Integrating structural heterogeneity, fiber orientation, and recruitment in multiscale ECM mechanics

https://doi.org/10.1016/j.jmbbm.2018.12.023Get rights and content

Abstract

Extracellular matrix (ECM) plays critical roles in establishing tissue structure-function relationships and controlling cell fate. However, the mechanisms by which ECM mechanics influence cell and tissue behavior remain to be elucidated since the events associated with this process span length scales from the tissue to molecular level. Entirely new methods are needed in order to better understand the multiscale mechanics of ECM. In this study, a multiscale experimental approach was established by integrating Optical Magnetic Twisting Cytometry (OMTC) with a biaxial tensile tester to study the microscopic (local) ECM mechanical properties under controlled tissue-level (global) loading. Adventitial layer of porcine thoracic artery was used as a collagen-based ECM. Multiphoton microscopy imaging was performed to capture the changes in ECM fiber structure during biaxial deformation. As visualized from multiphoton microscopy images, biaxial stretch induces gradual fiber straightening and the fiber families become evident at higher stretch levels. The OMTC measurements show that the local apparent storage and loss modulus increases with the global biaxial stretch, however there exists a complex interplay among local ECM mechanical properties, ECM structural heterogeneity, and fiber distribution and engagement. The phase lag does not change significantly with global biaxial stretch. Our results also show a much faster increase in global tissue tangent modulus compared to the local apparent complex modulus with biaxial stretch, indicating the scale dependency of ECM mechanics.

Introduction

Extracellular matrix (ECM) provides the principal avenue for mechanical and biochemical communications between tissue and cells. Cells actively sense the external mechanical forces applied through the ECM and also their surrounding local stiffness (Throm Quinlan et al., 2011). External mechanical forces and ECM stiffness have been shown to play important roles in regulating fundamental cellular morphology (Wells, 2008) and functions, such as proliferation (Wells, 2008; Schrader et al., 2011), differentiation (Engler et al., 2006; Even-Ram et al., 2006) and mobility (Pelham and Wang, 1997; Zaman et al., 2006). Many pathological conditions involve significant alterations of ECM mechanical properties (Baker et al., 2009; Janmey and Miller, 2011; Lu et al., 2012; Engler et al., 2008; Chaturvedi et al., 2010; Georges et al., 2007; Wells, 2008; Shkumatov et al., 2015; White, 2015). Understanding the translation of mechanical forces from the tissue to cellular level is crucial and a requisite bridge between research in the fields of cell and tissue mechanics. However, the force translation in ECM is inherently a multiscale process ranging from the macroscopic to microscopic level. The mechanical properties of ECM, known to have a 3D hierarchical structure, have been shown to be highly dependent on the scale of measurement (Aifantis et al., 2011; Li et al., 2017). As a result, the distribution of mechanical force in the ECM is highly dependent on the hierarchical structure and the local mechanical properties of the ECM (Pizzo et al., 2005; Mow et al., 1994).

The macro- and microscopic mechanical properties of ECM have been broadly studied. To investigate the macroscopic mechanical properties of collagen matrix, various experimental methods, such as uniaxial tension (Roeder et al., 2002; Feng et al., 2003; Duan and Sheardown, 2006; Van Oosten et al., 2016), biaxial tension (Jhun et al., 2009; Sander et al., 2009; Xu et al., 2011), and rheological tests (Vader et al., 2009; Xu et al., 2011; Kurniawan et al., 2012; Li and Zhang, 2014; Motte and Kaufman, 2012) have been performed. In microscopic mechanical measurements, controlled mechanical forces were applied using a variety of techniques including indentation (Wenger et al., 2007; Aifantis et al., 2011; Mckee et al., 2011), laser tracking microrheology (Velegol and Lanni, 2001; Parekh and Velegol, 2007; Sun et al., 2004; Shayegan and Forde, 2013), magnetic twisting cytometry (Leung, et al., 2007), and optical magnetic twisting cytometry (OMTC) (Li et al., 2017). These studies on the mechanical properties of ECM are focused on a single measurement scale. Advances in imaging techniques promote the integration of multiscale mechanical loading and imaging modalities to provide perspectives on load induced changes in ECM microstructure, cellular morphology and alignment (Bell et al., 2012; Throm Quinlan et al., 2011). An integrated mechanical loading-confocal microscopy system was developed and used to study three-dimensional (3D) mechanical strains in collagen matrix at multiple length scales in response to uniaxial (Roeder et al., 2004, Roeder et al., 2009) and biaxial stretching (Bell et al., 2012). To obtain local mechanical properties, local strains were measured, and the macro-scale mechanical properties of a substrate are usually used in the calculation of local traction forces (Franck et al., 2011; Toyjanova et al., 2014).

To date, there has been limited understanding of multiscale ECM mechanical properties. Several previous studies have attempted to measure the local mechanical properties of ECM and showed that the local mechanical properties of an interconnected network do not always match its global properties. Microscopic mechanical properties of ECM measured by laser trap microscopy (Velegol and Lanni, 2001) and AFM (Gautreau et al., 2006; Throm Quinlan and Billar, 2012)yielded consistently greater stiffness measurements than those obtained from macroscopic rheometry and uniaxial tensile tests. Significant differences in the mechanical properties of ECM were shown ranging from the macro to nano scales as Etendon < Efiber < Efibri (Aifantis et al., 2011). The microscopic mechanical properties were found to be less sensitive to changes in collagen concentration, as compared to the macroscopic mechanical properties (Li et al., 2017; Baniasadi and Minary-Jolandan, 2015). The scale of measurements was found to play an important role in multiscale ECM mechanical properties. The differences in multiple ECM mechanical properties emerge when the probe size is comparable to or smaller than the average pore size of the sample (Costa et al., 2003).

In the present study, we created a multiscale experimental approach that combines optical magnetic twisting cytometry (OMTC) and biaxial tensile testing techniques to study the changes in local ECM mechanical properties with controlled global biaxial mechanical loading. The biaxial tensile test considers the multi-axial loading state under physiological conditions and has shown promises in fully characterizing the anisotropic nature of soft biological tissues (Sacks and Sun, 2003; Nerurkar et al., 2010; Bell et al., 2012). By oscillating microscale ferromagnetic beads bound to collagen fibers, OMTC was used to measure the local mechanical properties of collagen matrix. OMTC allows the measurements of a population of beads and can effectively eliminate unbound or loosely bound beads (Fabry et al., 2001). Porcine adventitia layer was used as an ECM equivalent due to several reasons: 1) Porcine adventitia is the outmost layer of the artery and consists primarily of type I collagen fibers. 2) It is semi-transparent so it is possible to make optical measurements using OMTC. 3) Compared to collagen gel, it is more robust to work with when biaxial loading is applied. Multiphoton microscopy was also used to capture the structural changes of collagen fibers under tissue-level biaxial loading.

Section snippets

Sample preparation

Descending thoracic aortas of porcine (12 – 24 month old) were harvested at a local abattoir and transported to the laboratory on ice. Before experiments, the aortas were cleaned of adherent tissues and fat and rinsed in deionized water. All samples were taken from approximately the same longitudinal region of the aorta to avoid changes in mechanical properties along the longitudinal anatomic position (Zeinali-Davarani et al., 2015). The adventitial layer was carefully peeled from the porcine

Tissue-level mechanical properties and ECM structure

Fig. 2a shows the average Cauchy stress vs. stretch curves from biaxial tensile testing of the adventitia tissue sample, which is highly nonlinear with the longitudinal direction stiffening slightly earlier than the circumferential direction. To better compare the mechanical properties in the longitudinal and circumferential directions, tangent modulus was obtained by differentiating the Cauchy stress-stretch curves in Fig. 2a as dσ/dλ. As shown in Fig. 2b, a prominent increase in tangent

Discussion

In this study, a new experimental methodology was developed to study the changes of microscopic ECM mechanics under macroscopic loading. Specifically, OMTC was used to measure the local mechanical properties of the collagen network when subjected to controlled global biaxial mechanical loading. Our study suggests that it is important to consider the complex interplay among ECM fiber orientation, fiber engagement, and the tissue-level mechanical loading when understanding the scale dependency of

Conclusions

A novel experimental approach that allows the measurements of local ECM mechanical properties with controlled tissue-level biaxial mechanical loading was established and used to study the multiscale ECM mechanics of the collagen network in the adventitial layer of the arterial wall. This study reported several interesting findings of the role of ECM fiber network structure in determining the local ECM mechanical properties upon tissue-level mechanical loading. Our study shows that the ECM fiber

Acknowledgements

The authors would like to acknowledge the funding support from the National Science Foundation (CAREER 0954825) and National Institutes of Health (2R01HL098028).

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