Exogenous Collagen Crosslinking is Highly Detrimental to Articular Cartilage Lubrication.

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.

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

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.

Evidence of differential mass change rates between human breast cancer cell lines in culture.

Investigating the growth signatures of single cells will determine how cell growth is regulated and cell size is maintained. The ability to precisely measure such changes and alterations in cell size and cell mass could be important for applications in cancer and drug screening. Here, we measure the mass growth rate of individual benign (MCF-10A), non-invasive (MCF-7), and highly-invasive malignant (MDA-MB-231) breast cancer cells. A micro-patterning technique was employed to allow for the long-term growth of motile cells. Results show mass growth rates at 4.8%, 1.2%, and 2.8% for MCF-10A, MCF-7, and MDA-MB-231, demonstrating that normal cells have a higher mass growth rate than cancerous cells. All the cell lines show an increase in mass change rate indicating that the mass accumulation rate is exponential over a single cell cycle. The growth rates measured with our MEMS sensor are compared with doubling times obtained through conventional bulk analysis techniques, and exhibit excellent agreement.

Slowing DNA Transport Using Graphene-DNA Interactions.

Slowing down DNA translocation speed in a nanopore is essential to ensuring reliable resolution of individual bases. Thin membrane materials enhance spatial resolution but simultaneously reduce the temporal resolution as the molecules translocate far too quickly. In this study, the effect of exposed graphene layers on the transport dynamics of both single (ssDNA) and double-stranded DNA (dsDNA) through nanopores is examined. Nanopore devices with various combinations of graphene and Al2O3 dielectric layers in stacked membrane structures are fabricated. Slow translocations of ssDNA in nanopores drilled in membranes with layers of graphene are reported. The increased hydrophobic interactions between the ssDNA and the graphene layers could explain this phenomenon. Further confirmation of the hydrophobic origins of these interactions is obtained through reporting significantly faster translocations of dsDNA through these graphene layered membranes. Molecular dynamics simulations confirm the preferential interactions of DNA with the graphene layers as compared to the dielectric layer verifying the experimental findings. Based on our findings, we propose that the integration of multiple stacked graphene layers could slow down DNA enough to enable the identification of nucleobases.

Measuring physical properties of neuronal and glial cells with resonant microsensors.

Microelectromechanical systems (MEMS) resonant sensors provide a high degree of accuracy for measuring the physical properties of chemical and biological samples. These sensors enable the investigation of cellular mass and growth, though previous sensor designs have been limited to the study of homogeneous cell populations. Population heterogeneity, as is generally encountered in primary cultures, reduces measurement yield and limits the efficacy of sensor mass measurements. This paper presents a MEMS resonant pedestal sensor array fabricated over through-wafer pores compatible with vertical flow fields to increase measurement versatility (e.g., fluidic manipulation and throughput) and allow for the measurement of heterogeneous cell populations. Overall, the improved sensor increases capture by 100% at a flow rate of 2 µL/min, as characterized through microbead experiments, while maintaining measurement accuracy. Cell mass measurements of primary mouse hippocampal neurons in vitro, in the range of 0.1-0.9 ng, demonstrate the ability to investigate neuronal mass and changes in mass over time. Using an independent measurement of cell volume, we find cell density to be approximately 1.15 g/mL.

Enzymatic digestion does not compromise sliding-mediated 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 (µk<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 µk 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.

Viscoelastic polyacrylamide MR elastography phantoms with tunable damping ratio independent of shear stiffness.

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.

Micromechanical properties of hydrogels measured with MEMS resonant sensors.

Hydrogels have gained wide usage in a range of biomedical applications because of their biocompatibility and the ability to finely tune their properties, including viscoelasticity. The use of hydrogels on the microscale is increasingly important for the development of drug delivery techniques and cellular microenvironments, though the ability to accurately characterize their micromechanical properties is limited. Here we demonstrate the use of microelectromechanical systems (MEMS) resonant sensors to estimate the properties of poly(ethylene glycol) diacrylate (PEGDA) microstructures over a range of concentrations. These microstructures are integrated on the sensors by deposition using electrohydrodynamic jet printing. Estimated properties agree well with independent measurements made using indentation with atomic force microscopy.

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

Materials with the ability to change properties can expand the capabilities of in vitro models of biological processes and diseases as it has become increasingly clear that static, stiff materials with smooth surfaces fall short in recapitulating the in vivo cellular microenvironment. Here, we introduce a patterned material that can be rapidly stiffened and softened in situ 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 in vitro 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.

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

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.

Characterization of mass and swelling of hydrogel microstructures using MEMS resonant mass sensor arrays.

The use of hydrogels for biomedical engineering, and for the development of biologically inspired cellular systems at the microscale, is advancing at a rapid pace. Microelectromechanical system (MEMS) resonant mass sensors enable the mass measurement of a range of materials. The integration of hydrogels onto MEMS resonant mass sensors is demonstrated, and these sensors are used to characterize the hydrogel mass and swelling characteristics. The mass values obtained from resonant frequency measurements of poly(ethylene glycol)diacrylate (PEGDA) microstructures match well with the values independently verified through volume measurements. The sensors are also used to measure the influence of fluids of similar and greater density on the mass measurements of microstructures. The data show a size-dependent increase in gel mass when fluid density is increased. Lastly, volume comparisons of bulk hydrogels with a range polymer concentration (5% to 100% (v/v)) show a non-linear swelling trend.

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

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 in vitro. 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.

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

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-1. 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.

A microcantilever heater-thermometer with a thermal isolation layer for making thermal nanotopography measurements.

This paper presents a microcantilever having a microscale heater-thermometer fabricated from doped single crystal silicon that is mounted on a silicon nitride thermal isolation structure. The silicon nitride isolation structure is in turn connected to doped single crystal silicon legs. The cantilever fabrication, its characterization, and its application in thermal nanotopography measurements are presented in this work. The cantilever can reach temperatures over 600 degrees C with a heating power of 4 mW. The cantilever has a thermal resistance that exceeds 10(5) K W(-1) when away from a substrate. Making a contact-mode scan over a silicon calibration grating of height 20 nm, the cantilever has a topography reading sensitivity of 1.3 x 10(-4) nm(-1), and a topography reading resolution of about 7 pm Hz(-1/2). These performance characteristics compare extremely well to published ones for other kinds of cantilevers.

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

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.

Tunable and Reversible Substrate Stiffness Reveals a Dynamic Mechanosensitivity of Cardiomyocytes.

New directions in material applications have allowed for the fresh insight into the coordination of biophysical cues and regulators. Although the role of the mechanical microenvironment on cell responses and mechanics is often studied, most analyses only consider static environments and behavior, however, cells and tissues are themselves dynamic materials that adapt in myriad ways to alterations in their environment. Here, we introduce an approach, through the addition of magnetic inclusions into a soft poly(dimethylsiloxane) elastomer, to fabricate a substrate that can be stiffened nearly instantaneously in the presence of cells through the use of a magnetic gradient to investigate short-term cellular responses to dynamic stiffening or softening. This substrate allows us to observe time-dependent changes, such as spreading, stress fiber formation, Yes-associated protein translocation, and sarcomere organization. The identification of temporal dynamic changes on a short time scale suggests that this technology can be more broadly applied to study targeted mechanisms of diverse biologic processes, including cell division, differentiation, tissue repair, pathological adaptations, and cell-death pathways. Our method provides a unique in vitro platform for studying the dynamic cell behavior by better mimicking more complex and realistic microenvironments. This platform will be amenable to future studies aimed at elucidating the mechanisms underlying mechanical sensing and signaling that influence cellular behaviors and interactions.

Optomechanical microrheology of single adherent cancer cells.

There is a close relationship between the mechanical properties of cells and their physiological function. Non-invasive measurements of the physical properties of cells, especially of adherent cells, are challenging to perform. Through a non-contact optical interferometric technique, we measure and combine the phase, amplitude, and frequency of vibrating silicon pedestal micromechanical resonant sensors to quantify the "loss tangent" of individual adherent human colon cancer cells (HT-29). The loss tangent, a dimensionless ratio of viscoelastic energy loss and energy storage - a measure of the viscoelasticity of soft materials, obtained through an optical path length model, was found to be 1.88 ± 0.08 for live cells and 4.32 ± 0.13 for fixed cells, revealing significant changes (p < 0.001) in mechanical properties associated with estimated nanoscale cell membrane fluctuations of 3.86 ± 0.2 nm for live cells and 2.87 ± 0.1 nm for fixed cells. By combining these values with the corresponding two-degree-of-freedom Kelvin-Voigt model, we obtain the elastic stiffness and viscous loss associated with each individual cell rather than estimations from a population. The technique is unique as it decouples the heterogeneity of individual cells in our population and further refines the viscoelastic solution space.

Increased Afterload Augments Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model.

Sunitinib, a multitargeted oral tyrosine kinase inhibitor, used widely to treat solid tumors, results in hypertension in up to 47% and left ventricular dysfunction in up to 19% of treated individuals. The relative contribution of afterload toward inducing cardiac dysfunction with sunitinib treatment remains unknown. We created a preclinical model of sunitinib cardiotoxicity using engineered microtissues that exhibited cardiomyocyte death, decreases in force generation, and spontaneous beating at clinically relevant doses. Simulated increases in afterload augmented sunitinib cardiotoxicity in both rat and human microtissues, which suggest that antihypertensive therapy may be a strategy to prevent left ventricular dysfunction in patients treated with sunitinib.

Aligning Synthetic Hippocampal Neural Circuits via Self-Rolled-Up Silicon Nitride Microtube Arrays.

Directing neurons to form predetermined circuits with the intention of treating neurological disorders and neurodegenerative diseases is a fundamental goal and current challenge in neuroengineering. Until recently, only neuronal aggregates were studied and characterized in culture, which can limit information gathered to populations of cells. In this study, we use a substrate constructed of arrays of strain-induced self-rolled-up membrane 3D architectures. This results in changes in the neuronal architecture and altered growth dynamics of neurites. Hippocampal neurons from postnatal rats were cultured at low confluency (∼250 cells mm-2) on an array of transparent rolled-up microtubes (µ-tubes; 4-5 µm diameter) of varying topographical arrangements. Neurite growth on the µ-tubes was characterized and compared to controls in order to establish a baseline for alignment imposed by the topography. Compared to control substrates, neurites are significantly more aligned toward the 0° reference on the µ-tube array. Pitch (20-60 and 100 µm) and µ-tube length (30-80 µm) of array elements were also varied to investigate their impact on neurite alignment. We found that alignment was improved by the gradient pitch arrangement and with longer µ-tubes. Application of this technology will enhance the ability to construct intentional neural circuits through array design and manipulation of individual neurons and can be adapted to address challenges in neural repair, reinnervation, and neuroregeneration.

Mechanically dynamic PDMS substrates to investigate changing cell environments.

Mechanics of the extracellular matrix (ECM) play a pivotal role in governing cell behavior, such as cell spreading and differentiation. ECM mechanics have been recapitulated primarily in elastic hydrogels, including with dynamic properties to mimic complex behaviors (e.g., fibrosis); however, these dynamic hydrogels fail to introduce the viscoelastic nature of many tissues. Here, we developed a two-step crosslinking strategy to first form (via platinum-catalyzed crosslinking) networks of polydimethylsiloxane (PDMS) and then to increase PDMS crosslinking (via thiol-ene click reaction) in a temporally-controlled manner. This photoinitiated reaction increased the compressive modulus of PDMS up to 10-fold within minutes and was conducted under cytocompatible conditions. With stiffening, cells displayed increased spreading, changing from ∼1300 to 1900 µm2 and from ∼2700 to 4600 µm2 for fibroblasts and mesenchymal stem cells, respectively. In addition, higher myofibroblast activation (from ∼2 to 20%) for cardiac fibroblasts was observed with increasing PDMS substrate stiffness. These results indicate a cellular response to changes in PDMS substrate mechanics, along with a demonstration of a mechanically dynamic and photoresponsive PDMS substrate platform to model the dynamic behavior of ECM.