Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain

Abstract

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing.

Tough and tunable scaffold-hydrogel composite biomaterial for soft-to-hard musculoskeletal tissue interfaces

“…Our evaluation of toughness and mechanical performance of hydrogel insertions does not take into consideration the existence of preexisting flaws or tears. Fracture toughness describes a material's ability to resist crack propagation and, in the case of the native enthesis, involves microscale mechanisms such as fiber realignment and fiber densification (2,3,20,(42)(43)(44)(45). While studies measuring fracture toughness of triphasic biomaterials will be critical in optimization of mechanical performance for in vivo applications, the goal of the present study is to describe a novel biomaterial design paradigm.…”

Promoting effective tendon healing and remodeling

“…Consequently, we have established methods to evaluate 3 dimensional (3D) cell and ECM strains that can be used with whole tendon tissue in order to quantify the effect of the higher stiffness of healing MRL/MpJ tendon on the deformations experienced by the cells and thereby the biological outcome. 48 A custom Matlab program was developed to calculate 3D cell and matrix strain (Fig. 6).…”

The Micromechanical Environment of the Impinged Achilles Tendon Insertion

“…Our 3-D multiphoton elastography approach is innovative because we simultaneous mapped 3-D strains across multiple length scales including the local matrix, PCM and cell scales at a high resolution and in a single experiment. In particular, to independently assess local matrix strain, we incorporated the position information of all tracked cells before and after impingement to define the deformation gradient tensor 𝑭 and, in turn, the several key advantages compared to conventional 2-D approaches [15][16][17], or even certain 3-D approaches [35] used to assess microscale tissue strain patterns. Most of these studies applied digital volume correlation (DVC) [35] or other textural/correlationbased approaches to characterize strain patterns.…”

“…In particular, to independently assess local matrix strain, we incorporated the position information of all tracked cells before and after impingement to define the deformation gradient tensor 𝑭 and, in turn, the several key advantages compared to conventional 2-D approaches [15][16][17], or even certain 3-D approaches [35] used to assess microscale tissue strain patterns. Most of these studies applied digital volume correlation (DVC) [35] or other textural/correlationbased approaches to characterize strain patterns. However, these methods generally require large numbers of features (e.g., cells) to tabulate local displacements.…”

“…Our analysis has several key advantages compared to conventional 2-D approaches [15][16][17], or even certain 3-D approaches [35] used to assess microscale tissue strain patterns. Most of these studies applied digital volume correlation (DVC) [35] or other textural/correlationbased approaches to characterize strain patterns. However, these methods generally require large numbers of features (e.g., cells) to tabulate local displacements.…”