Introduction:
Age-related osteoporosis is characterized by reduced bone mass and increased marrow adiposity, leading to ~200 million fractures annually. A key pathogenic mechanism is the dysregulated lineage commitment of mesenchymal stem cells (MSCs), which preferentially differentiate into adipocytes rather than osteoblasts. We previously identified Tribble Homolog 3 (Trb3) as a critical molecular regulator of MSC fate determination. Here, we engineered Trb3-enriched exosome mimetics (EM-Trb3) and evaluated their therapeutic potential for restoring bone regeneration in osteoporotic conditions.

Methods:
EM-Trb3 was generated using an extrusion-based approach from small-molecule-treated MSCs to enrich endogenous Trb3. Vesicle morphology, size distribution, and Trb3 expression were characterized by TEM, NTA, and ELISA. Osteogenic and adipogenic differentiation of MSCs treated with EM-Trb3 was assessed by various molecular approaches. To enhance bone targeting, EM-Trb3 was metabolically conjugated with alendronate (EM-Trb3-ALD), and bone-binding capacity was evaluated using Nile red-labeled vesicles. Therapeutic efficacy was assessed in osteoporotic mice following systemic administration of EM-Trb3-ALD, with bone regeneration quantified by micro-CT and histological analyses.

Results:
EM-Trb3 exhibited typical extracellular vesicle morphology and significantly elevated Trb3 levels. In vitro, EM-Trb3 enhanced osteogenic differentiation, evidenced by increased osteogenic gene expression, ALP activity, and mineral deposition, while suppressing adipogenesis through downregulation of adipogenic markers. In vivo, systemic administration of high-dose EM-Trb3-ALD significantly enhanced bone regeneration compared to EM alone, as demonstrated by micro-CT and histological evaluation. Mechanistically, EM-Trb3-mediated effects were associated with activation of Wnt/β-catenin signaling.

Conclusion:
Trb3-enriched EMs effectively restore MSC osteogenic potential and enhance bone regeneration in osteoporotic models. This strategy represents a promising targeted therapeutic approach for osteoporosis treatment.