Materials Today Bio

Controlled delivery of growth factors and viable cells remains a significant challenge in bone tissue engineering. In this study, a 3D-printed hydrogel scaffold system was developed for the co-delivery of bone morphogenetic protein-9 (BMP-9) and preosteoblasts to enhance bone regeneration. The system consisted of a 3D-printed base scaffold containing BMP-9-coated calcium sulfate (CaS) microparticles and a photocurable hydrogel coating layer encapsulating viable cells. The scaffold design exploited electrostatic interactions between BMP-9 and gelatin matrices by incorporating gelatin type B in the base scaffold and gelatin type A in the coating layer. Differences in the isoelectric points of these gelatin types were utilized to regulate protein binding and release. Release studies demonstrated that CaS microparticles alone exhibited rapid burst release, with nearly 80% of BMP-9 released within 24 h. Encapsulation of BMP-9 coated CaS particles in the 3D-printed scaffolds reduced the release rate, while the addition of the coating layer significantly improved sustained release, limiting BMP-9 release to approximately 50-60% by day 5. Bioactivity studies showed enhanced cell attachment in BMP-9 containing scaffolds compared with controls. Live/Dead cytotoxicity assays demonstrated high cell viability (>80%) within the coating layer over the culture period, confirming that the encapsulation and photocuring processes did not adversely affect cell survival. Cell proliferation and differentiation were further evaluated using WST-1 and alkaline phosphatase assays. The results demonstrate that electrostatic interactions governed by gelatin type selection can regulate BMP-9 release while maintaining high cell viability, providing a promising platform for growth factors and cell delivery in bone tissue engineering.

Biomimetic tubular scaffolds hold great promise for tackling unmet clinical needs thanks to their biocompatibility and recapitulation of cellular microenvironments, conferring the ability to promote regeneration. Potential applications include small-diameter vascular implants and grafts for airway repair, for which no viable off-the-shelf solutions currently exist. The tubular materials (4 and 8 mm internal and external diameters) presented here consist purely of type I collagen, contain no chemical crosslinkers, and reproduce the multi-scale architecture of the native tissue including the presence of collagen fibrils. A novel two-step protocol provides materials with distinct concentric layers. A porous external structure, obtained by means of ice templating combined with collagen topotactic fibrillogenesis, favours oriented cell colonization. A smooth and much less porous internal layer provides mechanical and water-tightness properties relevant for in vivo implantation and promotes the formation of an endothelial monolayer under both static and flow conditions. The compliance of the double-layered materials under physiological pressure is close to that of piglet carotid arteries. The materials are also determined to be sufficiently flexible to provide the ability to perform ex vivo anastomosis with bronchi, although the relatively low value of suture retention strength remains a limitation for in vivo suturing.

Pluripotent stem cells represent a potentially unlimited cell source for the fabrication of human bioartificial tissues to study and treat degenerative conditions such as type 1 diabetes. Alginate is widely used for mammalian cell immobilization and the primary hydrogel studied for pancreatic islet encapsulation. Rheological properties of alginate solutions or fully gelled forms are unsuitable as support matrix for embedded 3D printing. We describe partially gelled self-healing alginate formulations tuned for embedded 3D printing. Perfusable multi-plane hierarchical networks branching into 10 parallel channels, obtained by 3D printing of Pluronic F127 into the alginate support, show high fidelity to computer-assisted models. Therapeutic {beta}-cell doses (40x106 cells/mL) within centimeter-thick perfusable constructs remained viable for at least 1 week of culture under flow, with rapid insulin secretion detected upon glucose challenges. Stem cell-derived islet clusters cultured in 5-channel contructs for 25 days differentiated towards functional insulin-expressing cells. We describe a novel approach to generate cm-scale perfusable endocrine pancreatic constructs using sacrificial embedded 3D printing into alginate. This approach offers an adaptable platform to engineer perfusable cm-scale functional endocrine pancreatic tissues and potentially other vascularized bioartificial tissues.

Central nervous system (CNS) disease or injury might be treated by implanted devices, tissue regenerative scaffolds, or drug delivery platforms. However, inflammatory CNS responses limit these interventions and may worsen outcomes following damage to the CNS. Via the foreign body reaction (FBR), macrophages and glial cells trigger a "glial scar" around implants, reducing device performance, scaffold regenerative ability, or drug delivery potential. Previous studies have shown that stiffness of CNS implants significantly affects glial encapsulation, but few studies have investigated materials that truly match brain tissue stiffness. Porous precision-templated scaffolds (PTS) with uniform, interconnected, 40 {micro}m pores have shown favorable healing outcomes and a reduced FBR in numerous soft and hard tissue applications. To quantify the effects of both hydrogel compliance (stiffness) and pore size on glial encapsulation, we implanted poly(2-hydroxyethyl methacrylate-co-glycerol methacrylate) (pHEMA/GMA) PTS of varying stiffness and pore size for 4 weeks in rat brain. We observed reduced astrocyte encapsulation around PTS compared to solid hydrogel rods, reduced pro-inflammatory macrophage polarization for softer hydrogels versus stiffer hydrogels, and the presence of neuronal markers and neurogenesis within the pores. Utilizing soft, precision-porous hydrogels could provide a strategy for mitigating glial scarring and improving implant-based CNS treatments.

In vitro models are increasingly critical for interrogating cancer biology and therapeutic response, however, accurately recapitulating the tumor microenvironment (TME) remains a persistent challenge, particularly in head and neck cancers (HNC) characterized by complex cell-matrix interactions and heterogeneity. Current models often lack independent tunability of biochemical and biophysical cues, limiting systematic investigation of microenvironmental cues in a high-throughput format. Here, we establish a 3D droplet-based bioprinting platform for the fabrication of customizable in vitro TME models using poly(ethylene glycol) (PEG) hydrogels. Human HNC cell lines (FaDu and 2A3) with differing HPV statuses were bioprinted into PEG matrices spanning physiologically relevant stiffnesses (0.7-4.8 kPa) and compositions, including non-functionalized PEG and peptide-functionalized PEG (PEGfnc: RGD, YIGSR, CNYYSNS) and cultured for 7 days. Cluster growth, cell viability, and cluster morphology were assessed across multiple time points, matrix compositions, and matrix stiffnesses. Proliferation and endpoint phenotype expression were visualized using confocal microscopy through immunofluorescence. Results indicated enhanced cell viability in PEGfnc matrices, compared to non-functionalized matrices, while effect of matrix stiffness was less prominent. Median cluster size reached 40-50 m by day 7, and linear mixed-effects modeling identified how changes in cluster surface area, volume, and tumoroid complexity varied with cell type, matrix, and stiffness. By decoupling and systematically varying key TME parameters, this approach provides a robust and scalable framework for dissecting tumor-matrix interactions and advancing physiologically relevant in vitro models for cancer research and therapeutic screening.

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.

Neurotransmitters in the gut play a vital role in human health and neuroscience, and their real-time monitoring is essential for understanding underlying physiological mechanisms. However, bioelectronic systems capable of measuring neurotransmitters in vivo at the anatomical site of interest remain underdeveloped and largely depend on bulky, off-the-shelf electronic components, thereby constraining the development of systems that are both practical and minimally invasive. Here, we report a miniature ingestible pill that is capable of real-time in vivo sensing of two key neurotransmitters: serotonin (5-HT) and dopamine (DA). The system incorporates a fully printed three-electrode-based electrochemical sensor for neurotransmitter sensing and a custom application-specific integrated circuit (ASIC) that integrates all major functional blocks on a single chip, enabling a platform for fully wireless monitoring of gut neurotransmitters. The pill, measuring 5.8 mm in diameter and 19 mm in length, supports multiple electrochemical sensing techniques, including amperometry and voltammetry, with only 42 A of average current consumption. We demonstrate the ingestible platform through in vivo studies in rat animal models, enabling real-time monitoring of gut neurotransmitters.

Extracellular matrix (ECM) mechanical properties regulate tissue homeostasis and disease progression, with persistent ECM stiffening serving as a hallmark of fibrosis; yet, the early transition from healthy to diseased tissue remains poorly understood. Dynamic three-dimensional (3D) tissue models that capture early-stage stiffening are needed to investigate cellular responses during disease initiation. This work presents an innovative platform for studying cell responses in 3D environments undergoing active matrix stiffening. A bioinspired synthetic ECM incorporates collagen-mimetic peptides and employs sequential, non-terminal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to enable controlled increases in matrix stiffness over physiologically relevant timescales. Alternating polymer incubations produce a 2.5-fold increase in storage modulus over 72 hours, modeling the mechanical transition from healthy to early-stage fibrotic lung tissue. Live-cell reporter fibroblasts enable real-time monitoring of alpha-smooth muscle actin (SMA) expression, revealing significant upregulation during matrix stiffening that remains transient and difficult to detect via traditional endpoint assays. Active stiffening also modulates fibroblast motility, transiently increasing migration speed while persistently enhancing directional persistence. Complementary computational reaction-diffusion modeling provides mechanistic insight into modulus gradient formation and reaction kinetics. This versatile toolbox enables investigation of early mechanobiological responses to matrix stiffening and may aid identification of markers of fibrotic disease onset.

Maintaining a confluent, antithrombotic endothelium on cardiovascular biomaterial surfaces remains a major barrier to long-term hemocompatibility, as endothelial cells (ECs) rapidly denude under supraphysiological shear in prosthetic devices. Here, we hypothesized that mesoscale surface geometry ([~]100-200 {micro}m) could reorganize near-wall hemodynamics, preserving endothelial coverage and function under extreme shear. Engineered microtrenches were introduced onto an implant biomaterial to generate spatially defined shear environments. Under supraphysiological near-wall shear ([~]250 dyn/cm{superscript 2}), microtrenched geometries created attenuated shear and vorticity gradients. Endothelial monolayers were sustained in these flow domains for 120 hours, whereas flat controls rapidly denuded. Endothelial retention in 22.5{degrees} angled trenches increased dramatically, from an EC of 33 to 101 dyn/cm{superscript 2}. 45{degrees} angled trenches further increased endothelial shear resistance to an EC of 207 dyn/cm{superscript 2}. Endothelial monolayers demonstrated collective mechano-adaptation to ultra-high shear through VE-cadherin junction thickening and coordinated cytoskeletal and nuclear alignment. Mechanoadapted monolayers exhibited increased eNOS expression correlated with local shear and elevated nitrite production (45{degrees}: 50.4 {+/-} 6.1 {micro}M; 22.5{degrees}: 35.7 {+/-} 3.3 {micro}M; 0{degrees}: 28.4 {+/-} 6.8 {micro}M). In contrast, interfaces with abrupt shear transitions or elevated rotational flow exhibited reduced coverage, junctional thinning, and re-emergence of VCAM-1 and PAI-1, indicating inflammatory and pro-thrombotic activation. Structural, functional, and inflammatory readouts exhibited peak responses within a shared shear-vorticity regime. Multivariate regression identified shear-vorticity coupling as the dominant predictor of endothelial persistence, with optima clustering within a mechanical range ({approx}0.8-2.9 x 10 dyn{middle dot}cm-{superscript 2}{middle dot}s-{superscript 1}). These findings establish geometry-driven modulation of near-wall flow as a predictive, material-agnostic strategy for endothelialization and vasoprotection of high-shear cardiovascular implants.

Precision-cut lung slices (PCLS) retain the native cells and extracellular matrix that contribute to the structural and functional integrity of lung tissue. This technique enables the study of cell-matrix interactions and is particularly useful for pre-clinical pharmacological studies. More specifically, PCLS are widely used to model the complex pathophysiology of pulmonary fibrosis, an uncurable and progressive interstitial lung disease. Current ex vivo pulmonary fibrosis models expose PCLS to pro-fibrotic biochemical cues over a short timeframe (hours to days) and quickly collect samples for analysis due to viability concerns. This condensed timeline is a limitation to understanding chronic disease mechanisms. To extend the utility of ex vivo pulmonary fibrosis models, PCLS were embedded in engineered hydrogels and exposed to pro-fibrotic biochemical and biophysical cues. Hydrogel-embedded PCLS maintained greater than 80% total cell viability over 3 weeks in culture. Gene expression patterns in samples exposed to pro-fibrotic cues matched trends measured in human fibrotic lung tissue. Finally, treatment with Nintedanib, a Food and Drug Administration approved pulmonary fibrosis drug, moderately reduced fibroblast activation and influenced epithelial cell differentiation. Collectively, these results show that hydrogel-embedded PCLS models of pulmonary fibrosis extend our ability to study fibrotic processes ex vivo and, when applied to human tissues, present a new approach methodology for studying lung disease and treatment.

Extracellular matrix (ECM) proteins assemble to form a heterogeneous connective scaffold that supports cells. Physical interactions between cells and the matrix regulate cellular behaviors and influence subsequent tissue construction. However, there is a lack of fundamental understanding regarding the contributions of individual native ECM proteins to the matrix. This gap arises from the need for nanoscopic characterization, which operates on a much smaller length scale than typical assessments in cell and tissue cultures, as well as in tissue reconstruction and clinical implantation. This study aims to systematically investigate how individual ECM proteins affect lipid membranes structurally and mechanically, and how these influences regulate cell migration. Results from Langmuir isotherm analysis, X-ray reflectivity measurements, and cell scratch assays demonstrate that strong collagen adsorption on the membrane surface disrupts lipid packing. However, its rigid network provides a sturdy scaffold for cell adhesion, thereby enhancing cell attachment and promoting cell migration. In contrast, elastin has a minimal structural or mechanical impact on the membrane during both adsorption and compression, but it benefits cells by facilitating migration and reducing the risk of infection. Fibronectin, on the other hand, exhibits complex mechanical responses to compression, characterized by significant structural rearrangements that occur during adsorption. This strong interaction with the membrane can result in excessively high adhesion forces, ultimately limiting cell motility. These findings lay the foundation for the design of artificial scaffolds that can manipulate cellular responses, a critical step toward advancing regenerative medicine and tissue engineering. SignificanceFabricating extracellular matrix (ECM) scaffolds from cells offers advantages over traditional approaches, such as decellularized tissues, which face donor limitations, and artificial scaffolds, which may hinder cellular communication. However, the slow harvesting process of cell-derived ECM has limited its clinical applications. This research is part of a larger mission to engineer ECM prescaffolds on lipid carriers tailored to cell requirements, enhancing ECM production and regulating cell behavior. The first step involves systematically analyzing the structural and mechanical effects of ECM on lipid membranes and how these effects regulate cellular behavior. This work confirms distinct characteristics of ECM proteins, advancing fundamental understanding of cell-matrix interactions and paving the way for scaffold engineering.

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

The endothelialization of organ-on-chip platforms and vascular implants is often limited by slow cell attachment and unstable monolayer formation. This work presents a scalable workflow that imprints micro- and nano-gratings into elastin-like recombinamer (ELR)-based hydrogels, enabling rapid endothelial cell capture and accelerating monolayer formation within 14 days. Three gelatin-ELR formulations are engineered, with {superscript 1}H-NMR confirming incorporation of sequences designed to modulate bioactivity (ELR1: inert; ELR2: uPA-responsive; ELR3: RGD-adhesive). ELR incorporation generates fibrillar microstructures and enhances mechanical performance, yielding elastic-dominant networks suitable for high-fidelity pattern transfer and stable culture. Using this library, the combined effects of ELR bioactivity and groove geometry on human iPSC-derived endothelial cells (iPSC-ECs) are systematically evaluated. In a 15-minute attachment assay, patterned ELR composites markedly improve cell retention compared to gelatin, with ELR2 on [~]350 nm and [~]4 {micro}m grooves performing best, consistent with controlled, cell-mediated interfacial remodeling. This early advantage persists, as ELR2 and ELR3 hydrogels support rapid alignment and reach confluence by day 14, whereas gelatin remains sub-confluent. Cytoskeletal analysis confirms F-actin alignment. By combining enhanced early capture with protease-regulated remodeling, ELR2 identifies a favorable design window. These results establish a materials design framework linking programmable ELR chemistry with surface topography to engineer endothelial interfaces, providing a versatile platform for vascular biomaterials and microphysiological systems.

Living organisms achieve adaptive actuation through the seamless integration of neural motor control circuitry and proprioceptive feedback. While biohybrid robotics aims to replicate these capabilities by merging engineered muscle with synthetic scaffolds, the field remains limited by interfaces that lack the efficiency and closed-loop regulation of natural neuromuscular systems. Here, we introduce a biohybrid muscle actuator system featuring a bioelectronic interface based on soft poly(3,4-ethylenedioxythiophene) (PEDOT) fibers for stimulation and sensing. These fibers conformally couple to muscle tissues, eliciting robust contractions at voltages as low as 1 V--requiring ultra-low power (0.376 {+/-} 0.034 mW) and preserving long-term tissue viability. By leveraging the independent addressability of these fibers, we demonstrate selective actuation of individual muscle units to achieve precise spatiotemporal control of a two-muscle-powered walking biohybrid robot, reaching a locomotion speed of 5.43 {+/-} 0.79 mm/min. When configured as strain sensors, the fibers exhibit a high gauge factor of 155.45 {+/-} 6.59 and resolve contractile displacements within tens of micrometers. We demonstrate that this sensing modality can be integrated into a closed-loop controller to autonomously modulate stimulation based on real-time feedback, significantly mitigating muscle fatigue (p = 0.038) during continuous operation. This work establishes a versatile platform for efficient actuation and intrinsic feedback sensing, providing a blueprint for efficient, autonomous, and adaptive biohybrid machines. SummarySoft conductive fibers enable a bioelectronic interface for low-power actuation and closed-loop control in biohybrid robots.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Engineering three-dimensional neuronal tissues with defined architecture and functional connectivity remains a critical challenge for applications in disease modeling, drug discovery, and regenerative medicine. Recently, a variety of fabrication methods have arisen, such as bioprinting or manual assembly of organoids, but often struggle with scalability, reproducibility, or maintaining cell viability. Here, two scaffold-free acoustic levitation bioreactors are introduced: one optimized for the culture of uniform neuronal spheroids, and another designed for the structuration of assembloids composed of distinct neuronal identities. Using acoustic standing waves, these platforms enable the contactless manipulation of cells and aggregates, facilitating the formation of highly viable functionally mature spheroids. This study shows that both striatal and cortical cell aggregates formed in acoustic levitation self-organize into spheroids within 24 hours and remain viable up to 10 days under these particular culture conditions without medium renewal. These neuro-spheroids demonstrate healthy development with increased growth and typical terminal differentiation and synaptic maturation. Moreover, concentric cortico-striatal assembloids were successfully structured and cultivated using optimized acoustofluidic chips. Offering versatile and scalable tools for engineering complex neuronal networks, acoustic levitation reveals itself as an innovative approach to 3D neuronal tissue modeling, with broad implications for bioengineering, regenerative medicine and fundamental neuroscience research.

Curvature provides essential mechanical cues for epithelial cells, playing a key role in cell differentiation and morphology. Repeatable manufacture of precisely controlled curvature in soft hydrogel materials is therefore essential to study epithelial mechanobiology and function. Multiphoton (MP) based biofabrication holds promise due to its high resolution and three-dimensional design flexibility. Here, we leverage MPs advantages while increasing print speed to develop two complementary tools based on replica molding and multiphoton ablation. These can provide scalable hydrogel curvatures with tunable surface properties relevant for epithelial tissue engineering. In replica molding, MP prints are transferred into PDMS used to pattern centimeter scale arrays in hydrogels. In multiphoton ablation, hydrogels are locally degraded to generate precisely controlled curvatures and surface topography. With both methods, we repeatably guide epithelial cells into alveolar and duct-like shapes. Concave alveolar-like surfaces are shown to enhance the formation of thicker epithelial layers. We observe that surface properties, controlled by both tools, could enhance cytoskeletal organization. Using these biofabrication techniques, individual effects of curvature, surface properties, hydrogel composition, and bulk stiffness on epithelial cells can be studied. Both approaches offer high curvature control and throughput, providing a viable alternative to traditional 3D culture and other printing methods.

Accelerating the development of enzymatic degradation of polyesters such as poly(ethylene terephthalate) (PET) and poly(butylene terephthalate) (PBT) requires a rapid and parallelizable detection method. We developed a protein-based biosensor for the fast and accurate quantification of the PET and PBT degradation product, terephthalate (TPA), which we named TPAsense. Engineering TPAsense required overcoming low thermal stability and aggregation of the initial construct by introducing stabilizing mutations without disrupting the binding affinity to TPA. The sensor performance was validated by screening for the PBT degrading activity of a Leaf-branch Compost Cutinase (LCC) mutant library and comparing with liquid chromatography data. TPAsense detects nanomolar concentrations of TPA enabling shorter incubation times for screening workflows. In addition, a comparative analysis of PETase and PBTase kinetics was performed with TPAsense. Finally, we demonstrated the detection of PET microplastic in samples from a wastewater treatment plant by combining the biosensor and a PETase. TPAsense offers a platform to accelerate PETase and PBTase development for plastic waste recycling and detection of microplastic in the environment.

Understanding how airborne particulates disrupt the human alveolar barrier requires in vitro systems that accurately replicate its composition and function. We present a biodegradable lung alveoli-on-a-chip that reproduces the architecture and physiology of the human air-blood interface using a porous poly(lactic-co-glycolic acid) (PLGA) membrane positioned between epithelium and endothelium under air-liquid interface (ALI) culture. The membrane, fabricated by porogen-assisted nonsolvent-induced phase separation, exhibited >50 % porosity, [~]2 {micro}m thickness, and mechanical compliance over 100-fold higher than conventional Transwell inserts, closely resembling the native interstitium. During co-culture, gradual PLGA degradation was compensated by cell-secreted extracellular-matrix (ECM) proteins such as collagen IV and laminin, forming a self-remodeling barrier that maintained integrity for at least 11 days. The platform supported stable epithelial-endothelial co-culture, high transepithelial electrical resistance, and physiologically relevant permeability. To demonstrate its utility, the chip was used to assess pulmonary toxicity of four types of waste-combustion-derived particulates, including rubber, plastic bags, plastic bottles, and textile fibers, delivered apically under ALI conditions. All combustion products reduced cell viability, increased hydrogen-peroxide release, and elevated {gamma}-H2AX expression, indicating oxidative and genotoxic stress, while disrupting barrier permeability. Rubber combustion particles elicited the most severe toxicity, causing the greatest loss of viability, accumulation of reactive oxygen species, and formation of DNA double-strand breaks. Together, these results establish a biodegradable, ECM-remodeling lung alveoli-on-a-chip as a physiologically relevant platform for investigating source-specific particulate toxicity and alveolar-barrier pathophysiology. By bridging environmental exposure models with human-relevant lung biology, this system provides a quantitative and translatable tool for evaluating respiratory risks and therapeutic interventions.