Compare Two Different Methods On Decellularization Of SIS-ECM Mesh

BackgroundThe abdominal wall hernia cause by the congenital or acquired abdominal wall defect is a problem that surgeon must be confronted with.About more than 990,000 abdominal wall hernia repair surgical procedures performed in the USA alone every year.Compared to traditional sutured repair,hernia repair with meshes could obtain a more satisfactory postoperative effect,and tension-free repair with prosthetic mesh can reduce the risk of recurrence by 50 to 75%,to achieve satisfactory outcomes,mesh repairs are adopted by more and more abdominal surgeons.Currently,tension-free repair with mesh is regarded as the gold standard for hernia repair,and mesh repair has replaced direct sutures.This increased mesh use has resulted in accelerated mesh production,with more than 200 mesh types available for hernia repair in the United States at present,and about 1 million copies of the patch applied in hernia repair each year.The current clinical application of the patch types mainly include synthetic polymer materials,and Biomaterials,Biomaterials include animal-derived materials and human-derived materials.Small intestinal submucosa(SIS)is a readily available,abundant ECM material derived from the submucosa of the porcine small intestine.Now as a regenerative material is widely used in clinical and preclinical areas,it helps to repair the damage and defect where organism is hurt.The mechanism to cure body damage is contributed to its function of induct stem cells come to the defected or damaged place and reborn it with self tissues.Also using ECM to regenerate tissue can avoid chronic stimulations.Although decellularized matrix is wonderful in tissue regeneration,there are also some defects,like acute inflammatory response,seroma,the answers which cause those problems are now reported on some magazines,the residuum of xenogenous components,like the cell membrane fragments,DNA section,which is relevant by the methods and reagents used in decellularize matrix,so it is much more important to invent a new,better protocol to make biological materials.ObjectiveAcellular extracellular matrix scaffold has catch a lots of attention in research and application recent years,however,complicated steps of decellularization,limited acellular degree and the "excessive" acelluar,make the biological materials failed to make full use in clinic area.in this experiment we select porcine small intestinal submucosa as the research material,the physical and mechanical treated(U-SIS)as the control group,shake(S-SIS)and perfusion(P-SIS)two ways to acellular cells as the experiment groups,compare the residual DNA content,biomechanical changes,and residual bioactive factor,in order to explore a more optimized,more desirable way to make extracellular matrix scaffold.Methods(1)Preparation of porcine small intestinal submucosa: The porcine small intestine was obtained and harvested from Shanghai Covidien clinical trial base(around 100 Kg at 6 months)within 4 h of sacrifice.The jejunum were only chose as experimental materials.Simple mechanical treated intestinal submucosa(U-SIS)as the control group.0.1% peracetic acid 4% ethanol mixed solution shook intestinal submucosa S-SIS and 0.1% peracetic acid 4% ethanol mixed solution perfusion intestinal submucosa P-SIS as the experiment groups.All the samples were frozen-dried with a lyophilizer for 24 h,sealed into hermetic packages,and then sterilized by ethylene oxide.(2)Observing the microscopic three-dimensional structure of acellular extracellular matrix and evaluating the decellular degree: detected of three kinds of acellular materials content of DNA,HE staining and scanning electron microscopy to evaluate the acellular degree of those extracellular matrix.(3)Evaluated the biomechanical of those biomaterials: three kinds of materials were respectively measure by tensile strength,material permeability and expansion breaking force.(4)Evaluated content of biological active factor: compare the VEGF,TGF-?,b-FGF contents of three kinds of materials.(2)The mechanism of perfusionTwo kinds of bioreactors were designed for studying on the mechanism of perfusion.1.Connect a piece of sis on a bioreactor which was used to detect the penetrated effects of decellularization.Both sides were tightened with nuts to avoid the PAA solution flowing out.The Specific operation protocol was similar to the perfusion and after decellularization,the SIS-ECM was lyophilized and sterilized,then extracted the content of DNA to evaluate the acellular degree.2.Connect a piece of sis on a bioreactor which was used to detect the flushing effects of acellular.Both sides were tightened with nuts to avoid the PAA solution flew out.The Specific operation protocol was similar to the perfusion and after decellularization,the SIS-ECM was lyophilized and sterilized,then extracted the content of DNA to evaluate the acellular degree.Results(1)DNA content: the content of U-SIS was 13766.49 + 1628.32 ng/mg,the content of S-SIS was 4856.58 + 151.17 ng/mg,the content of P-SIS was 2009.71 + 242.50 ng/mg.The results show that both protocols can reduce the DNA content of porcine small intestinal submucosa(P<0.05),and the perfusion protocol is a better way to decellularize,it is much better than shaking protocol,the difference was statistically significant(P=0.000).(2)biomechanical testing: 1.Uniaxial tensile testing result: U-SIS: 4.43±0.3 N/cm;S-SIS: 3.43±0.3 N/cm;P-SIS: 3.82±0.5 N/cm,compared with the control group,the tensile strength of S-SIS group and P-SIS group all decreased significantly,and the difference was statistically significant(P<0.05),P-SIS group compared with the S-SIS group the tensile strength is slightly larger,but without statistical significance(P=0.173).2.Water vapour transmission rate testing: U-SIS: 19680±3614.53 g/? d,S-SIS:33792±5036.80 g/? d,P-SIS:33398.4±6491.25 g/? d.Compared with the control group,the Water vapour transmission rate of P-SIS group and S-SIS group increased significantly(P<0.05),but there was no significant difference between S-SIS and P-SIS(P=0.904).3.Burst strength testing: U-SIS: 23.54 + 1.93 PSI;S-SIS: 17.34 + 1.36 PSI;P-SIS: 23.62 + 1.92 PSI.S-SIS group has the lowest bursting force among the three groups,and the differences were statistically significant(P<0.05),the bursting force of P-SIS slightly larger than the U-SIS,but without statistical significance(P=0.944).(3)growth factor content results: 1.b-FGF content: U-SIS: 7.45 ± 0.77pg/mg;S-SIS: 4.44 ± 0.60 pg/mg;P-SIS: 5.34 ± 0.40 pg/mg,compared with the control group,the content of b-FGF of S-SIS and P-SIS all decreased,and with a significant difference(P<0.05),in P-SIS group the content of b-FGF was higher than S-SIS group,and have statistical significance(P=0.037).2.TGF-? content: U-SIS: 10.47 ± 0.29pg/mg;S-SIS: 4.71±0.80 pg/mg;P-SIS: 9.28 ± 0.59 pg/mg;compared to the U-SIS,the TGF-? content in other two groups were all decreased.The amount of TGF-? in U-SIS and P-SIS have statistical difference(P=0.021).And the TGF-? content of P-SIS was higher than S-SIS,with a statistical difference(P=0.000).3.VEGF content: U-SIS: 70.50± 1.33pg/mg;S-SIS: 11.29 ± 0.13 pg/mg;P-SIS: 22.74 ± 1.37 pg/mg,compared with the control group,the VEGF content of S-SIS and P-SIS were shapely decreased,with a statistical difference(P<0.05),and the VEGF content of S-SIS is the smallest,the VEGF content of P-SIS was two folds than S-SIS,and with a statistical difference(P=0.000)H&E Staining:H&E Staining of thin section of U-SIS showing several intact cells and nuclei attached on the surface of the matrix scaffold.But after treated with perfused or shooked protocols,the acellular biological scaffold basically cannot see the cellular components,and the structure of P-SIS was more tighter than S-SIS.SEM analysis of SIS specimens at different acellular methods:The surface of the U?SIS shows a lot of cells and cellular debris,but the structure of collagenous fibers gather closely.The SIS?ECM made by shook method can still see some cells and cellular debris on the surface of the material.And the collagenous fibers gather much Loose than U?SIS.The mucosal surface of P?SIS shows only extracellular matrix,no cells and cellular debris left on it.And the protein fibers gather much closer than S?SIS.MechanismsThe pressure group DNA content: 2629.63 + 52.74ng/mg,the irrigating group DNA content: 2773.16 + 74.71 ng/mg,DNA content of pressure group is smaller than washing group,and was statistically significant(P=0.028),but DNA content of each group was high than perfusion group,with statistically significant(P<0.05).Conclusion Using shook method to make extracellular matrix is the most general way known to lots of person,in this experiment we developed a new method to avoid some shortcomings of shock acellular extracellular matrix production.1.The new method can avoided the tangles between each sheet of SIS-ECM,which can affect the effect of decellularization;The new method can avoid the constantly changed acellular reagent in shocking procedure,and avoid the cross contamination of bacteria,and perfusion protocol can make the production process more mechanized,it can save a lot of manpower.2.The new method can keep the ultrastructure of the extracellular matrix,and had the much better decellular result,so it minimize the transplant rejection.3.The new decellularization method kept the biomechanical stabilities,and bursting force is higher than the old method.4.The new method can keep more biological activity factor,provide a better environment for tissue remodeling.So it provided a better development direction for SIS-ECM production.5.The mechanism of perfuse protocol is depend on the infiltration and the water erosion effect.From the above experimental results show that the perfusion method is a novel,optimized,and desirable acellular method to development direction for the production of high biological activity,high mechanical strength biomaterials.