Physicochemically guided modular assembly of a multi-appendage skin model via 3D bioprinting

Abstract Recapitulating the structural and functional complexity of human skin, particularly the diverse appendages such as hair follicles and sweat glands, remains a formidable challenge in regenerative medicine. While three-dimensional (3D) bioprinting offers precise spatial control, directing stem cells toward specific appendage lineages within a printed construct requires a sophisticated integration of microenvironmental cues. In this study, we present a modular biofabrication strategy that leverages the synergy between biomechanically matched hydrogel stiffness and tissue-specific biochemical extracts to direct the differentiation of mesenchymal stem cell spheroids. Quantitative mapping of native mouse skin via atomic force microscopy revealed distinct mechanical niches, characterized by a compliant perifollicular dermis (4.58 ± 0.63 kPa) and a significantly stiffer peri-glandular microenvironment (9.43 ± 1.49 kPa). Guided by these physiological benchmarks, we engineered gelatin methacryloyl hydrogels with tunable moduli to serve as bioinspired physical scaffolds. Complementary biochemical signals were derived from newborn mouse plantar dermis and dorsal dermis, which proteomic analysis identified as being enriched in bone morphogenetic protein and Sonic Hedgehog signaling components, respectively. Our results demonstrate that while matrix stiffness alone initiates epithelial commitment, the precise pairing of physical and biochemical cues is essential for lineage-specific maturation. These lineage-committed modules were spatially assembled via modular 3D bioprinting into an integrated multi-appendage skin model that maintained high cell viability and phenotypic stability during long-term culture. Our work provides a scalable approach for fabricating complex heterotypic tissues and serves as a versatile platform for disease modeling and drug testing.