Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.

Effect of Geometry and Orientation on the Tensile Properties and Failure Mechanisms of Compliant Suture Joints.

Compliant sutures surrounded by stiff matrices are present in biological armors and carapaces, providing enhanced mechanical performance. Understanding the mechanisms through which these sutured composites achieve outstanding properties is key to developing engineering materials with improved strength and toughness. This article studies the impact of suture geometry and load direction on the performance of suture joints using a two-stage reactive polymer resin that enables facile photopatterning of mechanical heterogeneity within a single polymer network. Compliant sinusoidal sutures with varying geometries are photopatterned into stiff matrices, generating a modulus contrast of 2 orders of magnitude. Empirical relationships are developed connecting suture wavelength and amplitude to composite performance under parallel and perpendicular loading conditions. Results indicate that a greater suture interdigitation broadly improves composite performance when loading is applied perpendicular to suture joints but has deleterious effects when loading is applied parallel to the joint. Investigations into the failure mechanisms under perpendicular loading highlight the interplay between suture geometry and crack growth stability after damage initiation occurs. Our findings could enable a framework for engineering composites and bio-inspired structures in the future.

Multimorphic Materials: Spatially Tailoring Mechanical Properties via Selective Initiation of Interpenetrating Polymer Networks.

Access to multimaterial polymers with spatially localized properties and robust interfaces is anticipated to enable new capabilities in soft robotics, such as smooth actuation for advanced medical and manufacturing technologies. Here, orthogonal initiation is used to create interpenetrating polymer networks (IPNs) with spatial control over morphology and mechanical properties. Base catalyzes the formation of a stiff and strong polyurethane, while blue LEDs initiate the formation of a soft and elastic polyacrylate. IPN morphology is controlled by when the LED is turned "on", with large phase separation occurring for short time delays (≈1-2 min) and a mixed morphology for longer time delays (>5 min), which is supported by dynamic mechanical analysis, small angle X-ray scattering, and atomic force microscopy. Through tailoring morphology, tensile moduli and fracture toughness can be tuned across ≈1-2 orders of magnitude. Moreover, a simple spring model is used to explain the observed mechanical behavior. Photopatterning produces "multimorphic" materials, where morphology is spatially localized with fine precision (<100 µm), while maintaining a uniform chemical composition throughout to mitigate interfacial failure. As a final demonstration, the fabrication of hinges represents a possible use case for multimorphic materials in soft robotics.

Perilacunar bone tissue exhibits sub-micrometer modulus gradation which depends on the recency of osteocyte bone formation in both young adult and early-old-age female C57Bl/6 mice.

Osteocytes resorb and replace bone local to the lacunar-canalicular system (LCS). However, whether osteocyte remodeling impacts bone quality adjacent to the LCS is not understood. Further, while aging is well-established to decrease osteocyte viability and truncate LCS geometry, it is unclear if aging also decreases perilacunar bone quality. To address these questions, we employed atomic force microscopy (AFM) to generate nanoscale-resolution modulus maps for cortical femur osteocyte lacunae from young (5-month) and early-old-age (22-month) female C57Bl/6 mice. AFM-mapped lacunae were also imaged with confocal laser scanning microscopy to determine which osteocytes recently deposited bone as determined by the presence of fluorochrome labels administered 2d and 8d before euthanasia. Modulus gradation with distance from the lacunar wall was compared for labeled (i.e., bone forming) and non-labeled lacunae in both young and aged mice. All mapped lacunae showed sub-microscale modulus gradation, with peak modulus values 200-400 nm from the lacunar wall. Perilacunar modulus gradations depended on the recency of osteocyte bone formation (i.e., the presence of labels). For both ages, 2d-labeled perilacunar bone had lower peak and bulk modulus compared to non-labeled perilacunar bone. Lacunar length reduced with age, but lacunar shape and size were not strong predictors of modulus gradation. Our findings demonstrate for the first time that osteocyte perilacunar remodeling impacts bone tissue modulus, one contributor to bone quality. Given the immense scale of the LCS, differences in perilacunar modulus resulting from osteocyte remodeling activity may affect the quality of a substantial amount of bone tissue.

Permanent and reversibly programmable shapes in liquid crystal elastomer microparticles capable of shape switching.

Reversibly programmable liquid crystal elastomer microparticles (LCEMPs), formed as a covalent adaptable network (CAN), with an average diameter of 7 µm ± 2 µm, were synthesized via a thiol-Michael dispersion polymerization. The particles were programmed to a prolate shape via a photoinitiated addition-fragmentation chain-transfer (AFT) exchange reaction by activating the AFT after undergoing compression. Due to the thermotropic nature of the AFT-LCEMPs, shape switching was driven by heating the particles above their nematic-isotropic phase transition temperature (TNI). The programmed particles subsequently displayed cyclable two-way shape switching from prolate to spherical when at low or high temperatures, respectively. Furthermore, the shape programming is reversible, and a second programming step was done to erase the prolate shape by initiating AFT at high temperature while the particles were in their spherical shape. Upon cooling, the particles remained spherical until additional programming steps were taken. Particles were also programmed to maintain a permanent oblate shape. Additionally, the particle surface was programmed with a diffraction grating, demonstrating programmable complex surface topography via AFT activation.

Light-Activated Stress Relaxation, Toughness Improvement, and Photoinduced Reversal of Physical Aging in Glassy Polymer Networks.

A covalent adaptable network (CAN) with high glass transition temperature (Tg ), superior mechanical properties including toughness and ductility, and unprecedented spatio-temporally controlled dynamic behavior is prepared by introducing dynamic moieties capable of reversible addition fragmentation chain transfer (RAFT) into photoinitiated copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)-based networks. While the CuAAC polymerization yields glassy polymers composed of rigid triazole linkages with enhanced toughness, the RAFT moieties undergo bond exchange leading to stress relaxation upon light exposure. This unprecedented level of stress relaxation in the glassy state leads to numerous desirable attributes including glassy state photoinduced plasticity, toughness improvement during large deformation, and even photoinduced reversal of the effects of physical aging resulting in the rejuvenation of mechanical and thermodynamic properties in physically aged RAFT-CuAAC networks that undergo bond exchange in the glassy state. Surprisingly, when an allyl-sulfide-containing azide monomer (AS-N3 ) is used to form the network, the network exhibits up to 80% stress relaxation in the glassy state (Tg  - 45 °C) under fixed displacement. In situ activation of RAFT during mechanical loading results in a 50% improvement in elongation to break and 40% improvement in the toughness when compared to the same network without light-activation of RAFT during the tensile testing.

Controlled Growth of Polyamide Films atop Homogenous and Heterogeneous Hydrogels using Gel-Liquid Interfacial Polymerization.

Controlled growth of crosslinked polyamide (PA) thin films is demonstrated at the interface of a monomer-soaked hydrogel and an organic solution of the complementary monomer. Termed gel-liquid interfacial polymerization (GLIP), the resulting PA films are measured to be chemically and mechanically analogous to the active layer in thin film composite membranes. PA thin films are prepared using the GLIP process on both a morphologically homogeneous hydrogel prepared from poly(2-hydroxyethylmethacrylate) (PHEMA) and a phase-separated, heterogeneous hydrogel prepared from poly(acrylamide) (PAAm). Two monomer systems are examined: trimesoyl chloride (TMC) reacting with m-phenylene diamine (MPD) and TMC reacting with piperazine (PIP). Unlike the self-limiting growth behavior in TFC membrane fabrication, diffusion-limited, continuous growth of the PA films is observed, where both the thickness and roughness of the PA layers increase with reaction time. A key morphological difference is found between the two monomer systems using the GLIP process: TMC/MPD produces a ridge-and-valley surface morphology whereas TMC/PIP produces nodule/granular structures. The GLIP process represents a unique opportunity to not only explore the pore characteristics (size, spacing, and continuity) on the resulting structure and morphology of interfacially polymerized thin films, but also a method to modify the surface of (or encapsulate) hydrogels.

Monitoring Fast, Voxel-Scale Cure Kinetics via Sample-Coupled-Resonance Photorheology.

Photopolymerizable materials are the focus of extensive research across a variety of fields ranging from additive manufacturing to regenerative medicine. However, poorly understood material mechanical and rheological properties during polymerization at the relevant exposure powers and single-voxel length-scales limit advancements in part performance and throughput. Here, a novel atomic force microscopy (AFM) technique, sample-coupled-resonance photorheology (SCRPR), to locally characterize the mechano-rheological properties of photopolymerized materials on the relevant reaction kinetic timescales, is demonstrated. By coupling an AFM tip to a photopolymer and exposing the coupled region to a laser, two fundamental photopolymerization phenomena: (1) timescales of photopolymerization at high laser power and (2) reciprocity between photodose and material properties are studied. The ability to capture rapid kinetic changes occurring during polymerization with SCRPR is demonstrated. It is found that reciprocity is only valid for a finite range of exposure powers in the verification material and polymerization is highly localized in a low-diffusion system. After polymerization, in situ imaging of a single polymerized voxel is performed using material-appropriate topographic and nanomechanical modalities of the AFM while still in the as-printed environment.

Enabling Applications of Covalent Adaptable Networks.

The ability to behave in a fluidlike manner fundamentally separates thermoset and thermoplastic polymers. Bridging this divide, covalent adaptable networks (CANs) structurally resemble thermosets with permanent covalent crosslinks but are able to flow in a manner that resembles thermoplastic behavior only when a dynamic chemical reaction is active. As a consequence, the rheological behavior of CANs becomes intrinsically tied to the dynamic reaction kinetics and the stimuli that are used to trigger those, including temperature, light, and chemical stimuli, providing unprecedented control over viscoelastic properties. CANs represent a highly capable material that serves as a powerful tool to improve mechanical properties and processing in a wide variety of polymer applications, including composites, hydrogels, and shape-memory polymers. This review aims to highlight the enabling material properties of CANs and the applied fields where the CAN concept has been embraced.

A readily programmable, fully reversible shape-switching material.

Liquid crystalline (LC) elastomers (LCEs) enable large-scale reversible shape changes in polymeric materials; however, they require intensive, irreversible programming approaches in order to facilitate controllable actuation. We have implemented photoinduced dynamic covalent chemistry (DCC) that chemically anneals the LCE toward an applied equilibrium only when and where the light-activated DCC is on. By using light as the stimulus that enables programming, the dynamic bond exchange is orthogonal to LC phase behavior, enabling the LCE to be annealed in any LC phase or in the isotropic phase with various manifestations of this capability explored here. In a photopolymerizable LCE network, we report the synthesis, characterization, and exploitation of readily shape-programmable DCC-functional LCEs to create predictable, complex, and fully reversible shape changes, thus enabling the literal square peg to fit into a round hole.

Publisher Correction: Bistable and photoswitchable states of matter.

The original version of this Article contained errors in Fig. 3. In Fig. 3a, the word 'fluid' in grey was incorrectly given as 'solid' in green, below that, 'solid' in green was previously 'fluid' in grey. Also, the label on the arrow incorrectly read 'TMG (1 mol%) HABI-Cl (3 mol%) 455 nm, 1 min'; the correct version reads 'TMG (1 mol%) HABI-O-n-oct (4 mol%) 455 nm, 4 min'. In the accompanying legend, the word 'photobase' was originally incorrectly given as 'photoacid'. Additionally, in Fig. 3b, the label on the central image was 'As is: solid', rather than the correct 'As is: fluid'. This has been corrected in both the PDF and HTML versions of the Article.

Bistable and photoswitchable states of matter.

Classical materials readily switch phases (solid to fluid or fluid to gas) upon changes in pressure or heat; however, subsequent reversion of the stimulus returns the material to their original phase. Covalently cross-linked polymer networks, which are solids that do not flow when strained, do not change phase even upon changes in temperature and pressure. However, upon the addition of dynamic cross-links, they become stimuli responsive, capable of switching phase from solid to fluid, but quickly returning to the solid state once the stimulus is removed. Reported here is the first material capable of a bistable switching of phase. A permanent solid to fluid transition or vice versa is demonstrated at room temperature, with inherent, spatiotemporal control over this switch in either direction triggered by exposure to light.

Mechanochemical Effects on Extracellular Signal-Regulated Kinase Dynamics in Stem Cell Differentiation.

Understanding how key signaling molecules are coregulated by biochemical agents and physical stimuli during stem cell differentiation is critical but often lacking. Due to the important role of extracellular signal-regulated kinase (ERK), this study has examined its temporal dynamics to determine the coregulation of mechanochemical cues on ERK phosphorylation for smooth muscle cell (SMC) differentiation. To assess ERK1/2 activity, a fluorescence resonance energy transfer-based biosensor was transfected into mesenchymal stem cells. The influences of nanopatterned substrates, growth factors, and drugs on ERK activities were related to their effects on SMC differentiation. Results revealed that nanopatterned substrates significantly increased ERK activity in cells, overriding ERK response from administered biochemical factors. The nanopatterned substrates reduced expression of SMC markers after a 48-h biochemical treatment, except for the combination with ERK inhibitor PD98059 treatment, which enhanced expression of mature SMC marker MYH11. Immunofluorescent staining for focal adhesion proteins, vinculin and zyxin, indicated no significant differences in vinculin cluster distribution or dimension, while the location of zyxin changed from adhesion sites of cell periphery on nonpatterned substrate to actin filaments on nanopatterned substrate. The zyxin-reinforced stress fibers likely enhanced the cytoskeletal tension to increase ERK dynamics. Collectively, results suggest that physical stimuli play a dominating role in initial ERK signaling and early-stage differentiation through focal adhesion changes, and the capability of monitoring signaling events in real time could be exploited to guide the engineering of cell microenvironment.

Implementation of two distinct wavelengths to induce multistage polymerization in shape memory materials and nanoimprint lithography.

Here, a process is introduced for forming dual stage thiol-Michael/acrylate hybrid networks photocured by two different wavelengths, demonstrating its use in nanoimprint lithography (NIL) and shape memory materials. Initiated with a visible light sensitive photobase and a UV-sensitive radical initiator, thiol-Michael-acrylate hybrid polymerizations were programmed to proceed sequentially and orthogonally, with base-catalyzed thiol-Michael photopolymerization as the first stage and radical mediated acrylate photopolymerization as the second stage. By regulating the photopolymerization formulations, i.e. thiol-to-acrylate ratios, initiator loadings and irradiation conditions, a series of materials with highly tunable mechanical performance was achieved, with ultimate Tg values ranging from 23 to 70 °C. With a photopatternable first stage and a readily reconfigurable second stage, its implementation in nanoimprint lithography (NIL) enabled surface features on the scale of 10 nm to be formed on a photopatterned substrate. Additionally, the dual stage polymer results in a relatively homogenous polymer network with a narrow glass transition temperature (Tg), which enables rapid response in applications as shape memory materials, with shape-fixity values above 95% and shaperecovery values above 99%. With its unique photocuring process and programmable mechanical properties, the two color light controlled photopolymerization can be exploited as a useful tool in a wide range of materials science applications.

Dynamic Covalent Chemistry at Interfaces: Development of Tougher, Healable Composites through Stress Relaxation at the Resin-Silica Nanoparticles Interface.

The interfacial region in composites that incorporate filler materials of dramatically different modulus relative to the resin phase acts as a stress concentrator and becomes a primary locus for composite failure. A novel adaptive interface (AI) platform formed by coupling moieties capable of dynamic covalent chemistry (DCC) is introduced to the resin-filler interface to promote stress relaxation. Specifically, silica nanoparticles (SNP) are functionalized with a silane capable of addition fragmentation chain transfer (AFT), a process by which DCC-active bonds are reversibly exchanged upon light exposure and concomitant radical generation, and copolymerized with a thiol-ene resin. At a fixed SNP loading of 25 wt%, the toughness (2.3 MJ m-3) is more than doubled and polymerization shrinkage stress (0.4 MPa) is cut in half in the AI composite relative to otherwise identical composites that possess a passive interface (PI) with similar silane structure, but without the AFT moiety. In situ activation of the AI during mechanical loading results in 70% stress relaxation and three times higher fracture toughness than the PI control. When interfacial DCC was combined with resin-based DCC, the toughness was improved by 10 times relative to the composite without DCC in either the resin or at the resin-filler interface.

Influence of support-layer deformation on the intrinsic resistance of thin film composite membranes.

It is commonly believed that the overall permeation resistance of thin film composite (TFC) membranes is dictated by the crosslinked, ultrathin polyamide barrier layer, while the porous support merely serves as the mechanical support. Although this assumption might be the case under low transmembrane pressure, it becomes questionable under high transmembrane pressure. A highly porous support normally yields under a pressure of a few MPa, which can result in a significant level of compressive strain that may significantly increase the resistance to permeation. However, quantifying the influence of porous support deformation on the overall resistance of the TFC membrane is challenging. In particular, it is difficult to determine the deformation/strain of the membrane during active separation. In this study, we use nanoimprint lithography (NIL) to achieve precise compressive deformation in commercial TFC membranes. By adjusting the NIL conditions, membranes were compressed to strain levels up to 60%. SEM and AFM measurements showed that the compression had minimal impact on the barrier-layer surface morphology and total surface area with most of the deformation occurring in the support layer. DI water permeation measurements revealed that the water flux reduction decreases with an increase of strain level. Most significantly, the intrinsic membrane resistance showed negligible changes at strain levels lower than 30%-40%, but increased exponentially at higher strain levels, reaching 250%-500% of pristine (unstrained) membrane values. Using a resistance-in-series model, the strain dependency of the TFC membrane resistance can be described.

Formation of a Crack-Free, Hybrid Skin Layer with Tunable Surface Topography and Improved Gas Permeation Selectivity on Elastomers Using Gel-Liquid Infiltration Polymerization.

Surface modifications of elastomers and gels are crucial for emerging applications such as soft robotics and flexible electronics, in large part because they provide a platform to control wettability, adhesion, and permeability. Current surface modification methods via ultraviolet-ozone (UVO) and/or O2 plasma, atomic layer deposition (ALD), plasmas deposition, and chemical treatment impart a dense polymer or inorganic layer on the surface that is brittle and easy to fracture at low strain levels. This paper presents a new method, based on gel-liquid infiltration polymerization, to form hybrid skin layers atop elastomers. The method is unique in that it allows for control of the skin layer topography, with tunable feature sizes and aspect ratios as high as 1.8 without fracture. Unlike previous techniques, the skin layer formed here dramatically improves the barrier properties of the elastomer, while preserving skin layer flexibility. Moreover, the method is versatile and likely applicable to most interfacial polymerization systems and network polymers on flat and patterned surfaces.

Light-Stimulated Permanent Shape Reconfiguration in Cross-Linked Polymer Microparticles.

Reconfiguring the permanent shape of elastomeric microparticles has been impossible due to the incapability of plastic deformation in these materials. To address this limitation, we synthesize the first instance of microparticles comprising a covalent adaptable network (CAN). CANs are cross-linked polymer networks capable of reconfiguring their network topology, enabling stress relaxation and shape changing behaviors, and reversible addition-fragmentation chain transfer (RAFT) is the corresponding dynamic chemistry used in this work to enable CAN-based microparticles. Using nanoimprint lithography to apply controllable deformations we demonstrate that upon light stimulation microparticles are able to reconfigure their shape to permanently fix large aspect ratios and nanoscale surface topographies.

Influences of Substrate Adhesion and Particle Size on the Shape Memory Effect of Polystyrene Particles.

Formulations and applications of micro- and nanoscale polymer particles have proliferated rapidly in recent years, yet knowledge of their mechanical behavior has not grown accordingly. In this study, we examine the ways that compressive strain, substrate surface energy, and particle size influence the shape memory cycle of polystyrene particles. Using nanoimprint lithography, differently sized particles are programmed into highly deformed, temporary shapes in contact with substrates of differing surface energies. Atomic force microscopy is used to obtain in situ measurements of particle shape recovery kinetics, and scanning electron microscopy is employed to assess differences in the profiles of particles at the conclusion of the shape memory cycle. Finally, finite element models are used to investigate the growing impact of surface energies at smaller length scales. Results reveal that the influence of substrate adhesion on particle recovery is size-dependent and can become dominating at submicron length scales.

Morphing metal-polymer janus particles.

The direct deformation and shape recovery of micron-sized polystyrene particles via nanoimprint lithography is reported. The recovery of the programmed PS particles can be utilized to create a range of smart Janus particles with contrasting properties in conductivity and topography, by use of metal-layer constrained recovery.