Results of an interlaboratory study on the working curve in vat photopolymerization.

The working curve informs resin properties and print parameters for stereolithography, digital light processing, and other photopolymer additive manufacturing (PAM) technologies. First demonstrated in 1992, the working curve measurement of cure depth vs radiant exposure of light is now a foundational measurement in the field of PAM. Despite its widespread use in industry and academia, there is no formal method or procedure for performing the working curve measurement, raising questions about the utility of reported working curve parameters. Here, an interlaboratory study (ILS) is described in which 24 individual laboratories performed a working curve measurement on an aliquot from a single batch of PAM resin. The ILS reveals that there is enormous scatter in the working curve data and the key fit parameters derived from it. The measured depth of light penetration Dp varied by as much as 7x between participants, while the critical radiant exposure for gelation Ec varied by as much as 70x. This significant scatter is attributed to a lack of common procedure, variation in light engines, epistemic uncertainties from the Jacobs equation, and the use of measurement tools with insufficient precision. The ILS findings highlight an urgent need for procedural standardization and better hardware characterization in this rapidly growing field.

Thermomechanical And Morphological Properties of Loligo Vulgaris Squid Sucker Ring Teeth.

Climate change is accelerating the increase of temperatures across the planet and resulting in the warming of oceans. Ocean warming threatens the survival of many aquatic species, including squids, and has introduced physiological, behavioral, and developmental changes, as well as physical changes in their biological materials composition, structure, and properties. Here, we characterize and analyze how the structure, morphology, and mechanical properties of European common squid Loligo vulgaris sucker ring teeth (SRT) are affected by temperature. SRT are predatory teethed structures located inside the suction cups of squids, which are used to capture prey, and are composed of semicrystalline structural proteins that give rise to high mechanical strength (GPa-range modulus). We observe here that this biological material reversibly softens with temperature, undergoing a glass transition at ∼35°C, to a MPa-range modulus. We analyze the SRT protein nanostructures as a function of temperature, as well as microscale and macroscale morphological changes, to understand their impact in the material properties. The results suggest that even small deviations from their habitat temperatures can result in significant softening of the material (up to 40% in modulus loss). Temperature changes following recent global climate trends and predictions might affect environmental adaptation in squid species, and pose emerging survival challenges to adapt to increasing ocean temperatures.

Programmable Tissue Folding Patterns in Structured Hydrogels.

Folding of mucosal tissues, such as the tissue within the epithelium of the upper respiratory airways, is critical for organ function. Studying the influence of folded tissue patterns on cellular function is challenging mainly due to the lack of suitable cell culture platforms that can recreate dynamic tissue folding in vitro. Here, a bilayer hydrogel folding system, composed of alginate/polyacrylamide double-network (DN) and hyaluronic acid (HA) hydrogels, to generate static folding patterns based on mechanical instabilities, is described. By encapsulating human fibroblasts into patterned HA hydrogels, human bronchial epithelial cells form a folded pseudostratified monolayer. Using magnetic microparticles, DN hydrogels reversibly fold into pre-defined patterns and enable programmable on-demand folding of cell-laden hydrogel systems upon applying a magnetic field. This hydrogel construction provides a dynamic culture system for mimicking tissue folding in vitro, which is extendable to other cell types and organ systems.

Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel.

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances. STATEMENT OF SIGNIFICANCE: Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.

Rapid magnetically directed assembly of pre-patterned capillary-scale microvessels.

Capillary scale vascularization is critical to the survival of engineered 3D tissues and remains an outstanding challenge for the field of tissue engineering. Current methods to generate micro-scale vasculature such as 3D printing, two photon hydrogel ablation, angiogenesis, and vasculogenic assembly face challenges in rapidly creating organized, highly vascularized tissues at capillary length-scales. Within metabolically demanding tissues, native capillary beds are highly organized and densely packed to achieve adequate delivery of nutrients and oxygen and efficient waste removal. Here, we adopt two existing techniques to fabricate lattices composed of sacrificial microfibers that can be efficiently and uniformly seeded with endothelial cells (ECs) by magnetizing both lattices and ECs. Ferromagnetic microparticles (FMPs) were incorporated into microfibers produced by solution electrowriting (SEW) and fiber electropulling (FEP). By loading ECs with superparamagnetic iron oxide nanoparticles (SPIONs), the cells could be seeded onto magnetized microfiber lattices. Following encapsulation in a hydrogel, the capillary templating lattice was selectively degraded by a bacterial lipase that does not impact mammalian cell viability or function. This work introduces a novel approach to rapidly producing organized capillary networks within metabolically demanding engineered tissue constructs which should have broad utility for the fields of tissue engineering and regenerative medicine.

Plasticized liquid crystal networks and chemical motors for the active control of power transmission in mechanical devices.

The miniaturization of mechanical devices poses new challenges in powering, actuation, and control since traditional approaches cannot be used due to inherent size limitations. This is particularly challenging in untethered small-scale machines where independent actuation of multicomponent and multifunctional complex systems is required. This work showcases the integration of self-powered chemical motors and liquid crystal networks into a powertrain transmission device to achieve orthogonal untethered actuation for power and control. Driving gears with a protein-based chemical motor were used to power the transmission system with Marangoni propulsive forces, while photothermal liquid crystal networks were used as a photoresponsive clutch to engage/disengage the gear system. Liquid crystal networks were plasticized for optimized photothermal bending actuation to break the surface tension of water and achieve reversible immersion/resurfacing at the air-water interface. This concept is demonstrated in a milliscale transmission gear system and offers potential solutions for aquatic soft robots whose powering and control mechanisms must be necessarily decoupled.

Transparent and Self-Healing Elastomers for Reconfigurable 3D Materials.

Transparent soft materials are widely used in applications ranging from packaging to flexible displays, wearable devices, and optical lenses. Nevertheless, soft materials are susceptible to mechanical damage, leading to functional failure and premature disposal. Herein, a transparent self-healing elastomer that is able to repair the polymer network via exchange reactions of dynamic disulfide bonds is introduced. Due to its self-healing ability, the mechanical properties of the elastomer can be recovered as well as its transparency after multiple cycles of abrasion and healing. The self-healing polymer is fabricated into 3D structures by folding or modular origami assembly of planar self-healing polymer sheets. The 3D polymer objects are employed as storage containers of solid and liquid substances, reactors for photopolymerization, and cuvettes for optical measurements (exhibiting superior properties to those of commercial cuvettes). These dynamic polymers show outstanding mechanical, optical, and recycling properties that could potentially be further adapted in adaptive smart packaging, reconfigurable materials, optical devices, and recycling of elastomers.

Functional Chemical Motor Coatings for Modular Powering of Self-Propelled Particles.

Inspired by the locomotion of semiaquatic insects, a variety of surface swimming microrobots propelled by surface tension Marangoni forces have been developed over the years. However, most Marangoni micromotor systems present limitations in their applications due to poor performance, short lifetime, low efficiency, and toxicity. We have developed a functional chemical motor coating consisting of protein microfilms with entrapped fuel to functionalize inactive substrates or particles. This motor material system generates large Marangoni propulsive forces with extremely small amounts of fuel due to a self-regulated fuel release mechanism based on dynamic nanostructural changes in the protein matrix, enhancing the lifetime and efficiency performance over other material systems and motors. These motor functional coatings offer great versatility as they can be coated on a wide array of substrates and materials across length scales, with opportunities as modular power sources for microrobots and small-scale devices. The synergy between the protein motor matrix and the chemical fuel enables the wider design of self-powered surface microrobots without previous limitations in their fabrication and performance, including the new design of hybrid microrobots with protein functional coatings as a modular power source.

Diffusive Dynamic Modes of Recombinant Squid Ring Teeth Proteins by Neutron Spectroscopy.

Stimuli-responsive structural proteins are emerging as promising biocompatible materials for a wide range of biological and nonbiological applications. To understand the physical properties of structural proteins and to replicate their performance in biosynthetic systems, there is a need to understand the molecular mechanisms and relationships that regulate their structure, dynamics, and properties. Here, we study the dynamics of a recombinant squid-inspired protein from Loligo vulgaris (Lv18) by elastic and quasielastic neutron scattering (QENS) to understand the connection between nanostructure, chain dynamics, and mechanical properties. Lv18 is a semicrystalline structural protein, which is plasticized by water above its glass transition temperature at 35 °C. Elastic scans revealed an increased protein chain mobility upon hydration, superimposed dynamic processes, and a decrease in dynamic transition temperatures. Further analysis by QENS revealed that while dry Lv18 protein dynamics are dominated by localized methyl group rotations, hydrated Lv18 dynamics are dominated by the confined diffusion of flexible chains within a ß-sheet nanocrystalline network (8 Å of confinement radius). Our findings establish a relationship between the segment block architecture of Lv18, the diffusive motions within the protein structure, and the mechanical properties of recombinant squid proteins, which will advance the molecular design of novel high-performance protein-inspired materials with tailored dynamics and mechanical properties.

Soft actuators for real-world applications.

Inspired by physically adaptive, agile, reconfigurable and multifunctional soft-bodied animals and human muscles, soft actuators have been developed for a variety of applications, including soft grippers, artificial muscles, wearables, haptic devices and medical devices. However, the complex performance of biological systems cannot yet be fully replicated in synthetic designs. In this Review, we discuss new materials and structural designs for the engineering of soft actuators with physical intelligence and advanced properties, such as adaptability, multimodal locomotion, self-healing and multi-responsiveness. We examine how performance can be improved and multifunctionality implemented by using programmable soft materials, and highlight important real-world applications of soft actuators. Finally, we discuss the challenges and opportunities for next-generation soft actuators, including physical intelligence, adaptability, manufacturing scalability and reproducibility, extended lifetime and end-of-life strategies.

High shear rate propulsion of acoustic microrobots in complex biological fluids.

Untethered microrobots offer a great promise for localized targeted therapy in hard-to-access spaces in our body. Despite recent advancements, most microrobot propulsion capabilities have been limited to homogenous Newtonian fluids. However, the biological fluids present in our body are heterogeneous and have shear rate-dependent rheological properties, which limit the propulsion of microrobots using conventional designs and actuation methods. We propose an acoustically powered microrobotic system, consisting of a three-dimensionally printed 30-micrometer-diameter hollow body with an oscillatory microbubble, to generate high shear rate fluidic flow for propulsion in complex biofluids. The acoustically induced microstreaming flow leads to distinct surface-slipping and puller-type propulsion modes in Newtonian and non-Newtonian fluids, respectively. We demonstrate efficient propulsion of the microrobots in diverse biological fluids, including in vitro navigation through mucus layers on biologically relevant three-dimensional surfaces. The microrobot design and high shear rate propulsion mechanism discussed herein could open new possibilities to deploy microrobots in complex biofluids toward minimally invasive targeted therapy.

Liquid-Crystal-Elastomer-Actuated Reconfigurable Microscale Kirigami Metastructures.

Programmable actuation of metastructures with predesigned geometrical configurations has recently drawn significant attention in many applications, such as smart structures, medical devices, soft robotics, prosthetics, and wearable devices. Despite remarkable progress in this field, achieving wireless miniaturized reconfigurable metastructures remains a challenge due to the difficult nature of the fabrication and actuation processes at the micrometer scale. Herein, microscale thermo-responsive reconfigurable metasurfaces using stimuli-responsive liquid crystal elastomers (LCEs) is fabricated as an artificial muscle for reconfiguring the 2D microscale kirigami structures. Such structures are fabricated via two-photon polymerization with sub-micrometer precision. Through rationally designed experiments guided by simulations, the optimal formulation of the LCE artificial muscle is explored and the relationship between shape transformation behaviors and geometrical parameters of the kirigami structures is build. As a proof of concept demonstration, the constructs for temperature-dependent switching and information encryption is applied. Such reconfigurable kirigami metastructures have significant potential for boosting the fundamental small-scale metastructure research and the design and fabrication of wireless functional devices, wearables, and soft robots at the microscale as well.

Task space adaptation via the learning of gait controllers of magnetic soft millirobots.

Untethered small-scale soft robots have promising applications in minimally invasive surgery, targeted drug delivery, and bioengineering applications as they can directly and non-invasively access confined and hard-to-reach spaces in the human body. For such potential biomedical applications, the adaptivity of the robot control is essential to ensure the continuity of the operations, as task environment conditions show dynamic variations that can alter the robot's motion and task performance. The applicability of the conventional modeling and control methods is further limited for soft robots at the small-scale owing to their kinematics with virtually infinite degrees of freedom, inherent stochastic variability during fabrication, and changing dynamics during real-world interactions. To address the controller adaptation challenge to dynamically changing task environments, we propose using a probabilistic learning approach for a millimeter-scale magnetic walking soft robot using Bayesian optimization (BO) and Gaussian processes (GPs). Our approach provides a data-efficient learning scheme by finding the gait controller parameters while optimizing the stride length of the walking soft millirobot using a small number of physical experiments. To demonstrate the controller adaptation, we test the walking gait of the robot in task environments with different surface adhesion and roughness, and medium viscosity, which aims to represent the possible conditions for future robotic tasks inside the human body. We further utilize the transfer of the learned GP parameters among different task spaces and robots and compare their efficacy on the improvement of data-efficient controller learning.

Zwitterionic 3D-Printed Non-Immunogenic Stealth Microrobots.

Microrobots offer transformative solutions for non-invasive medical interventions due to their small size and untethered operation inside the human body. However, they must face the immune system as a natural protection mechanism against foreign threats. Here, non-immunogenic stealth zwitterionic microrobots that avoid recognition from immune cells are introduced. Fully zwitterionic photoresists are developed for two-photon polymerization 3D microprinting of hydrogel microrobots with ample functionalization: tunable mechanical properties, anti-biofouling and non-immunogenic properties, functionalization for magnetic actuation, encapsulation of biomolecules, and surface functionalization for drug delivery. Stealth microrobots avoid detection by macrophage cells of the innate immune system after exhaustive inspection (>90 hours), which has not been achieved in any microrobotic platform to date. These versatile zwitterionic materials eliminate a major roadblock in the development of biocompatible microrobots, and will serve as a toolbox of non-immunogenic materials for medical microrobot and other device technologies for bioengineering and biomedical applications.

Biosynthetic self-healing materials for soft machines.

Self-healing materials are indispensable for soft actuators and robots that operate in dynamic and real-world environments, as these machines are vulnerable to mechanical damage. However, current self-healing materials have shortcomings that limit their practical application, such as low healing strength (below a megapascal) and long healing times (hours). Here, we introduce high-strength synthetic proteins that self-heal micro- and macro-scale mechanical damage within a second by local heating. These materials are optimized systematically to improve their hydrogen-bonded nanostructure and network morphology, with programmable healing properties (2-23 MPa strength after 1 s of healing) that surpass by several orders of magnitude those of other natural and synthetic soft materials. Such healing performance creates new opportunities for bioinspired materials design, and addresses current limitations in self-healing materials for soft robotics and personal protective equipment.

Self-Assembly of Topologically Networked Protein-Ti3C2Tx MXene Composites.

Hierarchical organization plays an important role in the stunning physical properties of natural and synthetic composites. Limits on the physical properties of such composites are generally defined by percolation theory and can be systematically altered using the volumetric filler fraction of the inorganic/organic phase. In natural composites, organic materials such as proteins that interact with inorganic filler materials can further alter the hierarchical order and organization of the composite via topological interactions, expanding the limits of the physical properties defined by percolation theory. However, existing polymer systems do not offer a topological parameter that can systematically modulate the assembly characteristics of composites. Here, we present a composite based on proteins and titanium carbide (Ti3C2Tx) MXene that manifests a topological network that regulates the organization, and hence physical properties, of these biomimetic composites. We designed, recombinantly expressed, and purified synthetic proteins consisting of polypeptides with repeating amino acid sequences (tandem repeats) that have the ability to self-assemble into topologically networked biomaterials. We demonstrated that the interlayer distance between MXene sheets can be controlled systematically by the number of tandem repeat units. We varied the filler fraction and number of tandem repeat units to regulate the in-plane and out-of-plane electrical conductivities of these composites. Once Ti3C2Tx MXene sheets are separated enough to facilitate formation of cross-links in our proteins with the number of tandem repeat units reaching 11, the linear I-V characteristics of the composites switched into nonlinear I-V curves with a distinct hysteresis for out-of-plane electron transport, while the in-plane I-V characteristics remained linear. This highlights the impact of synthetic protein templates, which can be designed to modulate electronic transport in composites both isotropically and anisotropically.

Highly Conductive Self-Healing Biocomposites Based on Protein Mediated Self-Assembly of PEDOT:PSS Films.

Composites of conducting polymers offer a broad spectrum of materials for interfacing electronic devices with biological systems. Particularly, material systems based on poly(styrenesulfonate) doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) have found applications in many bioelectronic devices as biosensitive transistors, controlled drug delivery media, and strain, temperature, and humidity sensors. The biocompatibility, intercoupled electronic and ionic conductivity, and air stable electrical properties render PEDOT:PSS based material systems indispensable for bioelectronics. However, these materials are commonly used in thin film form since freestanding films of pristine PEDOT:PSS are considered mechanically brittle compared to biological tissues, and unlike biological systems these conductive films cannot restore/heal their physical properties after excessive mechanical deformation. Here we report conductive biocomposites of PEDOT:PSS and tandem repeat proteins with the ability to self-heal once plasticized via water. The tandem repeat proteins acquired from squid ring teeth (SRT) induce structural effects on PEDOT:PSS including improved crystallinity and formation of fibrous network structures. These structural effects lead to electrical conductivity values reaching 120 S/cm for biocomposites with SRT protein concentrations below 20 wt %, which exceeds the conductivity of pristine PEDOT:PSS (∼100 S/cm). More importantly, tandem proteins facilitate consistent self-healing of freestanding biocomposites with SRT protein concentrations beyond 40 wt %. These robust biocomposites with high electrical conductivity and the ability to self-heal can find applications in numerous soft electronic systems spanning from implantable, transient, and epidermal electronics to electronic textiles.

Multifunctional and biodegradable self-propelled protein motors.

A diversity of self-propelled chemical motors, based on Marangoni propulsive forces, has been developed in recent years. However, most motors are non-functional due to poor performance, a lack of control, and the use of toxic materials. To overcome these limitations, we have developed multifunctional and biodegradable self-propelled motors from squid-derived proteins and an anesthetic metabolite. The protein motors surpass previous reports in performance output and efficiency by several orders of magnitude, and they offer control of their propulsion modes, speed, mobility lifetime, and directionality by regulating the protein nanostructure via local and external stimuli, resulting in programmable and complex locomotion. We demonstrate diverse functionalities of these motors in environmental remediation, microrobot powering, and cargo delivery applications. These versatile and degradable protein motors enable design, control, and actuation strategies in microrobotics as modular propulsion sources for autonomous minimally invasive medical operations in biological environments with air-liquid interfaces.

Squid-Inspired Tandem Repeat Proteins: Functional Fibers and Films.

Production of repetitive polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions have been an interest for the last couple of decades. Their molecular structure provides a rich architecture that can micro-phase-separate to form periodic nanostructures (e.g., lamellar and cylindrical repeating phases) with enhanced physicochemical properties via directed or natural evolution that often exceed those of conventional synthetic polymers. Here, we review programmable design, structure, and properties of functional fibers and films from squid-inspired tandem repeat proteins, with applications in soft photonics and advanced textiles among others.

Squid Ring Teeth-coated Mesh Improves Abdominal Wall Repair.

BACKGROUND: Hernia repair is a common surgical procedure with polypropylene (PP) mesh being the standard material for correction because of its durability. However, complications such as seroma and pain are common, and repair failures still approach 15% secondary to poor tissue integration. In an effort to enhance mesh integration, we evaluated the applicability of a squid ring teeth (SRT) protein coating for soft-tissue repair in an abdominal wall defect model. SRT is a biologically derived high-strength protein with strong mechanical properties. We assessed tissue integration, strength, and biocompatibility of a SRT-coated PP mesh in a first-time pilot animal study. METHODS: PP mesh was coated with SRT (SRT-PP) and tested for mechanical strength against uncoated PP mesh. Cell proliferation and adhesion studies were performed in vitro using a 3T3 cell line. Rats underwent either PP (n = 3) or SRT-PP (n = 6) bridge mesh implantation in an anterior abdominal wall defect model. Repair was assessed clinically and radiographically, with integration evaluated by histology and mechanical testing at 60 days. RESULTS: Cell proliferation was enhanced on SRT-PP mesh. This was corroborated in vivo by abdominal wall histology, dramatically diminished craniocaudal mesh contraction, improved strength testing, and higher tissue failure strain. There was no increase in seroma or visceral adhesion formation. No foreign body reactions were noted on liver histology. CONCLUSIONS: SRT applied as a coating appears to augment mesh-tissue integration and improve abdominal wall stability following bridged repair. Further studies in larger animals will determine its applicability for hernia repair in patients.