Chitosan nanocomposite scaffolds enable controlled 4-methoxycinnamic acid release to enhance bone regeneration.

Chitosan (CS)-based scaffolds are promising platforms for bone tissue engineering due to their biocompatibility and tunable drug delivery properties. Here, we report the development of 3D-printed polylactic acid scaffolds coated with polydopamine and loaded with CS nanoparticles encapsulating 4-methoxycinnamic acid (MCA), a hydrophobic polyphenolic compound with antioxidant and anti-inflammatory activities. Despite MCA's therapeutic potential, its application in bone regeneration has been limited by poor solubility and rapid clearance. We demonstrate that controlled MCA release from these CS scaffolds promotes osteogenesis and angiogenesis in vitro and in vivo. In mouse mesenchymal stem cells, MCA enhanced osteoblast differentiation at both cellular and molecular levels, with 20 µM identified as the optimal dose for mechanistic studies. Mechanistic analyses revealed activation of key osteogenic and angiogenic signaling pathways, including integrins, MAPKs, VEGFR2, IGF, and FGF2, in primary osteoblasts and endothelial cells. In a rat tibial defect model, MCA-loaded CS scaffolds substantially increased bone formation and vascularization, with micro-CT and histology confirming dense new bone deposition and a Ca:P ratio of 1.4, comparable to native bone. These findings establish a direct mechanistic link between CS-mediated controlled MCA delivery and activation of osteogenic and angiogenic pathways, offering a robust platform for scaffold-based bone tissue engineering strategies.