Fig. 3: LA@PA-TA@C hydrogel rescued epithelial regenerative ability and promoted mucociliary differentiation of epithelial organoids in vitro.

a The rabbit primary tracheal epithelial cells were co-cultured with different hydrogels (PA, PA-C, LA@PA-C, LA@PA-TA@C), 200 μM H₂O₂ was added to simulate the hyperactivated innate immune environment. Elements were created in BioRender. Yi, C. (2025) https://BioRender.com/m4mpwtc. b ZO-1 (white) and cell nuclei (DAPI, blue) IF staining of epithelial cells for tight junction marker expression after 3 days culture. Scale bar, 20 μm. Pink arrows represent empty areas after cell apoptosis. c quantitative analysis of the ZO-1 expression in b n = 4 samples per group, and the results were representative of 2 independent experiments. d–f Rabbit primary epithelial cells scratching assay under oxidative stress after 48 h. d IF staining of ZO-1 (marker of tight junctions, red), Ki67 (marker of proliferation, green), and cell nuclei (DAPI, blue). The circles represent empty areas after cell apoptosis. scale bar, 200 μm. e statistical analyses of the residual wound area in d. f quantitative analysis of the Ki67 expression in D. n = 4 samples per group in b, f, and the results were representative of 2 independent experiments. g–k qPCR revealed gene expression in primary epithelial cells after co-cultured with hydrogels under oxidative stress for 3days. g CK14, stem-related gene. h E-Cadherin, and i ZO-1 were tight junctions-related genes. j α-SMA, epithelial dedifferentiation-related gene. k Caspase 9, apoptosis-related gene. Group setting: Blank (no hydrogel and no H₂O₂), +200 μM H₂O₂ group (no hydrogel and add 200 μM H₂O₂). PA, PA-C, LA@PA-C, and LA@PA-TA@C represented groups co-cultured with corresponding hydrogels under 200 μM H₂O₂ environment_. n = 6 samples per group in g–k from two individual repeated experiments. l–o LA@PA-TA@C hydrogel rescued regenerative ability and promoted mucociliary differentiation of epithelial organoids. l the epithelial organoids culture model. The organoids were induced by rabbit primary tracheal epithelial cells on air-liquid interface (ALI) and were co-cultured with different hydrogels under oxidative stress for 21 days. Elements were created in BioRender. Yi, C. (2025) https://BioRender.com/m4mpwtc. m SEM images of organoids in different groups at 21 days. n staining for exhibiting organoid differentiation co-culture with PA hydrogel under oxidative stress for 21 days; o, co-culture with LA@PA-TA@C hydrogel under oxidative stress for 21 days. n, o gross morphology (HE staining), tight junction, TJ (ZO-1, green), mucociliogenic (PAS, purple; AC-Tub, green), and epithelial stemness (CK-14, red). Cell nuclei (DAPI, blue). Scale bar in (l–o), 20 μm; and 10 μm in o magnified regions. n = 3 samples per group in l–o, where each sample was from an individual experiments. Source data are provided as a Source Data file. Data are presented as mean ± SD. All error bars represent SD. p values calculated using ANOVA followed by Tukey’s post hoc test.