Functional Nanofiber Scaffolds Enabling Local Immunomodulation and Inhibition of Ectopic Bone Formation.

Heterotopic ossification (HO), the debilitating abnormal bone formation in soft tissues driven by complex inflammatory and osteogenic pathways, remains a major clinical challenge due to painful recurrence after surgery and the lack of effective, localized treatments circumvent systemic toxicity. Herein, we report a multifunctional electrospun nanofiber scaffold for sustained local delivery of ritonavir, revealing a previously underexplored role for this repurposed protease inhibitor in regulating mesenchymal lineage fate, osteogenesis, osteoclast activity, and immune signaling during HO progression. The scaffold sustainably released ritonavir for 28 days while maintaining high cell viability and mechanical integrity. Local ritonavir delivery suppressed chondrogenic differentiation by downregulating SOX9 and COL2A1 and biased cells toward a fibroblastic phenotype marked by elevated COL1A1 and α-SMA expression, disrupting the chondrogenic template required for ectopic bone formation. In parallel, ritonavir inhibits osteogenic differentiation, enhances osteoclast-mediated resorption, and promotes macrophage polarization toward an anti-inflammatory phenotype with reduced TNF-α and IL-6 secretion. In vivo, the ritonavir-eluting scaffold significantly attenuates ectopic bone formation in a tendon injury-associated HO model and promotes tendon functional recovery by providing mechanical stabilization to the injured tendon. Collectively, this work establishes localized ritonavir delivery as a multifunctional strategy integrating lineage control, bone remodeling regulation, and immunomodulation for HO prevention. This multifunctional therapeutic approach offers a clinically translatable solution for preventing aberrant bone formation and improving outcomes in trauma and orthopedic patients at risk of HO.