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Study on the Construction of Functional Cartilage by Silk Fibroin-chitosan Scaffolds Seeded with TGF-β1Gene Transfected BMSCs

Author: ZhangPeng
Tutor: WangWenLiang
School: Hebei Medical University
Course: Surgery
Keywords: Silk Fibroin Chitosan Cartilage tissue engineering Bonemarrow mesenchymal stem cells Gene transfection Induced differentiation
CLC: R684.3
Type: Master's thesis
Year: 2014
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Abstract


Objectives: Osteoarthritis is one of the diseases of serious impact onhuman life, due to the lack of blood supply in articular cartilage, once thedamage occur, its repair ability is very poor, and pathogenetic condition willgradually progress over time. Along with the increasing of elderly populationin the world and our country, incidence of osteoarthritis rising, bone jointdiseases tend to be younger at the same time, and the number is growing. Atpresent although the treatments of repairing cartilage defect have brought usnew hope, but there are still shortcomings, mainly include the need to removebone and cartilage from a donor area or drill in normal tissue, so cause newdamage for the donor area, and need at least2times surgery, due to the limiteddonor source at the same time, the above methods can’t completely meet theclinical demand, therefore, to seek an alternative osteochondral graft is a bigproblem needs to be solved urgently. The work is on the basis of reported inthe literature and our previous research, we used silk fibroin and chitosan asraw materials to make the scaffolds by freeze-drying methods, and then moveTGF-β1gene transfected BMSCs to inoculate onto the scaffolds as the seedcells to construct tissue-engineered cultivation model, and to secrete growthfactors, through autocrine and paracrine way, induce BMSCs differentiate intochondrocytes in the micro environment. The results of the study is of greatsignificance for repairing osteochondral defects, also provide some clinicalreferences for the treatment of osteoarthritis. Therefore, the research work isa meaningful exploration for the treatment and rehabilitation of related boneand cartilage diseases.Methods:1Use density gradient centrifugation method to separate rat BMSCs. 2Alizarin red staining and ALP staining were performed to observe thedegree of osteogenic differentiation of the osteogenic liquid-induced BMSCs.Sirius scarlet staining and toluidine blue staining were performed to observethe degree of chondrogenic differentiation of the chondrogenic liquid-inducedBMSCs.3Prepare silk fibroin-chitosan tissue engineering scaffolds byfreeze-drying method: silk fibroin and chitosan were in different proportionduring the preparation of three kinds of silk fibroin-chitosan scaffolds, Fouriertransform infrared spectroscopy was used to analyze the structures of saffolds;Measure the physical and chemical properties of each saffolds, including:density、porosity、soluble loss rate in hot water、modulus elasticity、bibulousrate and antibacterial performance; Observe the aperture size and shape ofscaffolds by SEM; MTS method was used to investigate the growth of thecells on the scaffolds; SEM was used to observe the adhesion of cells on theoptimal scaffold; Observe the morphology and osmotic growth of cells on theoptimal scaffolds by HE staining.4TGF-β1plasmid and its empty vector plasmid transfected the thirdgeneration of BMSCs by liposome, specific steps in accordance with theliposome lipofection2000instructions.5Tissue-engineered cultivation model: move each cell to inoculate ontothe scaffolds respectively. The experiment was divided into three groups:group A (TGF-β1plasmid transfection group) and group B (empty vectorplasmid transfection group) and group C (blank control group).6The expression levels of TGF-β1,Sox9and collⅡ mRNA weredetected by reverse transcription polymerase chain reaction (RT-PCR).7The Glycosaminoglycan (GAG) secretion of culture supernatantassayed with Alcian blue colorimetric method and the TGF-β1secretion ofculture supernatant was examined by enzyme-linked immnosorbentassay(ELISA).Results:1We can separate the rat BMSCs by density gradient centrifugation method successfully. When primary cells were inoculated, they werescattered in distribution and rounded, after24h some cells began to beadherent. Adherent cells were fusiform or polygonal,3-7d, cells appearedfusion growth, and showed colony-like proliferation,7-10d, can reach80-90%confluence, the growth rate of passaged cells were significantly accelerated,and showed a typical whirlpool-like growth. With the passage numberincreasing, the high purity of BMSCs were available. After3passages, themorphology of cells were more consistent.2Alizarin red staining、ALP staining、Sirius scarlet staining andtoluidine blue staining were performed to observe the multi-directionaldifferentiation potential of BMSCs. According to the results,the BMSCs thatwe obtained haved good multi-directional differentiation potential.3Fourier transform infrared spectroscopy proved that chitosan wasconstituted through sugar characteristic structure β1,4glycosidic bondconnecting to each other, so each group in the1154cm-1and897cm-1occurred characteristic absorption peak, for CS-SF scaffold materials, with theincreased content of chitosan, the absorption peak of1654cm-1and1540cm-1that are on behalf of αhelix/random coil structure constantlydecreased, and the absorption peak of1628cm-1and1516cm-1that arecorresponding to β fold structure increased, the above results showed that theaddition of chitosan modified the structure of silk fibroin, change from theαhelix/random coil structure to β fold structure; Density was respectively0.18±0.03,0.20±0.01,0.18±0.04(g/ml); Porosity was respectively87.36±2.15,77.82±1.37,72.22±1.37(%);Soluble loss rate in hot water wasrespectively:0,0,3.12±1.26(%);Modulus elasticity was respectively:8.35±2.64,15.50±1.64,12.55±3.37(KPa); Bibulous rate was respectively:1528.52±194.63,1078.22±100.52,1320.05±179.97(%); Antibacterialcircle diameter was respectively:18±1,22±2,20.33±1.53(mm); SEMresults showed the aperture size of group1was between50-250microns, wasrelatively consistent and the connectivity of pores was good, however thestructure of the other two groups was disorganized, the aperture size and the connectivity of pores was poor; Growth curve results: At1d, the number ofliving cells of group1was significantly higher copared to the other two groups(P <0.01),3d,5d and7d, the number of living cells in group1wasobviously higher than that of group2and group3(P <0.01); SEM showedthat cells were adherent on the optimal scaffold, with the increasing of culturedays, appeared more extracellular matrix around them. HE staining showedthat in the early culture phase BMSCs grew along the internal gap of theoptimal scaffold, with the increasing of culture days, adherent cells appearedcolony-like growth, we can clearly see the connection between cells; From theabove results we can confirme that the group1silk fibroin-chitosan scaffoldshave good cell compatibility, can promote BMSCs live and adherent, canprovide an open growth environment for cells, promote cells to grow internally,and ensure adequate nutrition and gas exchange.4When cells were transfected for48h, we detected the TGF-β1mRNAexpression level by reverse transcription polymerase chain reaction. RT-PCRresults showed that the TGF-β1mRNA expression level of group A is higherthan that of group B and group C (P <0.01).5Tissue-engineered cultivation models were respectively cultivated for3,6,9,12d in vitro.The results showed that at four time point, the secretion ofTGF-β1of group A was higher than group B and group C (P <0.05), at thesame time, the daily secretion of TGF-β1of group A showed a trend ofgradual decline, the daily secretion of TGF-β1of group B and group Calways showed a trend of lower expression.6Tissue-engineered cultivation models were respectively cultivated for3,6,9,12d in vitro.Results showed that at3,6,12d,the GAG content of culturesupernatant of group A was significantly higher that of group B and group C(P <0.01), at the same time, the daily production of GAG of Group A wasgradually rising, the daily production of GAG of group B and group C alwaysshowed a trend of lower expression. From the above results we can confirmethat the cells of group A appeared chondrocyte phenotype.7Tissue-engineered cultivation models were respectively cultivated for two weeks in vitro.Then we extracted total RNA. The expression levels ofSox9and collⅡmRNA were detected by reverse transcription polymerasechain reaction. The results showed that: group A appeared the expression ofcollagen type Ⅱ and Sox9mRNA, however group B and group C didn’tappeare the expression of collagen type Ⅱ and Sox9mRNA which are twokinds of chondrocytes specific marker genes.Conclusions:1We can separate the rat BMSCs with multi-directional differentiationpotential by density gradient centrifugation method successfully.2Silk fibroin and chitosan are natural biological material, raw materialavailability, good biocompatibility. we tried to mix silk fibroin with chitosanmechanically to improve their physical and chemical properties through thestrong hydrogen bonds and ionic bonds, and to develop silk fibroin-chitosanscaffolds which are suitable for cartilage tissue engineering.3TGF-β1plasmid and its empty vector plasmid transfected the thirdgeneration of BMSCs by liposome method successfully. Move each cells toinoculate onto the scaffolds respectively to construct tissue-engineeredcultivation model successfully. Each group was cultivated for two weeks, thenwe confirmed Group A haved good ability of chondrogenic differentiationfrom several aspects.

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CLC: > Medicine, health > Surgery > Orthopaedic Surgery ( movement system diseases,orthopedic surgery ) > Joint disease and injury > Arthritis
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