(271c) Quantifying Tissue-Induced Collagen Fiber Alignment in 3D Microfabricated Tissues

Introduction: The extracellular matrix (ECM) is a complex mixture of macromolecules that provides both biophysical and biochemical cues to cells and tissues. Cell-ECM interactions, which are known to drive the remodeling of the ECM, influence cell- and tissue-level behavior and are important in biological processes such as tissue morphogenesis and cancer progression [1, 2]. In particular, cell-induced collagen fiber alignment is widely observed, but the dynamics and mechanisms by which this alignment occurs remain unclear. Here, we quantified the dynamics of tissue-induced type-I collagen fiber alignment and evaluated the roles of contractility and proteolysis in the alignment process.

Materials and Methods: Three-dimensional (3D) microfabricated tissues were embedded in a matrix consisting of acid-extracted type-I bovine collagen and Matrigel using a previously developed soft-lithography approach [3]. 3D tissues were fabricated using functionally normal mouse mammary epithelial cells or breast cancer cells. Cytoskeletal contractility was manipulated using a Rho activator, a Rho kinase inhibitor, and a myosin II ATPase inhibitor. Proteolysis was inhibited using a broad-spectrum matrix metalloproteinase (MMP) inhibitor. Collagen fibers were visualized using confocal reflection microscopy, and fiber alignment was quantified from changes in the orientation of pixel intensity gradients over a period of 24 hours.

Results and Discussion: Tissue-induced collagen fiber alignment occurred rapidly and reached a plateau within 6 hours when ~50% of collagen fibers were aligned perpendicular to the tissue surface. During fiber alignment, a decrease in the tissue radius was observed, which indicated that tissue-exerted strain occurred coincident with the alignment process. Collagen alignment was not observed when tissues were treated with a Rho kinase or myosin II ATPase inhibitor. Tissues treated with a Rho activator showed an increase in both the rate and extent of collagen alignment. These results suggested that Rho-mediated cytoskeletal contractility has an important role in collagen alignment. We observed that inhibiting MMP activity did not affect the rate or extent of collagen fiber alignment, which further supported the idea that Rho-mediated cytoskeletal contractility is the dominant mechanism for collagen alignment. When comparing alignment in normal and cancer cells, we unexpectedly found that breast cancer cells, which have higher contractility but weaker cell-cell adhesions as compared to mammary epithelial cells, took longer to align the collagen fiber matrix. These results demonstrated that collective contractility is important for controlling the rate of collagen fiber alignment.

Conclusions: We quantified the dynamics of type-I collagen fiber alignment using 3D microfabricated tissues. Our results suggest that collective cellular contractility is the dominant mechanism that drives tissue-induced collagen fiber alignment. We found that tissues exerted strain on the surrounding collagen matrix and primarily used a mechanical mechanism to align collagen fibers.

References:

1. Provenzano PP et al. (2006) BMC Med 4(1):38.
2. Gjorevski N et al. (2015) Sci. Rep. 5:11458.
3. Piotrowski-Daspit AS et al. (2016) JoVE 113: e54283.