| Objectives In this study,PPDO and PLA were used for melt blending,and the most suitable ratio of PPDO/PLA was screened for 3D printing of pancreaticojejunostomy device through various in vitro and in vivo performance tests.Secondly,a double-layer drug coating is applied on the surface of the pancreaticojejunostomy device,so that it has dual functions of anti-tumor and promoting healing,and its anti-tumor and promoting healing effects and biosafety are verified through in vitro and in vivo experiments.The aim of this study is to explore the clinical application value of surface functionalized pancreaticojejunostomy device.Methods Part 1.The configuration of pancreaticojejunostomy device was Fused Filament Fabrication(FFF)3D printed by PPDO/PLA blended materials in different proportions.At the same time,the materials of the printing device are tested for surface microstructure identification,mechanical property testing,substance molecular structure identification,in vitro degradation properties,and surface hydrophilic properties of materials.The properties of different proportions of PPDO/PLA blends and their applicability in the preparation of pancreaticojejunostomy devices were compared.Part 2.The cytotoxicity of the materials were evaluated by CCK-8 experiment and live-dead staining experiment,and the cell adhesion properties on the surface of the material were observed by scanning electron microscope(SEM).The hemocompatibility of the material was verified by hemolysis experiment.In animal experiments,a 3D catheter was inserted into the distal duodenum of rats to establish an in situ degradation model in vivo.Venous blood was collected for hematological analysis on the 1st,3rd,7th,42 nd and 84 th days after operation.At the 6th and 12 th week,the intestinal wall around the catheter was taken for HE staining,IL-1β and TNF-α immunohistochemical staining,and the content of IL-1β and TNF-α in the surrounding intestinal wall was detected by Elisa method.Finally,the heart,liver,spleen,lung and kidney of rats were taken for HE staining to observe the toxicity of the material to important organs in the body after degradation and absorption.Part 3: Applied double-layer drug coating on the surface of pancreaticojejunostomy device.Using chitosan as the drug-loading medium,the outer layer of basic fibroblast growth factor coating(b FGF/CS)and the inner layer of nab-PTX/CS were constructed.Screening appropriate doses of drug coating combinations for cytotoxicity studies on normal fibroblasts(L929)and pancreatic cancer cells(Panc02).The surface morphology,mechanical properties changes and drug release kinetics of the drug-loaded coating devices were examined by scanning electron microscopy,energy dispersive analysis(EDS),mechanical experiments,degradation experiments and in vitro drug release experiments.In vitro cell experiments,CCK-8,live-dead staining,flow cytometric apoptosis assay,and cell drug uptake assay were used to verify the antitumor properties of the drug coating and the growth-promoting ability of fibroblasts.In animal experiments,a mouse subcutaneous residual tumor model was established,and disc samples with different drug coatings were implanted on the tumor surface.Blood was drawn after the 14 th postoperative day for biosafety assessment.The expression of Caspase-3 and Ki-67 was detected by immunohistochemistry and the expression of Caspase-3 and PCNA was detected by Western-Blot.The apoptosis of tumor tissue was analyzed by flow cytometry.Result Part 1.In this part of the study,the blend materials of 10%PPDO/PLA,30%PPDO/PLA,and 50% PPDO/PLA were synthesized,and the configuration of the pancreaticojejunostomy device was successfully printed by the 3D printing technology of fused filament fabrication(FFF),and successfully assembled,and the finished product has small error and high precision.The results of infrared spectroscopy and X-ray diffraction show that the peak positions and vibration frequencies of carbonyl(C=O)and ether bonds on the ester bond of PPDO/PLA with various ratios are significantly different,and the crystal structure is different.It is confirmed from the molecular structure that various ratios of PPDO/PLA were successfully synthesized and the thermal processing of 3D printing did not destroy the molecular structure of the blends.The results of thermogravimetric analysis showed that the higher the proportion of PPDO in the blends,the lower the initial decomposition temperature,but it was higher than the human body 37 ℃environment.The results of in vitro degradation experiments and mechanical experiments showed that with the increase of PPDO content,the degradation rate of PPDO/PLA was accelerated,and the Young’s modulus and tensile strength decreased.The in vitro degradation rate of 50% PPDO/PLA reached(46.04±5.79)% in 12 weeks.However,compared with pure PLA,it has the elastic modulus and controllable degradation performance closer to that of viscera.At the same time,as the proportion of PPDO increases,the hydrophilic properties of the material are better.Part 2.The cell cytotoxicity results showed that compared with the negative control,there was no significant difference in the cell viability of the extracts of different ratios of PPDO/PLA,and the cell viability in each group was higher than70%.Hemolysis experiments showed that PPDO/PLA blends had good blood compatibility.Cell adhesion experiments showed that compared with pure PLA,the higher the proportion of PPDO in the blends,the better the cell adhesion ability.The degradation trend in vivo was the same as in vitro,but the degradation rate in vivo was significantly faster than in vitro,and the in vivo degradation rate of 50%PPDO/PLA reached(46.04±5.79)% in 12 weeks.In terms of biocompatibility,there was no obvious pathological tissue damage and abnormality in the heart,liver,spleen,lung,kidney of SD rats in each group.The results of hematological indexes were all within the normal range.The immunohistochemical and ELISA results of IL-1β and TNF-α in the intestinal wall tissue showed that the inflammatory factors in the intestinal wall around the catheter in the early stage of 30% PPDO/PLA and50% PPDO/PLA were significantly increased compared with the control group,but with the degradation,the inflammatory response was lower than before,and no overexpression of inflammation was found.Part 3 : In this study,a pancreaticojejunostomy device containing a double-layer drug coating was successfully fabricated.The surface of the drug-coated device is pale yellow.SEM observation showed that the surface of the bare device without drug coating was rough,while the surface of the modified device was smoother.In addition,SEM and EDS detection results confirmed the presence of b FGF and nab-PTX drugs in the coating.The coating modification had no obvious effect on the mechanical and degradation properties of the material.The in vitro drug release results showed that in the double-layer b FGF/nab-PTX/CS coating,the early "burst release" effect of nab-PTX is avoided.Under the barrier of outer coating,the release of nab-PTX in inner layer began gradually on the 4th day,and reached the peak on the 10 th day,with a cumulative release of 84.1±0.5%.Drug uptake experiments showed that pancreatic cancer cells take up nab-PTX through endocytosis.In vitro cancer cell killing results indicated that 100 ng b FGF and 50 μg nab-PTX were the optimal doping doses for b FGF/nab-PTX/CS coatings.Live-dead staining,CCK-8,and flow-through apoptosis assays showed that the b FGF/nab-PTX/CS coating had better killing effect on cancer cell and lower toxicity to normal fibroblasts.It not only protects normal fibroblasts,but also inhibits the growth of tumor cells.In animal experiments,the results of western-blot and flow apoptosis assay and pathological detection verified that the b FGF/nab-PTX/CS coating has a good inhibitory effect on the growth of pancreatic cancer.Finally,hematological tests confirmed the good biocompatibility of the device.Conclusions(1)50% PPDO/PLA has good 3D printing performance and can be printed to complex configurations with good accuracy.At the same time,it has a suitable degradation rate to meet the time needs of the healing of pancreaticointestinal anastomosis,and the material has good cell adhesion property.Sum up,50% PPDO/PLA is a suitable option for the preparation of pancreaticojejunostomy device.(2)The double-drug-coated pancreaticojejunostomy device has sustained and controllable drug release kinetics,good anti-tumor properties and potential ability to promote fibroblast proliferation.In future clinical applications,it has the ability to support and repair the pancreaticojejunostomy,and at the same time,it can prevent the subsequent proliferation of residual tumor cells and reduce the possibility of local recurrence after pancreatic cancer surgery,which has certain application value. |
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