Spatiotemporal control of myofibroblast activation in acoustically-responsive scaffolds via ultrasound-induced matrix stiffening

Abstract

Hydrogels are often used to study the impact of biomechanical and topographical cues on cell behavior. Conventional hydrogels are designed a priori, with characteristics that cannot be dynamically changed in an externally controlled, user-defined manner. We developed a composite hydrogel, termed an acoustically-responsive scaffold (ARS), that enables non-invasive, spatiotemporally controlled modulation of mechanical and morphological properties using focused ultrasound. An ARS consists of a phase-shift emulsion distributed in a fibrin matrix. Ultrasound non-thermally vaporizes the emulsion into bubbles, which induces localized, radial compaction and stiffening of the fibrin matrix. In this in vitro study, we investigate how this mechanism can control the differentiation of fibroblasts into myofibroblasts, a transition correlated with substrate stiffness on 2D substrates. Matrix compaction and stiffening was shown to be highly localized using confocal and atomic force microscopies, respectively. Myofibroblast phenotype, evaluated by α-smooth muscle actin (α-SMA) immunocytochemistry, significantly increased in matrix regions proximal to bubbles compared to distal regions, irrespective of the addition of exogenous transforming growth factor-β1 (TGF-β1). Introduction of the TGF-β1 receptor inhibitor SB431542 abrogated the proximal enhancement. This approach providing spatiotemporal control over biophysical signals and resulting cell behavior could aid in better understanding fibrotic disease progression and the development of therapeutic interventions for chronic wounds. STATEMENT OF SIGNIFICANCE: Hydrogels are used in cell culture to recapitulate both biochemical and biophysical aspects of the native extracellular matrix. Biophysical cues like stiffness can impact cell behavior. However, with conventional hydrogels, there is a limited ability to actively modulate stiffness after polymerization. We have developed an ultrasound-based method of spatiotemporally-controlling mechanical and morphological properties within a composite hydrogel, termed an acoustically-responsive scaffold (ARS). Upon exposure to ultrasound, bubbles are non-thermally generated within the fibrin matrix of an ARS, thereby locally compacting and stiffening the matrix. We demonstrate how ARSs control the differentiation of fibroblasts into myofibroblasts in 2D. This approach could assist with the study of fibrosis and the development of therapies for chronic wounds.

Keywords: Acoustic droplet vaporization; Differentiation; Fibrin; Fibroblast; Myofibroblast; Phase-shift emulsion; Strain stiffening; Ultrasound.

Figure 1.

An acoustically-responsive scaffold (ARS), which consists of a phase-shift emulsion embedded within a hydrogel matrix, enables local modulation of stiffness using focused ultrasound. (A) A microfluidic chip was used to create emulsion by encapsulating a perfluoroheptane core in Pluronic F-68 surfactant. (B) The emulsion vaporizes into a gas bubble when ultrasound with a pressure exceeding the acoustic droplet vaporization threshold (i.e., P > P_ADV) is applied to the ARS. The generated bubble and its subsequent expansion due to in-gassing compacts the surrounding fibrin matrix, which causes stiffening. Normal human dermal fibroblasts (NHDFs) were plated on top of fibrin-based ARSs containing monodispersed emulsions either before (i.e., in situ) or after (i.e., ex situ) ultrasound exposure. (C) Representative images of NHDFs stained for nuclei (blue), F-actin (green), and α-SMA (red) on an ARS. The ultrasound-generated bubble is demarcated by a white circle (scale bars = 0.2 mm). The corresponding line plot of α-SMA intensity shows elevated signal proximal to the bubble.

Figure 2.

Acoustic droplet vaporization (ADV) resulted in local, radial compaction of fibrin surrounding the bubbles. Confocal microscopy images of acoustically-responsive scaffolds (ARSs) before (A) and 1-hour after (B) ADV. The fibrin matrix contained Alexa Fluor 647-labeled fibrinogen (fibrinogen₆₄₇). C) Maximum intensity projection confocal image of an ADV-generated bubble in an ARS. The region of interest (ROI) outlined in yellow dashed lines in (B) is enlarged in (D) and indicates significantly higher fibrinogen₆₄₇ intensity at the bubble-matrix interface. E) Line intensity measurements of fibrinogen₆₄₇ before and 1-hour after ADV are shown. The average intensity of fibrinogen₆₄₇ as a function of distance from the bubble interface (F) was computed by segmenting the matrix into ROIs, each having a length of 4 μm. The ROI most proximal to the bubble interface is outlined with red dashed lines in (D). Scale bar: 30 μm.

Figure 3.

Atomic force microscopy was used to characterize micromechanical properties of acoustically-responsive scaffolds (ARSs) after acoustic droplet vaporization (ADV). A) An optical image of an ARS post-ADV shows four regions defined as: cantilever with a 2 μm glass bead (I), region proximal to the ADV-bubble (II), region distal to the ADV-bubble (III), and a 50 μm × 50 μm area selected for indentation mapping (IV). B) A comparison of force-indentation responses in regions II & III, indicates a significant difference in stiffness. C) The Young’s modulus was mapped in region IV. Scale bar: 100 μm.

Figure 4.

Cell density and α-SMA expression in NHDFs were dependent on substrate stiffness and the presence of exogenous TGF-β1. A) Fluorescence images of NHDFs, which were stained for α-SMA, cultured on fibrinogen-coated PDMS substrates with different Young’s moduli and tissue culture plastic (TCP). Cells were cultured for 5 days in complete media with or without 5 ng/mL TGF-β1. Scale bar = 0.25 mm. B) Cell density was quantified based on DAPI staining. C) The level of α-SMA expression was quantified based on immunocytochemical staining. All data are represented as mean ± standard deviation (N = 3 plates per group with 3–5 fields of view per construct). Statistically significant differences (p < 0.05) between the same TGF-β1 conditions are denoted as follows: α vs. 1.5 kPa, β vs. 15 kPa, and γ vs. 28 kPa. A horizontal bar shows pairwise differences among +/− TGF-β1 conditions.

Figure 5.

α-SMA was elevated in NHDFs proximal to bubbles generated by acoustic droplet vaporization (ADV) compared to distal cells. A) Fluorescence images of NHDFs, which were stained for α-SMA, cultured on fibrin-based ARS. Cells were plated on ARS after exposure to ultrasound (i.e., ex situ exposure) or prior to exposure to ultrasound (i.e., in situ exposure, which generated ADV. Constructs were cultured for 5 days in complete media with or without 5 ng/mL TGF-β1. Bubbles, denoted by yellow circles, are observed in +ultrasound groups. Scale bar = 0.25 mm. B) Cell density was quantified based on DAPI staining. The level of α-SMA expression was quantified based on immunocytochemical staining for C) no ultrasound exposure, D) +ultrasound (ex situ exposure), and E) +ultrasound (in situ exposure) conditions. All data are represented as mean ± standard deviation (N = 3–5 ARSs per group with 3–5 fields of view per construct). For +ultrasound groups, cells were analyzed in regions proximal and distal to the bubbles, which are defined as regions within 0.1 mm and greater than 0.5 mm from the bubble edge, respectively. Statistically significant differences (p < 0.05) between the same TGF-β1 conditions are denoted as follows: δ vs. −ultrasound condition, ε vs. +ultrasound (ex situ exposure, proximal region), and ζ vs. +ultrasound (ex situ exposure, distal region). A horizontal bar shows pairwise differences.

Figure 6.

α-SMA levels increased in NHDFs cultured in low serum media compared to complete media. A) Fluorescent images of NHDFs, which were stained for α-SMA, cultured on fibrinogen-coated PDMS substrates with different Young’s moduli and tissue culture plastic (TCP). Cells were cultured for 3 days in starvation media. Scale bar = 0.25 mm. B) Cell density was determined based on DAPI staining. C) The level of α-SMA expression was quantified based on immunocytochemical staining. All data are represented as mean ± standard deviation (N = 3 plates per group with 3–5 fields of view per construct).

Figure 7.

Inhibition of TGF-β1 receptor abrogated the increase in α-SMA levels observed proximal to bubbles generated by acoustic droplet vaporization (ADV). A) Fluorescence images of NHDFs, which were stained for α-SMA, cultured on fibrin-based acoustically-responsive scaffolds (ARSs). Cells were plated on ARS after exposure to ultrasound (i.e., ex situ exposure) or prior to exposure to ultrasound (i.e., in situ exposure) to generate ADV. Constructs were cultured for 3 days in starvation media with or without 10 μM SB431542, an inhibitor of TGF-β1 receptor. Bubbles, denoted by yellow circles, are observed in +ultrasound groups. Scale bar = 0.25 mm. B) Cell density was determined by DAPI staining. The level of α-SMA expression was quantified based on immunocytochemical staining for C) no ultrasound exposure, D) +ultrasound (ex situ exposure), and E) +ultrasound (in situ exposure) conditions. All data are represented as mean ± standard deviation (N = 3–5 ARSs per group with 3–5 fields of view per construct). For +ultrasound groups, cells were analyzed in regions proximal and distal to the bubbles, which are defined as regions within 0.1 mm and greater than 0.5 mm from the bubble edge, respectively. A horizontal bar shows statistically significant differences (p < 0.05) between pairs.