Comparative study on different methods for preparing decellularized submucosal matrix materials of small intestine
Objective Three methods for preparing small intestinal submucosa(SIS)were applied to prepare SIS according to the current reported literatures.The effects of the three preparation methods on decellularization degree,structure,active ingredients and biocompatibility of SIS were compared.Methods SIS was prepared using Badylak's method,Abraham's method and Luo's method respectively.The degree of decellularization was detected with hematoxylin and eosin(HE)and 4',6-diamidino-2-phenylindole(DAPI)and residual DNA content detected with standard method;Proteomic analysis was applied to detect compo-nents of SIS,and scanning electron microscopy to detect structural changes;And co-culture the three types of SIS with bone marrow mesenchymal stem cells(BMSCs)to test cell compatibility.Quantitative data was pres-ented using an arithmetic mean±standard deviation table,and statistical significance was tested using analy-sis of variance.Results The HE and DAPI staining of SIS prepared by three methods all showed no visible nuclei to naked eye;The DNA content detection in SIS prepared by Badylak's,Abraham's and Luo's meth-od were 43.25,21.01 and 15.69 ng/mg ECM dry weight,respectively.Proteomic analysis results:644,717,and 135 protein types were detected in SIS-1,SIS-2,and SIS-3,respectively.There are 21 subtypes of collagen with the most types.The content of Collagen Ⅰ was the highest among all collagen types;and there were also fibronectin,proteoglycans,vascular endothelial growth factor(VEGF),fibroblast growth factor(FGF),transforming growth factor-β(TGF-β)and bone morphogenetic protein-2(BMP-2)within.SEM showed that the microstructures of all three types of SIS were damaged to a certain extent,with interrupted continuity of collagen fibers and disordered arrangement of collagen fibers;and BMSCs co-cultureed with all three types of SIS could maintained good morphology and proliferation activity.Conclusion The SIS pre-pared by three decellularization methods all meet the standards,retain most of the active ingredients and also good biocompatibility.The microstructure and bioactive components were all damaged to varying degrees.
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