Controlled release of microorganisms from engineered living materials.

Probiotics offer therapeutic benefits by modulating the local microbiome, the host immune response, and the proliferation of pathogens. Probiotics have the potential to treat complex diseases, but their persistence or colonization is required at the target site for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases clinically relevant doses of metabolically active probiotics in a sustained manner has been previously described. Here, we encapsulate stiff probiotic microorganisms within relatively less stiff hydrogels and show a generic mechanism where these microorganisms proliferate and induce hydrogel fracture, resulting in microbial release. Importantly, this fracture-based mechanism leads to microorganism release with zero-order release kinetics. Using this mechanism, small (∼1 µL) engineered living materials (ELMs) release >10 8 colony-forming-units (CFUs) of E. coli in 2 h. This release is sustained for at least 10 days. Cell release can be varied by more than three orders of magnitude by varying initial cell loading and modulating the mechanical properties of encapsulating matrix. As the governing mechanism of microbial release is entirely mechanical, we demonstrate controlled release of model Gram-negative, Gram-positive, and fungal probiotics from multiple hydrogel matrices. SIGNIFICANCE: Probiotics offer therapeutic benefits and have the potential to treat complex diseases, but their persistence at the target site is often required for effective treatment. Although probiotic persistence can be achieved by repeated delivery, no biomaterial that releases metabolically active probiotics in a sustained manner has been developed yet. This work demonstrates a generic mechanism where stiff probiotics encapsulated within relatively less stiff hydrogels proliferate and induce hydrogel fracture. This allows a zero-order release of probiotics which can be easily controlled by adjusting the properties of the encapsulating matrices. This generic mechanism is applicable for a wide range of probiotics with different synthetic matrices and has the potential to be used in the treatment of a broad range of diseases.

Reconfigurable Growth of Engineered Living Materials.

The growth of multicellular organisms is a process akin to additive manufacturing where cellular proliferation and mechanical boundary conditions, among other factors, drive morphogenesis. Engineers have limited ability to engineer morphogenesis to manufacture goods or to reconfigure materials comprised of biomass. Herein, a method that uses biological processes to grow and regrow magnetic engineered living materials (mELMs) into desired geometries is reported. These composites contain Saccharomyces cerevisiae and magnetic particles within a hydrogel matrix. The reconfigurable manufacturing process relies on the growth of living cells, magnetic forces, and elastic recovery of the hydrogel. The mELM then adopts a form in an external magnetic field. Yeast within the material proliferates, resulting in 259 ± 14% volume expansion. Yeast proliferation fixes the magnetic deformation, even when the magnetic field is removed. The shape fixity can be up to 99.3 ± 0.3%. The grown mELM can recover up to 73.9 ± 1.9% of the original form by removing yeast cell walls. The directed growth and recovery process can be repeated at least five times. This work enables ELMs to be processed and reprocessed into user-defined geometries without external material deposition.

Controlling shape morphing and cell release in engineered living materials.

Engineered living materials (ELMs) derive functionality from both a polymer matrix and the behavior of living cells within the material. The long-term goal of this work is to enable a system of ELM-based medical devices with both mechanical and bioactive functionality. Here, we fabricate multifunctional, stimuli-responsive ELMs comprised of acrylic hydrogel matrix and Escherichia coli. These ELMs undergo controlled changes in form and have a controlled release of bacteria from the composite. We hypothesize that the mechanical forces associated with cell proliferation within a covalently-crosslinked, non-degradable hydrogel are responsible for both phenomena. At constant cell loading, increased hydrogel elastic modulus significantly reduces both cell delivery and volume change associated with cell proliferation. ELMs that change volume over 100 % also result in ~106 colony forming units/mL in the growth medium over 2 h after 1 day of growth. At constant monomer feed ratios, increased cell loading leads to significantly increased cell delivery. Finally, these prokaryotic ELMs were investigated for their potential to deliver a probiotic that can reduce the proliferation of a uropathogen in vitro. Controlling the long-term delivery of bacteria could potentially be used in biomedical applications to modulate microbial communities within the human body.

Digitally Programmable Manufacturing of Living Materials Grown from Biowaste.

Material manufacturing strategies that use little energy, valorize waste, and result in degradable products are urgently needed. Strategies that transform abundant biomass into functional materials form one approach to these emerging manufacturing techniques. From a biological standpoint, morphogenesis of biological tissues is a "manufacturing" mode without energy-intensive processes, large carbon footprints, and toxic wastes. Inspired by biological morphogenesis, we propose a manufacturing strategy by embedding living Saccharomyces cerevisiae (Baker's yeast) within a synthetic acrylic hydrogel matrix. By culturing the living materials in media derived from bread waste, encapsulated yeast cells can proliferate, resulting in a dramatic dry mass and volume increase of the whole living material. After growth, the final material is up to 96 wt % biomass and 590% larger in volume than the initial object. By digitally programming the cell viability through UV irradiation or photodynamic inactivation, the living materials can form complex user-defined relief surfaces or 3D objects during growth. Ultimately, the grown structures can also be designed to be degradable. The proposed living material manufacturing strategy cultured from biowaste may pave the way for future ecologically friendly manufacturing of materials.

From Chaos to Control: Programmable Crack Patterning with Molecular Order in Polymer Substrates.

Cracks are typically associated with the failure of materials. However, cracks can also be used to create periodic patterns on the surfaces of materials, as observed in the skin of crocodiles and elephants. In synthetic materials, surface patterns are critical to micro- and nanoscale fabrication processes. Here, a strategy is presented that enables freely programmable patterns of cracks on the surface of a polymer and then uses these cracks to pattern other materials. Cracks form during deposition of a thin film metal on a liquid crystal polymer network (LCN) and follow the spatially patterned molecular order of the polymer. These patterned sub-micrometer scale cracks have an order parameter of 0.98 ± 0.02 and form readily over centimeter-scale areas on the flexible substrates. The patterning of the LCN enables cracks that turn corners, spiral azimuthally, or radiate from a point. Conductive inks can be filled into these oriented cracks, resulting in flexible, anisotropic, and transparent conductors. This materials-based processing approach to patterning cracks enables unprecedented control of the orientation, length, width, and depth of the cracks without costly lithography methods. This approach promises new architectures of electronics, sensors, fluidics, optics, and other devices with micro- and nanoscale features.

Stimuli-responsive engineered living materials.

Stimuli-responsive materials are able to undergo controllable changes in materials properties in response to external cues. Increasing efforts have been directed towards building materials that mimic the responsive nature of biological systems. Nevertheless, limitations remain surrounding the way these synthetic materials interact and respond to their environment. In particular, it is difficult to synthesize synthetic materials that respond with specificity to poorly differentiated (bio)chemical and weak physical stimuli. The emerging area of engineered living materials (ELMs) includes composites that combine living cells and synthetic materials. ELMs have yielded promising advances in the creation of stimuli-responsive materials that respond with diverse outputs in response to a broad array of biochemical and physical stimuli. This review describes advances made in the genetic engineering of the living component and the processing-property relationships of stimuli-responsive ELMs. Finally, the implementation of stimuli-responsive ELMs as environmental sensors, biomedical sensors, drug delivery vehicles, and soft robots is discussed.

4D-Printing of Photoswitchable Actuators.

Shape-switching behavior, where a transient stimulus induces an indefinitely stable deformation that can be recovered on exposure to another transient stimulus, is critical to building smart structures from responsive polymers as continue power is not needed to maintain deformations. Herein, we 4D-print shape-switching liquid crystalline elastomers (LCEs) functionalized with supramolecular crosslinks, dynamic covalent crosslinks, and azobenzene. The salient property of shape-switching LCEs is that light induces long-lived, deformation that can be recovered on-demand by heating. UV-light isomerizes azobenzene from trans to cis, and temporarily breaks the supramolecular crosslinks, resulting in a programmed deformation. After UV, the shape-switching LCEs fix more than 90 % of the deformation over 3 days by the reformed supramolecular crosslinks. Using the shape-switching properties, we print Braille-like actuators that can be photoswitched to display different letters. This new class of photoswitchable actuators may impact applications such as deployable devices where continuous application of power is impractical.

Anisotropic, porous hydrogels templated by lyotropic chromonic liquid crystals.

Approaches to control the microstructure of hydrogels enable the control of cell-material interactions and the design of stimuli-responsive materials. We report a versatile approach for the synthesis of anisotropic polyacrylamide hydrogels using lyotropic chromonic liquid crystal (LCLC) templating. The orientational order of LCLCs in a mold can be patterned by controlling surface anchoring conditions, which in turn patterns the polymer network. The resulting hydrogels have tunable pore size and mechanical anisotropy. For example, the elastic moduli measured parallel and perpendicular to the LCLC order are 124.9 ± 6.4 kPa and 17.4 ± 1.1 kPa for a single composition. The resulting anisotropic hydrogels also have 30% larger swelling normal to the LCLC orientation than along the LCLC orientation. By patterning the LCLC order, this anisotropic swelling can be used to create 3D hydrogel structures. These anisotropic gels can also be functionalized with extracellular matrix (ECM) proteins and used as compliant substrata for cell culture. As an illustrative example, we show that the patterned hydrogel microstructure can be used to direct the orientation of cultured human corneal fibroblasts. This strategy to make anisotropic hydrogels has potential for enabling patternable tissue scaffolds, soft robotics, or microfluidic devices.