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

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.