The Study On Biological Tissue Engineering Scaffolds

Objective:The scaffold material is an important foundation to build a tissue-engineered organ or tissue. Extracellular matrix retains the main structure and composition of the tissue, with the fine features of low immunogenicity, good mechanical properties and promoting cell growth and tissue regeneration.It is such an ideal scaffold material that it becomes a hot topic in tissue engineering. This study is aim to use the extracellular matrix obtained by decellularization to provide scaffolds for constructing tissue-engineering.Methods:1. The bovine pericardiums were treated with 5 methods,and they were divided into 6 groups.Group A:Fresh bovine pericardium; Group B:Trypsin-detergent group; Group C:Freeze-thaw-detergent 24h group; Group D:Freeze-thaw-detergent 48h group; Group E:Freeze-thaw-nuclease group; Group F:Detergent-nuclease group. Then by HE staining and Scanning Electron Microscope(SEM) to observe the effects of decellularization and fibrous changes among the 6 groups; By water content testing and mechanical testing to observe the changes in physical properties of the matrix,By detecting the DNA content of each group to determine the effect of decellularization qualitatively; By cytotoxicity test to detect the biocompatibility of bovine pericardium in each group, filtering out the best method to obtain acellular bovine pericardium.2. Treated the porcine derma with the selected best method, and used trypsin-SDS method as a comparison, observed the effects of decellularization and fibrous changes through HE staining and SEM.3. Modified the acellular bovine pericardium and acellular porcine derma with the crosslinking reagent EDC,detected the effect of crosslinking by mechanical performance test and vitro degradation test.Results:1. The results by HE staining and SEM showed that the 5 decellularization methods can all remove the cellular components of bovine pericardium effectively, while in Group A, group D and Group E, the fibers of bovine pericardium were damaged in different degree, however, in Group C and Group F, the fibers were in normal direction and arranged in fascicles, the matrix was well preserved. Compared with the fresh bovine pericardium group, the water content of each decellularized group was increased, while the DNA content decreased greatly, P<0.05, the difference was considered statistically significant; Compared with Group A, the largest tension of all decellularized group decreased except group E,P<0.05, the difference was statistically significant; The rupture tensile rate of Group B, D, E were lower than group A, P<0.05, and also the differences were statistically significant, while in Group C and Group F there were no statistically significant differences,P>0.05. And the elastic modulus of Group C and Group F were similar to Group A, P>0.05, without statistically significant difference, while the differences were statistically significant between Group B, Group D, Group E and Group A, P<0.05. The result of the cytotoxicity test shows that the cytotoxic level of Group B, C, D, E, F were 0.9,0.6,1.0,1.0,0.5 respectively, each group were qualified toxicity test, while Group C and Group F had the lowest toxicity.2. Treated the porcine derma with the method of Freeze-thaw-detergent 24h, the results by HE staining and SEM showed that this method can also remove the cellular components of porcine derma effectively,and the fibers was in normal direction and arranged in fascicles, the matrix was well preserved.3. After crosslinked with EDC, the results of mechanical properties testing showed that the largest tension, the rupture tensile rate, and the elastic modulus of the acellular bovine pericardium were 42.490N,0.169 and 83.709MPa respectively, while that of the uncrosslinked acellular bovine pericardium the data were 22.013N,0.226 and 44.323Mpa, P<0.05, the difference of mechanical properties was statistically significant. After 1 day, the degradation rate of the fresh bovine pericardium was 44.17%, the decellularized bovine pericardium was 57.89%,while the EDC crosslinked decellularized bovine pericardium was 2.33%. The degradation rate decreased obviously after crosslinking. This is also true for the porcine derma,the natural collagen sponge and the natural transparent collagen. Conclusions:1. Freeze-thaw-detergent 24h method and Detergent-nuclease method can remove the cell components of bovine pericardium successfully, while maintaining the major structural components and the histological and biological properties of bovine pericardium,and with low cytotoxicity.However,Freeze-thaw-detergent 24h method is more economical than Detergent-nuclease method and easier to operate. So the method freeze-thaw-detergent 24h can be the best choice to produce a decellularized bovine pericardium.2. Freeze-thaw-detergent 24h method is also a effective protocol to obtain acellular porcine derma.3. The mechanical properties and anti biodegrability of bovine pericardium were greatly improved after crosslinked with EDC, and the anti biodegrability of crosslinked porcine derma,natural collagen sponge and natural transparent collagen were also improved.