Research On The Standard Of Residual DNA And Immunogenicity In Acellular Tissue Matrix

Objective:This study produced ACTM biomaterials with different DNA,lipid and endotoxin residues by different methods and analyzed the correlations between the amount of DNA,lipid and endotoxin residues and the immunogenicity of ACTM in vitro,the host immune response,material reconstruction and tissue regeneration in the body.We aimed to confirm the key factor of immunogenicity of ACTM,and established new criteria of DNA residues in ACTM biomaterials.Therefore,this study was carried out in three steps.First,we produced and determined different DNA,lipid and endotoxin residues in the ACTM biomaterials.Second,we determined the immunogenicity of ACTM biomaterials with different DNA,lipid and endotoxin residues in vitro and in vivo experiment.Third,we established the partial-thickness abdominal defects in rat models and repaired the defects with the ACTM biomaterials with different DNA,lipid and endotoxin residues.Method:1.The preparation and test of ACTM biomaterials:(1)The ACTM biomaterials with different gradient DNA,lipid and endotoxin residues were prepared by different methods using porcine SIS and UBM.SIS-1 was decellularized with 0.1%PAA and 4%ethanol solution(ETOH).SIS-2 was decellularized with 0.1%PAA and 4%ETOH followed by chloroform/isopropanol(v/v,3:1).SIS-3 was decellularized with PAA-ETOH and chloroform/isopropanol followed by 0.05%sodium hydroxide(Na OH).UBM was decellularized with 0.1%PAA and 4%ETOH.SIS-PC was set up with submucosal layer of porcine small intestine without any decellularized treatment.SIS-NC was decellularized with EDTA-Na OH-HCl followed by Na Cl and PBS.(2)To determine the decellularization characteristics including cellular component residues,DNA,lipid,endotoxin residues,Alpha-Gal epitope removal rates,PAA residues,epoxy ethane residues and virus inactivated effects in the ACTM biomaterials.HE stainings were used to observe whether there is a cell residue.The amounts of DNA residues were quantified by the Pico Green assays.The Soxhlet extractions of lipid from each scaffold were carried out to quantify lipid residues.The amounts of endotoxin were measured by applying the chromogenic Limulus Amoebocyte Lysate(LAL)Endotoxin Assay Kits.The Alpha-Gal epitope removal rates were determined by inhibiting ELISA tests.The PAA residues in each group of materials were tested by PH meter.The amounts of epoxy ethane residues were determined by gas chromatography.The cell cultures were used to test the virus inactivated effect of the PAA treatment on each group.2.Determine the immuno-inflammatory responses of ACTM biomaterials with different DNA,lipid and endotoxin residues in vitro and in vivo experiments:(1)Immunoassay of ACTM biomaterials in vitro experiments included T cell proliferation,B cell proliferation,macrophage activation and cytokines(TNF-?and NO)production testing.(2)In vivo responses after intra-abdominal implantation in rat models.The inflammatory features included leukocyte and immunoglobulin in Peripheral blood.The numbers of neutrophils,lymphocytes and monocytes in the peripheral blood were determined by flow cytometry.Expression of immunoglobulin isotype(Ig A,Ig G2a and Ig M)in blood serum was detected with Mouse Immunoglobulin Isotyping Kits.The number of macrophages in peritoneal fluid was determined by flow cytometry.The levels of IL-4,IFN-?,TNF-?,and IL-10 in peritoneal fluid were measured by the Enhanced Sensitivity Flex Set.The weight of spleen was determined to calculate spleen index and the cells were stained with antibodies(CD3?CD19?CD4 and CD8)and confirmed by flow cytometric analysis.3.Repairing the partial-thickness abdominal defects in rat models with the ACTM patchs with different DNA,lipid and endotoxin residues:(1)To creat two partial-thickness abdominal defects in rats by operation.According to auto-control method,the defects in both sides were repaired with different ACTM patchs respectively.(2)Postoperative follow-up observation including general condition,the incidence of postoperative seroma formation,abdominal wall infection or bulging and mortality rate.(3)To observe the shrinkage of ACTM patchs.(4)The harvested tissues were respectively evaluated by histologic detections including HE staining and Masson trichrome staining and immunohistochemistry detection including CCR7?CD206?CD68?CD3?CD4?CD8 and CD19 to analyze the type of inflammatory cell infiltration,the type and proportion of lymphocytes and macrophages,the degradation of patchs,the regeneration of blood vessels and the regeneration of collagen fibers in the repair areas.Results:1.None cellular components including nuclei elements was observed under HE staining,and the Alpha-Gal epitope residue were all satisfactorily cleared away in all the prepared materials,except for the SIS-PC.There was no PAA residue in each group,and the epoxy ethane residues and virus inactivated effects were reduced to the level with no significant difference between the groups.The prepared SIS-1 showed statistically significant higher levels in the DNA,lipid and endotoxin residues in comparison with the SIS-2 or SIS-3(p<0.001),while the SIS-2 and SIS-3 were similar in the contents of DNA and lipid residues.The endotoxin residues in UBM were significantly lower than that of SIS-1 or SIS-2,but similar to that of SIS-3.The lipid and DNA residues did not differ significantly between UBM and SIS-1.By means of described decellularization methods,four types of porcine-derived biomaterials had been available on the basis of residue levels,i.e.SIS-1 with relatively high level in DNA,lipid and endotoxin,SIS-2 with mid DNA,low lipid and mid endotoxin,SIS-3 with low DNA,lipid and endotoxin,and UBM with high DNA,high lipid and low endotoxin.2.The assessment in vitro showed that the immuno-inflammatory responses were significantly higher in SIS-1 as compared with SIS-2,including lower levels of T or B lymphocyte transformation rate and higher levels of TNF-?and NO in vitro.As compared with SIS-2,SIS-3 significantly reduced the activation of T lymphocytes,B lymphocytes and macrophages,and the secretion of pro-inflammatory cytokines(TNF-?and NO)in the immunogenicity test in vitro.There was no significant difference between SIS-3 and UBM in the results of the immunogenicity and immune inflammatory responses in vitro,except for the one that T lymphocyte transformation rate in SIS-3significantly lower than that in UBM.3.The assessment in vivo showed that the immuno-inflammatory responses were significantly higher in SIS-1 as compared with SIS-2,including higher expression of TNF-?and IFN-?.The tissue-promoting cytokines,such as IL-4 and IL-10 in SIS-1 were significantly lower than those in SIS-2.The proportions of T lymphocytes in SIS-1 were significantly higher than those in SIS-2.And the lower proportions of CD4+T lymphocytes(Th)and higher CD8+T lymphocytes(CTL)in SIS-1 were shown in SIS-1as compared to SIS-2.In vivo immunogenicity,the numbers of macrophages from peritoneal fluid in SIS-2 were significantly higher than that in SIS-3(P<0.05).The secretions of pro-inflammatory cytokines,TNF-?and IFN-?,in SIS-2 were significantly higher than those in SIS-3.Meanwhile,the secretions of cytokines,IL-4 and IL-10,promoting tissue regeneration in SIS-2 were significantly lower than those in SIS-3.4.In vivo repair experiments in rat models,the incidences of seroma in SIS-1 were70%(21/30),and 40%(12/30)in SIS-2,while 90%(27/30)in SIS-PC.Meanwhile,no seroma occurred in any of animals in SIS-3 throughout the process,and the same in UBM and SIS-NC as well.SIS-3 and UBM showed no significant difference in shrinkage at each time point.However,the shrinkages in SIS-1 and SIS-2 were significantly higher than that of SIS-3 and UBM at 8 weeks after surgery(P<0.001),and SIS-1 was the most severely shrunk,accounting for only 57.1%of the original implanted area at 8 weeks after surgery.The severities of inflammatory cell infiltration in SIS-1 and SIS-2 were significantly higher than that of SIS-3 and UBM.The regenerations of the collagen fibers SIS-3 and UBM were better than that of SIS-1 and SIS-2.Macrophage infiltrations in the repair area in SIS-1 and SIS-2 were mainly M1 macrophages,and the proportions of M1macrophages and M1/M2 ratios in SIS-1 and SIS-2 were higher than that in SIS-3 and UBM.The T cell infiltrations in SIS-3 and UBM were mainly CD4~+(Th)cells,while in SIS-1 and SIS-2 predominant of CD8~+(CTL)cells,but the ratios of CD4~+/CD8~+in SIS-1and SIS-2 were much lower than that in SIS-3 or UBM.Conclusion:1.The different original tissue and different method of decell affected the potential immune response factors such as DNA,lipid and endotoxin residues in ACTM biomaterials.We harvested ACTM biomaterials with gradient DNA,lipid and endotoxin residues by different methods using porcine SIS and UBM.2.Immunoassay experiment of ACTM biomaterials in vitro and in vivo showed that the immunogenicities of ACTM biomaterials were unrelated to DNA residues and lipid,but were related to endotoxin residues.3.The results of the evaluation of the partial-thickness abdominal defects repair model further confirmed that endotoxin was the key factor affecting the early immunogenicity and immune inflammatory responses safety of ACTM.With the decreases of endotoxin residue,the incidences of early inflammatory response such as seroma and shrinkage were reduced,and the lower endotoxin materials implanted in the body could induce the regeneration of collagen fibers and promote tissue reconstruction in the repair area.4.DNA residues and lipid were not the key factors those affected the immune inflammatory response and immunogenicity in ACTM biomaterials.They did not affect the repairing results.We need to establish new DNA residues criteria of ACTM biomaterials.Endotoxin residues were closely related to ACTM immunogenicity,and endotoxin should be an important indicator of ACTM immunogenicity and security.Innovation point:This was the first study to confirm whether residual DNA in ACTM biomaterials is the key factor affecting the type of host-material response and immunogenicity.And we proposed that endotoxin was the determinant of host immuno-inflammatory response to porcine-derived biomaterials and should be an important indicator of ACTM immunogenicity and security in China and abroad.It was of great significance for the development of new acellular standards of ACTM.And it was important for the development and clinical application of ACTM biomaterials in China.