The properties of the extracellular matrix (ECM) have profound impact upon cell behaviour. As an abundant protein in mammals, collagen is a desirable base material to engineer an ECM tissue scaffold, but its structural weakness generally requires molecular crosslinking or incorporation of additional ECM-based macromolecules such as glycosaminoglycans. We have performed microscopic indentation to test collagen films under dry and aqueous conditions prepared with different levels of physical and chemical crosslinking. Our technique isolates intrinsic properties of the poro-viscoelastic matrix in a regime minimizing the influence of drainage hydrodynamics and allows direct measurement of the effect of hydrating a specific sample. A doubling of the effective stress-strain stiffness under crosslinking could be directly correlated to structural changes in X-ray diffraction spectra, while electron microscopy revealed possible fibril bridging mechanisms explaining observed toughness. Overall, an intrinsic viscoelastic stress-strain response of collagen under various conditions of cross-linking was observed for both dry and wet conditions, with the latter most affected by indentation rate. Under creep testing, a three order of magnitude increase in dynamic compliance and factor three reduction in relaxation time was found going from the dry to hydrated state. When fitted to a simple viscoelastic model, crosslinking showed a tendency to decrease relaxation time in both states, but reduced dynamic compliance only in the hydrated case. This suggests a reduced role of virtual crosslinks under hydration. This is the first study reporting consistent mechanical testing of dry and hydrated ECM-derived biomaterials, accessing the intrinsic material mechanics under in vivo-like conditions.

Statement of Significance

This manuscript presents new insights into the effect of crosslinking on mechanical properties of dry and hydrated collagen intended for tissue scaffolding applications. A novel microscopic indentation technique allowed testing of the poro-viscoelastic matrix isolated in a regime minimizing the influence of drainage hydrodynamics, so direct comparison of the effect of hydration on the intrinsic material behaviour to could be made. A variety of experimental techniques including X-ray diffraction, infrared spectroscopy, and scanning electron and atomic force microscopy were used to augment the mechanical testing. The results of creep testing were numerically analysed using a four-component viscoelastic model. This is the first mechanical testing of dry and hydrated ECM-derived biomaterials, accessing the intrinsic material mechanics under in vivo-like conditions.

中文翻译：

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交联和水合作用对粘弹性极限中的胶原蛋白透明质酸薄膜的微型平冲头压痕接触的影响。

细胞外基质（ECM）的性质对细胞行为具有深远的影响。作为哺乳动物中一种丰富的蛋白质，胶原蛋白是工程化ECM组织支架的理想基础材料，但是胶原蛋白的结构弱点通常需要分子交联或引入其他基于ECM的大分子，例如糖胺聚糖。我们已经进行了微观压痕测试，以在干燥和水性条件下测试胶原膜，这些条件是用不同水平的物理和化学交联制备的。我们的技术以最小化排水流体动力学影响的方式隔离了孔隙粘弹性基质的固有特性，并可以直接测量水合特定样品的效果。交联下有效应力-应变刚度的加倍可以直接与X射线衍射光谱中的结构变化相关，而电子显微镜显示可能的原纤维桥接机制解释了观察到的韧性。总的来说，在干燥和湿润条件下都观察到了胶原在各种交联条件下的固有粘弹性应力-应变响应，后者受压痕率的影响最大。在蠕变测试中，发现从干燥状态到水合状态，动态柔量增加了三个数量级，松弛时间减少了三倍。当拟合为简单的粘弹性模型时，交联在两种状态下均表现出减少弛豫时间的趋势，但仅在水合情况下降低了动态顺应性。这表明水合下虚拟交联的作用降低。这是第一项报道了对干燥和水合ECM衍生生物材料进行一致的机械测试的研究，该研究在以下条件下获得了固有的材料力学体内条件。

重要声明

该手稿提供了新的见解，旨在探讨交联对组织支架应用中干燥和水合胶原蛋白的机械性能的影响。一种新颖的微观压痕技术可以在最小化排水流体动力学影响的范围内测试孔隙粘弹性基质，因此可以直接比较水化作用对固有材料行为的影响。包括X射线衍射，红外光谱以及扫描电子和原子力显微镜在内的各种实验技术被用于增强机械测试。使用四组分粘弹性模型对蠕变测试的结果进行了数值分析。这是干燥和水合ECM衍生生物材料的首次机械测试，