanical property of the decellularized ligament scaffolds with cellculture was basically retained. Creating TEL by seeded transgenetic BMSCson decellularized rabbit ligament scaffolds was viable.