| Small intestinal submucosa(SIS)is a biological extracellular matrix(ECM)that primarily composed of collagen.SIS repair materials has good biocompatibility,suitable degradation,low or non-immunogenicity and certain mechanical strength.At present,SIS has been extensively used as scaffolds for tissue engineering applications of artery,bladder,intestinal tract and tendon.Cell-free porcine small intestinal submucosa hernia repair piece in our study was derived from porcine small intestinal submucosa,which was removed from cells,freezed-dried,sterilized.It is intended to repair the damage of hernia and anal fistula in clinical by long-term implantation.The aim of the present study was to detect the biological properties of biodegradable and immunotoxicity before clinical studies,offered the academic basis for safer and more effective appllications.(1)To reliably study the immunotoxicity of cell-free porcine small intestinal submucosa hernia repair piece,we used lipopolysaccharide(LPS)to optimize the immunostimulation model of rats and set up an immunostimulation group for the immunotoxicity study.Forty two Wistar rats were randomly divided intoseven groups: blank control group,sham-operated group,immunosuppression group,6h,12 h,24h,and 48 h after LPS administration group.After drug treatment,weight change,immune organ coefficient,hematological parameters,T-lymphcocyte subgroups analysis,lymphoproliferation assay,cytokines levels(IL-1? and TNF-?)were investigated.The results showed that the 6h and 12 h after LPS administration groups showed immune stimulant effect to a certain extent in immune organ coefficient,hematological parameters,T-lymphcocyte subgroups analysis,lymphoproliferation assay,and cytokines levels,and significant differences were observed compared with blank control group and sham-operated group(p<0.05).The 12 h after LPS administration group showed a more obvious immune stimulant effect.The immune stimulation phenomenon of the 24 h and 48 h after LPS administration groups was not obvious.The immunosuppression group showed a certain degree of the immunosuppression effect on the immune organ coefficient,hematological parameters,T-lymphcocyte subgroups analysis,lymphoproliferation assay,cytokines levels,which had marked differences compared with blank control group and sham-operated group(p<0.05).The above results indicated that the most obvious immune stimulant effect of rats was observed 12 h later after intraperitoneal injection of LPS,which can be applied in cell-free porcine small intestinal submucosa hernia repair piece immune toxicity studies as the immunostimulation model of rats.(2)To study the immunotoxicity of cell-free porcine small intestinal submucosa hernia repair piece.Eighty Wistar rats were randomly divided into five groups: high dose group,low dose group,sham-operated group,immunosuppression group,immunostimulation group.According to the study period,each group of sixteen rats were randomly divided into four experimentaloperation subgroups(n=4): the 1st,4th,8th,and 12 th week groups.After implantation treatment,weight change,hematological parameters,immune organ coefficient,histopathological examination,the killing abilities of NK cell,T-lymphcocyte subgroups analysis,lymphoproliferation assay,level of complement C3,concentration of immunoglobulin(IgG and IgM)and cytokine levels(IL-1?,IL-6 and TNF-?)were investigated.The results showed that at the1 st,4th,8th and 12 th week after cell-free porcine small intestinal submucosa hernia repair piece implantation into rats,compared with the sham-operated group,the high dose group and low dose group had no significant differences in weight change,hematological parameters,immune organ coefficient,histopathological examination,T-lymphcocyte subgroups analysis,lymphoproliferation assay,levels of cytokine and immunoglobulin(p>0.05).But compared with the sham-operated group,the high dose group and low dose group had some differences in the killing abilities of NK cell and level of complement C3.The immunostimulation group showed immune stimulant effect to a certain extent in hematological parameters,immune organ coefficient,histopathological examination,T-lymphcocyte subgroups analysis and lymphoproliferation assay,and significant differences were observed compared with other groups(p<0.05).The immunosuppression group showed a certain degree of the immunosuppression effect in the hematological parameters,immune organ coefficient,histopathological examination,T-lymphcocyte subgroups analysis and lymphoproliferation assay for B-and T-cell mitogen,which had marked differences compared with other groups(p<0.05).The above results indicated that there were no obvious immunotoxicity after the cell-free porcine small intestinal submucosa hernia repair piece were implanted into abdominal subcutaneous tissue of rats.(3)To analyze the biodegradation of the cell-free porcine small intestinal submucosa hernia repair piece by both in vitro and in vivo evaluation.In vitro degradation includes accelerated and real-time degradation.The accelerated degradation was carried out in 0.5mg/mL?collagenase with shocking.Then all the samples were taken out in different time points,and the degradation rate was measured.The results showed that the cell-free porcine small intestinal submucosa hernia repair piece was gradually degraded in 0.5mg/mL ?collagenase solution as time increasing.The degradation rate was 68% at 20 h of incubation,degradation rate was lower compared with COOK? hernia repair piece before 20 h of incubation,and it was equal after 20 h of incubation.At the96 h of incubation,their degradation rates were over 90%.The real-time degradation was carried out in 0.01 M PBS solution and let it sit.Then all the samples were taken out in different time points,and the degradation rate was measured.The results showed that the cell-free porcine small intestinal submucosa hernia repair piece was barely degraded at 30 days.Then it was gradually degraded as time increasing,and the degradation rate was over 90% at180 days.The subcutaneous implant test in vivo degradation was alike that in immunotoxicity.After the cell-free porcine small intestinal submucosa hernia repair piece was implanted into abdominal subcutaneous tissue of rats,the gross and histological observations of implantation were evaluated at the 1st,4th,8th,and 12 th week.The results showed that the cell-free porcine small intestinal submucosa hernia repair piece was continuously wrapped by connective tissue and was degraded and absorbed at the 1st,4th,and 8th week.It was completely osteointerated with connective tissue at the 12 th week.The histological observations showed that it was continuously osteointerated with connective tissue and inflammatory cells decreased at the 1st,4th,and 8th week.It wascompletely osteointerated with connective tissue at the 12 th week,few inflammatory cells were observed in a local parts and there was no obvious inflammatory lesions.The cell-free porcine small intestinal submucosa hernia repair piece was almost thoroughly degraded after 12 weeks of implantation.The results of degradations in vitro and in vivo indicated that the cell-free porcine small intestinal submucosa hernia repair piece has suitable degradation. |
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