| PartⅠ:The preparation and biocompatibility of injectable thermo-sensitive chitosan/Poly(vinyl alcohol) hydrogelObjective:To investigate the preparation conditions of thermosensitive chitosan/ PVA(Polyvinyl alcohol) hydrogel and evaluate the mechanical properties,spatial structure,infrared spectral characteristics and swelling degree,degradation rate and gel biocompatibility of these hydrogen.Methods:2% chitosan hydrochloride solution was transferred to a neutral PH and mixed with different proportions of PVA,then the gelation time and strength were observed,infrared spectral characteristics were analysised by infrared analyzers,spatial structures were observed under a scanning electron microscopy (SEM) after freeze-drying, the equilibrium swelling degree and the degradation rate of these different samples were tested.Then through sensitization experiments, systemic toxicity, cell toxicity and muscle implantation test to evaluate the biocompatibility of them.Results:The different ratios of chitosan and polyvinyl alcohol gels showed that with a larger proportion of PVA in the gel,the gelaton time were extended,the strength were increased, the network structure became dense,swelling degree turned smaller.Gel had no simulations to the SD rats skin through sensitization experiments,rabbits without systemic toxicity after excessive injection,the cytotoxicity were between grade 0 toⅠ.there were inflammatory infiltration and fibrous capsule formation of the early days after the gel implanted into muscle,with the time prolonged,inflammatory reaction disappeared and capsule thinning.The blood vessels grew into the gel and the gel were seen part of degradation after 12w implantation.All these results were certificated that the gel had good histocompatibility.Conclusion:Chitosan/PVA composite hydrogel with a thermosensitive characteristics,interpenetrating network structure,better mechanical strength,low cytotoxicity and good biocompatibility,It complys with the requirement of tissue engineering scaffold material.Part II:Rabbit BMSCs differentiation into chondrocytes induced by Ad-TGF-β1 in vitroObjective:To induce bone marrow mesenchymal stem cells differentiation into chondrogenic by Ad-TGF-β1 transfection.Methods:Rabbit bone marrow mesenchymal stem cells were isolated and cultured in vitro,to observe the cell’s growth state and growth curve.The morphological changes of the third passage of MSCs was observed after transfectection by Ad-hTGF-β1,and through toluidine blue staining detecting the expression of glycosaminoglycan in the cells after transfected at different time.RT-PCR methods to detect hTGF-β1 and Aggrecan mRNA expression in cells after transfection 1w,2w,3w,4w at different time points.Using Western blot method to analysis hTGF-β1 and Collagen II expression 2w after transfection and type II collagen immunohistochemistry to defect the type II collagen expression in the transfected cells.Results:There were some cells nesrosis at the early after transfection,then returned to normal in about 3d, toluidine blue staining were positive 2w after transfection and continue to express with the time.RT-PCR showed that mRNA of hTGFβ-1 and Aggrecan were expressed in the transfected cells at different time points,the expression of Aggrecan were increased and that of hTGF-β1 were reduced with the time.HTGF-β1 and Type II collagen protein were defected by Western blot methods in the transfected cells after 2 weeks and they were both negative in the empty virus infection and untransfected group.Type II collagen of transfected cells were positive by immunohistochemical staining and positive rate increased with the time.Conclusion:Bone marrow mesenchymal stem cells could express the process of chondrocyte phenotype by Ad-hTGFβ-1 transfected in vitro. Part III:Experiment study of rabbit articiular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cellsObjective:To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGFβ-1 transfected bone marrow mesenchymal stem cells.Methods:Bone marrow mesenchymal stem cells were isolated and cultured in vitro.The CS/PVA composite hydrogel were prepared as a three-dimensional scaffolds and to observe the activity of proliferation under laser confocal microscope The mesenchymal stem cells were tranfected by Ad-hTGF-β1 in vitro and the the positive rate of transfection was defected by cell immunofluorescence methods after Ad-hTGF-β1 transfected for one week.24 adult New Zealand white rabbits with full articular cartilage defects were randomly divided into four groups,each group had 6 animals,both hind limbs were used in the experiment.Group A:to repair the defects with hydrogel combined with transfected cells;Group B:to repair the defects with hydrogel combined with untransfected cells;Group C:to repair the defects with hydrogel only;Group D:blank control group.24 animals were sacrificed after 4 weeks,8 weeks and 16 weeks.The defect site and the surrounding normal cartilage tissue were obtained for gross observation and histological staining.Specimens and histological observation were used to evaluate the repairment effect after 16 weeks according to Wakitani’s score,which had statistically significant when P<0.05.Results:Rabbit BMSCs showed a high activity of proliferation.in the CS/PVA gel,when transfected by Ad-hTGF-β1,the positive rate was about 85.4% by cell immunofluorescence methods.Gross observation after 4 weeks,the defect sites of group A,B and C were filled with porcelain white gel and the filler combined with the surrounding articular surface of the femur closely. The articular surface were smooth and the filler had a significant boundaries with the normal tissure.The cartilage defect sites of group D were filled with old blood stasis block.After 8 weeks,the defect edge of group and group B were unclear with the normal tissure,the shape of cartilage defects were closely to the surrounding cartilage.the texture and color of group C had a widely difference with the cartilage around.and that of group D were filled with granulation in and around the bottom of the defect. After 16 weeks the defects of group A were repaired by cartilage-like tissue,the surface of these articulars were smooth and could not distinguish the normal articular cartilage and the defects.The cell arrangement and densities of regenerated cartilage were similar to normal cartilage,Safranin O staining were positive and typeⅡcollagen immunohistochemistry were positive.There was a significant difference according to Wakitani’s score compaired with other groups.Conciusion:CS/PVA gel is a kind of thermosensitive injectable scaffold.Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells. |
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