Evaluation Properties Of Bovine Pericardium Treated With Decellularization And Photooxidation

Objective:To find a good protocol of decellularization and dye-mediated photooxidation in bovine pericardium,evaluation the feasibility as a new biomaterial substitution in cardiac surgery.Methods:1.One hundred and thirty fresh bovine pericardium patches were divided into fresh group and other twelve groups(Ⅰ-ⅫⅠ)which were treated with different decellularization protocols(n=10).Morphologic and physiochemical properties of thirteen groups were studied,including collagen fibers observed by light microscope.2.Forty fresh bovine pericardium patches were divided into four groups including fresh group,decelluar bovine pericardium patches crosslinked by dye-mediated photo-oxidation 36h,48h and 60h(n=10). Morphologic and physiochemical properties of four groups,including heatshrinking temperature,tensile stress and the max elongation length were studied.3.Sixty fresh bovine pericardium patches were divided into four groups including fresh group,glutaraldehyde treated group, decellularization group,and decelluar tissue crosslinked by dye-mediated photo-oxidation(n=15).Heatshrinking temperature,tensile stress and the max elongation length were studied.One of the trial patches in each groups were implanted subcutaneously in the same SD rat,total 15 animals.All trial rats were sacrificed 8 weeks after implantation,and the specimens were retrieved.The morphologic properties of test specimen were evaluated by HE stain.The tissue calcium content was determined by flame atomic absorption spectrophotometer.The tissue calcium salt distribution was evaluated by Von Kossa calcium salts stain.Results:1.All bovine pericardium patches of decellularization groups except groupⅫⅠwere remained well.Some cells were observed in the bovine pericardium patches which were decellularized with 0.25%tritonX-100 and 0.025%trypsin for a short time.The breakage of collagen fibers were observed in the bovine pericardium which were decellularized with 20u/ml DNase-I and 0.2mg/ml RNase-A for a long time.The cell components in theⅫgroup of bovine pericardium patches was completely removed, the construction of matrix and the collagen fibers were maintained well.2.The heatshrinking temperature,tensile stress and the max elongation length of the decellularization group was lower than the fresh groups (P＜0.05).However,following further treatment with dye-mediated photo-oxidation,the mechanical properties increased,and related with photo-oxidation stabilization time.The mechanical properties of the decelluar tissue crosslinked by dye-mediated photo-oxidation 48h group was higher than the fresh groups(P＜0.05),however,this cannot be oberserved in decelluar tissue crosslinked with photo-oxidation 60h(P＞0.05).Compared with glutaraldehyde group,the heatshrinking temperature, tensile stress of the decelluar tissue crosslinked by dye-mediated photo-oxidation 48h group was lower(P＜0.05),but the max elongation length was higher(P＜0.05).3.Compared with other groups,the decelluar tissue crosslinked with photo-oxidation 48h group were soft and tenacious,its fibrous structure kept intact,the tissue degradation was slighter.The calcium salts density in total of the trial patches was significantly less than those of fresh group and glutaraldehyde group,and was similar to decellularization group.The calcium content of the decelluar tissue crosslinked by dye-mediated photo-oxidation 48h group was similar to the decellularization group,but was significantly lower than the other groups(P＜0.05).The Von Kossa calcium salts stain showed the tissue calcium salt distribution in the decellularization group and photo-oxidation 48h group were lower than the other groups.Conclusion:1.Good acellular bovine pericardium matrix can be made through a gentle multi-process decellularization protocols(0.25%tritonX-100 48h, 0.025%trypsin and 0.02%EDTA 30min,20u/ml DNase-I and 0.2mg/ml RNase-A 12h).2.The mechanical properties of acellular bovine pericardium increase significantly with photo-oxidation stabilization time and the properties of photo-oxidation 48h was more fine than others.3.Decelluar bovine pericardium patches crosslinked by dye-mediated photo-oxidation has good mechanical properties,can effectively prevent the calcification in vivo,maybe a new biomaterial substitution in cardiac surgery.