Cyclic mechanical loading of photopolymerized methacrylated hydrogels for probing interdependent effects of strain, stiffness, and substrate composition in pulmonary fibrogenesis

Abstract

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM), stiffening of the lung tissue, and impaired gas exchange. Our current understanding of fibrogenesis generally focuses on the individual roles of mechanical and biochemical stimuli in driving disease progression. However, many mechano-chemical pathways are interrelated, so dissecting the interactive effects of mechanical and biochemical signals is an important knowledge gap. To address this gap, we investigated lung fibroblast behavior on static and cyclically strained photopolymerizable hydrogels consisting of different ratios of methacrylated gelatin, methacrylated hyaluronan, and non-methacrylated gelatin to create substrates with tunable stiffness and chemistry, representative of both healthy and fibrotic lung ECM properties. We observed that higher stiffness gels amplified the impact of strain, resulting in distinct differences in expression of MMP1, CTGF, Rho/ROCK, and ECM deposition genes. Substrates with hyaluronan demonstrated a capacity to modulate strain-induced fibrogenic responses, suggesting a buffering effect of hyaluronan on fibrotic disease progression. Overall, our results highlight mechanotransductive changes in gene expression in response to substrate composition, stiffness, and cyclic mechanical strain. Through the controlled study of mechanical and biochemical cues, our findings contribute to a deeper understanding of the pathogenesis of pulmonary fibrosis.

Keywords: Fibrogenic response; GelMA; HAMA; In vitro lung model; Mechanobiology.

Fig. 1

Schematic of Published In Vitro Lung Systems with Cyclic Strain in Relation to Pathophysiological Conditions. Each data point represents an in vitro lung system with dynamic strain referenced in recently published review articles on in vitro lung models^–. Marker shapes denote the chemistry of the substrate on which the cells are cultured. Gray shading indicates the bioreactor operating space of the in vitro system presented here. Green and orange boxes represent strain and frequency typically associated with breathing during rest and exercise, respectively. FRC = Functional residual capacity. TLC = Total Lung Capacity.

Fig. 2

Hydrogel Compressive Modulus as a Function of Hydrogel Formulation. (A) GelMA: Gelatin compressive modulus decreases as the percentage of non-methacrylated gelatin (Gel) volume increases (n = 3 for each formulation). (B) Incorporating 10% HA (HAMA: GelMA: Gels) increases the compressive modulus compared to GelMA: Gels; modulus also decreases as the % Gel increases (n = 5 for each formulation). Reference values of high and low stiffness were chosen based on compressive modulus values reported for fibrotic and healthy lung tissue and are depicted by the grey and green horizontal lines, respectively.​ Linear regression parameters are shown in Table S1.

Fig. 3

Viability of NHLFs on Hydrogels. (A) Representative 10X merged fluorescent images from a Live/Dead assay, with live cells stained in blue and dead cells stained in green. Scale bars = 400 μm. B) Quantification of viability using the Live/Dead assay. TC – tissue culture plastic. Error bars represent mean ± SD for n = 5 images; *p ≤ 0.05.

Fig. 4

Morphology of NHLFs on Hydrogels. (A) Representative 20X phase contrast images of NHLFs showing morphology; scale bar = 200 μm. B) Quantification of NHLF aspect ratio on each substrate type. TC – tissue culture plastic. Error bars represent mean ± SD for n = 30 cells. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Fig. 5

Hydrogel Strain Fields. (A) Images of the ink-speckled hydrogel and sample grips at the midpoint of the deformation cycle, with color overlays representing strain amplitude at each location within the region of interest. B) The time-varying distribution of regional strains in the hydrogel region of interest over one cycle. Each data point represents a tracked speckle at that image frame. Arrow indicates timepoint of image used as reference to register other images.

Fig. 6

Principal component analysis with k-means clustering of gene expression of NHLFs. Gene expression was measured using RT-qPCR for NHLFs subjected to static or cyclical strain on different hydrogel formulations. Average 2^− ddCt data (n = 3) were dimensionally reduced to reveal important determinants of gene expression pattern.

Fig. 7

RT-qPCR results of select genes impacted by stiffness. NHLFs on GelMA: Gel showed significant changes in MMP1, ROCK1, YAP, TGF-b1, IL-6 and CTGF expression as a function of stiffness and strain. Statisitcal analysis performed with 2-way ANOVA, n = 3, mean ± SD. p-values are shown in Table S2.

Fig. 8

Heatmap of fibrogenic gene expression. Gene expression from static conditions was subtracted from relative gene expression of each experimental group shown to display direction of gene expression change in relationship to static conditions. n = 3 per condition.