Fabrication And Evaluation Of A Novel Xenogeneic Decellularized Costal Cartilage Graft

Objective : Due to the poor biocompatibility of synthetic prostheses,millions of rhinoplasty recipients are forced to choose autologous costal cartilage as grafts.Autologous costal cartilage has strong biocompatibility,but it suffers from many shortcomings including limited source,donor site injury(pain,restricted movement,thoracic deformity and chest wall scar),and prolonged operation time,which greatly limit its actual clinical application.The decellularized xenogeneic cost cartilage retaining natural extrdecellularized matrix and low immunogenicity is expected to be a promising alternative for autologous cost cartilage.However,the traditional cartilage decellularization method is mainly based on multiple cycles of detergents and repeated washing for a long time,which causes the loss of tissue components and the obvious damage of the mechanical structure,resulting in poor in vivo implantation result.In order to address these obstacles,this study prepared a novel type of decellularized xenogeneic costal cartilage with an improved decellularization method that combines osmotic pressure,freezing-thawing,enzymatic treatment,and mild detergent washing.The following assays were performed: 1.To evaluate the decellularization effect,histomorphological characteristics,bioactive component content and mechanical properties of the novel decellularized cost cartilage;2.To assess the cytotoxicity and cytocompatibility of the novel decellularized cost cartilage with in vitro experiments;3.To analyze the histocompatibility,inflammatory response and morphological maintenance of the new decellularized cost cartilage with adult New-Zealand white rabbits.Methods: 1.The novel xenogeneic costal cartilage(CDCC)was prepared by an improved decellularization method,and compared with decellularized costal cartilage prepared with tradition decellularization protocol(SDCC)and native costal cartilage(NCC).HE staining and DAPI staining were performed to determine the residual status of chondrocytes.After each group of DNA was extracted,quantitative detection was performed to evaluate the residual status of DNA,and the fragment size of residual DNA was detected by agarose gel electrophoresis experiment.In addition,quantitative analysis of ?-Gal epitopes is also carried out.The tissue morphology was analyzed by Safranin O staining and type II collagen immunohistochemical staining,and the surface microstructure of the material was observed by scanning electron microscope.The content of total collagen and GAGs were assessed by biochemical quantitative detection.The preservation of critical cytokines(b-FGF,TGF-?1 and TIMP-1)was analyzed by ELISA.The mechanical mechanics test was used to evaluate the mechanical properties of each group.2.Rat nasal septal chondrocytes were isolated and seeded with CDCC and SDCC scaffolds to analyze the biocompatibility of decellularized costal cartilage in each group by measuring the amount of DNA released in the medium.Rat chondrocytes were cultured in CDCC and SDCC extracts,and the cytotoxicity of each group of materials was evaluated by CCK-8 experiment.L929 fibroblasts were seeded on two groups of materials,and cell adhesion was observed under electron microscope.The possible systemic and local immune responses caused by the graft were evaluated by subcutaneous implantation experiments.3.Adult New Zealand rabbits were used as experimental animals,and randomly divided into two groups according to the grafts: the conventional decellularized costal cartilage group(SDCC)and the novel modified decellularized costal cartilage group(CDCC).At the 3rd and 6th months after surgery,the experimental rabbits were subjected to magnetic resonance analysis to evaluate the condition of the implants.HE,Safranin O and type II collagen immunohistochemical staining were analyzed to evaluate the implant morphology,degradation and inflammation reaction.The quantitative analysis of glycosaminoglycans was employed to evaluate the degree of degradation and absorption of the implant.Results: 1.The results of HE staining and DAPI staining showed that the decellularization effect of CDCC was the same as that of SDCC,and neither of them had visible cell membrane and nucleus residues.The DNA quantitative test results showed that after decellularization treatment,compared with the DNA content of NCC,both CDCC and SDCC decreased significantly(p<0.05),which reached the international standard below 50ng/mg,with no significant difference between them(p<0.05).The results of agarose gel electrophoresis showed that neither CDCC nor SDCC showed visible DNA bands,which reached the standard of no more than 200 bp DNA fragments.The quantitative detection results of ?-Gal epitope showed that the results of the CDCC group and the SDCC group were significantly lower than those of the NCC group(p<0.05).In immunohistochemical staining of type II collagen,NCC,SDCC and CDCC all showed brown-yellow collagen staining,and there was no significant difference.Safranin O staining showed that SDCC and CDCC were significantly lightly stained compared with NCC,but CDCC was significantly darker than SDCC.The quantitative test results of total collagen proved that the total collagen content of NCC,CDCC and SDCC groups had no significant difference(p>0.05).The quantitative analysis results of glycosaminoglycans showed that CDCC and SDCC were significantly lower than NCC(p<0.05),but the glycosaminoglycan content of CDCC was significantly higher than SDCC(p<0.05).ELISA results showed that the contents of b-FGF,TGF-?1 and TIMP-1in CDCC were significantly higher than SDCC(p<0.05).Scanning electron microscopy results show that compared with NCC,CDCC and SDCC retain intact collagen fibers,but the morphology of chondrocyte lacuna in CDCC is better than that of SDCC.The mechanical property analysis results show that,compared with NCC,the Young's modulus of CDCC is significantly higher than that of SDCC(p<0.05).2.After nasal septal chondrocytes were co-cultured with SDCC and CDCC,no significant DNA release was detected in the two groups of media,and their DNA content was significantly lower than that of the positive control group(p<0.05),with no significant difference between the groups and compared with the negative control(p>0.05).In addition,the CCK-8experiment results showed that at the three time points of 24 h,48h and 72 h,there is no significant difference between the OD450 values of CDCC and SDCC(p>0.05),and both were significantly higher than the positive control group(p<0.05).),with no significant difference when compared with the negative control group(p>0.05).In the experiment of subcutaneous implantation on the back of rats,the IL-2 and IFN-? of SDCC group and CDCC group were higher than that of the blank group on the 3rd and 7th day,and there was a significant statistical difference(p<0.05).On the 14 th day,the SDCC group was higher than the blank group,and there was a statistical difference(p<0.05),and there was no statistical difference between the CDCC group and the blank group(p>0.05).On the28 th day,there was no significant statistical difference in IL-2 and IFN-? between the blank group,SDCC group and CDCC group(p>0.05),while the expression of IL-4among the three groups were comparable(p>0.05).The results of local immunohistochemical staining of subcutaneous implantation showed that the number of CD11b(+)cells in SDCC and CDCC gradually increased between the 3rd and 7th days,and began to fall on the 14 th day.On the 28 th day,the staining of the graft area in the two groups was significantly lighter than before,and there was no significant difference between the two groups.Quantitative analysis of immunohistochemical stained sections showed that there was no statistical difference in the quantitative results between the two groups at 4 time points(p>0.05).3.In the experiment of subcutaneous implantation in the nose of New Zealand white rabbits,in the MRI images at 3 and 6 months after the operation,we can observe that the grafts in the SDCC group are significantly smaller,while the implants in the CDCC group remained stable morphology without significant changes.Liquefaction occurred around the graft in the SDCC group at the 3rd month after the operation,while the graft in the CDCC group was tightly connected to the surrounding tissues;at the 6th month after the operation,a liquefaction zone appeared in the SDCC center,indicating that the SDCC group was degraded.On the contrary,the grafts in the CDCC group maintained a uniform density without liquefaction or necrosis,and adhered closely to the surrounding tissues,forming a biological association.The results of HE staining showed that there was a large number of inflammatory cell infiltration in the SDCC group and degradation fissues in the central area at the 6th month after the operation.In the CDCC group,the inflammatory cells only appeared around the surface of the graft without significant degradation.In addition,inflammatory cell count analysis also confirmed that the number of inflammatory cells in the CDCC group was significantly lower than that in the SDCC group,and the difference was statistically significant(p<0.05).At 6 months after surgery,Safranin O staining showed that there were more GAGs in the CDCC group than in the SDCC group,especially in the peripheral part of the CDCC.In addition,the quantitative analysis of GAGs proved that the content of GAG in the CDCC group was significantly higher than that in SDCC6 months after implantation,and the difference was more significant than before implantation(p<0.01).The immunohistochemical staining results of type II collagen showed that the collagen structure of SDCC group was obviously destroyed,while the CDCC group had no obvious lysis and a small amount of new collagen tissue was seen in the superficial area of the graft.Conclusions: 1.The cellular antigen components in the novel decellularized xenogeneic cost cartilage prepared by the modified decellularization method are effectively eliminated,and compared with the decellularized cost cartilage prepared by the traditional method,various extrdecellularized matrixes are better preserved,and have better mechanical properties.2.The novel decellularized xenogeneic costal cartilage has good biocompatibility and no cytotoxicity.3.The novel decellularized xenogeneic cost cartilage could maintain its morphology,slow down the degradation rate and reduce inflammation,in comparison with conventional graft.