Supplementary Material for: The MAASWERP study: An international, comparative case study on measuring biomechanics of the aged murine aorta

Introduction: Arterial stiffening is a hallmark of vascular ageing, and unravelling its underlying mechanisms has become a central theme in the field of cardiovascular disease. While various techniques and experimental setups are accessible for investigating biomechanics of blood vessels both in vivo and ex vivo, comparing findings across diverse methodologies is challenging.
Methods: Arterial stiffness in the aorta of adult (5 months) and aged (24 months) wild-type C57Bl/6J mice was measured in vivo, after which ex vivo biomechanical evaluation was performed using the Rodent Oscillatory Tension Setup to study Arterial Compliance (ROTSAC; University of Antwerp, Belgium) and the DynamX setup (Maastricht University, The Netherlands). Stiffness of aortic tissue was measured in both absence an presence of activated smooth muscle cells (i.e., contraction). Measurements in both setups were conducted in parallel with matched protocols and identical buffers and chemicals.
Results: Overall, both methods revealed age-related increased aortic stiffness, although parameters of aortic mechanics showed different numerical values, suggesting that results are not directly interchangeable between methods. Surprisingly, smooth muscle cell contraction had opposing effects between the setups. Indeed, smooth muscle cell contraction increased arterial stiffness in the ROTSAC but decreased stiffness in the DynamX. These opposing effects could be attributed to how the two setups differentially load the collagen fibres in the arterial wall, ex vivo.
Conclusion: Overall, this study provided critical insights into how different experimental setups can influence the interpretation of aortic biomechanics, emphasizing the need for careful consideration and contextualization of results based on the methodology used.

引言：动脉僵硬度增加（Arterial stiffening）是血管老化的典型特征，揭示其潜在机制已成为心血管疾病领域的核心课题。尽管目前有多种技术和实验装置可用于在体（in vivo）和离体（ex vivo）研究血管生物力学，但跨不同方法学比较研究结果仍具挑战性。
方法：在体测量成年（5月龄）和老年（24月龄）野生型C57Bl/6J小鼠主动脉的动脉僵硬度，随后使用啮齿动物振荡张力动脉顺应性研究装置（Rodent Oscillatory Tension Setup to study Arterial Compliance，ROTSAC；比利时安特卫普大学）和DynamX装置（荷兰马斯特里赫特大学）进行离体生物力学评估。分别在活化平滑肌细胞缺失和存在（即收缩状态）的条件下测量主动脉组织的僵硬度。两种装置的测量均采用匹配的方案和相同的缓冲液及化学试剂并行开展。
结果：总体而言，两种方法均显示主动脉僵硬度随年龄增加而升高，但主动脉力学参数的数值存在差异，提示不同方法的结果不可直接互换。意外的是，平滑肌细胞收缩在两种装置中产生了相反的效应：在ROTSAC装置中，平滑肌细胞收缩增加了动脉僵硬度，而在DynamX装置中则降低了僵硬度。这些相反效应可归因于两种装置在离体条件下对动脉壁胶原纤维施加的负荷存在差异。
结论：本研究为不同实验装置如何影响主动脉生物力学结果的解读提供了关键见解，强调需基于所用方法对结果进行审慎考量和情境化分析。