| Part one: Study and analysis of in vitro properties of polyurethane meniscus tissue engineering scaffolds prepared by solvent melt method Objective:The tissue engineering scaffolds of polyurethane meniscus were prepared by solvent-melt method.On the basis of in vitro performance culture,the physical and chemical properties,biological properties,toxic effects and genetic effects of the scaffolds were discussed and analyzed,providing theoretical evidence for subsequent in vivo culture.Materials and Methods:According to the data of meniscus of adult New Zealand white rabbit,different components of polyurethane meniscus prosthesis scaffolds were prepared by solvent-melt method.The samples containing POSS were referred to as PB samples,while those containing methanol were referred to as B samples.Rabbit bone marrow mesenchymal stem cells were extracted by density gradient centrifugation and cultured in chondrogenic medium.The specific test items are as follows: select the optimal mechanical properties of the scaffold through the compression modulus test,and test its physical and chemical properties(wettability,porosity,degradation rate,mechanical strength);The cell affinity of polyurethane meniscus prosthesis scaffolds was detected by initial cell adhesion count and cck-8 proliferation test.Scanning electron microscopy,HE staining and immunohistochemical staining were used to detect the cellular compatibility of polyurethane meniscus prosthesis scaffolds.Rt-pcr was used to detect the expression level of genes related to fibrous cartilage.To evaluate the biological properties and functions of scaffolds in promoting the differentiation of rabbit bone marrow mesenchymal stem cells.Results:Physical properties of the scaffold: the research indicates that it is similar to the three-dimensional structure of the natural meniscus.The meniscus support prepared by the solvent-melt method of copper drum was milky white with smooth upper and lower surfaces.The porosity of sample B without end-sealing agent was relatively small on the whole.However,after the end-sealing agent was added,the meniscus support of PB was uniform on the whole with uniform pore size distribution.In the porosity analysis,sample B was less than sample PB,P=0.048,and the difference was statistically significant.In the compression modulus analysis,the value of PB sample was less than that of B sample,P=0.040,and the difference was statistically significant.In the pore size analysis,the B samples without POSS have smaller pore size distribution on the whole,the average pore size is 150.66 ± 13.45 microns,the overall pore size of the bracket is small,the interconnectivity of internal pore channels is not good.The pore diameter distribution of PB samples is uniform,the average pore diameter is 500.16 ± 20.34 microns.In the analysis of water absorption rate,the rising trend of sample B was worse than that of sample PB,and the P values of statistical data were all less than 0.05,indicating a statistically significant difference.Finally,the in vitro degradation rate was analyzed: the in vitro degradation rate of PB sample was lower than that of B sample,and the material was more stable.The statistical P values were 0.034,0.045 and 0.048 respectively,all less than 0.05,and the difference was statistically significant.Cell culture and morphological observation: the rabbit bone marrow mesenchymal stem cells initially extracted were round and in suspension.Cells in full training after 10 hours of rabbit bone marrow mesenchymal stem cells began to gradually adherent process,when the experiment to the 72 hours after inverted microscope can see sleep most of the stem cells have been successfully completed attached process,full training after 7 days of stem cells,can see the growth of the adherent cells remain single warrior,and was completely purified and present state of aggregation.The third generation of bone marrow mesenchymal stem cells showed strong vitality and proliferation ability under the induction conditions of their fibrous cartilage system,showing a biological behavior similar to that of meniscus cells,and the cells showed a tendency of adherent wall and mutual fusion and growth.Cell adhesion count analysis: the number of cells adsorbed by B sample on node scaffolds at different times was lower than that of PB sample,all P < 0.05,and the difference was statistically significant.Cck-8 proliferation experiment analysis: OD value of cell proliferation of B sample was lower than PB sample at different time node scaffolds,PB sample showed a faster way,P < 0.05,the difference was statistically significant.Cell compatibility test of scaffolds in vitro: HE staining analysis indicated that the number of cells stained with B sample was less than that of PB sample as a whole,and the number of cells adhered to PB sample was more.Sem analysis indicated that both PB sample scaffolds and B sample scaffolds had a certain degree of adhesion.The cells were polygonal or long spindle shaped for growth,and the cells were relatively flat at the flat place of the scaffolds,while the cells were more three-dimensional at the uneven place of the scaffolds.PB samples showed that the proportion of cell adhesion was larger.The results of immunohistochemical staining showed that the proportion of cells on the surface of sample B was less than that of PB samples,indicating that the adhesion point of cells on the surface was less and the density of cells on the surface of PB samples was higher.Finally,fluorescence tracking cell proliferation detection and analysis revealed that the overall number of cells in sample B was small,and the morphology was mainly small circle,with almost no fusion of cells.The number of cells and the fusion of cells can be clearly seen on the surface of PB samples,suggesting that the surface of PB samples is more suitable for the growth and fusion of cells.Gene expression of cells on meniscus scaffold: Study suggests COLIA1 concentration expressed as sample B(2.51 ± 0.45)< PB samples(4.30 ± 0.56),COLII expression trend for B sample(0.71 ± 0.05)< PB samples(2.32 ± 0.49),AGG expression trend for B sample(1.24 ± 0.12)< PB samples(3.12 ±0.43),the growth of cartilage important SOX9 gene analysis reflects the same trend in the process,express trend for B sample(0.34 ± 0.05)< PB samples(0.90 ±0.34),In the expression analysis of each genotype,the P value was less than 0.05,and the difference was statistically significant.Conclusions:The preparation of tissue engineering scaffold of polyurethane meniscus by solvent-melt method is practical and the material has good biocompatibility.The addition of end-sealing agent POSS samples showed better physical properties and biocompatibility,as well as a better cell growth and fusion environment in the process of in vitro cell culture,providing theoretical basis for subsequent in vivo culture.Part two: Solvent-melt method for the preparation of polyurethane meniscus tissue engineering scaffold in vivo performance analysisObjective:On the basis of the first part of the study,the biocompatibility and repair and regeneration of polyurethane meniscus tissue engineering scaffolds in animals were fully evaluated.Materials and Methods:To evaluate the biocompatibility of biomimetic scaffold materials in vivo in accordance with the national standard for biological evaluation of medical materials,the New Zealand white rabbit was used as the experimental animal to fully construct the animal model of meniscus injury in non-blood transport area.The New Zealand white rabbits were divided into the control group and the stent group,and the two groups of rats were sacrificed 12 weeks after the operation.The related items of the two groups of rats were tested as follows: gross specimen observation;Knee joint function score;Histological and immunohistochemical and biomechanical evaluations;Acute and chronic systemic toxicity assessment in the stent group;Systemic skin irritation in the stent group;Biomechanical evaluation,etc.Results:Postoperative gross observation: there was no abnormal phenomenon in the control group,and the growth diet of the rabbits was completely normal.Polyurethane stent group: white rabbits could normally stand within three weeks after surgery,and the affected limb presented periodic instability during walking.By 6 weeks after the surgery,they could almost walk normally.Both standing and walking showed normal characteristics,and instability still appeared in the process of running for a long time.All the rabbits in each group were given prophylactic antibiotic treatment 3 days after the surgery and the healing of the incision was fully observed.All the rabbits did not show the continuous redness,swelling and suppuration around the knee joint and other postoperative infections.The overall mental state of the white rabbits was good.Experimental results of acute systemic toxicity: the white rabbits in the control group and the stent group were in good overall condition,with no symptoms of peritoneal membrane irritation,dyspnea,tremor,etc.,and the rats in the stent group showed decreased activity and death.Intradermal stimulation experiment analysis: no skin erythema edema and other subcutaneous irritation symptoms were observed at all injection points of the white rabbits in the stent group.According to the relevant scoring criteria,the subcutaneous stimulation index of the polyurethane scaffold extract was 0.Meniscus injury repair analysis: no synovial hyperplasia and encapsulation was observed in the two groups of white rabbits implanted with scaffold materials due to host immune rejection.After 12 weeks of degradation and metabolism,the scaffold material has been fused with the surrounding meniscus tissue,which is difficult to be recognized by the naked eye.The polyurethane stent group can effectively promote and repair the meniscus injury process in large white rabbits.The meniscus recovery at the injury site is very similar to that of the control group,with relatively uniform tissue texture and the ability to withstand certain tension.Knee joint score analysis: the study indicated that the score of the control group was significantly higher than that of the stent group,with a P value of 0.040,and the difference was statistically significant.Histological evaluation: the repair of meniscus injury was relatively obvious in the cell scaffold group,which could effectively improve the platform support for tissue cells and thus effectively promote the repair and regeneration of meniscus tissue in the non-blood supply area.The results of HE staining showed that most meniscus injury areas in the stent group at 12 weeks after surgery had been filled with regenerated tissue,and the extension of blood vessels could be seen in some newly generated meniscus tissues.As a whole,the tissue septal was not very serious,and the effective fixation was comprehensive.Mechanical analysis of meniscus repair: the tensile elastic modulus of the stent group was 35.33± 6.70 MPa,while the normal meniscus of the control group was 38.90 ± 3.45 MPa.Conclusions:Polyurethane scaffolds have good biocompatibility in vivo,can better promote the normal blood supply tissue around the meniscus,and provide platform support for the normal growth of tissue cells,which has certain practical significance. |
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