Eva Ingeborg Reihs

Current synovial models fail to capture human‐relevant OA traits. This study develops a fully humanized, animal‐free synovial membrane model, mimicking OA synovial structure and molecular profile. Protocols for rheumatoid synovial micromasses are adapted for 3D biochip cultures of OA synoviocytes using TISSEEL fibrin and ELAREM lysate. Cell activity, mRNA expression, and structural changes are evaluated under varying hydrogel stiffness and cytokine exposure, with results compared to human OA and animal (equine and murine) synovial tissues. The animal‐free biochip protocols replicate synovial architecture successfully. Improved gene expression of reticular collagen III (COL3A1) is achieved with 50 mg mL ⁻¹ fibrinogen and 1% hPL. A 50 pg mL ⁻¹ TNF‐α and IL‐1β stimulus induced a pro‐fibrotic phenotype (COL1A1, COL3A1) distinct from the inflammatory response triggered by ng/mL dosages (IL6, MMP1, MMP3, and MMP13, vs the pg/mL model). The clinical relevance of the patient‐relevant OA synovial model is underscored by significant Yap1 overexpression, reflecting synovial hyperplasia from cell activation and inflammation. Yap1 distribution, as a biomarker (ctrl vs kOA tissue), is best replicated in the low‐dose pg/ml‐treated model. The tissue‐mimetic biochips provide a human‐relevant OA study platform offering patient‐relevant molecular insights into the structure‐function relationships of osteoarthritic synovial tissues while eliminating animal‐derived materials.

Can nonhuman animals be used for the benefit of humans in a scientifically and morally justified manner and, if yes, how? Based on our own experiences as scholars from various academic backgrounds, we argue that this question can only be answered as an interdisciplinary and international endeavor, considering insights from research ethics and animal ethics as well as scientific and legal aspects. The aim of this article is to contribute to the foundation of the emerging field of animal research ethics. In doing so, we describe the following seven phases of animal research experiments: ethical, legal and social presumptions (phase 0), planning (phase I), review (phase II), conduct of experiments (phase III), publication/dissemination (phase IV), further exploitation of results (phase V), and evaluation (phase VI). In total, 20 key ethical, legal, and practical challenges that an ethical framework for the use of animals in research needs to address are identified and analyzed. Finally, we characterize the following four meta-challenges and opportunities associated with animal research ethics as a field: (1) moral pluralism, (2) the integration of views and positions outside the laboratory, (3) international plurality of conduct, standards, and legal norms, and (4) interdisciplinary education.

Due to advances in additive manufacturing and prototyping, affordable and rapid microfluidic sensor-integrated assays can be fabricated using additive manufacturing, xurography and electrode shadow masking to create versatile platform technologies aimed toward qualitative assessment of acute cytotoxic or cytolytic events using stand-alone biochip platforms in the context of environmental risk assessment. In the current study, we established a nasal mucosa biosensing platform using RPMI2650 mucosa cells inside a membrane-integrated impedance-sensing biochip using exclusively rapid prototyping technologies. In a final proof-of-concept, we applied this biosensing platform to create human cell models of nasal mucosa for monitoring the acute cytotoxic effect of zinc oxide reference nanoparticles. Our data generated with the biochip platform successfully monitored the acute toxicity and cytolytic activity of 6 mM zinc oxide nanoparticles, which was non-invasively monitored as a negative impedance slope on nasal epithelial models, demonstrating the feasibility of rapid prototyping technologies such as additive manufacturing and xurography for cell-based platform development.

As common industrial by-products, airborne engineered nanomaterials are considered important environmental toxins to monitor due to their potential health risks to humans and animals. The main uptake routes of airborne nanoparticles are nasal and/or oral inhalation, which are known to enable the transfer of nanomaterials into the bloodstream resulting in the rapid distribution throughout the human body. Consequently, mucosal barriers present in the nose, buccal, and lung have been identified and intensively studied as the key tissue barrier to nanoparticle translocation. Despite decades of research, surprisingly little is known about the differences among various mucosa tissue types to tolerate nanoparticle exposures. One limitation in comparing nanotoxicological data sets can be linked to a lack of harmonization and standardization of cell-based assays, where (a) different cultivation conditions such as an air-liquid interface or submerged cultures, (b) varying barrier maturity, and (c) diverse media substitutes have been used. The current comparative nanotoxicological study, therefore, aims at analyzing the toxic effects of nanomaterials on four human mucosa barrier models including nasal (RPMI2650), buccal (TR146), alveolar (A549), and bronchial (Calu-3) mucosal cell lines to better understand the modulating effects of tissue maturity, cultivation conditions, and tissue type using standard transwell cultivations at liquid-liquid and air-liquid interfaces. Overall, cell size, confluency, tight junction localization, and cell viability as well as barrier formation using 50% and 100% confluency was monitored using trans-epithelial-electrical resistance (TEER) measurements and resazurin-based Presto Blue assays of immature (e.g., 5 days) and mature (e.g., 22 days) cultures in the presence and absence of corticosteroids such as hydrocortisone. Results of our study show that cellular viability in response to increasing nanoparticle exposure scenarios is highly compound and cell-type specific (TR146 6 ± 0.7% at 2 mM ZnO (ZnO) vs. ~90% at 2 mM TiO₂ (TiO₂) for 24 h; Calu3 93.9 ± 4.21% at 2 mM ZnO vs. ~100% at 2 mM TiO₂). Nanoparticle-induced cytotoxic effects under air-liquid cultivation conditions declined in RPMI2650, A549, TR146, and Calu-3 cells (~0.7 to ~0.2-fold), with increasing 50 to 100% barrier maturity under the influence of ZnO (2 mM). Cell viability in early and late mucosa barriers where hardly influenced by TiO₂ as well as most cell types did not fall below 77% viability when added to Individual ALI cultures. Fully maturated bronchial mucosal cell barrier models cultivated under ALI conditions showed less tolerance to acute ZnO nanoparticle exposures (~50% remaining viability at 2 mM ZnO for 24 h) than the similarly treated but more robust nasal (~74%), buccal (~73%), and alveolar (~82%) cell-based models.

As common industrial by-products, airborne engineered nanomaterials are considered important environmental toxicants to monitor due to their potential health risks to humans and animals. The main uptake routes of airborne nanoparticles are nasal and/or oral inhalation, which are known to enable the transfer of nanomaterials into the blood stream resulting in rapid distribution in the body. Consequently, mucosal barriers present in nose, buccal and lung have been identified and intensively studied as the key tissue barrier to nanoparticle translocation. Despite decades of research, surprisingly little is known about the differences among various mucosa tissue types to tolerate nanoparticle exposures. One limitation in comparing nanotoxicological data sets can be linked to a lack of harmonization and standardization of cell-based assays, where a) different cultivation conditions such as air-liquid interface or submerged cultures, b) varying barrier maturity and c) diverse media substitutes have been used. The current comparative nanotoxicological study therefore aims at analyzing the toxic effects of nanomaterials on four human mucosa barrier models including nasal (RPMI2650), buccal (TR146), alveolar (A549), and bronchial (Calu-3) mucosal cell lines to better understand the modulating effects of tissue maturity, cultivation conditions and tissue type using standard Transwell cultivations at liquid-liquid and air-liquid interfaces. Overall, cell size, confluency, tight junction localization, and cell viability as well as barrier formation using 50% and 100% confluency was monitored using trans-epithelial-electrical resistance (TEER) measurements and Presto Blue assays of immature (e.g. 5 days) and mature (e.g. 22 days) cultures in the presence and absence of corticosteroids such as hydrocortisone. Results of our study show that cellular responses to increasing nanoparticle exposures are highly cell type specific, where bronchial mucosal cell barriers models cultivated under ALI conditions showed less tolerance to acute ZnO nanoparticle exposures. Additionally, stronger toxicities are found using early mucosa barriers compared to later barrier models being maturated under air-liquid cultivation conditions.