Abstract: PURPOSE: To develop a highly porous hydrogel microsphere scaffold based on aqueous biphasic separation technology, aimed at enhancing its angiogenic potential in both in vitro and in vivo environments. METHODS: Porous hydrogels were prepared through phase separation. The porous structure was observed using confocal microscopy, and the microscopic pore structure of the material was examined using scanning electron microscopy(SEM). The mechanical strength of the material and the thermal stability of the hydrogel were measured. Porous hydrogel microspheres were fabricated using microfluidic technology. In vitro tests were performed to assess the biocompatibility and angiogenesis-promoting ability of the material. The porous hydrogel microspheres were implanted subcutaneously in mice, and histological staining was conducted to observe biocompatibility and vascular formation. RESULTS: A porous hydrogel system was successfully constructed. Confocal and SEM confirmed that after crosslinking of gelatin methacryloyl (GelMA), the selective removal of sodium alginate(SA) phase formed a network of interconnected pores, exhibiting the ability to bear stress and stability. CCK-8 assay and live/dead staining results demonstrated that the material exhibited excellent biocompatibility. The GelMA/SA material extract significantly promoted the formation of tubular structures in human umbilical vein endothelial cells(HUVECs). Subcutaneous implantation in mice showed that the porous hydrogel facilitated tissue ingrowth and localized angiogenesis. CONCLUSIONS: The phase separation-engineered porous hydrogel microsphere system, combining continuous pore structure and angiogenesis-promoting effects, provides a novel scaffold material for tissue engineering and regenerative repair.

Key words: Phase separation, Porous hydrogels, Angiogenesis, Tissue engineering and regeneration