Bioorthogonal “Click Chemistry” Bone Cement Enables Pro-Translational Spinal Fusion in a Large Animal Sheep Model

Spinal fusion is widely performed to treat instability of various spinal pathologies. Currently, the clinical gold standard is still autografts, which unfortunately are limited by donor site morbidity, restricted supply, and inconsistent outcomes. To overcome these challenges, we developed an organic-inorganic nanohybrid (click-ON) cement based on poly(propylene fumarate) (PPF) polymers cross-linked through the strain-promoted azide-alkyne cycloaddition (SPAAC) bioorthogonal click chemistry. This catalyst-free system cures rapidly in situ without external energy or toxic initiators, enabling safe and practical surgical handling. The cement was further reinforced with osteogenic nanohydroxyapatite (nHA) to improve osteoinductivity and microspheres releasing bioactive recombinant human bone morphogenetic protein 2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) to promote coupled osteogenesis and angiogenesis. In a sheep lumbar spinal fusion model, the click-ON cement was implanted with an injectable formulation in the interbody space and a moldable formulation in the posterolateral fusion site with clinically used autograft/rhBMP-2 as the positive control. Longitudinal CT imaging demonstrated fusion with bridging bone formation across both the interbody space and posterolateral region after 6 months. Histological analyses confirmed extensive new bone deposition, integration with host tissue, and vascular ingrowth within the cement, while immunohistochemical staining showed the colocalization of CD31 and alkaline phosphatase (ALP), indicating active angiogenesis and osteogenesis, respectively. The outcomes are comparable to the positive control, which are clinical gold standard bone grafts. Manual palpation further verified the mechanical stability of the fused segments in sheep with a bioactive click-ON cement. These results established click-chemistry-enabled PPF-based cement as a promising alternative to autografts, offering advantages in moldability, biological activity, and functional fusion outcomes.