Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models ­ An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from dif­ferent countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

Toxicity assessment of CeO2 and CuO nanoparticles at the air-liquid interface using bioinspired condensational particle growth.

CeO2 and CuO nanoparticles (NPs) are used as additives in petrodiesel to enhance engine performance leading to reduced diesel combustion emissions. Despite their benefits, the additive application poses human health concerns by releasing inhalable NPs into the ambient air. In this study, a bioinspired lung cell exposure system, Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was employed for evaluating the toxicity of aerosolized CeO2 and CuO NPs with a short duration of exposure (≤10 min vs. hours in other systems) and without exerting toxicity from non-NP factors. Human epithelial A549 lung cells were cultured and maintained within DAVID at the air-liquid interface (ALI), onto which aerosolized NPs were deposited, and experiments in submerged cells were used for comparison. Exposure of the cells to the CeO2 NPs did not result in detectable IL-8 release, nor did it produce a significant reduction in cell viability based on lactate dehydrogenase (LDH) assay, with a marginal decrease (10%) at the dose of 388 µg/cm2 (273 cm2/cm2). In contrast, exposure to CuO NPs resulted in a concentration dependent reduction in LDH release based on LDH leakage, with 38% reduction in viability at the highest dose of 52 µg/cm2 (28.3 cm2/cm2). Cells exposed to CuO NPs resulted in a dose dependent cellular membrane toxicity and expressed IL-8 secretion at a global dose five times lower than cells exposed under submerged conditions. However, when comparing the ALI results at the local cellular dose of CuO NPs to the submerged results, the IL-8 secretion was similar. In this study, we demonstrated DAVID as a new exposure tool that helps evaluate aerosol toxicity in simulated lung environment. Our results also highlight the necessity in choosing the right assay endpoints for the given exposure scenario, e.g., LDH for ALI and Deep Blue for submerged conditions for cell viability.

Bioinspired Hierarchical Carbon Structures as Potential Scaffolds for Wound Healing and Tissue Regeneration Applications.

Engineered bio-scaffolds for wound healing provide an attractive treatment option for tissue engineering and traumatic skin injuries since they can reduce dependence on donors and promote faster repair through strategic surface engineering. Current scaffolds present limitations in handling, preparation, shelf life, and sterilization options. In this study, bio-inspired hierarchical all-carbon structures comprising carbon nanotube (CNT) carpets covalently bonded to flexible carbon fabric have been investigated as a platform for cell growth and future tissue regeneration applications. CNTs are known to provide guidance for cell growth, but loose CNTs are susceptible to intracellular uptake and are suspected to cause in vitro and in vivo cytotoxicity. This risk is suppressed in these materials due to the covalent attachment of CNTs on a larger fabric, and the synergistic benefits of nanoscale and micro-macro scale architectures, as seen in natural biological materials, can be obtained. The structural durability, biocompatibility, tunable surface architecture, and ultra-high specific surface area of these materials make them attractive candidates for wound healing. In this study, investigations of cytotoxicity, skin cell proliferation, and cell migration were performed, and results indicate promise in both biocompatibility and directed cell growth. Moreover, these scaffolds provided cytoprotection against environmental stressors such as Ultraviolet B (UVB) rays. It was seen that cell growth could also be tailored through the control of CNT carpet height and surface wettability. These results support future promise in the design of hierarchical carbon scaffolds for strategic wound healing and tissue regeneration applications.

Evaluation of Human Performance Aiding Live Synthetically Engineered Bacteria in a Gut-on-a-Chip.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. Herein, we present a simplistic gut-chip model using common Caco2 and HT-29 cell lines to establish a dynamic human screening platform for a cortisol sensing tryptamine producing synbiotic for cognitive performance sustainment. The synbiotic, SYN, was engineered from the common probiotic E. coli Nissle 1917 strain. It had the ability to sense cortisol at physiological concentrations, resulting in the activation of a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. SYN was successfully cultivated within the gut-chip showing log-phase growth comparable to the wild-type strain. Tryptophan metabolism occurred quickly in the gut compartment when exposed to 5 µM cortisol, resulting in the complete conversion of bioavailable tryptophan into tryptamine. The flux of tryptophan and tryptamine from the gut to the vascular compartment of the chip was delayed by 12 h, as indicated by the detectable tryptamine in the vascular compartment. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, enabled host analyses of cellular viability and pro-inflammatory cytokine secretion, and succeeded in providing an efficacy test of a novel synbiotic. Organ-on-a-chip technology holds promise in aiding traditional therapeutic pipelines to more rapidly down select high potential compounds that reduce the failure rate and accelerate the opportunity for clinical intervention.

Textile-Integrated Liquid Metal Electrodes for Electrophysiological Monitoring.

Next generation textile-based wearable sensing systems will require flexibility and strength to maintain capabilities over a wide range of deformations. However, current material sets used for textile-based skin contacting electrodes lack these key properties, which hinder applications such as electrophysiological sensing. In this work, a facile spray coating approach to integrate liquid metal nanoparticle systems into textile form factors for conformal, flexible, and robust electrodes is presented. The liquid metal system employs functionalized liquid metal nanoparticles that provide a simple "peel-off to activate" means of imparting conductivity. The spray coating approach combined with the functionalized liquid metal system enables the creation of long-term reusable textile-integrated liquid metal electrodes (TILEs). Although the TILEs are dry electrodes by nature, they show equal skin-electrode impedances and sensing capabilities with improved wearability compared to commercial wet electrodes. Biocompatibility of TILEs in an in vivo skin environment is demonstrated, while providing improved sensing performance compared to previously reported textile-based dry electrodes. The "spray on dry-behave like wet" characteristics of TILEs opens opportunities for textile-based wearable health monitoring, haptics, and augmented/virtual reality applications that require the use of flexible and conformable dry electrodes.

Designer Extracellular Vesicles Modulate Pro-Neuronal Cell Responses and Improve Intracranial Retention.

Gene/oligonucleotide therapies have emerged as a promising strategy for the treatment of different neurological conditions. However, current methodologies for the delivery of neurogenic/neurotrophic cargo to brain and nerve tissue are fraught with caveats, including reliance on viral vectors, potential toxicity, and immune/inflammatory responses. Moreover, delivery to the central nervous system is further compounded by the low permeability of the blood brain barrier. Extracellular vesicles (EVs) have emerged as promising delivery vehicles for neurogenic/neurotrophic therapies, overcoming many of the limitations mentioned above. However, the manufacturing processes used for therapeutic EVs remain poorly understood. Here, we conducted a detailed study of the manufacturing process of neurogenic EVs by characterizing the nature of cargo and surface decoration, as well as the transfer dynamics across donor cells, EVs, and recipient cells. Neurogenic EVs loaded with Ascl1, Brn2, and Myt1l (ABM) are found to show enhanced neuron-specific tropism, modulate electrophysiological activity in neuronal cultures, and drive pro-neurogenic conversions/reprogramming. Moreover, murine studies demonstrate that surface decoration with glutamate receptors appears to mediate enhanced EV delivery to the brain. Altogether, the results indicate that ABM-loaded designer EVs can be a promising platform nanotechnology to drive pro-neuronal responses, and that surface functionalization with glutamate receptors can facilitate the deployment of EVs to the brain.

Bioscaffold Stiffness Mediates Aerosolized Nanoparticle Uptake in Lung Epithelial Cells.

In this study, highly porous, ultrasoft polymeric mats mimicking human tissues were formed from novel polyurethane soft dendritic colloids (PU SDCs). PU SDCs have a unique fibrillar morphology controlled by antisolvent precipitation. When filtered from suspension, PU SDCs form mechanically robust nonwoven mats. The stiffness of the SDC mats can be tuned for physiological relevance. The unique physiochemical characteristics of the PU SDC particles dictate the mechanical properties resulting in tunable elastic moduli ranging from 200 to 800 kPa. The human lung A549 cells cultured on both stiff and soft PU SDC membranes were found to be viable, capable of supporting the air-liquid interface (ALI) cell culture, and maintained barrier integrity. Furthermore, A549 cellular viability and uptake efficiency of aerosolized tannic acid-coated gold nanoparticles (Ta-Au) was found to depend on elastic modulus and culture conditions. Ta-Au nanoparticle uptake was twofold and fourfold greater on soft PU SDCs, when cultured at submerged and ALI conditions, respectively. The significant increase in endocytosed Ta-Au resulted in a 20% decrease in viability, and a 4-fold increase in IL-8 cytokine secretion when cultured on soft PU SDCs at ALI. Common tissue culture materials exhibit super-physiological elastic moduli, a factor found to be critical in analyzing nanomaterial cellular interactions and biological responses.

Examining cellular responses to reconstituted antibody protein liquids.

Protein ionic liquids (PIL) are a new class of biologic stabilizers designed to protect the functionality and extend the shelf-life of biotechnological and therapeutic agents making them more readily available, and resistant to austere environments. Protein biorecognition elements such as monoclonal antibodies are commonly utilized therapeutics that require the robust stabilization offered by PILs, but biocompatibility remains an important issue. This study has focused on characterizing the biocompatibility of an antibody based PIL by exposing multiple cells types to a cationized immunoglobulin suspended in an anionic liquid (IgG-IL). The IgG-IL caused no significant alterations in cellular health for all three cell types with treatments < 12.5 µg/mL. Concentrations ≥ 12.5 µg/mL resulted in significant necrotic cell death in A549 and HaCaT cells, and caspase associated cell death in HepG2 cells. In addition, all cells displayed evidence of oxidative stress and IL-8 induction in response to IgG-IL exposures. Therapeutic Ig can be utilized with a wide dose range that extends into concentrations we have found to exhibit cytotoxicity raising a toxicity concern and a need for more extensive understanding of the biocompatibility of IgG-ILs.

Characterization of an engineered live bacterial therapeutic for the treatment of phenylketonuria in a human gut-on-a-chip.

Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.

In Vitro Aerosol Exposure to Nanomaterials: From Laboratory to Environmental Field Toxicity Testing.

Exposure to nanomaterials (NMs) is inevitable, requiring robust toxicological assessment to understand potential environmental and human health effects. NMs are favored in many applications because of their small size; however, this allows them to easily aerosolize and, subsequently, expose humans via inhalation. Toxicological assessment of NMs by conventional methods in submerged cell culture is not a relevant way to assess inhalation toxicity of NMs because of particle interference with bioassays and changes in particokinetics when dispersed in medium. Therefore, an in vitro aerosol exposure chamber (AEC) was custom designed and used for direct deposition of NMs from aerosols in the environment to the air-liquid interface of lung cells. Human epithelial lung cell line, A549, was used to assess the toxicity of copper, nickel, and zinc oxide nanopowders aerosolized by acoustic agitation in laboratory study. Post optimization, the AEC was used in the field to expose the A549 cells to NM aerosols generated from firing a hand gun and rifle. Toxicity was assessed using nondestructive assays for cell viability and inflammatory response, comparing the biologic effect to the delivered mass dose measured by inductively coupled plasma-mass spectrometry. The nanopowder exposure to submerged and ALI cells resulted in dose-dependent toxicity. In the field, weapon exhaust from the M4 reduced cell viability greater than the M9, while the M9 stimulated inflammatory cytokine release of IL-8. This study highlights the use of a portable chamber with the capability to assess toxicity of NM aerosols exposed to air-liquid interface in vitro lung cell culture.

Cancer cell aggregate hypoxia visualized in vitro via biocompatible fiber sensors.

To fully understand biological behavior in vitro often dictates that oxygen be reported at either a local or a cellular level. Oxygen sensors based on the luminescent quenching of a specific form of electrospun fiber were developed for measurement of both gaseous and dissolved oxygen concentrations. Electrospinning was used to fabricate "core-shell" fiber configurations in which oxygen-sensitive transition-metal porphyrin complexes are embedded in an optically clear, gas permeable polycarbonate polymer 'core' while polycaprolactone provided a protective yet biocompatible 'shell'. By taking advantage of the resulting high sensitivity and fast response of electrospun core-shell fiber sensors, we were able to locate and image hypoxic regions in contact with aggregates of glioblastoma cells. Nanoscale, biomimetic sensors containing oxygen-sensitive porphyrins are particularly well suited to biological applications. These 'smart' nanofiber based sensors do not consume oxygen, their mechanical and chemical characteristics can be finely tuned allowing tailoring of biocompatibility and microstructure. Core-shell nanofiber oxygen sensing fibers could provide real-time assessments of tumor cell response to pharmacological innovations designed to target hypoxic regions driving new knowledge and technological advancement.

Biocompatible electrospun polymer blends for biomedical applications.

Blends of natural and synthetic polymers have received considerable attention as biomaterials due to the potential to optimize both mechanical and bioactive properties. Electrospinning of biocompatible polymers is an efficient method producing biomimetic topographies suited to various applications. In the ultimate application, electrospun scaffolds must also incorporate drug/protein delivery for effective cell growth and tissue repair. This study explored the suitability of a ternary Polymethylmethacrylate-Polycaprolactone-gelatin blend in the preparation of electrospun scaffolds for biomedical applications. Tuning the blend composition allows control over scaffold mechanical properties and degradation rate. Significant improvements were observed in the mechanical properties of the blend compared with the individual components. In order to study drug delivery potential, triblends were impregnated with the model compound Rhodamine-B using sub/supercritical CO2 infusion under benign conditions. Results show significantly distinct release profiles of the impregnated dye from the triblends. Specific factors such as porosity, degradation rate, stress relaxation, dye-polymer interactions, play key roles in impregnation and release. Each polymer component of the triblends shows distinct behavior during impregnation and release process. This affects the aforementioned factors and the release profiles of the dye. Careful control over blend composition and infusion conditions creates the flexibility needed to produce biocompatible electrospun scaffolds for a variety of biomedical applications.

Recrystallization improves the mechanical properties of sintered electrospun polycaprolactone.

BACKGROUND: Resorbable electrospun polycaprolactone (PCL) scaffolds for tissue reconstruction can provide physicians with an "off the shelf" product tailored to the patient's specific tissue architecture. However, many tissue-engineering platforms do not possess the necessary long-term mechanical stability needed to properly support tissue development. OBJECTIVE: Sintering has been explored as a means of altering the properties of electrospun PCL. However, crystallinity-driven changes in mechanical properties following thermal treatment have not been previously investigated. METHODS: PCL nanofibers were produced by electrospinning and subsequently thermally sintered (at 55, 56 and 58°C) to enhance their long-term mechanical integrity in response to representative biological milieux. RESULTS: Scaffolds initially sintered at 56°C displayed 6-fold increases in compressive strength and 3-fold increases in modulus, while displaying 10-fold increases in energy dissipation with increasing sintering temperature. Sintering just below the Tm resulted in amorphization of the 55°C sample as indicated by the 20-fold lower XRD peak intensities. Although crystallinity is suppressed, the polymer chains likely retain chain alignment from electrospinning and are apparently highly susceptible to recrystallization. After only 1d PBS exposure, the 55°C samples recover a substantial fraction of the as-spun crystallinity; 7d of exposure fully restores as-spun peak intensities. The mechanical properties of all three (55, 56, or 58°C) scaffolds displayed peak values of compressive strength and modulus following 7d exposure. CONCLUSION: In contrast with the current state-of-the-art which assumes that tissue engineering scaffolds only grow weaker following exposure, in these scaffolds maximum values of compressive strength and modulus were observed after 7d of aqueous immersion. This suggests that polymeric recrystallization can be used to increase or optimize mechanical properties in vitro/in vivo. Scaffolds that increase their mechanical integrity during biological exposures constitute a new pathway enabling clinical advances.

Media-based effects on the hydrolytic degradation and crystallization of electrospun synthetic-biologic blends.

Tissue engineering scaffold degradation in aqueous environments is a widely recognized factor determining the fate of the associated anchorage-dependent cells. Electrospun blends of synthetic polycaprolactone (PCL) and a biological polymer, gelatin, of 25, 50, and 75 wt% were investigated for alterations in crystallinity, microstructure and morphology following widely used in vitro biological exposures. To our knowledge, the effects of these different aqueous-based biological media compositions on the degradation of these blends have never been directly compared. X-ray diffraction (XRD) analysis exposed that differences in PCL crystallinity were observed following exposures to phosphate buffered solution (PBS), Dulbecco's modified eagle medium (DMEM) cell culture media, and DI water following 7 days of exposure at 37 °C. XRD data suggested that in vitro medium exposures aid in providing chain mobility and rearrangement due to hydrolytic degradation of the gelatin phase, allowing previously constrained, poorly crystalline PCL regions to achieve more intense reflections resulting in the presence of crystalline peaks. The dry, as-spun modulus of relatively soft 100 % PCL fibers was approximately 10 % of any gelatin-containing composition. Tensile testing results indicate that hydrated gelatin containing scaffolds on average had a fivefold increase in elongation compared to as-spun scaffolds. After 24-h of aqueous exposure, the elastic modulus decreased in proportion to increasing gelatin content. After 1 day of exposure, the 75 and 100 % gelatin compositions largely ceased to display measurable values of modulus, elongation or tensile strength due to considerable hydrolytic degradation. On a relative basis, common aqueous in vitro medium exposures (deionized water, PBS, and DMEM) resulted in significantly divergent amounts of crystalline PCL, overall microstructure and fiber morphology in the blended compositions, subsequently 'shielding' scaffolds from significant changes in mechanical properties after 24-h of exposure. Understanding electrospun PCL-gelatin scaffold dynamics in different aqueous-based cell culture medias enables the ability to tailor scaffold composition to 'tune' degradation rate, microstructure, and long-term mechanical stability for optimal cellular growth, proliferation, and maturation.