摘要
目的:评价不同去细胞方法制备的猪主动脉瓣支架的组织学、生物力学特性及组织相容性,寻找更合适的去细胞心脏瓣膜支架制备方法。方法:分别采用3种不同的去细胞方法对新鲜猪主动脉瓣叶及带瓣主动脉管道进行脱细胞处理:0.01%胰蛋白酶+1%Triton+核酸酶组用含有0.01%胰蛋白酶-1%Triton和核酸酶的去细胞溶液37℃持续震荡24h;0.01%胰蛋白酶8h+1%脱氧胆酸(DCA)+核酸酶组先用0.01%胰蛋白酶37℃持续震荡消化处理8h,终止胰酶后加入含有1%DCA和核酸酶的去细胞溶液37℃持续震荡24h;1%DCA+核酸酶32h组:用含有1%DCA和核酸酶的去细胞溶液37℃持续震荡32h;同时以PBS漂洗的新鲜瓣叶作为对照组。通过H-E染色、扫描电镜和透射电镜观察评价去细胞效果。测定瓣叶弹性模量、最大抗张强度、应力伸长比及断裂伸长比以评价各组瓣叶的生物力学特性。大鼠皮下包埋实验评价去细胞支架的免疫源性、体内炎性反应及改建情况。结果:0.01%胰蛋白酶8h+1%DCA+核酸酶组细胞去除完全,优于0.01%胰蛋白酶+1%Triton+核酸酶组和1%DCA+核酸酶32h组。所制备瓣膜支架的弹性模量及最大抗张强度大于0.01%胰蛋白酶+1%Triton+核酸酶组。应力伸长比及断裂伸长比则小于0.01%胰蛋白酶+1%Triton+核酸酶组,与新鲜瓣叶无明显差异。体内埋植实验炎症反应轻微,宿主成纤维细胞能够长入支架并对其进行改建。结论:短时间低浓度胰蛋白酶与1%DCA和核酸酶相结合的去细胞法方便有效,既能完全去除猪带瓣主动脉管道的细胞成分,使免疫源性显著下降;同时瓣叶的生物力学特性保持稳定,可为受体细胞化组织工程心脏瓣膜的提供可靠的支架材料。
Objective： To compare the histological, immunological, and biomechanical characteristics of decellularized porcine aortic valve scaffold created by 3 different decellularization protocols and to search for a more suitable technique for creating aeellular tissue-engineered cardiac valve conduit. Methods： Porcine aortic valve leaflets and whole aortic roots were deeellularized by 3 different protocols. Decellularization procedure in group Ⅰ involved treatment with 0.01% trypsin, 1% Triton, and nuclease for 24 h; that in group Ⅱ involved treatment with 0.01% trypsin （8 h）, 1% DCA, and nuclease for 24 h; and that in group Ⅲ involved treatment with 1% DCA and nuclease for 32 h. All the treatments were conducted during continuous shaking at 37℃. Porcine aortic valve leaflets and whole aortic roots treated with PBS were taken as control. The deeellularization efficiencies of each protocol were assessed by H-E staining, scanning electron microscopy, and transmission electron microscopy. The biomechanical features of the acellular valve matrices were examined by stress-strain tests and tensile strength tests. The immunogenicity and inflarfimatory responses of the decellularized matrices, valve leaflets, and aortic wall were investigated by subcutaneous implantation of them in rats. Results： The native ceils in porcine aortic valve leaflets and aortic roots were completely removed in group Ⅱ , which was superior to group Ⅰ and Ⅲ. The values of elasticity modulus and ultimate tensile strength （UTS） of groupⅡ were greater than those in group Ⅰ （[5.77±0. 951 MPa vs [4.15±1.13] MPa and [7.82± 1.51] MPa vs [4.65±0.85] MPa, respectively; P〈0.05）. The extension ratios at 1.5 MPa and at rupture in group Ⅱ were less than those in group Ⅰ （[0.33±0.04] vs [-0.41±0. 091 and [-0.45±0. 021 vs [-0.60±0. 06]; P〈0.05）, but the extension ratio at rupture was similar to that of fresh porcine aortic valves （ [0.45±0.02] vs （[-0.46±0.03]）. Histological analysis showed only slight inflammatory responses in grouplland the host cells grew into the matrix, rebuilding the acellular matrices gradually. Conclusion： Decellularization using 8-hour pretreatment with 0. 01% trypsin, followed by 24 hours incubation with 1% DCA plus nuclease is effective and convenient; it not only removes the cells but also decreases the immunogenicity of the aortic valve matrices, making the product an excellent material for tissue-engineered cardiac valve conduit.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2007年第1期8-12,共5页
Academic Journal of Second Military Medical University
关键词
组织工程
心脏瓣膜
人工
去细胞
猪主动脉瓣膜支架
tissue engineering
heart valve prosthesis
decellularization
porcine aortic valve scaffolds
