Study On The Prevention Of Postoperative Adhesion Scar Formation By A New Type Of Nanofiber Membranes

Purpose:This project designed a new type of electrospun nanofiber membrane to prevent postoperative adhesion scar formation.It is planned to evaluate its performance in preventing postoperative adhesion scar formation through multiple angles such as material physical and chemical characterization,in vitro cell experiment and in vivo animal experiment.And further explore the potential mechanism of its function.Materials and Methods:1.Preparation and physicochemical characterization of PLGA-PVP-PC electrospinning nanofiber membrane(1)Synthesis of PLGA-PVP by cobalt 60 irradiation grafting(2)Prepare 12% PLGA,12% PLGA-PVP,15% PLGA-PVP,18%PLGA-PVP,PLGA-PVP-PC1,PLGA-PVP-PC2,PLGA-PVP-PC3 electrospinning solution respectively,and Electrospinning nanofiber membranes with different components and different concentrations are prepared.Observe the properties of each group of spinning solutions and compare them;explore the most suitable parameters for each group of electrospinning,and compare the stability of the electrospinning process.(3)Perform physical and chemical characterization of each group of electrospun nanofiber membranes,including gross observation,scanning electron microscope observation,infrared spectroscopy test,thermogravimetric analysis test,water contact angle test,water absorption test,in vitro degradation test,etc.And screen out the most suitable materials based on the results.2.Evaluation of cell compatibility of PLGA-PVP-PC electrospinning nanofiber membrane and exploration of its mechanism to prevent postoperative scar adhesion formationBy extracting rabbit fibroblasts as the research object,based on the electrospun nanofiber membrane material,the biological safety and the inhibitory effect on fibroblast deposition,infiltration and proliferation are verified through in vitro cell experiments,and its possible potential is explored Mechanisms.(1)Extraction,isolation,culture and identification of rabbit fibroblasts;(2)Measure the cytotoxicity of the 72-hour extract of each group of materials by CCK-8,and the value-added curve of 1,3,and 5 days of cocultivation of cells and materials;(3)Live/Dead fluorescent staining to observe the inhibition of rabbit fibroblast proliferation by each group of materials;(4)Load the DCFH-DA reactive oxygen probe to each group of cells,and measure the average fluorescence intensity by flow cytometry;(6)Using lipopolysaccharide to induce mouse macrophages to mimic the inflammatory response in vitro,and detect the expression of TNF-α and IL-10 in each group.(7)Test the gene expression of each group of materials and the cell coculture system by rt-PCR,and explore the relative expression of different materials on rabbit fibroblasts TGF-β1,Smad3,Smad7,COL1A1,COL3A1.3.Evaluation of the efficacy of PLGA-PVP-PC electrospun nanofiber membrane in the prevention of postoperative adhesion scar formation in animal lumbar laminectomy models(1)New Zealand rabbits were used as the research object to establish a lumbar laminectomy model,with no material inserted,commercial Inteerceed material inserted,and PLGA-PVP-PC2 material inserted,and the materials were taken at 1,2,and 4 weeks respectively.And conduct a series of evaluations.(2)Evaluate its morphology by gross observation,H&E staining,and Sirius scarlet picric acid staining.(3)Through TGF-β1,TNF-α,IL-6 immunohistochemical staining,evaluate the effects of each group of materials on inhibiting fibroblast proliferation and regulating the level of inflammatory response.(4)Evaluate the effect of each group of materials on the anti-oxidation performance by testing the activity of SOD and GSH-px.(5)Evaluate the role of each group of materials in regulating the balance of the fibrinolytic system by detecting the expression of t PA and PAI-1 genes.Result:1.Preparation and physicochemical characterization of PLGA-PVP-PC electrospinning nanofiber membraneSuccessfully prepared 12% PLGA,12% PLGA-PVP,15% PLGA-PVP,18% PLGA-PVP,PLGA-PVP-PC1,PLGA-PVP-PC2 groups of electrospun nanofiber membranes.12% PLGA has no obvious bead formation,the fiber diameter is(468.68±139.02)nm,and the fiber diameter is evenly distributed.12%PLGA-PVP shows some bead formation,the fiber diameter is(465.34±145.09)nm;15%PLGA-PVP shows no obvious bead formation,the fiber thickness is uniform,the fiber diameter is(462.05±135.96);18%concentration group nanofiber Scanning electron microscopy of the membrane shows that the fiber thickness is not uniform,and the average fiber diameter is(423.93±135.96).It can be seen that 15% PLGA-PVP is the best concentration group;PLGA-PVP-PC1 has no obvious bead formation,the fiber thickness is not uniform,and the average fiber diameter It is(649.61±137.37)nm;PLGAPVP-PC2 has no obvious bead formation,the fiber thickness is not uniform,and the average fiber diameter is(669.64±146.70)nm.The average water contact angle of the PLGA group was(112.08±4.15)°,which was in a hydrophobic state;and after PVP grafting,the water contact angle of the PLGA-PVP group was significantly improved,which was(31.24±2.58)°,which was in a hydrophilic state;Due to the presence of PC,the water contact angles of the PLGA-PVP-PC1 group and PLGA-PVP-PC2 group were(9.22±1.69)° and(2.58±0.81)°,respectively,showing a super-hydrophilic state.It can be seen from the figure that,due to the difference in hydrophilicity of the nanofiber membranes of each group,there is also a big difference in the water absorption rate.The water absorption rate of the PLGA group was42.39%,the water absorption rate of the PLGA-PVP group was 69.47%,the water absorption rate of the PLGA-PVP-PC1 group was 90.96%,and the water absorption rate of the PLGA-PVP-PC2 group was 96.17%.Due to the difference in hydrophilicity and water absorption of each group of nanofiber membranes,the degradation tendency in vitro is also different.At 1 week,the residual weight rates of PLGA,PLGA-PVP,PLGA-PVP-PC1,and PLGAPVP-PC2 were 98.16%,88.90%,78.42%,and 75.71% respectively;at 2 weeks,the residual weight rates of the four groups of materials were: 90.58%,79.23%,66.76%,59.20%;the residual weight rates of the four groups of materials at 3weeks were 84.30%,71.38%,51.44,41.70%;at 4 weeks,the residual weight rates of the four groups of materials were 82.64%,67.15%,45.26%,34.09%.2.Evaluation of cell compatibility of PLGA-PVP-PC electrospinning nanofiber membrane and exploration of its mechanism to prevent postoperative scar adhesion formationSuccessfully extracted rabbit fibroblasts.The CCK-8 extract test showed that PLGA-PVP-PC1 and PLGA-PVP-PC2 have good biocompatibility and no obvious toxicity.The co-culture experiment of CCK-8 cells and materials showed that the PLGA group and PLGA-PVP group can promote the growth of rabbit fibroblasts on the material,while the PLGA-PVP-PC1 group and PLGA-PVP-PC2 group can inhibit the growth of rabbit fibroblasts on the material.On growth.Live/Dead fluorescent staining shows that the surface has good inhibition of fibroblast deposition and proliferation.The flow cytometry of each group of samples mounted on the DCFH-DA probe confirms that the PLGA-PVP-PC electrospinning nanometer Fibrous membrane has a certain effect in reducing reactive oxygen species;the rt-PCR results of each group suggest that PLGA-PVP-PC1 and PLGA-PVP-PC2 may inhibit adhesion and adhesion by regulating the TGFβ1/Smad signaling pathway and downregulating the expression of COL1A1 and COL3A1 genes.The role of scar formation.3.Evaluation of the efficacy of PLGA-PVP-PC electrospun nanofiber membrane in the prevention of postoperative adhesion scar formation in animal lumbar laminectomy modelsInteerceed commercial membranes and PLGA-PVP-PC electrospun nanofiber membranes have better performance in preventing postoperative adhesion scar formation than the blank control group.Among them,in the established 1,2,4 week group epidural adhesion scar formation model,both can inhibit the formation of adhesion and scar to a certain extent,while the adhesion produced by PLGA-PVP-PC electrospinning nanofiber membrane Less,scar fibrosis is lighter,collagen secretion is less,especially when the Interceed anti-adhesion membrane is almost degraded after 2 weeks and cannot function as a physical barrier,PLGA-PVP-PC electrospun nanofiber membrane can be better Give full play to its excellent performance.At each time point,compared with Interceed anti-adhesion membrane and blank control group,PLGA-PVP-PC electrospun nanofiber membrane can significantly inhibit the expression of TGF-β1 and reduce the expression of TNF-α and IL-6 inflammatory factors,Significantly enhance the activity of SOD,GSH-px to play an antioxidant role,and significantly up-regulate the expression of t PA gene,significantly down-regulate the expression of PAI-1gene to play a role in regulating the balance of the fibrinolytic system.Conclusion:This subject designed a new type of electrospun nanofiber membrane to prevent postoperative adhesion scar formation,and successfully screened,prepared,and measured its physical and chemical characteristics.From in vitro to in vivo,it was developed from multiple perspectives in preventing postoperative adhesions.Biological safety.The material has the following characteristics:(1)Combining traditional PLGA with N-vinylpyrrolidone,phosphatidylcholine and other materials,while maintaining its good biocompatibility,it greatly improves its hydrophilicity,anti-oxidation performance and fiber-forming properties.Inhibition of cell proliferation;(2)The degradation time of the material is appropriate,and it can meet the time required for repairing the damaged part,and avoid the chronic inflammation caused by long-term degradation;(3)The material is soft in nature and thin in thickness,which not only avoids the adjacent tissue damage caused by the material being too hard,but also avoids the compression of the nerve tissue during the filling period;(4)The surface of the material has lubricating properties and also forms a super-hydrophilic layer,which plays a role in preventing fibrin deposition and fibroblast infiltration from a new perspective;(5)The material can effectively reduce the local deposition of fibrin and inhibit the infiltration,proliferation and synthesis of extracellular matrix by fibroblasts.It may play a role in the following ways: through the antioxidant effect of phosphatidylcholine,it reduces the oxidative stress response caused by reactive oxygen species and tissue ischemia;it has good anti-inflammatory properties and reduces the level of inflammatory response;Effectively regulate the balance of fibrinolytic system,promote fibrin dissolution and reduce fibrin deposition here.At the same time,the three can also interact and influence each other,further reducing the formation of postoperative adhesion scars.(6)The material is expected to become a new generation of products for preventing postoperative adhesion scar formation.