Evaluation Of Biosecurity Evaluation Of Tissue-engineered Decellularized Tracheal Scaffolds-cells Complexes

OBJECTIVE1.To produce tissue-engineered decellularized tracheal scaffolds-cells complexes by using genipin cross-linked sodium deoxycholate-enzymatic decellularization method and Trixon-100-enzymatic decellularization method planted autologous BMMSCs respectively.2.To evaluate biological safety of tissue-engineered decellularized tracheal scaffolds complexes from the acute toxicity, cell toxicity, the ability of vascularization, immune inflammatory reaction of Residual antigenic substance, and the degree of the complexes degradation and absorption.3.Through the evaluation of biological safety of tissue-engineered decellularized tracheal scaffolds complexes, to provide experimental basis for fabricating a kind of substitute can be closer to the native trachea.METHODS1.Fabrication of tissue-engineered decellularized tracheal scaffolds-cells complexes.(1) Preparation of cells:Using a healthy New Zealand rabbit(2month old,1.2kg) as donator of BMMSCs, we required BMMSCs of the third generation with originally culturing and subculturing.(2) Preparation of decellularized tracheal scaffolds:Using5healthy New Zealand rabbits(4month old,2.5kg) as donors of tracheal scaffolds, we decellularize the scaffolds and obtain decellularized tracheal scaffolds by using genipin cross-linked sodium deoxycholate-enzymatic decellularization method and Trixon-100-enzymatic decellularization method respectively.(3) Preparation of decellularized tracheal scaffolds-cells complexes:the BMMSCs were grown in the above two kinds of tracheal stent surface and develop.(4)Group of Experiments:we put decellularized tracheal scaffolds-cells complexes by using genipin cross-linked sodium deoxycholate-enzymatic decellularization method planted autologous BMMSCs(5×104cells/ml) as the group A, and put decellularized tracheal scaffolds-cells complexes by using Trixon-100-enzymatic decellularization method planted autologous BMMSCs as the group B.2.Through the following five kinds of methods to test biological security of tissue-engineered decellularized tracheal scaffolds-cells complexes.(1)Group A culture immersion fluid, group B culture immersion fluid and negative group (physiological saline) was injected into rats’s abdominal cavity to test the toxicity of decellularized tracheal scaffolds-cells complexes against the rats.(2)Growth and proliferation of BMMSCs of group A, group B, the negative control group (tetrafluoroethylene-hexafluoropropylene copolymer) and positive control group (polyvinyl fluoride) were detected by applying MTT method.(3) Adhesive growth of BMMSCs of the two experimental groups were observed by SEM (scanning electron microscopy).(4) The angiogenic properties of group A, group B and negative group (group A by infiltration of glutaraldehyde) and positive group(gelatin sponge) were evaluated by CAM(the chicken embryo chorioallantoic menbrane) assay.(5) we applied the original white rabbit of BMMSCs donor as transplant recipient,then the two experimental groups were transplanted into subcutaneous of recipient on both sides of the spine of the recipient respectively.decellularized tracheal scaffolds-cells complexes were gained at7th,14th,28th day after transplantation by operation,and, processed morphological observation and HE staining, to evaluate the immune inflammatory response,as well as decellularized tracheal scaffolds-cells complexes before transplantation.RESULTS1. The activity, feed and defecation of three groups of mice are normal, weight increase ((p>0.05)) and respiratory frequency change ((p>0.05)) were considered statistical insignificance,after culture immersion fluid of decellularized tracheal scaffolds-cells complexes injected into mice abdominal cavity.2. Cell morphological observation showed BMMSCs planted on the two experimental groups grew and stick well, no obvious difference.3. MTT assay cell growth curve showed that cell growth of the negative control group was best, while positive group’s was worst,and even group B’s was better than group A, there had on visible difference.4. SEM showed:BMMSCs of the two groups were adhesive outside of scaffold completely, cell layed each other, connected closely.5. Macroscopic observations of CAM assay showed that group A,group B and positive group surrounded by nutrient allantoic vessels.Nutrient allantoic vessels surrouding postive group were most.Group A also had new vessels,Some new vessels crawled in the undersurface of decellularized tracheal scaffolds-cells complexes. No new vessels were observed aroud negative group.6. Histological evaluation and HE staining of four continuously times(before transplantation,7th,14th and28th day after transplantation) comparation:Group A:the color were still kermesinus,the hardness did not decreased,still maintained high elasticity, had no obvious inflammation, the structure of each layer of the the complexes was complete without degradation.Group B became pompadour, had heavier inflammation,the structure were chaotic, then gradually soft and degradative.CONCLUSION1. The results of acute toxicity test and cytotoxicity test show that Both group A and group B hadn’t acute toxicity test and cytotoxicity, could promote cell proliferation and the new blood vessel formation of scaffold, and vascularization ability of group A is more stronger.2.After in vivo transplantation, the ability of slight inflammation and degradation of group A is more advantageous to transplant long-term survival and function in vivo.