Atomic Force Microscopy Nanoindentation Research Articles

Mediation of Cartilage Matrix Degeneration and Fibrillation by Decorin in Post-traumatic Osteoarthritis.

To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (Dcni KO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1β (IL-1β) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1β and release of sGAGs were quantified. In both Dcn-/- and Dcni KO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1β, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Non-destructive quantitative analysis of nano-mechanics of aged Xuan paper

Xuan paper, also named rice paper, was invented in the Tang Dynasty around 700 AD. Due to its softness, flexibility, fine texture and excellent durability, Xuan paper has been one of the most carriers for a large number of extraordinary artworks since its invention. The mechanical properties of Xuan paper will be varied over time, which has a significant impact on its application and preservation. In this study, for the first time, we employed atomic force microscopy nanoindentation measurement, a simple and nondestructive technique to quantitatively analyze the nanomechanical properties of Xuan paper. Combined with other characterizations, the property of Xuan paper under different aging times was investigated. It is expected that our results should lead to a better understanding of Xuan Paper as ancient materials with superior properties. The current results should provide useful information on the identification and protection of paper-based artifacts.

A Laboratory Model for Virus Particle Nanoindentation

ABSTRACT Self-organization is ubiquitous in biology, with viruses providing an excellent illustration of bioassemblies being much more than the sum of their parts. Following nature's lead, molecular self-assembly has emerged as a new synthetic strategy in the past 3 decades or so. Self-assembly approaches promise to generate complex supramolecular architectures having molecular weights of 0.5 to 100 MDa and collective properties determined by the interplay between structural organization and composition. However, biophysical methods specific to mesoscopic self-assembly, and presentations of the challenges they aim to overcome, remain underrepresented in the educational laboratory curriculum. We present here a simple but effective model for laboratory instruction that introduces students to the world of intermolecular forces and virus assembly, and to a cutting-edge technology, atomic force microscopy nanoindentation, which is able to measure the mechanical properties of single virus shells in vitro. In addition, the model illustrates the important idea that, at nanoscale, phenomena often have an inherent interdisciplinary character.

Effect of AFM Nanoindentation Loading Rate on the Characterization of Mechanical Properties of Vascular Endothelial Cell.

Vascular endothelial cells form a barrier that blocks the delivery of drugs entering into brain tissue for central nervous system disease treatment. The mechanical responses of vascular endothelial cells play a key role in the progress of drugs passing through the blood–brain barrier. Although nanoindentation experiment by using AFM (Atomic Force Microscopy) has been widely used to investigate the mechanical properties of cells, the particular mechanism that determines the mechanical response of vascular endothelial cells is still poorly understood. In order to overcome this limitation, nanoindentation experiments were performed at different loading rates during the ramp stage to investigate the loading rate effect on the characterization of the mechanical properties of bEnd.3 cells (mouse brain endothelial cell line). Inverse finite element analysis was implemented to determine the mechanical properties of bEnd.3 cells. The loading rate effect appears to be more significant in short-term peak force than that in long-term force. A higher loading rate results in a larger value of elastic modulus of bEnd.3 cells, while some mechanical parameters show ambiguous regulation to the variation of indentation rate. This study provides new insights into the mechanical responses of vascular endothelial cells, which is important for a deeper understanding of the cell mechanobiological mechanism in the blood–brain barrier.

Anodic Aluminum Oxide Nanopore Template-Assisted Fabrication of Nanostructured Poly(vinyl alcohol) Hydrogels for Cell Studies.

The effect of systematically varied mechanical properties and nano- and microscale surface topography on the adhesion and proliferation of human pancreatic cancer cells on fibronectin-functionalized poly(vinyl alcohol) (PVA) hydrogels was studied to understand the impact of these properties of the cell microenvironment on cell attachment and spreading. The mechanical properties of PVA, as assessed by atomic force microscopy (AFM) nanoindentation, were varied by the number of freezing-thawing cycles in the physical cross-linking process used for the generation of the hydrogels. Nano- and micropatterned hydrogel surfaces exposing nanosized PVA pillars and cuboids were fabricated by replicating ordered cylindrical nanopores of anodic aluminum oxide (AAO) and poly(dimethylsiloxane) (PDMS) templates, respectively. Softer PVA hydrogels, functionalized covalently with fibronectin, showed enhanced cell adhesion and proliferation of PaTu 8988t cells in comparison to stiffer hydrogels. In addition, PaTu 8988t cells favored the nanopatterned surfaces over micropatterned and flat hydrogels.

Atomic Simulation of Nanoindentation on the Regular Wrinkled Graphene Sheet.

Surface landscapes have vague impact on the mechanical properties of graphene. In this paper, single-layered graphene sheets (SLGS) with regular wrinkles were first constructed by applying shear deformation using molecular dynamics (MD) simulations and then indented to extract their mechanical properties. The influence of the boundary condition of SLGS were considered. The wrinkle features and wrinkle formation processes of SLGS were found to be significantly related to the boundary conditions as well as the applied shear displacement and velocity. The wrinkling amplitude and degree of wrinkling increased with the increase in the applied shear displacements, and the trends of wrinkling wavelengths changed with the different boundary conditions. With the fixed boundary condition, the degree of graphene wrinkling was only affected when the velocity was greater than a certain value. The effect of wrinkles on the mechanical characterization of SLGS by atomic force microscopy (AFM) nanoindentation was finally investigated. The regular surface wrinkling of SLGS was found to weaken the Young’s modulus of graphene. The Young’s modulus of graphene deteriorates with the increase in the degree of regular wrinkling.

Electrical and Elastic Properties of Individual Single‐Layer Nb4C3Tx MXene Flakes

Abstract2D carbides and nitrides (MXenes) are widely recognized for their exceptional promise for numerous applications. However, physical property measurements of their individual monolayers remain very limited despite their importance for revealing the intrinsic physical properties of MXenes. The first mechanical and electrical measurements of individual single‐layer flakes of Nb4C3Tx MXene, which are prepared via an improved synthetic method are reported. Characterization of field‐effect transistor devices based on individual single‐layer Nb4C3Tx flakes shows an electrical conductivity of 1024 ± 165 S cm−1, which is two orders of magnitude higher than the previously reported values for bulk Nb4C3Tx assemblies, and an electron mobility of 0.41 ± 0.27 cm2 V−1 s−1. Atomic force microscopy nanoindentation measurements of monolayer Nb4C3Tx membranes yield an effective Young's modulus of 386 ± 13 GPa, assuming a membrane thickness of 1.26 nm. This is the highest value reported for nanoindentation measurements of solution‐processable 2D materials, revealing the potential of Nb4C3Tx as a primary component for various mechanical applications. Finally, the agreement between the mechanical properties of 2D Nb4C3Tx MXene and cubic NbC suggests that the extensive experimental data on bulk carbides could be useful for identifying new MXenes with improved functional characteristics.

A Soft Mechanical Phenotype of SH-SY5Y Neuroblastoma and Primary Human Neurons Is Resilient to Oligomeric Aβ(1-42) Injury.

Aggregated amyloid beta (Aβ) is widely reported to cause neuronal dystrophy and toxicity through multiple pathways: oxidative stress, disrupting calcium homeostasis, and cytoskeletal dysregulation. The neuro-cytoskeleton is a dynamic structure that reorganizes to maintain cell homeostasis in response to varying soluble and physical cues presented from the extracellular matrix (ECM). Due this relationship between cell health and the ECM, we hypothesize that amyloid toxicity may be directly influenced by physical changes to the ECM (stiffness and dimensionality) through mechanosensitive pathways, and while previous studies demonstrated that Aβ can distort focal adhesion signaling with pathological consequences, these studies do not address the physical contribution from a physiologically relevant matrix. To test our hypothesis that physical cues can adjust Aβ toxicity, SH-SY5Y human neuroblastoma and primary human cortical neurons were plated on soft and stiff, 2D polyacrylamide matrices or suspended in 3D collagen gels. Each cell culture was exposed to escalating concentrations of oligomeric or fibrillated Aβ(1-42) with MTS viability and lactate dehydrogenase toxicity assessed. Actin restructuring was further monitored in live cells by atomic force microscopy nanoindentation, and our results demonstrate that increasing either matrix stiffness or exposure to oligomeric Aβ promotes F-actin polymerization and cell stiffening, while mature Aβ fibrils yielded no apparent cell stiffening and minor toxicity. Moreover, the rounded, softer mechanical phenotype displayed by cells plated onto a compliant matrix also demonstrated a resilience to oligomeric Aβ as noted by a significant recovery of viability when compared to same-dosed cells plated on traditional tissue culture plastic. This recovery was reproduced pharmacologically through inhibiting actin polymerization with cytochalasin D prior to Aβ exposure. These studies indicate that the cell-ECM interface can modify amyloid toxicity in neurons and the matrix-mediated pathways that promote this protection may offer unique targets in amyloid pathologies like Alzheimer's disease.

Nano-indentation reveals a potential role for gradients of cell wall stiffness in directional movement of the resurrection plant Selaginella lepidophylla

As a physical response to water loss during drought, inner Selaginella lepidophylla stems curl into a spiral shape to prevent photoirradiation damage to their photosynthetic surfaces. Curling is reversible and involves hierarchical deformation, making S. lepidophylla an attractive model with which to study water-responsive actuation. Investigation at the organ and tissue level has led to the understanding that the direction and extent of stem curling can be partially attributed to stiffness gradients between adaxial and abaxial stem sides at the nanoscale. Here, we examine cell wall elasticity to understand how it contributes to the overall stem curling. We compare the measured elastic moduli along the stem length and between adaxial and abaxial stem sides using atomic force microscopy nano-indentation testing. We show that changes in cortex secondary cell wall development lead to cell wall stiffness gradients from stem tip to base, and also between adaxial and abaxial stem sides. Changes in cortical cell wall morphology and secondary cell wall composition are suggested to contribute to the observed stiffness gradients.

Multi-scale mechanical investigation of articular cartilage suffered progressive pseudorheumatoid dysplasia

BackgroundProgressive pseudorheumatoid dysplasia is a rare skeletal dysplasia mainly caused by abnormal autosomal recessive inheritance. Although the main function of cartilage is mechanical support and the characteristics of this disease is the degradation of AC, previous studies on it had been mainly focused on clinical and genetic aspects and the mechanical behavior of the cartilage affected by PPRD is still ambiguous. In this study, we investigate the mechanics and structure of the cartilage suffered disease at multi-scale, from individual chondrocytes to the bulk-scale tissue. MethodsDepth-sensing indenter were employed to investigate the mechanics of cartilage; we performed atomic force microscope nanoindentation to investigate the cell mechanics and scanning electron microscopy were used to explore the structure feature and chemical composition. FindingsThe elastic modulus of chondrocytes harvested from cartilage suffered from progressive pseudorheumatoid dysplasia is significantly higher than from normal cartilage, same trend were also found in tissue level. Moreover, denser collagen meshwork and matrix calcification were also observed. InterpretationThe elastic modulus of cartilage should closely related to its denser structure and the calcification, and may potentially be an indicator for clinical diagnosis. The stiffening of chondrocytes during PPRD progression should play a rather important role in its pathogenesis.

Linearization of Recurrent-Neural-Network- Based Models for Predictive Control of Nano-Positioning Systems Using Data-Driven Koopman Operators

Recent studies have shown that the nonlinear dynamics of nano-positioning systems (e.g., piezo-electric actuators (PEAs)) can be accurately captured by recurrent neural networks (RNNs). One direct application of this technique is PEA system control for precision positioning: linearize the nonlinear RNN model and then apply model predictive control (MPC). However, due to the linearization approach commonly used (e.g., Taylor series), the control bandwidth and the control performance are quite limited as the obtained linear system is only guaranteed to be accurate within small neighborhood of the linearization point. To address this issue, we propose a Koopman operator-based approach for linearization and then use the obtained linear parameter-varying model for predictive control. This linearization scheme can significantly decrease the overall approximation error within the MPC prediction horizon, and thus, lead to improved tracking performance. The proposed approach was validated through two applications-trajectory tracking of PEA, and deformation control of polymers during atomic force microscope nano-indentation.

X-ray Tomography, AFM and Nanoindentation Measurements for Recyclability Analysis of 316L Powders in 3D Printing Process

Recyclability of the leftover metallic powder within the Additive manufacturing (3D printing) process has been less systematically investigated by the research groups although it is a usual practice in most academic and industrial laboratories to reuse the leftover powders for subsequent printing cycles. A better understanding of these mechanisms will assist in optimizing the number of times the recycled powder can be reused in the process to reduce the powder waste. We have recently focused on characterization of recycled powders left in the powder bed after the powder bed fusion process and evaluated the extent of porosity in the powder particles. X-ray computing tomography technique (XCT) has been used to analyze the concentration of porosity, inclusions and dendrites induced inside the recycled powder particles and compared that to the fresh counterparts. The XCT resolution of 2 µm was set to separately scan the powder badges for 3 hours. A roughly 10% more porosity has been calculated in reused powder particles (in at least 10 times reused power). Atomic Force Microscopy (AFM) was used to measure the roughness of the surface of powder particles which shows average roughness of 4.29 nm and 5.49 nm for the virgin and recycled powders, respectively. Nanoindentation measurement were also applied on a number of locations of the particles to compare the hardness of the virgin and recycled powders. For example, the recycled powder shows smaller a hardness of 207 GPa and an effective modulus of 9.60 GPa (average values) compared to 237 GPa and 9.87 GPa (average values) for it’s virgin counterpart which can be correlated to porosities created beneath the surface. Nanoindentation was also applied on (micro and nano) polished surface of the particles under a force of 250 µN for up to 10 seconds. The stainless Steel 316L powder has been the material under study with the powder particles of average size 50 µm which were analyzed using Xradia XCT, Bruker Dimension ICON AFM and Bruker HYSITRON TI Nanoindentation systems. Further investigation is ongoing to correlate the mechanical properties of the manufactured parts to the microstructure and chemical compositions of the virgin and recycled powders.

AFM nanoindentation reveals decrease of elastic modulus of lipid bilayers near freezing point of water

Cell lipid membranes are the primary site of irreversible injury during freezing/thawing and cryopreservation of cells, but the underlying causes remain unknown. Here, we probe the effect of cooling from 20 °C to 0 °C on the structure and mechanical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using atomic force microscopy (AFM) imaging and AFM-based nanoindentation in a liquid environment. The Young’s modulus of elasticity (E) at each temperature for DPPC was obtained at different ionic strengths. Both at 20 mM and 150 mM NaCl, E of DPPC bilayers increases exponentially –as expected–as the temperature is lowered between 20 °C and 5 °C, but at 0 °C E drops from the values measured at 5 °C. Our results support the hypothesis that mechanical weakening of the bilayer at 0 °C is produced by structural changes at the lipid-fluid interface.

Determination of the linear elastic regime in AFM nanoindentation experiments on cells

Atomic force microscopy nanoindentation method has been extensively used for the determination of cells’ mechanical properties. The data processing is usually performed using contact mechanics models at the linear elastic regime such as the Hertz model. It should be noted that the cells’ response can be considered as linearly elastic only for a limited range of indentation depths. Although there are in literature proposed values of the maximum allowed nanoindentation depth for most of the cell types, these values have been mostly derived empirically. As a result, the use of these empirically obtained findings can result in significant errors regarding Young’s modulus calculations. A large number of measurements, using spherical and conical indenters, showed errors in the range of 4−10% with respect to the Young’s modulus calculation. In this paper, a new mathematical approach is proposed that enables the identification of the optimum load - indentation data range for the application of the Hertz contact theory. The results presented in this paper indicate that the optimum choice of the maximum indentation depth can vary greatly even in the same sample. For example, 64 load indentation curves on the same cell using a spherical indenter showed that the maximum indentation depth is (553 ± 43)nm (mean value ± standard deviation). As a consequence, the determination of a specific maximum indentation depth for indentation experiments on a specific cell will lead to significant errors regarding Young’s modulus values. Hence, the selection of the linear elastic regime must be performed individually for each load—indentation curve to accurately determine the cell’s mechanical response.

Fabrication and Characterization of Pectin Hydrogel Nanofiber Scaffolds for Differentiation of Mesenchymal Stem Cells into Vascular Cells.

Despite significant progress over the past few decades, creating a tissue-engineered vascular graft with replicated functions of native blood vessels remains a challenge due to the mismatch in mechanical properties, low biological function, and rapid occlusion caused by restenosis of small diameter vessel grafts (<6 mm diameter). A scaffold with similar mechanical properties and biocompatibility to the host tissue is ideally needed for the attachment and proliferation of cells to support the building of engineered tissue. In this study, pectin hydrogel nanofiber scaffolds with two different oxidation degrees (25 and 50%) were prepared by a multistep methodology including periodate oxidation, electrospinning, and adipic acid dihydrazide crosslinking. Scanning electron microscopy (SEM) images showed that the obtained pectin nanofiber mats have a nano-sized fibrous structure with 300-400 nm fiber diameter. Physicochemical property testing using Fourier transform infrared (FTIR) spectra, atomic force microscopy (AFM) nanoindentations, and contact angle measurements demonstrated that the stiffness and hydrophobicity of the fiber mat could be manipulated by adjusting the oxidation and crosslinking levels of the pectin hydrogels. Live/Dead staining showed high viability of the mesenchymal stem cells (MSCs) cultured on the pectin hydrogel fiber scaffold for 14 days. In addition, the potential application of pectin hydrogel nanofiber scaffolds of different stiffness in stem cell differentiation into vascular cells was assessed by gene expression analysis. Real-time polymerase chain reaction (RT-PCR) results showed that the stiffer scaffold facilitated the differentiation of MSCs into vascular smooth muscle cells, while the softer fiber mat promoted MSC differentiation into endothelial cells. Altogether, our results indicate that the pectin hydrogel nanofibers have the capability of providing mechanical cues that induce MSC differentiation into vascular cells and can be potentially applied in stem cell-based tissue engineering.

Co-Blend Application Mode of Bulk Fill Composite Resin.

Objective: To evaluate the effect of a new application method of bulk-fill flowable composite resin material on bond-strength, nanoleakage, and mechanical properties of dentine bonding agents. Materials and methods: Sound extracted human molars were randomly divided into: manufacturer’s instructions (MI), manual blend 2 mm (MB2), and manual blend 4 mm (MB4). Occlusal enamel was removed and flattened, dentin surfaces were bonded by Prime & Bond universal (Dentsply and Optibond FL, Kerr). For the MI group, adhesives were applied following the manufacturer’s instructions then light-cured. For MB groups, SDR flow+ bulk-fill flowable composite resin was applied in 2- or 4-mm increment then manually rubbed by a micro brush for 15 s with uncured dentine bonding agents and the mixture was light-cured. Composite buildup was fabricated incrementally using Ceram.X One, Dentsply nanohybrid composite resin restorative material. After 24-h water storage, the teeth were sectioned to obtain beams of about 0.8 mm2 for 24-h and thermocycled micro-tensile bond strength at 0.5 mm/min crosshead speed. Degree of conversion was evaluated with micro-Raman spectroscopy. Contraction gaps at 24 h after polymerization were evaluated and atomic force microscopy (AFM) nano-indentation processes were undertaken for measuring the hardness across the interface. Depth of resin penetration was studied using a scanning electron microscope (SEM). Bond strength data was expressed using two-way ANOVA followed by Tukey’s test. Nanoindentation hardness was separately analyzed using one-way ANOVA. Results: Factors “storage F = 6.3” and “application F = 30.11” significantly affected the bond strength to dentine. For Optibond FL, no significant difference in nanoleakage was found in MI/MB4 groups between baseline and aged specimens; significant difference in nanoleakage score was observed in MB2 groups. Confocal microscopy analysis showed MB2 Optibond FL and Prime & Bond universal specimens diffusing within the dentine. Contraction gap was significantly reduced in MB2 specimens in both adhesive systems. Degree of conversion (DC) of the MB2 specimens were numerically more compared to MS1 in both adhesive systems. Conclusion: Present study suggests that the new co-blend technique might have a positive effect on bond strengths of etch-and-rinse adhesives to dentine.

Nanometer-precision non-local deformation reconstruction using nanodiamond sensing

Spatially resolved information about material deformation upon loading is critical to evaluating mechanical properties of materials, and to understanding mechano-response of live systems. Existing techniques may access local properties of materials at nanoscale, but not at locations away from the force-loading positions. Moreover, interpretation of the local measurement relies on correct modeling, the validation of which is not straightforward. Here we demonstrate an approach to evaluating non-local material deformation based on the integration of nanodiamond orientation sensing and atomic force microscopy nanoindentation. This approach features a 5 nm precision in the loading direction and a sub-hundred nanometer lateral resolution, high enough to disclose the surface/interface effects in the material deformation. The non-local deformation profile can validate the models needed for mechanical property determination. The non-local nanometer-precision sensing of deformation facilitates studying mechanical response of complex material systems ranging from impact transfer in nanocomposites to mechano-response of live systems.

A simplified approach for the determination of fitting constants in Oliver–Pharr method regarding biological samples

The atomic force microscopy (AFM) nanoindentation regarding biological samples is a challenging procedure. Biological samples at the nanoscale can be considered as purely elastic materials under the condition that the indentation depth is very small and the indenter is smooth. However, the indenters that are commonly used are pyramidal and in several cases the indentation depths are big comparing to the dimensions of the tip apex. Hence, pyramidal indenters usually cause a permanent damage to the sample. In this case, the best model that can be applied for the data processing is the Oliver–Pharr model which takes into account the elastic–plastic behavior of the sample. The Oliver–Pharr model is based on the fitting of the unloading load—indentation data to a power law equation. In this paper a simplified procedure which ensures the accurate fitting of the unloading load—indentation data to the Oliver–Pharr model is presented and validated on experimental data obtained from a human glioma cell line. It should be noted that the proposed method can be also applied for the data fitting in the case of purely elastic response.

A minimal continuum representation of a transverse isotropic viscoelastic pulp fibre based on micromechanical measurements

We propose a minimum input mechanical model of pulp fibres. A pulp fibre can be idealised as a complex compound bar, microfibrils render its behaviour anisotropic. The objective of this work is twofold: (i) develop an anisotropic viscoelastic material model of a pulp fibre that shows the essential characteristics with a minimum number of material parameters and (ii) establish suitable experiments with pulp fibres for obtaining these parameters.The model consists of an additive combination of elastic potentials for an isotropic and an anisotropic part. Viscoelasticity is implemented by a generalised Maxwell model. We suggest an incompressible treatment of the material to relate experiments and material parameters in a well-defined manner. By this means, the calibration of the parameters is performed with only two micromechanical experiments, atomic force microscopy nanoindentation on the fibre’s surface and uniaxial tensile tests. We advocate this strategy for certain applications, such as in paper network simulations or fibres in a dry state, when the fibre’s compressibility is of minor importance. The results of this analysis show a good performance of the proposed model applied to pulp fibres.

Facile tuning of the mechanical properties of a biocompatible soft material

Herein, we introduce a method to locally modify the mechanical properties of a soft, biocompatible material through the exploitation of the effects induced by the presence of a local temperature gradient. In our hypotheses, this induces a concentration gradient in an aqueous sodium alginate solution containing calcium carbonate particles confined within a microfluidic channel. The concentration gradient is then fixed by forming a stable calcium alginate hydrogel. The process responsible for the hydrogel formation is initiated by diffusing an acidic oil solution through a permeable membrane in a 2-layer microfluidic device, thus reducing the pH and freeing calcium ions. We characterize the gradient of mechanical properties using atomic force microscopy nanoindentation measurements for a variety of material compositions and thermal conditions. Significantly, our novel approach enables the creation of steep gradients in mechanical properties (typically between 10–100 kPa/mm) on small scales, which will be of significant use in a range of tissue engineering and cell mechanosensing studies.