Alginate-Based cryogenic Bi-nozzle 3D printed hierarchical porous scaffold for accelerating granulation tissue regeneration in exposed bone wounds

The exposed bone wound is defined as a kind of wound with bone denuded of periosteum and the deficiency of full-thickness skin and muscles. The wound have no matrix microenvironment and is prone to infection and, hence, hard to heal. Granulation tissue is significant during wound healing, providing a matrix and resisting infection. 3D printing is adopted to fabricate dermis and epidermis regeneration. However, the porosity of most 3D-printed scaffolds is low, leading to a poor drug-loading capacity. To address these limitations, this study developed an alginate-based hierarchical porous scaffold fabricated via cryogenic bi-nozzle 3D printing, enabling dual-factor spatial delivery of VEGF and LL-37 to simultaneously promote angiogenesis and immunomodulation. The alternating dual nozzle printing allows the scaffolds to carry two biological factors with spatial distribution while maintaining the structure integrity. In vitro studies demonstrated enhanced tube formation and endothelial migration, while in vivo implantation in rabbit exposed bone wounds revealed accelerated granulation tissue regeneration and re-epithelialization. The drug-loaded scaffolds can induce the tube formation of vascular endothelial cells, assist inflammatory cells in resisting infection, and ultimately form a fully functional granulation tissue. These results demonstrate the potential clinical applicability of this immunomodulatory dual-factor scaffold for challenging exposed bone wound repair. This graphical abstract illustrates the fabrication and healing mechanism of an alginate-based hierarchical porous scaffold for treating exposed bone wounds. The Clinical Problem: The challenge of an exposed bone wound is depicted, characterized by a lack of soft tissue, persistent inflammation, and poor vascularization. Our Engineering Solution: The scaffold is fabricated via cryogenic bi-nozzle 3D printing. One nozzle delivers VEGF-laden bioink to promote angiogenesis, while the other delivers LL-37-laden bioink for antimicrobial activity and immunomodulation. The low-temperature printing process creates a hierarchical porous structure (macro-pores from printing and micro-pores inside fibers via cryogenization), enabling high drug loading and sustained release. Multimodal Healing Mechanisms: Upon implantation, the scaffold orchestrates the wound microenvironment through: Spatially Guided Angiogenesis: VEGF release induces robust blood vessel formation from the wound edge. Accelerated Immune Resolution: LL-37, combined with the scaffold's structure, promotes a decisive shift in macrophage polarization from the pro-inflammatory M1 phenotype (CD86+) to the pro-healing M2 phenotype (CD206+). The Functional Outcome: The synergistic effects of enhanced vascularization and a mitigated inflammatory response lead to the rapid formation of fully functional granulation tissue, which covers the exposed bone and facilitates complete wound healing.