Sierad Leslie Neil, Shaw Eliza Laine, Bina Alexander, Brazile Bryn, Rierson Nicholas, Patnaik Sourav S, Kennamer Allison, Odum Rebekah, Cotoi Ovidiu, Terezia Preda, Branzaniuc Klara, Smallwood Harrison, Deac Radu, Egyed Imre, Pavai Zoltan, Szanto Annamaria, Harceaga Lucian, Suciu Horatiu, Raicea Victor, Olah Peter, Simionescu Agneta, Liao Jun, Movileanu Ionela, Harpa Marius, Simionescu Dan Teodor
1 Biocompatibility and Tissue Regeneration Laboratories, Department of Bioengineering, Clemson University , Clemson, South Carolina.
2 Tissue Bioengineering Laboratory, Department of Agricultural and Biological Engineering, Mississippi State University , Starkville, Mississippi.
Tissue Eng Part C Methods. 2015 Dec;21(12):1284-96. doi: 10.1089/ten.TEC.2015.0170.
There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for whole-root decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open-close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality.
各个年龄段的患者都迫切需要活体瓣膜置换物。可以通过组织工程构建这样的结构，考虑到主动脉根部独特的结构和生物学特性。主动脉瓣根部由几种不同的组织组成，必须对每个节段和组件进行仔细的结构和功能考量。先前的研究表明，浸泡技术不足以实现全根部去细胞化，主动脉壁节段尤其难以去细胞化。本研究的目的是开发一种压差梯度灌注系统，该系统既要严格到足以使主动脉根壁去细胞化，又要温和到足以保持瓣叶的完整性。新鲜猪主动脉根部已接受使用去污剂和酶的各种灌注去细胞化方案处理，并将结果与浸泡去细胞化的根部进行比较。使用细胞分析（细胞核和基质染色的组织学以及DNA分析）、生物力学（双轴和弯曲试验）和生理性心脏瓣膜生物反应器测试（通过开闭周期的先进图像分析和几何孔面积测量）等互补方法，定义了评估每个根节段（瓣叶、肌肉、窦、壁）去细胞化完整性、组织完整性和瓣膜功能的成功标准。用优化方法处理的完全脱细胞猪根部呈现出保留的宏观结构和微观基质成分，这转化为保守的各向异性力学性能，包括在主动脉血流和压力条件下测试时的弯曲性能和出色的瓣膜功能。本研究强调了以下几点的重要性：（1）使去细胞化方法适应特定的目标组织；（2）与仅依靠组织学相比，结合多种细胞分析方法；（3）开发相关的瓣膜特异性力学测试；（4）对瓣膜功能进行体外测试。
