Harnessing 3D Printing and Electrospinning for Multiscale Hybrid Patches Mimicking the Native Myocardium.

Engineered cardiac tissues show potential for regenerative therapy in ischemic heart disease. Yet, selection of soft biomaterials for scaffold manufacturing is primarily influenced by empirical and compositional factors, raising concerns about arrhythmic risks due to poor electrophysiological integration. Addressing this, we developed multiscale hybrid myocardial patches mimicking native myocardium's structural and biomechanical attributes, utilizing 3D printing and electrospinning techniques. We compared three patch types: pure silicone and silicone-poly(lactic-co-glycolic acid) (PLGA) with random (S-PLGA-R) and aligned (S-PLGA-A) fibers. S-PLGA-A patches with fiber orientation angles of 95-115° are achieved by applying a secondary electrical field using two parallel aluminum enhancers. With bulk and localized moduli of 350-750 and 13-20 kPa resembling the native myocardium, S-PLGA-A patches demonstrate a sarcomere length of 2.1 ± 0.2 µm, ≥50% higher strain motions and diastolic phase, and a 50-70% slower rise of calcium handling compared to the other two patches. This enhanced maturation and improved synchronization phenomena are attributed to efficient force transmission and reduced stress concentration due to mechanical similarity and linear propagation of electrical signals. This study presents a promising strategy for advancing regenerative cardiac therapies by harnessing the capabilities of 3D printing and electrospinning, providing a proof-of-concept for their effectiveness.

Frictional resistance and delamination mechanisms in 2D tungsten diselenide revealed by multi-scale scratch andin-situobservations.

Friction phenomena in two-dimensional (2D) materials are conventionally studied at atomic length scales in a few layers using low-load techniques. However, the advancement of 2D materials for semiconductor and electronic applications requires an understanding of friction and delamination at a few micrometers length scale and hundreds of layers. To bridge this gap, the present study investigates frictional resistance and delamination mechanisms in 2D tungsten diselenide (WSe2) at 10µm length and 100-500 nm depths using an integrated atomic force microscopy (AFM), high-load nanoscratch, andin-situscanning electron microscopic (SEM) observations. AFM revealed a heterogenous distribution of frictional resistance in a single WSe2layer originating from surface ripples, with the mean increasing from 8.7 to 79.1 nN as the imposed force increased from 20 to 80 nN. High-loadin-situnano-scratch tests delineated the role of the individual layers in the mechanism of multi-layer delamination under an SEM. Delamination during scratch consists of stick-slip motion with friction force increasing in each successive slip, manifested as increasing slope of lateral force curves with scratch depth from 10.9 to 13.0 (× 103) Nm-1. Delamination is followed by cyclic fracture of WSe2layers where the puckering effect results in adherence of layers to the nanoscratch probe, increasing the local maximum of lateral force from 89.3 to 205.6µN. This establishment of the interconnectedness between friction in single-layer and delamination at hundreds of layers harbors the potential for utilizing these materials in semiconductor devices with reduced energy losses and enhanced performance.

Quantum Dots on a String: In Situ Observation of Branching and Reinforcement Mechanism of Electrospun Fibers.

Immobilization of quantum dots (QDs) on fiber surfaces has emerged as a robust approach for preserving their functional characteristics while mitigating aggregation and instability issues. Despite the advancement, understanding the impacts of QDs on jet-fiber evolution during electrospinning, QDs-fiber interface, and composites functional behavior remains a knowledge gap. The study adopts a high-speed imaging methodology to capture the immobilization effects on the QDs-fiber matrix. In situ observations reveal irregular triangular branches within the QDs-fiber matrix, exhibiting distinctive rotations within a rapid timeframe of 0.00667 ms. The influence of FeQDs on Taylor cone dynamics and subsequent fiber branching velocities is elucidated. Synthesis phenomena are correlated with QD-fiber's morphology, crystallinity, and functional properties. PAN-FeQDs composite fibers substantially reduced (50-70%) nano-fibrillar length and width while their diameter expanded by 17%. A 30% enhancement in elastic modulus and reduction in adhesion force for PAN-FeQDs fibers is observed. These changes are attributed to chemical and physical intertwining between the FeQDs and the polymer matrix, bolstered by the shifts in the position of C≡N and C═C bonds. This study provides valuable insights into the quantum dot-fiber composites by comprehensively integrating and bridging jet-fiber transformation, fiber structure, nanomechanics, and surface chemistry.

Improving the development of human engineered cardiac tissue by gold nanorods embedded extracellular matrix for long-term viability.

A myocardial infarction (MI), commonly called a heart attack, results in the death of cardiomyocytes (CMs) in the heart. Tissue engineering provides a promising strategy for the treatment of MI, but the maturation of human engineered cardiac tissue (hECT) still requires improvement. Conductive polymers and nanomaterials have been incorporated into the extracellular matrix to enhance the mechanical and electrical coupling between cardiac cells. Here we report a simple approach to incorporate gold nanorods (GNRs) into the fibrin hydrogel to form a GNR-fibrin matrix, which is used as the major component of the extracellular matrix for forming a 3D hECT construct suspended between two flexible posts. The hECTs made with GNR-fibrin hydrogel showed markers of maturation such as higher twitch force, synchronous beating activity, sarcomere maturation and alignment, t-tubule network development, and calcium handling improvement. Most importantly, the GNR-hECTs can survive over 9 months. We envision that the hECT with GNRs holds the potential to restore the functionality of the infarcted heart.

Freeze casting to engineer gradient porosity in hydroxyapatite-boron nitride nanotube composite scaffold for improved compressive strength and osteogenic potential.

Graded porosity plays a crucial role in scaffolds for bone tissue engineering as it facilitates vital processes such as nutrient diffusion, cellular infiltration, and tissue integration. This paper explores the utilization of freeze casting (FC) as a technique to generate composite scaffolds comprising hydroxyapatite (HA) reinforced with 1D-boron nitride nanotubes (BNNTs) featuring graded porosity and improved compressive strength. Comparative studies were conducted using FC at room and sub-zero temperatures to assess the influence of temperature gradient and heat transfer rate on the production of gradient and aligned porosity in HA-BNNT composites. The FC process with a prolonged thermal gradient facilitated the creation of aligned pores in the HA-BNNT, exhibiting a wide distribution of 60% porosity ranging from 1 to 30 µm. Adding high strength 1 vol% BNNT reinforcement resulted in a remarkable 50% enhancement in compressive strength compared to the control sample. Osteoblasts seeded on the HA-BNNT substrate exhibited significantly higher alkaline phosphate activity, indicating accelerated mineralization compared to the control sample. Gradient porosity and wide pore distribution in the HA-BNNT scaffolds promoted osteogenic activities. Overall, the demonstrated FC processing technique and BNNT addition hold great potential for developing functional and biomimetic scaffolds that can effectively promote tissue regeneration, leading to improved clinical outcomes in bone tissue engineering applications.

Electrically and Thermally Triggered Three-Dimensional Graphene-Foam-Reinforced Shape Memory Epoxy Composites.

Shape memory polymer (SMP) epoxy composites have attracted significant attention due to their easy processing, lightweight nature, and ability to recover strain. However, their limited recovery rate and inferior mechanical properties have hindered their functional applications. This research explores the potential of three-dimensional (3D) graphene foam (GrF) as a highly efficient reinforcement for SMP epoxy composites. We demonstrated that the incorporation of a mere 0.13 wt.% GrF into mold-cast SMP epoxy leads to a 19% increase in the glass transition temperature (Tg). To elucidate the reinforcing mechanism, we fabricated and extensively analyzed composites with varying weight percentages of GrF. The GrF-based SMP epoxy composite exhibits a 57% increase in thermal conductivity, measuring 0.296 W mK-1 at 70 °C, due to the interconnected 3D graphene network within the matrix. Notably, this composite also demonstrates remarkable electrical conductivity, making it suitable for dual-triggering applications. The GrF-SMP epoxy composite achieves a maximum shape recovery ratio and a significant 23% improvement in the recovery rate, effectively addressing the issue of slow recovery associated with SMPs. We investigated the effect of switching temperatures on the shape recovery rate. We identified the optimal triggering temperature to initiate shape recovery for epoxy SMP and GrF-epoxy SMP as thermal energy equivalent to Tg + 20 °C. Additionally, we fabricated a bird-shaped composite using GrF reinforcement, which showcases self-healing capabilities through the crack opening and closure and serves as a tangible demonstration of the transformative potential of the composite. These GrF-epoxy SMP composites, responsive to stimuli, hold immense promise for diverse applications, such as mechanical systems, wearable sensors, morphing wings, foldable robots, and antennas.

Understanding spatiotemporal mechanical behavior, viscoelasticity, and functions of stem cell-derived cardiomyocytes.

Understanding myocytes' spatiotemporal mechanical behavior and viscoelasticity is a long-standing challenge as it plays a critical role in regulating structural and functional homeostasis. To probe the time-dependent viscoelastic behaviors of cardiomyocytes with cross-linked polymer networks, we measure stem cell-derived cardiomyocyte's (hiPSC-CM) deformation, adhesion, and contractility using atomic force microscopy (AFM) nanoindentation, fluidic micropipette, and digital image correlation (DIC). Our results show a cytoplasm load of 7-14 nN, a de-adhesion force of 0.1-1 nN, and an adhesion force between two hiPSC-CMs of 50-100 nN with an interface energy of 0.45 pJ. Based on the load-displacement curve, we model its dynamic viscoelasticity and discover its intimate associations with physiological properties. Cell detaching and contractile modeling demonstrate cell-cell adhesion and beating related strains manifesting viscoelastic behavior, highlighting viscoelasticity plays the primary role in governing hiPSC-CM spatiotemporal mechanics and functions. Overall, this study provides valuable information about the mechanical properties, adhesion behaviors, and viscoelasticity of single hiPSC-CM, shedding light on mechanical-structure relationships and their dynamic responses to mechanical stimuli and spontaneous contraction.

Bridging the Gap in Ashby's Map for Soft Material Properties for Tissue Engineering.

Ashby's map's role in rationally selecting materials for optimal performance is well-established in traditional engineering applications. However, there is a major gap in Ashby's maps in selecting materials for tissue engineering, which are very soft with an elastic modulus of less than 100 kPa. To fill the gap, we create an elastic modulus database to effectively connect soft engineering materials with biological tissues such as the cardiac, kidney, liver, intestine, cartilage, and brain. This soft engineering material mechanical property database is created for widely applied agarose hydrogels based on big-data screening and experiments conducted using ultra-low-concentration (0.01-0.5 wt %) hydrogels. Based on that, an experimental and analysis protocol is established for evaluating the elastic modulus of ultra-soft engineering materials. Overall, we built a mechanical bridge connecting soft matter and tissue engineering by fine-tuning the agarose hydrogel concentration. Meanwhile, a soft matter scale (degree of softness) is established to enable the manufacturing of implantable bio-scaffolds for tissue engineering.

High throughput screening system for engineered cardiac tissues.

Introduction: Three dimensional engineered cardiac tissues (3D ECTs) have become indispensable as in vitro models to assess drug cardiotoxicity, a leading cause of failure in pharmaceutical development. A current bottleneck is the relatively low throughput of assays that measure spontaneous contractile forces exerted by millimeter-scale ECTs typically recorded through precise optical measurement of deflection of the polymer scaffolds that support them. The required resolution and speed limit the field of view to at most a few ECTs at a time using conventional imaging. Methods: To balance the inherent tradeoff among imaging resolution, field of view and speed, an innovative mosaic imaging system was designed, built, and validated to sense contractile force of 3D ECTs seeded on a 96-well plate. Results: The system performance was validated through real-time, parallel contractile force monitoring for up to 3 weeks. Pilot drug testing was conducted using isoproterenol. Discussion: The described tool increases contractile force sensing throughput to 96 samples per measurement; significantly reduces cost, time and labor needed for preclinical cardiotoxicity assay using 3D ECT. More broadly, our mosaicking approach is a general way to scale up image-based screening in multi-well formats.

Dynamic Control of Contractile Force in Engineered Heart Tissue.

Three-dimensional engineered heart tissues (EHTs) derived from human induced pluripotent stem cells (iPSCs) have become an important resource for both drug toxicity screening and research on heart disease. A key metric of EHT phenotype is the contractile (twitch) force with which the tissue spontaneously beats. It is well-known that cardiac muscle contractility - its ability to do mechanical work - depends on tissue prestrain (preload) and external resistance (afterload). OBJECTIVES: Here, we demonstrate a technique to control afterload while monitoring contractile force exerted by EHTs. METHODS: We developed an apparatus that can regulate EHT boundary conditions using real-time feedback control. The system is comprised of a pair of piezoelectric actuators that can strain the scaffold and a microscope that can measure EHT force and length. Closed loop control allows dynamic regulation of effective EHT boundary stiffness. RESULTS: When controlled to switch instantaneously from auxotonic to isometric boundary conditions, EHT twitch force immediately doubled. Changes in EHT twitch force as a function of effective boundary stiffness were characterized and compared to twitch force in auxotonic conditions. CONCLUSION: EHT contractility can be regulated dynamically through feedback control of effective boundary stiffness. SIGNIFICANCE: The capacity to alter the mechanical boundary conditions of an engineered tissue dynamically offers a new way to probe tissue mechanics. This could be used to mimic afterload changes that occur naturally in disease, or to improve mechanical techniques for EHT maturation.

Membrane Remodeling of Human-Engineered Cardiac Tissue by Chronic Electric Stimulation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show immature features, but these are improved by integration into 3D cardiac constructs. In addition, it has been demonstrated that physical manipulations such as electrical stimulation (ES) are highly effective in improving the maturation of human-engineered cardiac tissue (hECT) derived from hiPSC-CMs. Here, we continuously applied an ES in capacitive coupling configuration, which is below the pacing threshold, to millimeter-sized hECTs for 1-2 weeks. Meanwhile, the structural and functional developments of the hECTs were monitored and measured using an array of assays. Of particular note, a nanoscale imaging technique, scanning ion conductance microscopy (SICM), has been used to directly image membrane remodeling of CMs at different locations on the tissue surface. Periodic crest/valley patterns with a distance close to the sarcomere length appeared on the membrane of CMs near the edge of the tissue after ES, suggesting the enhanced transverse tubulation network. The SICM observation is also supported by the fluorescence images of the transverse tubulation network and α-actinin. Correspondingly, essential cardiac functions such as calcium handling and contraction force generation were improved. Our study provides evidence that chronic subthreshold ES can still improve the structural and functional developments of hECTs.

Localized Nanoindentation Paradigm for Revealing Sutured Tissue Interface Mechanics and Integrity.

This study investigates the nanoindentation technique to elucidate the quasi-static and dynamic stress response at the wounded and sutured tissue interface. In vitro modeling and wound healing analysis enable an understanding of sutured tissue interface integrity, modulus, and stability using an artificial abdominal wall model. Sutured tissues with simple interrupted suturing (SIS) demonstrated a 35-40% higher modulus than simple continuous suturing (SCS). High-density suturing with a suture space of 2.5 mm exhibited a 2-fold higher modulus than low-density suturing with a suture space of 5 mm. The elastic modulus of the sutured pad immersed in deionized water was ∼70-95% of the dry condition. The dynamic stress data indicate that long-term body motions-triggered stress instability at the wound interface was affected by suturing style and density. The pivotal factors determining wound healing are quasi-static and dynamic modulus at the sutured interface, which is intimately associated with patient pain, wound complications, healing speed, and blood flow. The proposed method and data are an original approach to addressing wound healing, contributing to patient well-being and identifying, interpreting, and breaking the drawn-out debates in the suturing field.

Revealing nanomechanical deformation at the interface and degradation in all-thin-film inorganic electrochromic devices.

Recently, progress in electrochromic (EC) devices has been made in optimizing electrode and device configurations and performance. However, the ion insertion/de-insertion induced charge transfer (CT) nanomechanical effect has remained unexplored, i.e., repetitive electrode size changes at the nanoscale and stress/strain generated during electrochemical cycling, which is the focus of this work due to its intimate correlation with the elastic and plastic deformation at the interface. Considering the intervalence electrons, excellent electrochemical kinetics, and dramatic color changes, tungsten oxide (WO3) and nickel oxide (NiO) films are configured as the EC cathode and anode materials, respectively, within a full device. Upon extended cycles (>10 000), the void generation and delamination that occurred at the interface account for performance decay. Encouraged by the findings, nanoindentation mechanical tests and electrical kelvin probe force microscopy were employed to investigate the CT induced effects at the interface. There is a dramatic increase of up to 45% in the elastic Young's modulus in colored/charged WO3 at ∼40 mC cm-2. The correlation between CT and synergistic mechanical effect is interpreted by the Lippman equation. Interestingly, despite the charged state (colored; lithiated) with a relatively flat morphology bringing an ∼3.4 times higher electrostatic surface potential, the electrical work function unexpectedly decreases, arising from the dominant effect of the dipole layer potential over the chemical potential. The interatomic cohesive energy and equilibrium distance increase bury the seeds for mechanical deformation in the long run. This work provides fundamental insights into electro-chemo mechanics and interdisciplinary concerted interfacial effects at the nano/atomic level. The dependence of surface potential, stress, work function, and cohesive energy on electrochemical kinetics has been interpreted.

Corrosion Behavior of 1D and 2D Polymorphs of Boron Nitride Ceramic.

This study reports a fundamental electrochemical study to understand the corrosion behavior of 1D bulk, free-standing 1D boron nanotube (BNNT) buckypaper and compare it with a sintered 2D hBN nanoplatelet (BNNP) pellet. Tafel analysis indicates that 1D BNNT has superior corrosion resistance with a lower corrosion rate of 0.0026 mils per year (mpy). 2D BNNP, although having similar chemistry to 1D BNNT, resulted in an increased (40 times) corrosion rate of 0.107 mpy. The higher surface area and aspect ratio of BNNT drastically influenced the corrosion kinetics. The scientific outcomes will enable the better design of novel hBN-based corrosion-resistant materials.

Direct Observation of Adhesion and Mechanical Behavior of a Single Poly(lactic-co-glycolic acid) (PLGA) Fiber Using an In Situ Technique for Tissue Engineering.

Nanometer- and submicrometer-sized fiber have been used as scaffolds for tissue engineering, because of their fundamental load-bearing properties in synergy with mechano-transduction. This study investigates a single biodegradable poly(lactic-co-glycolic acid) (PLGA) fiber's load-displacement behavior utilizing the nanoindentation technique coupled with a high-resolution in situ imaging system. It is demonstrated that a maximum force of ∼3 µN in the radial direction and displacement of at least 150% of fiber diameter should be applied to acquire the fiber's macroscopic mechanical properties for tissue engineering. The adhesion behavior of a single fiber is captured using a high-resolution camera. The digital image correlation (DIC) technique is adopted to quantify the adhesion force (∼25 µN) between the fiber and the tip. Adhesion force has also been quantified for the fiber after immersing in phosphate-buffered saline (PBS) to mimic the bioenvironment. A 4-fold increase in adhesion force after PBS treatment was observed due to water penetration and hydrolysis on the fiber's surface. A high similarity between mechanical properties of a single fiber and native tissues (elastic modulus of 10-25 kPa) and superior adhesion force (25-107.25 µN) was observed, which is excellent for promoting cell-matrix communication. Overall, this study examines the mechanics of a single fiber using innovative indentation and imaging processing techniques, disclosing its profound and striking roles in tissue engineering.

Directional dependence on concomitant pressure and volume increases during left ventricular filling.

Myocardial infarction continues to be a leading cause of mortality and morbidity globally. A major challenge post-myocardial infarction is scar tissue growth, which eventually can lead to heart failure. Cardiovascular regenerative strategies to minimize scar tissue growth and promote cardiac tissue formation are currently being actively pursued via the development of cardiac patches. However, the patch must have viscoelastic properties that mimic healthy cardiac tissues to facilitate proper cardiac patch-to-cell communications. To this end, we investigated the tissue microstructure and the stress relaxation properties of the porcine left ventricle (LV) along its long and short axes using a nanoindentation technique. We found significantly higher collagen density along the long axis than the short axis (p < 0.05). We then identified a much more rapid stress relaxation along the porcine LV's short axis compared to its long axis during the diastolic filling timeframe. Therefore, these findings show that concomitant LV pressure and volume increases from blood filling during diastole are directional dependent, with its short axis responsible for increase in LV volume and the long axis responsible for increase in LV pressure. These directional-dependent stress relaxation properties are essential in the design of structurally, bio-mimetic cardiac patches to support cardiac function and regeneration.

Engineering a living cardiac pump on a chip using high-precision fabrication.

Biomimetic on-chip tissue models serve as a powerful tool for studying human physiology and developing therapeutics; however, their modeling power is hindered by our inability to develop highly ordered functional structures in small length scales. Here, we demonstrate how high-precision fabrication can enable scaled-down modeling of organ-level cardiac mechanical function. We use two-photon direct laser writing (TPDLW) to fabricate a nanoscale-resolution metamaterial scaffold with fine-tuned mechanical properties to support the formation and cyclic contraction of a miniaturized, induced pluripotent stem cell-derived ventricular chamber. Furthermore, we fabricate microfluidic valves with extreme sensitivity to rectify the flow generated by the ventricular chamber. The integrated microfluidic system recapitulates the ventricular fluidic function and exhibits a complete pressure-volume loop with isovolumetric phases. Together, our results demonstrate a previously unexplored application of high-precision fabrication that can be generalized to expand the accessible spectrum of organ-on-a-chip models toward structurally and biomechanically sophisticated tissue systems.

Meso/macro-scale ultra-soft materials' mechanical property evaluation device and testbed.

Ultra-soft materials find applications in biomedical devices, sensors and actuators, robotics, and wearable electronics. The mechanical properties of soft materials are often determined using nanoindentation and atomic force microscope techniques, which provide localized properties at a small-scale length. There is a need to evaluate the meso/macro-scale properties of ultra-soft materials to develop integrated devices made of the same. Metallic probes in the existing macroscale equipment cannot be used as they can pierce through the soft materials and fail to capture the associated adhesion forces, resulting in inaccurate values. This study has developed a meso/macro-scale mechanical testing platform to characterize ultra-soft materials accurately. This probe can be adapted to any mechanical testing load frame with a small load cell to capture the adhesion forces during the approach and detachment segments of soft materials' indentation. The integrated camera with the probe enables overcoming the challenge of surface detection and capturing the pull-on and pull-off events. Indentation tests on soft materials with varying stiffness (e.g., high-fat yogurt, chicken breast, aloe Vera, toothpaste, gelatin, and a chocolate bar) were conducted using a 10 mm stiff flat-end polymer probe. A variation of the Johnson-Kendall-Roberts technique was adopted to account for adhesion forces and compute stiffness. Our results suggest that the novel device and methodology can measure mechanical stiffness in the extensive range of 0.5 kPa to a few MPa with high reproducibility at the macro-scale length. The validation was carried out using a commercially available nanoindenter for soft materials.

A Facile and Scalable Approach in the Fabrication of Tailored 3D Graphene Foam via Freeze Drying.

One of the challenges in the processing of advanced composite materials with 2D reinforcement is their extensive agglomeration in the matrix. 3D architecture of 2D graphene sheets into a Graphene Foam (GrF) assembly has emerged as an effective way to overcome agglomeration. The highly reticulated network of branches and nodes of GrF offers a seamless pathway for photon and electron conduction in the matrix along with improved mechanical properties. 3D GrF nano-filler is often fabricated by chemical vapor deposition (CVD) technique, which demands high energy, slow deposition rate, and restricting production to small scale. This work highlights freeze-drying (FD) technique to produce 3D graphene nanoplatelets (GNP) foam with a similar hierarchical structure to the CVD GrF. The FD technique using water as the main chemical in 3D GNP foam production is an added advantage. The flexibility of the FD in producing GNP foams of various pore size and morphology is elucidated. The simplicity with which one can engineer thermodynamic conditions to tailor the pore shape and morphology is presented here by altering the GNP solid loading and mold geometry. The FD 3D GNP foam is mechanically superior to CVD GrF as it exhibited 1280 times higher elastic modulus. However, thermal diffusivity of the FD GNP foam is almost 0.5 times the thermal diffusivity of the CVD GrF due to the defects in GNP particles and pore architecture. The versatility in GNP foam scalability and compatibility to form foam of other 1D and 2D material systems (e.g., carbon nanotubes, boron nitride nanotubes, and boron nitride nanoplatelets) brings a unique dimensionality to FD as an advanced engineering foam development process.

A Method to Quantify Tensile Biaxial Properties of Mouse Aortic Valve Leaflets.

Understanding aortic valve (AV) mechanics is crucial in elucidating both the mechanisms that drive the manifestation of valvular diseases as well as the development of treatment modalities that target these processes. Genetically modified mouse models have become the gold standard in assessing biological mechanistic influences of AV development and disease. However, very little is known about mouse aortic valve leaflet (MAVL) tensile properties due to their microscopic size (∼500 µm long and 45 µm thick) and the lack of proper mechanical testing modalities to assess uniaxial and biaxial tensile properties of the tissue. We developed a method in which the biaxial tensile properties of MAVL tissues can be assessed by adhering the tissues to a silicone rubber membrane utilizing dopamine as an adhesive. Applying equiaxial tensile loads on the tissue-membrane composite and tracking the engineering strains on the surface of the tissue resulted in the characteristic orthotropic response of AV tissues seen in human and porcine tissues. Our data suggest that the circumferential direction is stiffer than the radial direction (n = 6, P = 0.0006) in MAVL tissues. This method can be implemented in future studies involving longitudinal mechanical stimulation of genetically modified MAVL tissues bridging the gap between cellular biological mechanisms and valve mechanics in popular mouse models of valve disease.