Biomimetic biphasic microsphere preparation based on the thermodynamic incompatibility of glycosaminoglycan with gelatin methacrylate for hair regeneration

Hair follicle (HF) tissue engineering is promising for hair loss treatment especially for androgenetic alopecia. Physiologically, the initiation of HF morphogenesis relies on the interactions between hair germ mesenchymal and epithelial layers. To simulate this intricate process, in this study, a co-flowing microfluidic-assisted technology was developed to produce dual aqueous microdroplets capturing growth factors and double-layer cells for subsequent use in hair regeneration. Microspheres, called G/HAD, were generated using glycosaminoglycanbased photo-crosslinkable biological macromolecule (HAD) shells and gelatin methacrylate (GelMA) cores to enclose mesenchymal cells (MSCs) and mouse epidermal cells (EPCs). The findings indicated that the glycosaminoglycan-based HAD shells display thermodynamic incompatibility with GelMA cores, resulting in the aqueous phase separation of G/HAD cell spheres. These G/HAD microspheres exhibited favorable characteristics, including sustained growth factor release and wet adhesion properties. After transplantation into the dorsal skin of BALB/c nude mice, G/HAD cell microspheres efficiently induced the regeneration of HFs. This approach enables the mass production of approximately 250 dual -layer microspheres per minute. Thus, this dual -layer microsphere fabrication method holds great potential in improving current hair regeneration techniques and can also be combined with other tissue engineering techniques for various regenerative purposes.