This study presents a synthetic biology-based approach to develop bacterial extracellular vesicles (BEVs) displaying bone morphogenetic protein-2 (BMP-2) and CXCR4 to treat osteoporosis (OP). The researchers constructed a recombinant probiotic Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4 (ECN-pClyA-BMP-2-CXCR4), which overexpresses BMP-2 and CXCR4 on the surface of BEVs. These engineered BEVs, termed BEVs-BC, were isolated and tested for their ability to target bone marrow mesenchymal stem cells (BMSCs) and promote osteogenic differentiation while inhibiting adipogenic differentiation. The BEVs-BC showed strong bone-targeting ability in vivo, as demonstrated by biophotonic imaging, and significantly improved bone density and structure in ovariectomized (OVX) mice. The BEVs-BC also activated the BMP/SMAD signaling pathway, which is crucial for osteoblast differentiation and bone formation. The study further confirmed the safety and biocompatibility of BEVs-BC through in vivo toxicity assays, showing no significant organ toxicity or adverse effects. The results indicate that BEVs-BC, constructed using synthetic biology, offer a promising and effective strategy for the treatment of OP, with potential for industrial application. The study highlights the potential of synthetic biology in developing targeted and efficient therapeutic strategies for bone diseases.This study presents a synthetic biology-based approach to develop bacterial extracellular vesicles (BEVs) displaying bone morphogenetic protein-2 (BMP-2) and CXCR4 to treat osteoporosis (OP). The researchers constructed a recombinant probiotic Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4 (ECN-pClyA-BMP-2-CXCR4), which overexpresses BMP-2 and CXCR4 on the surface of BEVs. These engineered BEVs, termed BEVs-BC, were isolated and tested for their ability to target bone marrow mesenchymal stem cells (BMSCs) and promote osteogenic differentiation while inhibiting adipogenic differentiation. The BEVs-BC showed strong bone-targeting ability in vivo, as demonstrated by biophotonic imaging, and significantly improved bone density and structure in ovariectomized (OVX) mice. The BEVs-BC also activated the BMP/SMAD signaling pathway, which is crucial for osteoblast differentiation and bone formation. The study further confirmed the safety and biocompatibility of BEVs-BC through in vivo toxicity assays, showing no significant organ toxicity or adverse effects. The results indicate that BEVs-BC, constructed using synthetic biology, offer a promising and effective strategy for the treatment of OP, with potential for industrial application. The study highlights the potential of synthetic biology in developing targeted and efficient therapeutic strategies for bone diseases.