Poster Q2 - Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

Introduction: Substrate-associated cues, such as mechanical (stiffness) and topographic (patterning), profoundly influence cellular response. Knowing the specific perturbations necessary to dissect multiple cues that drive migration requires a specific understanding of the underlying processes, specifically with the ability to drive a single cue or multiple cues in tandem. The direction, timescale and combination of these mechanical effects on emergent cellular responses are understudied. Tools to examine how time-varying substrate-associated stimuli drive physiological and pathological processes can unlock the next level of mechanobiological insight. Here, we use a micro-patterned magnetorheological elastomer that can be rapidly stiffened and softened in response to an external magnetic field to investigate the effects of neonatal rat cardiomyocyte orientation and alignment with respect to differences in mechanical (stiffness) and contact-guided (topographic) stimulation. By integrating dynamic control of mechanical stiffness that can be temporally tuned and reversed, we can rigorously test the effects of load by first conditioning under the same conditions and then acutely changing in-vitro biomechanics to properly mimic clinically relevant phenomenology, such as myocardial infarction.

Materials and

Methods: Micro-patterned magnetorheological elastomers (MREs) were prepared by a two-step molding process. To begin a PDMS (10:1, Sylgard 184) stamp was created by molding against a glass diffraction gradient with features 3 µm in height. The stamps were then functionalized with trichloro -(1H, 1H, 2H, 2H-perfluorooctyl) silane (Sigma Aldrich). Next, the prepolymer MRE mixture was prepared by combining Sylgard 184 (10:1) with Sylgard 527 (1:1) at a 30:1 (184:527) ratio and then subsequently combined with carbonyl iron particles at a 50wt% with the prepolymer solution. The MRE mixture was then poured into a 35 mm dish and partially cured in an oven at 60 °C for 40 minutes, at which a drop of the MRE mixture was added to the stamp and applied to the surface of the partially cured MREs and allowed to fully cure in the oven overnight. The devices were then sterilized with 70% ethanol and then coated with fibronectin (10µg/mL) in PBS for 1 hr at 37 °C to promote cell adhesion to the device. Neonatal rat cardiomyocytes were isolated and then seeded onto the devices either with or without magnets applied for 24 hours of culture and then conditions were switched leading to an acute change in substrate stiffness. (Fig. 1A). After the initial static culture period and 3 or 24 hours post-stiffening or softening the cells were fixed and stained to measure the angle and degree of alignment of the cardiomyocytes on both patterned and unpatterned MREs.

Results, Conclusions, and Discussions: In this work we investigated the effect of acutely changing substrate stiffness in conjunction with or without micropatterning on the alignment of cardiomyocytes. We cultured neonatal rat cardiomyocytes on both patterned and flat substrates and either stiffened (increased stiffness) or softened (decreased stiffness) that substrate. We found striking differences in alignment, where CMs qualitatively appear the most aligned on the soft patterned substrate and the most disorganized on the stiff unpatterned surface. This highlights the importance of simultaneously considering multiple cues to drive neonatal rat cardiomyocyte alignment toward a physiologically relevant phenotype. We quantified cell alignment on matrix stiffening and softening events using changes in alignment angle (mean vector angle) and degree of alignment (mean vector length). First, the alignment angle (−90° < θ < 90°) represents the angle of cells with respect to the horizontal axis. Visual inspection of the alignment angle on the unpatterned surfaces for the stiffening and softening events suggests a wide distribution of the angles between -90° and 90°, and a bias of the angle distribution toward 0° on the patterned surfaces (Fig. 1C). This observation is confirmed by t-tests between groups for both the stiffening and softening events. The values for the degree of alignment range from 0 to 1, where a value of 1 indicates greater horizontal alignment. The cardiomyocytes on the patterned substrates with matrix stiffening revealed a decrease in the degree of alignment with time compared to the control. Whereas the softening matrix showed an increase in the degree of alignment with time compared to the control (Fig. 1C). These observations were confirmed by ANOVAs and the subsequent Tukey’s post-hoc tests, which found significance between the temporal patterned substrates and the control. Patterned cell culture substrates have been present for decades, however, what makes our platform novel is the ability to dynamically tune substrate stiffness (mechanical guidance) in addition to the micropatterning (topographical guidance). This platform allows for studying dynamic processes such as morphogenesis, angiogenesis, or fibrosis that were previously limited to substrates with static stiffness or unpatterned surfaces .

Acknowledgements (Optional): This research was partly supported by the Delaware Center for Musculoskeletal Research COBRE (P20 GM139760), with grants from the National Institute of General Medical Science from the National Institutes of Health and the State of Delaware.