Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues’ contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues’ Young’s moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues’ properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues’ contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues’ Young’s moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues’ properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues’ contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues’ Young’s moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues’ properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships

Effects of Geometry on the Mechanics and Alignment of Three-Dimensional Engineered Microtissues

The structure and stiffness of the extracellular matrix (ECM) in living tissues play a significant role in facilitating cellular functions and maintaining tissue homeostasis. However, the wide variation and complexity in tissue composition across different tissue types make comparative study of the impact of matrix architecture and alignment on tissue mechanics difficult. Here we present a microtissue-based system capable of controlling the degree of ECM alignment in 3D self-assembled fibroblast-populated collagen matrix, anchored around multiple elastic micropillars. The pillars provide structural constraints, control matrix alignment, enable measurement of the microtissues’ contractile forces, and provide the ability to apply tensile strain using magnetic particles. Utilizing finite element models (FEMs) to parametrize results of mechanical measurements, spatial variations in the microtissues’ Young’s moduli across different regions were shown to be correlated with the degree of ECM fiber alignment. The aligned regions were up to six times stiffer than the unaligned regions. The results were not affected by suppression of cellular contractile forces in matured microtissues. However, comparison to a distributed fiber anisotropic model shows that variations in fiber alignment alone cannot account for the variations in the observed moduli, indicating that fiber density and tissue geometry also play important roles in the microtissues’ properties. These results suggest a complex interplay between mechanical boundary constraints, ECM alignment, density, and mechanics and offer an approach combining engineered microtissues and computational modeling to elucidate these relationships

MechanicalTestingData

Compressed file containing raw force data of the cyclic loading experiments

TEM Images

Compressed file containing TEM images of en face and transverse section of dense disorganized collagen substrate after cyclic loadin

Statistical comparison of mean collagen anisotropy at different depths for the superior-temporal peripapillary scleral quadrant (two-tailed t-tests).

<p>Significance is indicated at three probability levels</p><p>* p < 0.05</p><p>** p < 0.01</p><p>*** p < 0.001.</p><p>Statistical comparison of mean collagen anisotropy at different depths for the superior-temporal peripapillary scleral quadrant (two-tailed t-tests).</p

Matrix modulus <i>μ</i>, parameters of the exponential fiber model (<i>α</i>, <i>β</i>) and fiber stiffness (4<i>αβ</i>) obtained by global optimization.

<p>On average, both matrix and fiber stiffness were larger for the damaged glaucoma group compared to the undamaged glaucoma group and larger for the undamaged glaucoma group compared to the normal group. Grade 0 represents ≤ 10% axon loss, 1 means 10–25% axon loss, 2 means 25–50%, and 3 means 50–75% axon loss.</p

Donor information for the normal and diagnosed glaucoma scleras subjected to inflation testing and WAXS measurement of the collagen fiber structure.

<p>Grade 0 corresponded to an optic nerve with less than 10% of axon loss (normal appearance), grade 1 was 10% to 25% axon loss (mild damage), grade 2 was 25% to 50% axon loss (intermediate damage), and grade 3 was 50% to 75% axon loss (severe damage). In the specimen name, F stands for female, M for male, C for Caucasian, AA for African American, r for right, and l for left. Left/right eyes from the same donor are indicated with the same symbol in the specimen name.</p

Statistical comparison of mean collagen anisotropy at different depths for the inferior-nasal peripapillary scleral quadrant (two-tailed t-tests).

<p>Significance is indicated at three probability levels</p><p>* p < 0.05</p><p>** p < 0.01</p><p>*** p < 0.001.</p><p>Statistical comparison of mean collagen anisotropy at different depths for the inferior-nasal peripapillary scleral quadrant (two-tailed t-tests).</p