Microenvironment-responsive living hydrogel containing engineered probiotic for treatment of massive bone defects

Self-activating and microenvironment-responsive biomaterials for tissue regeneration would address the escalating need for bone grafting, but remain challenging. The emergence of microbial living therapeutics offers vast potential in regenerative medicine, as genetically engineered probiotics possess efficient stimuli-responsiveness and tunable biological functions. Here, using elevated endogenous nitric oxide (NO) signals as a biological trigger in bone fracture injuries, a Living Responsive Regenerative Medicine (LRRM) strategy for <i>in situ</i> bone defect repair through real-time controlled release of bone morphogenetic protein-2 (BMP2) is proposed. The <i>Escherichia coli</i> Nissle 1917 (EcN) strain, genetically engineered to sense NO signals and correspondingly produce and secrete BMP2, was firstly encapsulated in gelatin methacryloyl (GelMA) microspheres and then embedded in a bulky hyaluronic acid methacryloyl (HAMA) hydrogel to form a living hydrogel device that circumvents immune attack and prevents bacterial leakage. <i>In vivo</i> multiple bone defect models demonstrated the efficacy of the living hydrogel in enhancing the maturation of bone callus, promoting neovascularization, and facilitating full-thickness bone union. Strategic incorporation of engineered probiotics and the bilayer-structured encapsulation system may emerge as an effective and microenvironment-responsive medicine approach for tissue regeneration.