Construction Of Versatile Decellularized Tissue Engineering Scaffolds From Natural Tissues

Both natural extracellular matrix and synthetic cellular matrix are important components in tissue damage and repair.The natural acellular scaffold provides a natural three-dimensional structure,excellent physical and mechanical properties,and good biocompatibility.However,due to the complexity of chemical composition and three-dimensional structure of natural tissues,the application of natural acellular scaffolds is uncertain.The purpose of this study is to explore the feasibility of constructing universal tissue engineering scaffolds by comparing the physical and chemical properties and biocompatibility of acellular scaffolds from different tissue sources,so as to provide new ideas for tissue engineering research.In this study,liver,lung and brain tissues of mice were firstly selected as the materials for the preparation of decellularized tissue engineering scaffolds.The physical freeze-thaw cycle and a series of acellular reagents including Triton X-100,sodium deoxycholate(SDC),sodium dodecyl sulfate(SDS)and Trypsin/EDTA were used to conduct the modular decellularized treatment.According to tthe modular decellularized treatment.,decellularized liver scaffold(DLIS),decellularized lung scaffold(DLUS)and decellularized brain scaffold(DBRS)were prepared at the same time.Then,the physical and chemical characteristics of the acellular scaffolds were studied by H&E staining,DAPI fluorescence staining,Masson staining,and AB-PAS staining,in order to observe the degree of cell removal and scaffold morphology of DLIS,DLUS and DBRS.The contents of DNA,glycosaminoglycan and collagen in the decellularized scaffolds were determined by spectrophotometry to further study the chemical components of the scaffolds.Finally,three different sources of decellularized scaffolds were seeded by HepG2 cells(human liver cancer cell line),A549 cells(human lung cancer cell line)and SH-SY5 Y cells(human neuroblastoma cell line).The growth and proliferation of these three kinds of cells in different tissue scaffolds were detected by CCK-8 method to explore the biocompatibility of decellularized scaffolds.The research results are as follows:(1)The effectiveness of preparation of decellularized scaffolds: After decellularized treatment,the liver,lung and brain tissues of the mice changed from dark to light in color,from dense to loose in texture,and finally became a milky white translucent structure.Morphological and structural analysis was performed on the treated tissues.H&E staining showed that there were few purply-dyed nuclei in all three tissues,and DAPI fluorescence staining showed few fluorescein-colored nuclei.Qualitative and quantitative analysis of nuclear DNA was carried out.Agarose electrophoresis showed that no obvious DNA bands were found in the three tissues after treatments,and fluorescence quantitative method detected only a small amount of DNA in the tissues after the three treatments,with the DNA removal rate reaching98.1 to 99.7 percent.In summary,after decellularized treatment in this experiment,all three tissues have effectively removed the cell components.(2)Morphology and chemical composition of decellularized scaffolds: Masson staining and aixian blue-glycogen staining showed that after acellular treatment,collagen and glycosaminoglycan(GAG)components in the extracellular matrix of the three kinds of tissues were more retained,and the three-dimensional scaffold structure was still basically intact.The quantitative results showed that after decellularized treatment,collagen was retained 53.11% in DLIS,63.67% in DLUS and 57.24% in DBRS.According to the dry weight of the acellular scaffold,the relative content of collagen was 37.1±9.48 ?g/mg in DLIS,134.21±19.16 ?g/mg in DLUS,and 55.54±10.22 ?g/mg in DBRS.GAG was retained 35.75% in DLIS,31.20% in DLUS,and15.34% in DBRS.The relative content of GAG was 5.85±0.92 ?g/mg in DLIS,3.44±0.95 ?g/mg in DLUS,and 4.57±0.90 ?g/mg in DBRS.The above results showed that the three kinds of scaffolds prepared by the stereotypical acellular treatment in this experiment all retained good three-dimensional porous structure,and the retention and composition of collagen and GAG in the three scaffolds were close to each other.(3)Cytocompatibility of decellularized scaffolds: three different cell lines of HepG2 cells,A549 cells and SH-SY5 Y cells were seeded into DLIS,DLUS and DBRS respectively,and they can all survive in the three kinds of decellularized scaffolds.CCK-8 method was used to detect the cell viability.It was found that during the 7-day culture period,the activity of three different cell lines increased gradually,and the cells grown in the acellular scaffold were more vigorous than those grown in the ordinary plane culture.On the other hand,The results of co-culture of three decllularized scaffolds with different cells are universal and versatile.That is to say,DLIS could simultaneously promote the growth of HepG2 cells,A549 cells and SH-SY5 Y cells,and DLUS and DBRS could also simultaneously promote the growth of these three kinds of cells.These results indicate that the cellular compatibility of these three different types of decellularized scaffolds is characterized by versatility.In this study,three kinds of decellularized scaffolds were successfully prepared by using different natural tissues.By detecting and comparing the structure and chemical composition of the three kinds of decellularized scaffolds,it was found that the chemical composition and morphological structure of the three kinds of scaffolds were similar.Cytocompatibility studies have confirmed that these three kinds of decellularized scaffolds are versatile in the acceptance of cells from different sources,which indicates that it is technically feasible to prepare general-purpose tissue engineering scaffolds suitable for all kinds of tissue cells.