Cryogenic 3D printing scaffolds achieve efficient bone regeneration by sequentially modulating the immune microenvironment through synergistic gas-mediated signaling.

Currently, the repair of large-sized critical bone defects remains a major clinical challenge, often compromised by implant-induced inflammation and inadequate vascularization. Here, we report a novel biomimetic composite scaffold (MnLA/HBP) fabricated via cryogenic 3D printing, designed to achieve strength requirements through controlled cryogenic printing and freeze-drying processes, whilst sequentially orchestrate bone healing through a synergistic gasotransmitter therapy. The scaffold was engineered to co-deliver carbon monoxide (CO) and nitric oxide (NO) prodrugs (MnCO and l-Arginine) in an inflammation-responsive manner. In vitro, the MnLA/HBP scaffold effectively reprogrammed macrophages from a pro-inflammatory (M1) to a pro-regenerative (M2) phenotype by simultaneously inhibiting the NF-κB pathway and activating the Nrf2 pathway. This established an immunotolerant microenvironment that subsequently promoted angiogenesis. The pro-angiogenic effect was driven by the complementary activation of the MAPK and PI3K-Akt pathways, culminating in the potent amplification of VEGF signaling Finally, the synergistic CO/NO signaling activated the sGC-cGMP-PKG axis, significantly promoting osteogenic differentiation. In a rat critical-sized calvarial defect model, the MnLA/HBP scaffold demonstrated superior bone regeneration efficacy compared to single-drug or blank controls. Our findings present a sophisticated "immune microenvironment reprogrammer" that integrates anti-inflammatory, pro-angiogenic, and osteogenic functions through intelligent gas synergy, offering a highly promising strategy for repairing complex bone defects.