Lumbrokinase-Loaded Gelma Hydrogel Promotes Vascularized Bone Regeneration

The effective treatment of critical-sized bone defects demands a coordinated interplay between osteogenesis and angiogenesis. We incorporated lumbrokinase (LK) into gelatin methacryloyl (GelMA) hydrogel to enable controlled release of LK, thus enhancing vascularized bone regeneration in critical-sized bone defects. We constructed 3-mm-diameter critical-sized cranial defects (control, GelMA, and LK-loaded GelMA groups) in 6-week-old male C57/BL6 mice and euthanized them after 8 weeks. The quantity and morphology of new bone were analyzed by micro-computed tomography, hematoxylin and eosin (H&E), Masson’s trichrome staining. The in vivo capacity of angiogenesis was analyzed by immunofluorescence staining, and biocompatibility was analyzed by H&E staining of important organs. For in vitro experiments, material characterization of LK-loaded GelMA hydrogel and its biocompatibility were assessed. The osteogenesis promotion capacity was determined by alkaline phosphatase (ALP), alizarin red staining (ARS), and qPCR. The angiogenesis promotion capacity was assessed by qPCR, transwell, scratch, and angiogenesis tests. Inflammatory regulation was determined by qPCR, reactive oxygen species (ROS) staining, and conditioned culture. In vivo experiments found that LK-loaded GelMA hydrogel increased relative bone growth surface area, bone volume/tissue volume, and the relative fluorescence intensity of endomucin and CD31. In vitro experiments proved that LK-loaded GelMA promoted osteoblast differentiation, migration, angiogenesis, and M2 macrophage polarization. Our studies found that LK-loaded GelMA exhibited in vivo and in vitro osteogenic, angiogenic, and inflammatory regulatory capacity, demonstrating its potential for clinical application in critical-sized bone defects.