牦牛牛心包在抗钙化心脏瓣膜中的应用研究

Application of yak pericardium in anti-calcification heart valves

目的探讨牦牛牛心包用作抗钙化心脏瓣膜材料在经导管主动脉瓣置换术应用中的可行性。方法以黄牛牛心包和商品化的赛诺产品作对照,先对牦牛和黄牛牛心包进行脱细胞处理,然后分别用苏木精-伊红(HE)染色和DNA检测试剂盒定性和定量分析脱细胞效果。采用戊二醛和京尼平两种交联剂分别对上述脱细胞牛心包进行交联,在检测力学性能、热力学稳定性和生物相容性的基础上,将经交联处理的脱细胞牛心包埋植于幼年Wistar大鼠皮下2~4周,取材后进行组织学染色和组织钙化情况分析。结果经脱细胞处理后的牦牛牛心包几乎不可见细胞核,DNA含量由(0.90±0.13)μg/mg降低至(0.09±0.02)μg/mg,脱细胞后DNA含量远低于赛诺产品(P<0.001)。牦牛脱细胞牛心包及戊二醛、京尼平交联牦牛脱细胞牛心包的力学强度均大于相应的黄牛牛心包对照组(均P<0.05),其中戊二醛交联牦牛脱细胞牛心包的最大应力最高[(8.44±2.61) MPa],高于赛诺产品组[(7.92±1.81) MPa](P<0.05)。交联前后的牦牛脱细胞牛心包的热皱缩温度均略高于黄牛脱细胞牛心包,但差异均无统计学意义(均P>0.05)。经戊二醛或京尼平交联后,牦牛脱细胞牛心包的溶血率和细胞增殖率与黄牛脱细胞牛心包相近(均P>0.05),但皮下埋植材料组织再生能力劣于黄牛脱细胞牛心包,钙化程度略高于黄牛脱细胞牛心包(均P>0.05),皮下埋植4周戊二醛交联组的钙含量为32.62~65.49 μg/mg。结论牦牛牛心包较黄牛牛心包具有更优异的力学性能和热力学稳定性,其生物相容性也可满足经导管主动脉瓣置换术的要求。

Objective To explore the feasibility of the application of yak pericardium as anti-calcified heart valve materials in transcatheter aortic valve replacement (TAVR). Cattle pericardium and commercial Sino products were used as controls. Methods Yak and cattle pericardium was decellularized, and then the acellular extent was qualitatively and quantitatively analyzed with hematoxylin-eosin (HE) staining and DNA detection kit respectively. Two cross-linking agents, i.e. glutaraldehyde and genipin, were used to cross-link the acellular pericardium, respectively. After testing the mechanical property, thermodynamic stability and biocompatibility of the pericardium, the cross-linked pericardium was subcutaneously implanted in juvenile Wistar rats. After 2~4 weeks of the implantation, histological staining and tissue calcification analysis were conducted. Results The nuclei of the yak pericardium were almost invisible after decellularized treatment, and the DNA content decreased from (0.90±0.13)μg/mg to (0.09±0.02)μg/mg that was significantly lower than that of Sino product (P<0.001). The mechanical strength of the acellular yak pericardium, glutaraldehyde or genipin cross-linked yak pericardium was higher than that of the cattle pericardium (all P<0.05). The maximum stress value, obtained in the glutaraldehyde cross-linked yak pericardium, was (8.44±2.61) MPa, which was higher than (7.92±1.81) MPa of the Sino product (P<0.05). The shrinkage temperature of non- and cross-linked yak pericardium was slightly higher than that of cattle pericardium, but difference was not statistically significant (all P>0.05). For the two cross-linked yak pericardium, the hemolysis rate and cell proliferation ratio was similar with that of cattle pericardium (all P>0.05), the tissue regeneration ability of subcutaneous implantations was inferior, and the calcification level was higher than that of cattle pericardium with an no statistically significant difference (all P>0.05). After 4 weeks of the subcutaneously implantation, the calcium content of the glutaraldehyde cross-linked pericardium was 32.62~65.49 μg/mg. Conclusions Yak pericardium has better mechanical properties and thermodynamic stability than cattle pericardium, and the biocompatibility can meet the requirements of transcatheter aortic valve replacement.