Multifunctional NGF-loaded PLGA microsphere-PCL/GelMA scaffold enables coordinated osteo-neuro-vascular regeneration via PI3K/AKT pathway activation

Large bone defects, particularly in craniofacial regions, remain a major clinical challenge in orthopaedics and oral and maxillofacial surgery due to limited donor availability, immune rejection, and poor healing associated with conventional grafts. To address this problem, we developed a multifunctional composite scaffold by incorporating nerve growth factor (NGF)-loaded PLGA microspheres into a PCL/GelMA matrix, aiming for sustained NGF release and coordinated osteogenesis, angiogenesis, and neurogenesis. In vitro experiments demonstrated that the NGF-loaded scaffold significantly promoted osteogenic differentiation of dental pulp stem cells, as evidenced by increased alkaline phosphatase activity, mineralized nodule formation, and upregulated expression of RUNX2 , ALPL , COL1A1 , and BGLAP . In a rat critical-sized cranial defect model, the NGF-loaded scaffold promoted robust new bone formation, with increased bone volume fraction, trabecular thickness, and trabecular number compared to controls. Simultaneously, the NGF-loaded scaffold up-regulated the expression of vascular markers, including CD31 and α-SMA, and neural marker NF200, thus establishing a neurovascular-osteogenic regenerative microenvironment. Transcriptomic and western blot analysis further revealed activation of the PI3K/AKT pathway, upregulation of osteogenic transcription factors, such as Smad1/5 and Runx2 , and downregulation of osteoclast-associated genes. In contrast, scaffolds treated with the NGF pathway inhibitor Ro 08-2750 showed limited regenerative outcomes. Collectively, this study presents a neurotrophic factor-based scaffold system that supports multi-lineage regeneration and offers a promising strategy for the clinical repair of complex bone defects. • A biomimetic PLGA/PCL/GelMA scaffold was developed for sustained NGF delivery. • The scaffold enhances osteogenesis via upregulation of ALP, OCN, and COL-1 expression. • Coordinated neurogenesis and angiogenesis are simultaneously promoted by NGF release. • PI3K/AKT pathway activation mediates osteo–neuro–vascular regeneration. • The multifunctional scaffold shows translational potential for complex tissue repair.