去细胞组织工程同种心脏瓣膜的构建

目的 全面评价去细胞组织工程同种心脏瓣膜的生物学、生物力学及免疫学特性。方法采用低渗液-去污剂[0.5％去氧胆酸(DOA),1％DOA,1％Triton]-核酸酶去细胞法,测定了,47例去细胞同种心脏瓣膜去细胞前后组织学、免疫学、组织厚度、组织含水量、热皱缩温度、组织基因组DNA含量、胶原蛋白含量、应力应变曲线、破坏强度及组织伸长比变化。结果组织学检查1％DOA 24 h组完整地去除了同种心脏瓣膜、管壁及肌肉组织中所有的细胞成分,保留了完整的细胞外基质;免疫组织化学证实白细胞DR(HLA-DR)抗原表达明显下降;组织基因组DNA含量显著下降(瓣叶下降91.14％,管壁下降91.53％);与去细胞前相比,只有管壁组织的含水量(去细胞前75.4±1.8,去细胞后82.0±0.7,P<0.05)明显增加,组织厚度、瓣叶组织含水量、热皱缩温度、应力应变曲线、破坏强度及组织伸长比差异无显著意义。结论低渗液-去污剂(1％DOA)-核酸酶去细胞法方便有效,去细胞组织工程同种心脏瓣膜的生物学及生物力学特性稳定,免疫性显著下降,符合机体的要求,为受体细胞化组织工程心脏瓣膜的研制提供了可靠的天然的纤维支架材料。

Decellularized human tissue engineering aortic valves conduit

OBJECTIVE: To explore the suitable method to create a decellularized human tissue engineering homograft aortic valves conduit and to study its biologic, immol/Lunological, and biomechanical properties. METHODS: Human homograft aortic valves conduits donated by healthy adult males undergoing brain death preserved in liquid nitrogen were treated with pH 8.0 hypotonic Tris buffer for 12 hours; then pH 8.0 isotonic buffer with 0.5% DOA, 1% DOA or 1% Triton for 12 hours-24 h; l and then pH 7.6 isotonic buffer with DNAase 200 micro g/ml, RNAase 20 micro g/ml for 2 hours. the histology, HLA-DR antigen, water content, thinkness, denaturation temperature, DNA content, Collagen Contents, stress-strain, destroying stress were examined. RESULTS: In comparison with the standard cryopreserved human homograft aortic valves conduit, the valves, wall and muscle of the homograft aorta treated by 1% DOA for 24 hours were decellularized completely, and the 3-dimensional network structure of elastic fibers and collagenous fibers remained intact. Immol/Lunohistochemistry showed a remarkable decrease of expression of tissue genome DNA contents, a decrease by 91.14% in the valves, and by 91.53% in the wall, and a remarkable decrease of the expression of HLA-DR antigens. However, the water content in the decellular aortic wall was increased significantly (75.4 +/- 1.8 vs 82.0 +/- 0.7, P < 0.05). No significant differences were found in the denaturation temperature, stress-strain, destroying stress parameter, and tissue extension ratio in the decellular human homograft aortic valves conduits. CONCLUSION: The decellularization method by isotonic buffer with 1% DOA-DNAase is effective. The decellularized human homograft aortic valves conduits are unaltered in biologic, biomechanical, and physical properties with lowered immol/Lunogenicity, It can be used as an ideal valve for patients or as a homograft stent for developing tissue engineering valve by host recellularization.