Janvier Adam J, Pendleton Emily G, Mortensen Luke J, Green Daniel C, Henstock James R, Canty-Laird Elizabeth G
Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, UK.
Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA.
J Tissue Eng. 2022 Oct 31;13:20417314221130486. doi: 10.1177/20417314221130486. eCollection 2022 Jan-Dec.
Tendon is predominantly composed of aligned type I collagen, but additional isoforms are known to influence fibril architecture and maturation, which contribute to the tendon's overall biomechanical performance. The role of the less well-studied collagen isoforms on fibrillogenesis in tissue engineered tendons is currently unknown, and correlating their relative abundance with biomechanical changes in response to cyclic strain is a promising method for characterising optimised bioengineered tendon grafts. In this study, human mesenchymal stem cells (MSCs) were cultured in a fibrin scaffold with 3%, 5% or 10% cyclic strain at 0.5 Hz for 3 weeks, and a comprehensive multimodal analysis comprising qPCR, western blotting, histology, mechanical testing, fluorescent probe CLSM, TEM and label-free second-harmonic imaging was performed. Molecular data indicated complex transcriptional and translational regulation of collagen isoforms I, II, III, V XI, XII and XIV in response to cyclic strain. Isoforms (XII and XIV) associated with embryonic tenogenesis were deposited in the formation of neo-tendons from hMSCs, suggesting that these engineered tendons form through some recapitulation of a developmental pathway. Tendons cultured with 3% strain had the smallest median fibril diameter but highest resistance to stress, whilst at 10% strain tendons had the highest median fibril diameter and the highest rate of stress relaxation. Second harmonic generation exposed distinct structural arrangements of collagen fibres in each strain group. Fluorescent probe images correlated increasing cyclic strain with increased fibril alignment from 40% (static strain) to 61.5% alignment (10% cyclic strain). These results indicate that cyclic strain rates stimulate differential cell responses via complex regulation of collagen isoforms which influence the structural organisation of developing fibril architectures.
肌腱主要由排列整齐的I型胶原蛋白组成，但已知其他异构体也会影响纤维结构和成熟度，这有助于肌腱的整体生物力学性能。目前尚不清楚研究较少的胶原蛋白异构体在组织工程化肌腱的原纤维形成中所起的作用，将它们的相对丰度与循环应变引起的生物力学变化相关联，是表征优化的生物工程肌腱移植物的一种有前景的方法。在本研究中，将人间充质干细胞（MSCs）在含有3%、5%或10%循环应变的纤维蛋白支架中以0.5Hz培养3周，并进行了包括qPCR、蛋白质免疫印迹、组织学、力学测试、荧光探针共聚焦激光扫描显微镜（CLSM）、透射电子显微镜（TEM）和无标记二次谐波成像的综合多模态分析。分子数据表明，I、II、III、V、XI、XII和XIV型胶原蛋白异构体在循环应变作用下存在复杂的转录和翻译调控。与胚胎肌腱形成相关的异构体（XII和XIV）在hMSCs形成新肌腱的过程中沉积，这表明这些工程化肌腱是通过某种发育途径的重演形成的。用3%应变培养的肌腱中位纤维直径最小，但抗应力能力最高，而在10%应变下培养的肌腱中位纤维直径最大，应力松弛率最高。二次谐波产生揭示了每个应变组中胶原纤维的不同结构排列。荧光探针图像显示，随着循环应变从40%（静态应变）增加到61.5%对齐（10%循环应变），纤维排列增加。这些结果表明，循环应变率通过对胶原蛋白异构体的复杂调控刺激不同的细胞反应，从而影响发育中的纤维结构的组织结构。
