micropatterned-magneto-rheological-elastomers-to-drive-changes

Micropatterned Magneto-Rheological Elastomers to Drive Changes in Cardiomyocyte Alignment

Substrate-associated cues, such as mechanical and topographic, profoundly influence cellular response. However, much of the foundational research employs ...

University of Delaware

Nemours/Alfred I. duPont Hospital for Children

Biophysical studies on single cells have linked cell mechanics to physiology, functionality and disease. Evaluation of mass and viscoelasticity versus cell cycle can provide further insights into cell cycle progression and the uncontrolled proliferation of cancer. Using our pedestal microelectromechanical systems resonant sensors, we have developed a non-contact interferometric measurement technique that simultaneously tracks the dynamic changes in the viscoelastic moduli and mass of adherent colon (HT-29) and breast cancer (MCF-7) cells from the interphase through mitosis and then to the cytokinesis stages of their growth cycle. We show that by combining three optomechanical parameters in an optical path length equation and a two-degree-of-freedom model, we can simultaneously extract the viscoelasticity and mass as a function of the nano-scaled membrane fluctuation of each adherent cell. Our measurements are able to discern between soft and stiff cells across the cell cycle and demonstrated sharp viscoelastic changes due to cortical stiffening around mitosis. Cell rounding before division can be detected by measurement of mechanical coupling between the cells and the sensors. Our measurement device and method can provide for new insights into the mechanics of single adherent cells versus time.

Simultaneous time-varying viscosity, elasticity, and mass measurements of single adherent cancer cells across cell cycle.

The mechanical environment of the myocardium has a potent effect on cardiomyocyte form and function, yet an understanding of the cardiomyocyte responses to extracellular stiffening remains incomplete. We therefore employed a cell culture substrate with tunable stiffness to define the cardiomyocyte responses to clinically relevant stiffness increments in the absence of cell-cell interactions. When cultured on substrates magnetically actuated to mimic the stiffness of diseased myocardium, isolated rat adult cardiomyocytes exhibited a time-dependent reduction of sarcomere shortening, characterized by slowed contraction and relaxation velocity, and alterations of the calcium transient. Cardiomyocytes cultured on stiff substrates developed increases in viscoelasticity and microtubule detyrosination in association with early increases in the α-tubulin detyrosinating enzyme vasohibin-2 (Vash2). We found that knockdown of Vash2 was sufficient to preserve contractile performance as well as calcium transient properties in the presence of extracellular substrate stiffening. Orthogonal prevention of detyrosination by overexpression of tubulin tyrosine ligase (TTL) was also able to preserve contractility and calcium homeostasis. These data demonstrate that a pathologic increment of extracellular stiffness induces early, cell-autonomous remodeling of adult cardiomyocytes that is dependent on detyrosination of α-tubulin.

Extracellular stiffness induces contractile dysfunction in adult cardiomyocytes via cell-autonomous and microtubule-dependent mechanisms.

Materials with the ability to change properties can expand the capabilities of  models of biological processes and diseases as it has become increasingly clear that static, stiff materials with smooth surfaces fall short in recapitulating the  cellular microenvironment. Here, we introduce a patterned material that can be rapidly stiffened and softened  in response to an external magnetic field through the addition of magnetic inclusions into a soft silicone elastomer with topographic surface patterning. This substrate can be used for cell culture to investigate short-term cellular responses to dynamic stiffening or softening and the interaction with topography that encourages cells to assume a specific morphology. We investigated short-term cellular responses to dynamic stiffening or softening in human ventricular cardiac fibroblasts. Our results indicate that the combination of dynamic changes in stiffness with and without topographic cues induces different effects on the alignment and activation or deactivation of myofibroblasts. Cells cultured on patterned substrates exhibited a more aligned morphology than cells cultured on flat material; moreover, cell alignment was not dependent on substrate stiffness. On a patterned substrate, there was no significant change in the number of activated myofibroblasts when the material was temporally stiffened, but temporal softening caused a significant decrease in myofibroblast activation (50% to 38%), indicating a competing interaction of these characteristics on cell behavior. This material provides a unique  platform to observe the time-dependent dynamics of cells by better mimicking more complex behaviors and realistic microenvironments for investigating biological processes, such as the development of fibrosis.

Biomimetic Substrate to Probe Dynamic Interplay of Topography and Stiffness on Cardiac Fibroblast Activation.

We report tuning of the moduli and surface roughness of magnetorheological elastomers (MREs) by varying applied magnetic field. Ultrasoft MREs are fabricated using a physiologically relevant commercial polymer, Sylgard 527, and carbonyl iron powder (CIP). We found that the shear storage modulus, Young's modulus, and root-mean-square surface roughness are increased by ~41×, ~11×, and ~11×, respectively, when subjected to a magnetic field strength of 95.5 kA m. Single fit parameter equations are presented that capture the tunability of the moduli and surface roughness as a function of CIP volume fraction and magnetic field strength. These magnetic field-induced changes in the mechanical moduli and surface roughness of MREs are key parameters for biological applications.

Magnetic field tuning of mechanical properties of ultrasoft PDMS-based magnetorheological elastomers for biological applications.

Ultrasoft magnetorheological elastomers (MREs) offer convenient real-time magnetic field control of mechanical properties that provides a means to mimic mechanical cues and regulators of cells . Here, we systematically investigate the effect of polymer stiffness on magnetization reversal of MREs using a combination of magnetometry measurements and computational modeling. Poly-dimethylsiloxane-based MREs with Young's moduli that range over two orders of magnitude were synthesized using commercial polymers Sylgard 527, Sylgard 184, and carbonyl iron powder. The magnetic hysteresis loops of the softer MREs exhibit a characteristic pinched loop shape with almost zero remanence and loop widening at intermediate fields that monotonically decreases with increasing polymer stiffness. A simple two-dipole model that incorporates magneto-mechanical coupling not only confirms that micrometer-scale particle motion along the applied magnetic field direction plays a defining role in the magnetic hysteresis of ultrasoft MREs but also reproduces the observed loop shapes and widening trends for MREs with varying polymer stiffnesses.

The effect of polymer stiffness on magnetization reversal of magnetorheological elastomers.

Engineered cardiac microtissues were fabricated using pluripotent stem cells with a hypertrophic cardiomyopathy associated c. 2827 C>T; p.R943x truncation variant in myosin binding protein C (MYBPC3). Microtissues were mounted on iron-incorporated cantilevers, allowing manipulations of cantilever stiffness using magnets, enabling examination of how in vitro afterload affects contractility. MYPBC3 microtissues developed augmented force, work, and power when cultured with increased in vitro afterload when compared with isogenic controls in which the MYBPC3 mutation had been corrected (MYPBC3(ed)), but weaker contractility when cultured with lower in vitro afterload. After initial tissue maturation, MYPBC3 CMTs exhibited increased force, work, and power in response to both acute and sustained increases of in vitro afterload. Together, these studies demonstrate that extrinsic biomechanical challenges potentiate genetically-driven intrinsic increases in contractility that may contribute to clinical disease progression in patients with HCM due to hypercontractile MYBPC3 variants.

Biomechanical Impact of Pathogenic MYBPC3 Truncation Variant Revealed by Dynamically Tuning In Vitro Afterload.

Healthy articular cartilage is a remarkable bearing material optimized for near-frictionless joint articulation. Because its limited self-repair capacity renders it susceptible to osteoarthritis (OA), approaches to reinforce or rebuild degenerative cartilage are of significant interest. While exogenous collagen crosslinking (CXL) treatments improve cartilage's mechanical properties and increase its resistance to enzymatic degradation, their effects on cartilage lubrication remain less clear. Here, we examined how the collagen crosslinking agents genipin (GP) and glutaraldehyde (GTA) impact cartilage lubrication using the convergent stationary contact area (cSCA) configuration. Unlike classical configurations, the cSCA sustains biofidelic kinetic friction coefficients (μk) via superposition of interstitial and hydrodynamic pressurization (i.e., tribological rehydration). As expected, glutaraldehyde- and genipin-mediated CXL increased cartilage's tensile and compressive moduli. Although net tribological rehydration was retained after CXL, GP or GTA treatment drastically elevated μk. Both healthy and "OA-like" cartilage (generated via enzymatic digestion) sustained remarkably low μk in saline- (≤0.02) and synovial fluid-lubricated contacts (≤0.006). After CXL, μk increased up to 30-fold, reaching values associated with marked chondrocyte death in vitro. These results demonstrate that mechanical properties (i.e., stiffness) are necessary, but not sufficient, metrics of cartilage function. Furthermore, the marked impairment in lubrication suggests that CXL-mediated stiffening is ill-suited to cartilage preservation or joint resurfacing.

Exogenous Collagen Crosslinking is Highly Detrimental to Articular Cartilage Lubrication.

Articular cartilage's remarkable low-friction properties are essential to joint function. In osteoarthritis (OA), cartilage degeneration (e.g., proteoglycan loss and collagen damage) decreases tissue modulus and increases permeability. Although these changes impair lubrication in fully depressurized and slowly slid cartilage, new evidence suggests such relationships may not hold under biofidelic sliding conditions more representative of those encountered in vivo. Our recent studies using the convergent stationary contact area (cSCA) configuration demonstrate that articulation (i.e., sliding) generates interfacial hydrodynamic pressures capable of replenishing cartilage interstitial fluid/pressure lost to compressive loading through a mechanism termed tribological rehydration. This fluid recovery sustains in vivo-like kinetic friction coefficients (µ<0.02 in PBS and <0.005 in synovial fluid) with little sensitivity to mechanical properties in healthy tissue. However, the tribomechanical function of compromised cartilage under biofidelic sliding conditions remains unknown. Here, we investigated the effects of OA-like changes in cartilage mechanical properties, modeled via enzymatic digestion of mature bovine cartilage, on its tribomechanical function during cSCA sliding. We found no differences in sliding-driven tribological rehydration behaviors or µ between naïve and digested cSCA cartilage (in PBS or synovial fluid). This suggests that OA-like cartilage retains sufficient functional properties to support naïve-like fluid recovery and lubrication under biofidelic sliding conditions. However, OA-like cartilage accumulated greater total tissue strains due to elevated strain accrual during initial load application. Together, these results suggest that elevated total tissue strains-as opposed to activity-mediated strains or friction-driven wear-might be the key biomechanical mediator of OA pathology in cartilage. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) decreases cartilage's modulus and increases its permeability. While these changes compromise frictional performance in benchtop testing under low fluid load support (FLS) conditions, whether such observations hold under sliding conditions that better represent the joints' dynamic FLS conditions in vivo is unclear. Here, we leveraged biofidelic benchtop sliding experiments-that is, those mimicking joints' native sliding environment-to examine how OA-like changes in mechanical properties effect cartilage's natural lubrication. We found no differences in sliding-mediated fluid recovery or kinetic friction behaviors between naïve and OA-like cartilage. However, OA-like cartilage experienced greater strain accumulation during load application, suggesting that elevated tissue strains (not friction-driven wear) may be the primary biomechanical mediator of OA pathology.

Enzymatic digestion does not compromise sliding-mediated cartilage lubrication.

Physiologically modeled test samples with known properties and characteristics, or phantoms, are essential for developing sensitive, repeatable, and accurate quantitative MRI techniques. Magnetic resonance elastography (MRE) is one such technique used to estimate tissue mechanical properties, and it is advantageous to use phantoms with independently tunable mechanical properties to benchmark the accuracy of MRE methods. Phantoms with tunable shear stiffness are commonly used for MRE, but tuning the viscosity or damping ratio has proven to be difficult. A promising candidate for MRE phantoms with tunable damping ratio is polyacrylamide (PAA). While pure PAA has very low attenuation, viscoelastic hydrogels have been made by entrapping linear polyacrylamide strands (LPAA) within the PAA network. In this study, we evaluate the use of LPAA/PAA gels as physiologically accurate phantoms with tunable damping ratio, independent of shear stiffness, via MRE. Phantoms were made with 15.3 wt% PAA while the LPAA concentration ranged from 4.5 wt% to 8.0 wt%. MRE was performed at 9.4 T with 400 Hz vibration on all phantoms revealing a strong, positive correlation between damping ratio and LPAA content (p < 0.001). There was no significant correlation between shear stiffness and LPAA content, confirming a constant PAA concentration yielded constant shear stiffness. Rheometry at 10 Hz was performed to verify the damping ratio of the phantoms. Nearly identical slopes for damping ratio versus LPAA content were found from both MRE and rheometry (0.0073 and 0.0075 respectively). Ultimately, this study validates the adaptation of polyacrylamide gels into physiologically-relevant MRE phantoms to enable testing of MRE estimates of damping ratio.

Article	Year
Simultaneous time-varying viscosity, elasticity, and mass measurements of single adherent cancer cells across cell cycle. Scientific reports\| PubMed ID: 32733047	2020
Extracellular stiffness induces contractile dysfunction in adult cardiomyocytes via cell-autonomous and microtubule-dependent mechanisms. Basic research in cardiology\| PubMed ID: 36006489	2022
Biomimetic Substrate to Probe Dynamic Interplay of Topography and Stiffness on Cardiac Fibroblast Activation. ACS omega\| PubMed ID: 36816659	2023
Magnetic field tuning of mechanical properties of ultrasoft PDMS-based magnetorheological elastomers for biological applications. Multifunctional materials\| PubMed ID: 36860552	2021
The effect of polymer stiffness on magnetization reversal of magnetorheological elastomers. APL materials\| PubMed ID: 36861033	2022
Biomechanical Impact of Pathogenic MYBPC3 Truncation Variant Revealed by Dynamically Tuning In Vitro Afterload. Journal of cardiovascular translational research\| PubMed ID: 36877449	2023
Exogenous Collagen Crosslinking is Highly Detrimental to Articular Cartilage Lubrication. Journal of biomechanical engineering\| PubMed ID: 38323667	2024
Enzymatic digestion does not compromise sliding-mediated cartilage lubrication. Acta biomaterialia\| PubMed ID: 38428511	2024
Viscoelastic polyacrylamide MR elastography phantoms with tunable damping ratio independent of shear stiffness. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 38537609	2024

Elise A. Corbin

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