4D Printed Protein-AuNR Nanocomposites with Photothermal Shape Recovery.

4D printing is the 3D printing of objects that change chemically or physically in response to an external stimulus over time. Photothermally responsive shape memory materials are attractive for their ability to undergo remote activation. While photothermal methods using gold nanorods (AuNRs) have been used for shape recovery, 3D patterning of these materials into objects with complex geometries using degradable materials has not been addressed. Here, we report on the fabrication of 3D printed shape memory bioplastics with photo-activated shape recovery. Protein-based nanocomposites based on bovine serum albumin (BSA), poly (ethylene glycol) diacrylate and gold nanorods were developed for vat photopolymerization. These 3D printed bioplastics were mechanically deformed under high loads, and the proteins served as mechanoactive elements that unfolded in an energy-dissipating mechanism that prevented fracture of the thermoset. The bioplastic object maintained its metastable shape-programmed state under ambient conditions. Subsequently, up to 99% shape recovery was achieved within 1 min of irradiation with near-infrared light. Mechanical characterization and small angle X-ray scattering (SAXS) analysis suggest that the proteins mechanically unfold during the shape programming step and may refold during shape recovery. These composites are promising materials for the fabrication of biodegradable shape-morphing devices for robotics and medicine.

Light-Induced Living Polymer Networks with Adaptive Functional Properties.

The advent of covalent adaptable networks (CANs) through the incorporation of dynamic covalent bonds has led to unprecedented properties of macromolecular systems, which can be engineered at the molecular level. Among the various types of stimuli that can be used to trigger chemical changes within polymer networks, light stands out for its remote and spatiotemporal control under ambient conditions. However, most examples of photoactive CANs need to be transparent and they exhibit slow response, side reactions, and limited light penetration. In this vein, it is interesting to understand how molecular engineering of optically active dynamic linkages that offer fast response to visible light can impart "living" characteristics to CANs, especially in opaque systems. Here, the use of carbazole-based thiuram disulfides (CTDs) that offer dual reactivity as photoactivated reshuffling linkages and iniferters under visible light irradiation is reported. The fast response to visible light activation of the CTDs leads to temporal control of shape manipulation, healing, and chain extension in the polymer networks, despite the lack of optical transparency. This strategy charts a promising avenue for manipulating multifunctional photoactivated CANs in a controlled manner.

Engineered Living Material Bioreactors with Tunable Mechanical Properties using Vat Photopolymerization.

3D-printed engineered living materials (ELM) are promising bioproduction platforms for agriculture, biotechnology, sustainable energy, and green technology applications. However, the design of these platforms faces several challenges, such as the processability of these materials into complex form factors and control over their mechanical properties. Herein, ELM are presented as 3D-printed bioreactors with arbitrary shape geometries and tunable mechanical properties (moduli and toughness). Poly(ethylene glycol) diacrylate (PEGDA) is used as the precursor to create polymer networks that encapsulate the microorganisms during the vat photopolymerization process. A major limitation of PEGDA networks is their propensity to swell and fracture when submerged in water. The authors overcame this issue by adding glycerol to the resin formulation to 3D print mechanically tough ELM hydrogels. While polymer concentration affects the modulus and reduces bioproduction, ELM bioreactors still maintain their metabolic activity regardless of polymer concentration. These ELM bioreactors have the potential to be used in different applications for sustainable architecture, food production, and biomedical devices that require different mechanical properties from soft to stiff.

Additive Manufacturing of Engineered Living Materials with Bio-augmented Mechanical Properties and Resistance to Degradation.

Engineered living materials (ELMs) combine living cells with polymeric matrices to yield unique materials with programmable functions. While the cellular platform and the polymer network determine the material properties and applications, there are still gaps in our ability to seamlessly integrate the biotic (cellular) and abiotic (polymer) components into singular material, then assemble them into devices and machines. Herein, we demonstrated the additive-manufacturing of ELMs wherein bioproduction of metabolites from the encapsulated cells enhanced the properties of the surrounding matrix. First, we developed aqueous resins comprising bovine serum albumin (BSA) and poly(ethylene glycol diacrylate) (PEGDA) with engineered microbes for vat photopolymerization to create objects with a wide array of 3D form factors. The BSA-PEGDA matrix afforded hydrogels that were mechanically stiff and tough for use in load-bearing applications. Second, we demonstrated the continuous in situ production of L-DOPA, naringenin, and betaxanthins from the engineered cells encapsulated within the BSA-PEGDA matrix. These microbial metabolites bioaugmented the properties of the BSA-PEGDA matrix by enhancing the stiffness (L-DOPA) or resistance to enzymatic degradation (betaxanthin). Finally, we demonstrated the assembly of the 3D printed ELM components into mechanically functional bolts and gears to showcase the potential to create functional ELMs for synthetic living machines.

A tripartite microbial co-culture system for de novo biosynthesis of diverse plant phenylpropanoids.

Plant-derived phenylpropanoids, in particular phenylpropenes, have diverse industrial applications ranging from flavors and fragrances to polymers and pharmaceuticals. Heterologous biosynthesis of these products has the potential to address low, seasonally dependent yields hindering ease of widespread manufacturing. However, previous efforts have been hindered by the inherent pathway promiscuity and the microbial toxicity of key pathway intermediates. Here, in this study, we establish the propensity of a tripartite microbial co-culture to overcome these limitations and demonstrate to our knowledge the first reported de novo phenylpropene production from simple sugar starting materials. After initially designing the system to accumulate eugenol, the platform modularity and downstream enzyme promiscuity was leveraged to quickly create avenues for hydroxychavicol and chavicol production. The consortia was found to be compatible with Engineered Living Material production platforms that allow for reusable, cold-chain-independent distributed manufacturing. This work lays the foundation for further deployment of modular microbial approaches to produce plant secondary metabolites.

Gene expression dynamics in input-responsive engineered living materials programmed for bioproduction.

Engineered living materials (ELMs) fabricated by encapsulating microbes in hydrogels have great potential as bioreactors for sustained bioproduction. While long-term metabolic activity has been demonstrated in these systems, the capacity and dynamics of gene expression over time is not well understood. Thus, we investigate the long-term gene expression dynamics in microbial ELMs constructed using different microbes and hydrogel matrices. Through direct gene expression measurements of engineered E. coli in F127-bisurethane methacrylate (F127-BUM) hydrogels, we show that inducible, input-responsive genetic programs in ELMs can be activated multiple times and maintained for multiple weeks. Interestingly, the encapsulated bacteria sustain inducible gene expression almost 10 times longer than free-floating, planktonic cells. These ELMs exhibit dynamic responsiveness to repeated induction cycles, with up to 97% of the initial gene expression capacity retained following a subsequent induction event. We demonstrate multi-week bioproduction cycling by implementing inducible CRISPR transcriptional activation (CRISPRa) programs that regulate the expression of enzymes in a pteridine biosynthesis pathway. ELMs fabricated from engineered S. cerevisiae in bovine serum albumin (BSA) - polyethylene glycol diacrylate (PEGDA) hydrogels were programmed to express two different proteins, each under the control of a different chemical inducer. We observed scheduled bioproduction switching between betaxanthin pigment molecules and proteinase A in S. cerevisiae ELMs over the course of 27 days under continuous cultivation. Overall, these results suggest that the capacity for long-term genetic expression may be a general property of microbial ELMs. This work establishes approaches for implementing dynamic, input-responsive genetic programs to tailor ELM functions for a wide range of advanced applications.

Triplet Fusion Upconversion for Photocuring 3D-Printed Particle-Reinforced Composite Networks.

High energy photons (λ < 400 nm) are frequently used to initiate free radical polymerizations to form polymer networks, but are only effective for transparent objects. This phenomenon poses a major challenge to additive manufacturing of particle-reinforced composite networks since deep light penetration of short-wavelength photons limits the homogeneous modification of physicochemical and mechanical properties. Herein, the unconventional, yet versatile, multiexciton process of triplet-triplet annihilation upconversion (TTA-UC) is employed for curing opaque hydrogel composites created by direct-ink-write (DIW) 3D printing. TTA-UC converts low energy red light (λmax  = 660 nm) for deep penetration into higher-energy blue light to initiate free radical polymerizations within opaque objects. As proof-of-principle, hydrogels containing up to 15 wt.% TiO2 filler particles and doped with TTA-UC chromophores are readily cured with red light, while composites without the chromophores and TiO2 loadings as little as 1-2 wt.% remain uncured. Importantly, this method has wide potential to modify the chemical and mechanical properties of complex DIW 3D-printed composite polymer networks.

Enhancing long-term storage and stability of engineered living materials through desiccant storage and trehalose treatment.

Engineered living materials (ELMs) have broad applications for enabling on-demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast-laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1-year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ-CT and confocal microscopy indicate that cells form spherical colonies within F127-BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on-demand, cold-chain independent bioproduction.

Extending BigSMILES to non-covalent bonds in supramolecular polymer assemblies.

As a machine-recognizable representation of polymer connectivity, BigSMILES line notation extends SMILES from deterministic to stochastic structures. The same framework that allows BigSMILES to accommodate stochastic covalent connectivity can be extended to non-covalent bonds, enhancing its value for polymers, supramolecular materials, and colloidal chemistry. Non-covalent bonds are captured through the inclusion of annotations to pseudo atoms serving as complementary binding pairs, minimal key/value pairs to elaborate other relevant attributes, and indexes to specify the pairing among potential donors and acceptors or bond delocalization. Incorporating these annotations into BigSMILES line notation enables the representation of four common classes of non-covalent bonds in polymer science: electrostatic interactions, hydrogen bonding, metal-ligand complexation, and π-π stacking. The principal advantage of non-covalent BigSMILES is the ability to accommodate a broad variety of non-covalent chemistry with a simple user-orientated, semi-flexible annotation formalism. This goal is achieved by encoding a universal but non-exhaustive representation of non-covalent or stochastic bonding patterns through syntax for (de)protonated and delocalized state of bonding as well as nested bonds for correlated bonding and multi-component mixture. By allowing user-defined descriptors in the annotation expression, further applications in data-driven research can be envisioned to represent chemical structures in many other fields, including polymer nanocomposite and surface chemistry.

Prototype Development of a Temperature-Sensitive High-Adhesion Medical Tape to Reduce Medical-Adhesive-Related Skin Injury and Improve Quality of Care.

Medical adhesives are used to secure wound care dressings and other critical devices to the skin. Without means of safe removal, these stronger adhesives are difficult to painlessly remove from the skin and may cause medical-adhesive-related skin injuries (MARSI), including skin tears and an increased risk of infection. Lower-adhesion medical tapes may be applied to avoid MARSI, leading to device dislodgement and further medical complications. This paper outlines the development of a high-adhesion medical tape designed for low skin trauma upon release. By warming the skin-attached tape for 10-30 s, a significant loss in adhesion was achieved. A C14/C18 copolymer was developed and combined with a selected pressure-sensitive adhesive (PSA) material. The addition of 1% C14/C18 copolymer yielded the largest temperature-responsive drop in surface adhesion. The adhesive film was characterized using AFM, and distinct nanodomains were identified on the exterior surface of the PSA. Our optimized formulation yielded 67% drop in adhesion when warmed to 45 °C, perhaps due to melting nanodomains weakening the adhesive-substrate boundary layer. Pilot clinical testing resulted in a significant decrease in pain when a heat pack was used for removal, giving an average pain reduction of 66%.

Methacrylated Bovine Serum Albumin and Tannic Acid Composite Materials for Three-Dimensional Printing Tough and Mechanically Functional Parts.

Nature uses proteins as building blocks to create three-dimensional (3D) structural components (like spiderwebs and tissue) that are recycled within a closed loop. Furthermore, it is difficult to replicate the mechanical properties of these 3D architectures within synthetic systems. In the absence of biological machinery, protein-based materials can be difficult to process and can have a limited range of mechanical properties. Herein, we present an additive manufacturing workflow to fabricate tough, protein-based composite hydrogels and bioplastics with a range of mechanical properties. Briefly, methacrylated bovine-serum-albumin-based aqueous resins were 3D-printed using a commercial vat photopolymerization system. The printed structures were then treated with tannic acid to introduce additional non-covalent interactions and form tough hydrogels. The hydrogel material could be sutured and withstand mechanical load, even after immersion in water for 24 h. Additionally, a denaturing thermal cure could be used to virtually eliminate rehydration of the material and form a bioplastic. To highlight the functionality of this material, a bioplastic screw was 3D-printed and driven into wood without damage to the screw. Moreover, the 3D-printed constructs enzymatically degraded up to 85% after 30 days in pepsin solution. Thus, these protein-based 3D-printed constructs show great potential for biomedical devices that degrade in situ.

Direct-Ink Write 3D Printing Multistimuli-Responsive Hydrogels and Post-Functionalization Via Disulfide Exchange.

Herein, we describe a multi-stimuli-responsive hydrogel that can be 3D printed via a direct-ink write process to afford cross-linked hydrogel networks that can be post-functionalized with thiol-bearing molecules. Poly(alkyl glycidyl ether)s with methacrylate groups at their termini were synthesized and self-assembled into hydrogels with three key stimuli-responsive behaviors necessary for extrusion based 3D printing: a sol-gel temperature response, shear-thinning behavior, and the ability to be photochemically crosslinked. In addition, the chemically crosslinked hydrogels demonstrated a temperature dependent swelling consistent with an LCST behavior. Pyridyl disulfide urethane methacrylate (PDS-UM) monomers were introduced into the network as a thiol-reactive handle for post-functionalization of the hydrogel. The reactivities of these hydrogels were investigated at different temperatures (5, 25, 37 °C) and swelling statuses (as-cured versus preswollen) using glutathione as a reactive probe. To illustrate the versatility of the platform, a number of additional thiol-containing probes such as proteins, polymers, and small molecules were conjugated to the hydrogel network at different temperatures, pH's, and concentrations. In a final demonstration of the multi-stimuli-responsive hydrogel platform, a customized DIW 3D printer was used to fabricate a printed object that was subsequently conjugated with a fluorescent tag and displayed the ability to change in size with environmental temperature.

Photocross-Linked Antimicrobial Amino-Siloxane Elastomers.

Mechanically robust bulk antimicrobial polymers are one way to address disease transmission via contaminated surfaces. Here, we demonstrate the visible light photo-oxidative cross-linking of amine-containing PDMS using a single-component, solvent-free system where amines have a dual role as antimicrobial functionalities and cross-linking sites. Rose Bengal, a xanthene dye used as a fluorescent stain, is thermally reacted with the polymer to give a solvent-free liquid siloxane that can generate reactive singlet oxygen upon aerobic green light irradiation, coupling the amine functionalities into imine cross-links. Photorheological experiments demonstrate that light intensity is the largest kinetic factor in the photo-oxidative curing of these polymers. Room temperature irradiation under an ambient atmosphere results in free-standing elastic materials with mechanical properties that depend on the amount of Rose Bengal present. An ultimate elongation strain of 117% and Young's modulus of 2.15 MPa were observed for the highest dye loading, with both mechanical properties found to be higher than those for the same solution-based dye amounts. We demonstrate that the solvent-free nature of the material can be exploited to generate 3D structures using low-temperature deposition as well as direct-write patterning and photolithography on glass substrates. The antimicrobial activity was investigated, with the cross-linked material demonstrating greater efficacy against E. coli (Gram negative) compared with MRSA (Gram positive) bacterial strains and inducing complete cell lysis of incubated CHO-K1 mammalian cells, demonstrating applicability as a mechanically robust single-component antimicrobial elastomer.

3D-Printed Bioplastics with Shape-Memory Behavior Based on Native Bovine Serum Albumin.

Bio-based plastics that can supplant petroleum-derived materials are necessary to meet the future demands of sustainability in the life cycle of plastic materials. While there are significant efforts to develop protein-based plastic materials for commercial use, their application is limited by poor processability and limitations in mechanical performance. Here, we present a bovine serum albumin (BSA)-based resin for stereolithographic apparatus (SLA) 3D printing that affords bioplastic objects with shape-memory behavior. We demonstrate that the native conformation of these globular proteins is largely retained in the 3D-printed constructs and that each protein molecule possesses a "stored length" that could be revealed during mechanical deformation (extension or compression) of the 3D bioplastic objects. While the plastically deformed objects could retain this state for an indefinite period of time, heating the object or submerging in water allowed it to return to its original 3D-printed shape.

Mechanoactivation of Color and Autonomous Shape Change in 3D-Printed Ionic Polymer Networks.

Stimuli-responsive materials can enhance the field of three-dimensional (3D) printing by generating objects that change shape in response to external cues. While temperature and pH are common inputs for initiating a response in a 3D-printed object, there are few examples of using a mechanical input to afford a response. Herein, we report a suite of mechanochromic ionic liquid gel inks that can be used to fabricate 3D-printed objects that use a single mechanoactivation event to elicit both a mechanochromic response and an autonomous shape change. Direct-ink write 3D printing was used to deposit ionic liquid gel inks to create multimaterial objects that underwent a predetermined mechanoactivated shape change (mechanomorphic) when the sample was pulled and then released. When spiropyran was incorporated into the inks, the onset of spiropyran isomerization into its purple merocyanine form occurred at strains dependent upon the particular ion gel ink formulation. We suggest that the color onset could be used as a simple indicator for when the strain required to achieve a predetermined change in shape has been reached, potentially serving as real-time visual cue for the user. Such morphing 3D-printed structures with integrated instructions have potential application to areas including stowable structures as well as multiresponsive autonomous components and sensors.

Bioproduced Proteins On Demand (Bio-POD) in hydrogels using Pichia pastoris.

Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production.

Metabolism Control in 3D-Printed Living Materials Improves Fermentation.

The three-dimensional (3D) printing of cell-containing polymeric hydrogels creates living materials (LMs), offering a platform for developing innovative technologies in areas like biosensors and biomanufacturing. The polymer material properties of cross-linkable F127-bis-urethane methacrylate (F127-BUM) allow reproducible 3D printing and stability in physiological conditions, making it suitable for fabricating LMs. Though F127-BUM-based LMs permit diffusion of solute molecules like glucose and ethanol, it remains unknown whether these are permissible for oxygen, essential for respiration. To determine oxygen permissibility, we quantified dissolved oxygen consumption by the budding yeast-laden F127-BUM-based LMs. Moreover, we obtained data on cell-retaining LMs, which allowed a direct comparison between LMs and suspension cultures. We further developed a highly reliable method to isolate cells from LMs for flow cytometry analysis, cell viability evaluation, and the purification of macromolecules. We found oxygen consumption heavily impaired inside LMs, indicating that yeast metabolism relies primarily on fermentation instead of respiration. Applying this finding to brewing, we observed a higher (3.7%) ethanol production using LMs than the traditional brewing process, indicating improved fermentation. Our study concludes that the present F127-BUM-based LMs are useful for microaerobic processes but developing aerobic bioprocesses will require further research.

3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix.

3D bioprinting is a powerful technique for engineering tissues used to study cell behavior and tissue properties in vitro. With the right formulation and printing parameters, bioinks can provide native biological and mechanical cues while allowing for versatile 3D structures that recapitulate tissue-level organization. Bio-based materials that support cellular adhesion, differentiation, and proliferation - including gelatin, collagen, hyaluronic acid, and alginate - have been successfully used as bioinks. In particular, decellularized extracellular matrix (dECM) has become a promising material with the unique ability to maintain both biochemical and topographical micro-environments of native tissues. However, dECM has shown technical limitations for 3D printing (3DP) applications posed by its intrinsically low mechanical stability. Herein, we report hydrogel bioinks composed of partially digested, porcine cardiac decellularized extracellular matrix (cdECM), Laponite-XLG nanoclay, and poly(ethylene glycol)-diacrylate (PEG-DA). The Laponite facilitated extrusion-based 3DP, while PEG-DA enabled photo-polymerization after printing. Improving upon previously reported bioinks derived from dECM, our bioinks combine extrudability, shape fidelity, rapid cross-linking, and cytocompatibility in a single formulation (> 97% viability of encapsulated human cardiac fibroblasts and > 94% viability of human induced pluripotent stem cell derived cardiomyocytes after 7 days). The compressive modulus of the cured hydrogel bioinks was tunable from 13.4-89 kPa by changing the concentration of PEG-DA in the bioink formulation. Importantly, this span of mechanical stiffness encompasses ranges of tissue stiffness from healthy (compressive modulus ~5-15 kPa) to fibrotic (compressive modulus ~30-100 kPa) cardiac tissue states. The printed constructs demonstrated shape fidelity, adaptability to different printing conditions, and high cell viability following extrusion and photo-polymerization, highlighting the potential for applications in modeling both healthy and fibrotic cardiac tissue.

Physical Confinement Impacts Cellular Phenotypes within Living Materials.

Additive manufacturing allows three-dimensional printing of polymeric materials together with cells, creating living materials for applications in biomedical research and biotechnology. However, an understanding of the cellular phenotype within living materials is lacking, which is a key limitation for their wider application. Herein, we present an approach to characterize the cellular phenotype within living materials. We immobilized the budding yeast Saccharomyces cerevisiae in three different photo-cross-linkable triblock polymeric hydrogels containing F127-bis-urethane methacrylate, F127-dimethacrylate, or poly(alkyl glycidyl ether)-dimethacrylate. Using optical and scanning electron microscopy, we showed that hydrogels based on these polymers were stable under physiological conditions, but yeast colonies showed differences in the interaction within the living materials. We found that the physical confinement, imparted by compositional and structural properties of the hydrogels, impacted the cellular phenotype by reducing the size of cells in living materials compared with suspension cells. These properties also contributed to the differences in immobilization patterns, growth of colonies, and colony coatings. We observed that a composition-dependent degradation of polymers was likely possible by cells residing in the living materials. In conclusion, our investigation highlights the need for a holistic understanding of the cellular response within hydrogels to facilitate the synthesis of application-specific polymers and the design of advanced living materials in the future.

Cell-Laden Hydrogels for Multikingdom 3D Printing.

Living materials are created through the embedding of live, whole cells into a matrix that can house and sustain the viability of the encapsulated cells. Through the immobilization of these cells, their bioactivity can be harnessed for applications such as bioreactors for the production of high-value chemicals. While the interest in living materials is growing, many existing materials lack robust structure and are difficult to pattern. Furthermore, many living materials employ only one type of microorganism, or microbial consortia with little control over the arrangement of the various cell types. In this work, a Pluronic F127-based hydrogel system is characterized for the encapsulation of algae, yeast, and bacteria to create living materials. This hydrogel system is also demonstrated to be an excellent material for additive manufacturing in the form of direct write 3D-printing to spatially arrange the cells within a single printed construct. These living materials allow for the development of incredibly complex, immobilized consortia, and the results detailed herein further enhance the understanding of how cells behave within living material matrices. The utilization of these materials allows for interesting applications of multikingdom microbial cultures in immobilized bioreactor or biosensing technologies.