Large bone defects are difficult to repair because conventional scaffolds often ignite excessive inflammation and fail to build a functional vascular network. A successful repair requires a precise sequence which includes calming inflammation, growing blood vessels, and finally forming new bone. However, most materials target only one step.

In a study published in Biomaterials, a team of researchers led by Prof. LIN Jinxin from Fujian Institute of Research on the Structure of Matter of the Chinese Academy of Sciences engineered a biomimetic composite scaffold (MnLA/HBP) that delivers two gasotransmitters, carbon monoxide (CO) and nitric oxide (NO), in an inflammation‑responsive manner.

Researchers fabricated the scaffold using cryogenic three-dimensional (3D) printing. Low temperature preserved the activity of heat‑sensitive gas prodrugs: manganese carbonyl (MnCO) as a CO donor and L‑arginine as an NO donor. The scaffold features a highly interconnected porous structure and a compressive strength of about 13 MPa, matching human cancellous bone. It releases CO and NO only when triggered by reactive oxygen species (H₂O₂) in the inflammatory microenvironment, avoiding premature leakage and off‑target toxicity.

In vitro mechanistic studies revealed that CO simultaneously inhibited the NF‑κB pathway and activated the Nrf2 pathway, reprogramming macrophages from a pro‑inflammatory M1 phenotype to a pro‑regenerative M2 phenotype, which dramatically reduced inflammatory cytokines and elevated anti‑inflammatory markers. Meanwhile, NO activated the MAPK/ERK and PI3K‑Akt pathways in endothelial cells, promoting cell migration and tube formation. The two gases synergistically activated the sGC‑cGMP‑PKG axis in osteoblasts, significantly up‑regulating osteogenic genes and enhancing matrix mineralization.

The scaffold was tested in a rat critical‑sized calvarial defect model. After 12 weeks, micro‑CT analysis showed that the MnLA/HBP scaffold achieved a bone volume/tissue volume ratio of over 60%, far outperforming single‑drug scaffolds (MnCO/HBP or L‑Arg/HBP) and blank controls. Histological staining confirmed robust new bone formation, dense collagen deposition, and mature mineralized tissue. Immunohistochemistry revealed strong expression of osteocalcin and osteopontin in the MnLA/HBP group. Importantly, no toxicity was detected in major organs, confirming excellent biocompatibility.

This study establishes an "immune‑vascular‑osteogenic" sequential therapy paradigm through gasotransmitter synergy. The cryogenic 3D printing strategy not only preserves the activity of labile gas prodrugs but also allows precise control over scaffold architecture. The MnLA/HBP scaffold offers a promising platform for treating critical‑sized bone defects.