The Application Of Nano-gene Engineering And Temperature-sensitive Hydrogel Scaffold In Tissue Engineering Cartilage

Part one Isolation,Purification and Identification of Rat Bone Marrow Mesenchymal Stem Cells(RMSCs)Objective Rat bone marrow mesenchymal stem cells(RMSCs)were isolated,purified,cultured and identified in vitro to provide materials for further experiments.Methods1.Separation and Purification of RMSCs Rat bone marrow was extracted from the hind leg bone of 5-6 weeks old Sprague Dawley(SD)rats.The bone marrow was diluted with D-Hanks solution for full incorporation and centrifuged for 5 minutes at 800 rpm/min.The supernatant was discarded and the cells were gently dispersed into single cell suspension with DMEM.The suspension was slowly added to the surface of density lymphocyte isolation medium(?:1.077g/cm-3)and centrifuged with 3000 rpm/min for 20 minutes.Monocyte layer was taken out and washed twice with D-Hanks solution.The cells were further cultured in D MEM flask containing 20% fetal bovine serum(FBS).2.Identification of RMSCs Cell viability was measured by trypan blue assay.The culture flask was placed in a cell culture box at 37 ? and 5% CO2.After 48 hours,the culture medium was replaced and the uncoated cells were discarded.The cell status was observed under inverted phase contrast microscope every day.0.25% trypsinase was digested and passaged when the cell fusion reached the bottom of the culture bottle.The third generation RMSCs cells were identified by flow cytometry and directed differentiation in vitro.Results1.Morphological observation:After inoculation,RMSCs were distributed at the bottom of the culture bottle,round,bright cytoplasm and good refraction.After 24 hours,a single cell began to adhere to the wall,enlarged and the cytoplasm extended outward,similar to fibroblasts.After 48 hours,the adherent cells were spindle,triangle,fan and round in shape.Five days later,the cells formed dispersed colonies.2.Flow cytometry:Flow cytometry showed that the positive rates of CD29 and CD44 were 95.34%and 85.12% respectively,while the negative rates of CD34 and CD45 were 4.69%and 5.12% respectively.MTT growth curve showed that the cells proliferated faster and grew steadily.3.Directional differentiation in vitro: In the experiment of induced adipogenesis,lipid droplets in cells were identified to agglomerate,which proved that directional adipogenesis was better.Induced osteoblasts were stained with Alizarin red,and more calcium nodules were observed in cells,which proved that directional osteogenesis was better.Induced chondrocytes were stained with Collagen II-FITC,and the cells were green under green fluorescence microscope,which proved that directional chondrogenesis was betterConclusion RMSCs with high purity,rapid proliferation and stable biological characteristics can be obtained by combining density gradient centrifugation and adherence screening,which provides ideal seed cells for further experiments.Part two Preparation of cationic nanoliposomes and Sox9 gene transfectionObjective To investigate the feasibility of transfecting Sox9 gene into RMSCs by nanocationic liposome and its expression.Methods1.The fabrication and characterization of cationic liposomes Cationic liposomes were prepared by thin film dispersion method.In short,350 mg 2,3-dioleoyl-propyl(2,3-Dioleoyloxy-propyl)-trimethylammonium(DOTAP)was accurately weighed and dissolved in 1 ml chloroform and mixed with 370 mg of dioleoyl-sn-glycero-3-phospholine(DOPC).The total volume of the mixture was diluted to 10 m L with chloroform and whirled for 10 minutes.DOTAP and DOPC were dispersed on the wall of the tube by using a rotating evaporator to evaporate the solvent at 50 ?.After drying,evaporation continued for 1 hour to remove residual solvents.The lipid membrane was prepared into powder and dissolved in 4ml water under intense stirring to form cationic liposomes.The preparation was further characterized by transmission electron microscopy(TEM)and dynamic laser scattering(DLS).A certain amount of cationic liposomes was diluted to 10 m L with water.The diluent was added to the sample pool for determination.The particle size and Zeta potential were measured on the basis of the number of particles.Each sample was measured six times and the average value was obtained.A certain amount of cationic liposomes was diluted to 10 m L by adding water.The diluent was put in a small beaker for 10 minutes,followed by negative staining with 3% phosphotungstic acid.The negative staining solution was absorbed onto the copper mesh,and the excess staining solution was absorbed by filter paper for 30 minutes.The particles were deposited on the copper mesh.The particle size and morphology were observed by TEM.All measurements were carried out at 25 ?.2.Cationic Liposome Mediates Differentiation Induced by RSMCs of Transcription Factor Sox9 The third generation RMSCs cultured in vitro were used for subsequent experiments.Firstly,Sox9 plasmid was fully mixed with cationic liposome solution(without FBS)at room temperature for 20 minutes.The mixture was then added to a6-well plate containing RMSCs and cultured in a 37 ?,5% CO2 incubator.Four hours later,the culture medium was replaced completely,and the culture was continued for subsequent detection and identification.Cell transfection was divided into three groups:(1)experimental group: transfection group containing Sox9 plasmid by cationic liposome;(2)control group 1: only containing the same amount of cationic liposome;(3)control group 2: transfection group containing Sox 9plasmid by Lipofectamine 2000 on the market.After 6 days of culture,the cells were identified by immunochemistry.3.Detection of Sox9 gene expression For the above experiment,after 7 days of continuous culture,the supernatant was collected and the expression and duration of Sox9 in RMSCs were determined by enzyme-linked immunosorbent assay(ELISA).SPSS statistical software was used for statistical analysis.Results1.Identification of cationic liposomes The average particle size of cationic liposomes was 85.76 ± 3.48 nm,zeta potential were 15.76 ± 2.1 m V,which indicated that the cationic liposomes had monodisperse properties of nanoparticles.TEM of cationic liposomes has the same results as DLS,and has the expected uniform spherical characteristics.2.Morphological changes in chondrocyte differentiation of RMSCs After 7 days of transfection of RMSCs with Sox9 gene,the cell processes became longer,the cell body became wider and the refractive index increased.The growth curve was described by MTT method.It was found that the proliferation rate of cells transfected with Sox9 gene was faster,the growth condition was good,and the toxicity was less.3.Detection of Sox9 gene expression in RMSCs by ELISA The expression of Sox9 protein by ELISA showed that the transfection efficiency of cationic liposomes was higher than that of commercial liposomes,and the optimal transfection concentration was guaranteed when the plasmid concentration was 100 ng/pore.Conclusion The nanocationic liposomes prepared by thin film dispersion method can successfully transfect RMSCs with cartilage-like phenotype,and the expression of Sox9 gene is stable.Part three Construction of thermosensitive hydrogel scaffoldsObjective Chitosan hydrogel scaffolds with temperature sensitivity were constructed in vitro.Methods Chitosan was added into 0.1 mol/L hydrochloric acid solution to form solution A with final concentration of 2%(w/v)and sodium beta-glycerophosphate solution with volume fraction of 50%(w/v).A series of hydrogels of different proportions were prepared by mixing the two solutions and mixing them in different proportions(v/v).The temperature sensitivity of the hydrogels was tested at 37 ?.The morphology was observed by SEM,and the mechanical properties,swelling properties,in vitro release ability,in vitro degradation degree and in vitro proliferation were tested.Results1.The formation of chitosan hydrogels with different ratios was observed at 37 ?.Experiments show that the ratio of chitosan solution to sodium beta-glycerophosphate solution is different and the gelling time is different.With the increase of sodium beta-glycerophosphate content,the gelling time decreases from 30 minutes to 3minutes.2.SEM observation: the pore structure of gel scaffolds is uniform and dense,and a good porosity(85%)can simulate the extracellular matrix environment of cell growth.3.Mechanical compression test: With the increase of the proportion of sodium beta-glycerophosphate,the mechanical strength of hydrogel scaffolds also increases.4.Gel swelling test: with the increase of beta glycerin phosphate,the swelling degree of hydrogel scaffolds decreased.5.Gel release and in vitro degradation test: the experimental results showed that when the ratio of chitosan solution to glycerol sodium phosphate solution was 5:1,the gel scaffold had a higher cumulative release and less degradation in vitro.6.In vitro gel proliferation test: the lower the hydrogel concentration,the higher the cell proliferation rate.Conclusion Different proportion of thermosensitive hydrogel scaffolds can be successfully prepared by using chitosan and sodium beta-glycerophosphate.When the solution ratio is 5:1,the hydrogel scaffolds are the most stable and more in line with the requirements of tissue engineering scaffolds.Part four Construction of tissue engineered cartilage by RMSCs transfected of Sox9 gene with nano-gene engineering and thermosensitive hydrogel scaffoldObjective The tissue engineered cartilage was constructed in nude mice by RMSCs transfected of Sox9 gene with nano-gene engineering and thermosensitive hydrogel scaffold.Methods The RMSCs cells transfected for 2 weeks were mixed with thermosensitive chitosan hydrogel and subcutaneously injected into the back of nude mice.In vivo experiments in nude mice were divided into four groups: RMSCs chitosan-hydrogel complex(group A),RMSCs chitosan-hydrogel complex(group B),RMSCs(group C)and chitosan hydrogel(group D)without transfection of Sox9 gene.Four weeks after feeding in SPF environment,the tissue of subcutaneous injection site of nude mice was observed to observe the formation of cartilage.Hematoxylin-eosin staining(HE),Safranine O staining and collagen II and IHC staining were used to identify the cartilage.Western-Blot was used to further identify the cartilage.Results After 4 weeks of feeding,nude mice were sacrificed and the injection sites were dissected.Similar slice-like tissue structure was found in group A and group B.The appearance of group B was similar to that of translucent cartilage,while that of group A was similar to that of fibrous tissue,while no similar tissue structure was found in group C and group D.HE staining showed that there was no complete chondrocyte morphology in group A,while cell aggregation was observed in group B,similar to chondrocyte expression;Safranin O staining showed that there was no normal chondrocyte tissue in group A,and there was no significant difference in cell morphology and arrangement between group B and normal chondrocyte;collagen II and IX immunohistochemical staining showed that there was no obvious chondrocyte formation in group A and no obvious chondrocyte formation in group B.Chondrocyte formation was observed.After dewaxing,the cytoplasm was brown and the nucleus was vacuolar.Conclusion The tissue engineered cartilage was successfully constructed in nude mice using RMSCs transfected with Sox9 gene and thermosensitive hydrogel scaffolds.The scaffolds can be decomposed and absorbed,which provides a new method for cartilage repair.