| Objective1.To studied the cytotoxicity of electrospinning polyvinylidene fluoride（PVDF）material mesh which based on three-dimensional printing technology to mouse fibroblastcells（L929cells），and compared the cytotoxicity to polypropylene mesh.2.To set up the method of separating human vaginal epithelial cell（shVECs）from the vaginaltissue，and the way of proliferation in vitro. To study the biologic characteristics of hVECs invitro and the cytotoxicity and of polyvinylidene fluoride material mesh by humanvaginal epithelial cells.And compared the cytotoxicity to polypropylene mesh.Methods1. Conducting cytotoxicity test according to ISO10993evaluation standards.(1)Evaluation ofcell morphology and metabolic features:this study includes four groups: group A (blankcontrol group,complete culture medium)，group B (the positive control group leachingsolution of latex gloves）， group C (the negative control group leaching solution ofpolypropylene) and group D(leaching solution of PVDF）.Inverted phase contrast microscopeobserved cell morphology,when using each material leaching solution cultivate L929cells24h,48h,72h. Tetrazole compounds MTS to determine each absorbance values, andcalculate the relative growth rate (FGR) to toxicity grading.(2)Evaluation of the cell apoptosis:calculating Percentage of normal cells and late stage apoptotic and dead cells by detecting cellapoptosis when using each material leaching solution cultivate L929cells72h.(3)Cellular compatibility evaluation:The growth of cell was observed by scanning electronmicroscopy (SEM) on1d，3d and7d of cell cultivating on PVDF mesh.2.Taking patients，anterior wall vaginal tissue，who had colporrhaphia anterior-posterior forpelvic organ prolapse (POP). The vaginal tissue digested by Dispase II enzymes for thenight at4degrees to separated epithelium and lamina propria, and the epithelium cell inoculated to the keratinocytes in serum free medium,which was digested by trypsin.Vaginal epithelial cell morphology and growth characteristics was observed by invertedmicroscope, drawn growth curve by The first generation of cell and indentied by Cytokeratinimmunohistochemical staining.Conducting vitro biocompatibility test of PVDF for vaginalepithelial cell according to ISO10993evaluation standards.Inverted phase contrastmicroscope observed cell morphology,when using each material leaching solution cultivateL929cells24h,48h,72h. Tetrazole compounds MTS to determine each absorbance values,and calculate RGR to toxicity grading. Evaluation of the cell apoptosis: Flow cytometryinstrument detected cell apoptosis when using each material leaching solution cultivatevaginal epithelium cells72h.Cellular compatibility evaluation:The growth of cell wasobserved by scanning electron microscopy (SEM) on1d，3d and7d of cell cultivating onPVDF mesh.Ruselts1. It was basic consistent of L929cell morphology,adherent state, and proliferation at24h,48h and72h in group A, group C, group D,which had normal cells form with fusiform orirregular triangle, sticked a wall grew well. It had a large number of dead cells and cell rupturein group B,which cell layer was almost completely destroyed and presented severe toxicityto L929cell. MTS measured absorbance of four groups in three time: compared with Agroup, group C and group D cytotoxic level were1and0~1, and group B cell cytotoxicitylevel was4.Group A, group C, group D absorbance values comparison between any twomeans there was no statistically significant difference, which difference was statisticallysignificant compared with group B(P <0.05). Flow cytometry tested cell apoptosis prompt:cells survival rate of four groups respectively were (92.43±1.24)%,(3.66±0.25)%,(92.23±0.64)%and (92.8±0.89)%, and late stage apoptotic and dead cells were （4.32±1.14）%,（95.06±1.33）%,（3.57±1.15）%,（3.85±1.08）%.Group A, group C, group D absorbancevalues comparison between any two means there was no statistically significant difference,which difference was statistically significant compared with group B(P <0.05).SEM shewthat the cells gradually increased and spaced to fiber material to deep into with timeextension. 2.The Primary vaginal epithelial cells began to stick wall24hour or so.Growth rate wassignificantly increased after four days, and primitive cells commonly7to10days couldwere sub-cultured. The cells after passage basically completed stick wall after24h, couldbe passed new generation about five day to seven day to extend.Vaginal epithelial cellswere polygonal with uniform morphology, which refraction sex was good.Cells extend the cellaging after five generations. Cytokeratin antibody immunohistochemical staining waspositive.3.It was basic consistent of vaginal epithelial cells morphology,adherent state, andproliferation at24h,48h and72h in group A, group C, group D,which were polygonal withuniform morphology, and sticked a wall grew well. It had a large number of dead cells andcell rupture in group B,which cell layer was almost completely destroyed and presentedsevere toxicity to vaginal epithelial cells. MTS measured absorbance of four groups in threetime: compared with A group, group C and group D cytotoxic level were1and0~1, and groupB cell cytotoxicity level was4.Group A, group C, group D absorbance values comparisonbetween any two means there was no statistically significant difference, which difference wasstatistically significant compared with group B (P <0.05). Flow cytometry tested cellapoptosis prompt: cells survival rate of four groups respectively were （78.73±3.）%,（6.8±2.14）%,（76.57±5.86）%and（78.43±2.35）%, and group A, group C, group Dcomparison between any two means there was no statistically significant difference, whichdifference was statistically significant compared with group B (P <0.05). Late stage apoptoticand dead cells were （3.36±1.38）%,（80.43±0.61）%,（7.8±0.61）%and（4.12±0.44）%，which group C higher than that of group A and group D, and there was statistically significantdifference (P <0.05).SEM shown that the cells gradually increased and spaced to fibermaterial to deep into with time extension.Conclusion1.Dispase II enzymes combination with the pancreatic enzyme digestion method couldobtained high purity vaginal epithelial cells.2.PVDF material patch had no cytotoxicity for L929cells and human vaginal epithelial cells.3.Human vaginal epithelial cells had more sensitive than L929cells for toxicity of pelvic floor repair material. |
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