Tunable Mesoscopic Collagen Island Architectures Modulate Stem Cell Behavior

Abstract

The extracellular matrix is the biophysical environment that scaffolds mammalian cells in the body. The main constituent is collagen. In physiological tissues, collagen network topology is diverse with complex mesoscopic features. While studies have explored the roles of collagen density and stiffness, the impact of complex architectures remains not well-understood. Developing in vitro systems that recapitulate these diverse collagen architectures is critical for understanding physiologically relevant cell behaviors. Here, methods are developed to induce the formation of heterogeneous mesoscopic architectures, referred to as collagen islands, in collagen hydrogels. These island-containing gels have highly tunable inclusions and mechanical properties. Although these gels are globally soft, there is regional enrichment in the collagen concentration at the cell-scale. Collagen-island architectures are utilized to study mesenchymal stem cell behavior, and it is demonstrated that cell migration and osteogenic differentiation are altered. Finally, induced pluripotent stem cells are cultured in island-containing gels, and it is shown that the architecture is sufficient to induce mesodermal differentiation. Overall, this work highlights complex mesoscopic tissue architectures as bioactive cues in regulating cell behavior and presents a novel collagen-based hydrogel that captures these features for tissue engineering applications.

Keywords: biomaterials; cell-extracellular matrix interactions; collagen; tissue engineering.

Figure 1 -. Tunable Collagen Island architectures mimic tissue topology.

(a) Collagen architecture in tissue is diverse. Images are representative of normal (top) and diseased (bottom) breast, liver, and lung tissue. Scale bar is 20 um. (b) Schematic detailing collagen island assembly protocol. Made with Biorender.com. (c) Collagen island architecture schematic as a function of shear frequency and architecture imaged with confocal microscopy (stained collagen in cyan) and scanning electron microscopy to visualize macro, meso, and nanoscale. Scale bar in the second row is 100 μm. Scale bars in the third row and corresponding inset are 50 μm and 30 μm, respectively. Scale bar in the fourth row is 20 μm. (d) Pore area distribution for each island architecture. Right of dotted line indicates frequency of larger pore areas and therefore sparser collagen. (e) Cross sectional distribution of collagen architecture in a randomly selected location on confocal images. (f) Island area measured from confocal imaging, mean ± sem. (g) Young’s modulus of island gels measured from shear rheometry. N ≥ 3 independent gels, mean ± sd. *, p < 0.05; **, p < 0.01; ***p < 0.0001 by one-way ANOVA.

Figure 2 -. Mechanical properties of collagen islands are tunable.

(a) Strain sweeps of island gels. Differential shear modulus K as function of shear strain. Corresponding rate of strain stiffening (dK/dE) and maximum shear modulus for each architecture. N ≥ 3, mean ± sem, (b) Average normalized stress relaxation tests at different strains. Blue is 60%, red is 30%, and purple is 10% strain. Black line indicates decreasing half max relaxation time as hold strain increases. (c) τ_1/2 values for each island architecture at each given strain. While τ_1/2 decreases with increasing strain, there is no significant change in τ_1/2 values as a result of island architecture introduction. N ≥ 3, mean ± sd. * p < 0.05 by one-way ANOVA.

Figure 3 -. Cell contractile behavior is modulated by heterogeneous architecture.

(a) Schematic of gel compaction assay. (b) Representative timelapse and (c) zoomed in images of MSCs in smaller gel volumes over 12 hours. White arrows indicate plastic gel tracks formed between cells. Yellow arrows indicate island architecture movement. Scalebar for (b) is 500 μm and for (c) is 150 μm. (d) Representative images and kymograph for MSCs cultured in (i) normal or (ii) mid shear island gels. Yellow arrow indicates dynamic protrusion and white lines indicate island architecture movement. Scale bar is 30 μm. (e) Gel compaction for MSCs (6x10⁵ cells/ml) for varied island architectures over 7 days. N ≥ 3, mean ± sem. (f) 7 day time course of MSCs cultured in each island architecture . (g) Quantified gel compaction area at days 1,2,3 and 7 for each island gel. N ≥ 3, mean ± sd. Magenta is stained collagen and cyan is MSC-LifeAct cells. * p < 0.05; **, p < 0.01; ***p < 0.0001 by one-way ANOVA.

Figure 4 -. MSC migratory and differentiation behaviors are modulated by island architecture.

(a) Representative 3D track reconstructions for cell migration in island architectures. (b) Mean squared displacements, mean speeds, sphericity, and migration track length for migrating cells. N = 3, n > 200 cells. (c) Representative images of alkaline phosphatase staining (blue), indicating early osteogenic differentiation, for MSC cultured in island gels for 7 days. Scale bar is 200 μm. (d) Corresponding ALP (+) fraction of cells. n ≥ 3 images from 3 independent replicates. (e) Representative images of RUNX2 (green), DAPI (cyan), and F-actin (magenta) of MSCs cultured in island gels for 7 days. (f) Corresponding RUNX2 nuclear to cytoplasmic ratio of cells cultured in each island gel. N=3, n > 53 cells. Scale bar is 30 microns. mean ± sd * p < 0.05; **, p < 0.01; ***p < 0.0001 by one-way ANOVA.

Figure 5 -. iPSCs undergo mesodermal transitions in island architecture.

(a) Representative images of iPSCs grown in normal or mid shear islands in co-gels with either 20% Matrigel by volume or 50% Matrigel by volume over 7 days. Scale bar is 200 μm. (b) Circularity and solidity of iPSC aggregates over 7 days and significantly decreases in matrigel: island co-gels. (c) Quantified solidity, circularity, and % aggregates with low circularity (< 0.7) after 7 days of culture. N = 3, n > 5 aggregates, mean ± sd (d) Representative immunofluorescent images of 50% Matrigel: Normal and 50% Matrigel:Island gels stained for DAPI (cyan), F-actin (magenta), Nanog (yellow) and ZO-1 (green) after 7 days of culture. iPSCs cultured in island co-gels lose their pluripotency and apical-basal polarity indicated by loss of Nanog and ZO-1, respectively. (e) After 7 days of culture, iPSCs show mesodermal differentiation in 50% Matrigel:Island gels indicated by nuclear localization of (left) Snail and (right) Brachyury (insets). (f) Corresponding nuclear to cytoplasmic ratio of SNAIL (left) and Brachyury (right). Scale bar is 100 μm. Inset scale bar is 30 μm. mean ± sd * p < 0.05; **, p < 0.01; ***p < 0.001; ***p < 0.0001 by one-way ANOVA.