PLGA/PDLLA core–shell submicron spheres sequential release system: Preparation, characterization and promotion of bone regeneration in vitro and in vivo

Core–shell polymer spheres of submicron diameter are a promising vehicle for sequential delivery of angiogenic and osteogenic growth factors to bone defect sites, to simulate the orchestrated temporal sequence of angiogenesis and osteogenesis. To achieve a homogeneous distribution pattern in the scaffold matrix and avoid fast biological clearance in vivo, attention should be paid to the particle size of the spheres, using a modified coaxial electrospraying technique, we prepared core–shell spheres about 1 μm in diameter using the polymers poly-(d,l-lactide) in the shell and poly(l-lactide-co-glycolic acid) in the core, and loaded them with VEGF and BMP-2, growth factors that stimulate angiogenesis and osteogenesis, respectively. PLGA/PDLLA controlled sequential delivery profiles, including an initial burst release of VEGF and a sustained release behavior of BMP-2 from the VEGF//BMP-2 spheres, were obtained. The VEGF and BMP-2 released from the spheres maintained their bioactivity; VEGF could enhance the proliferation of endothelial cells and BMP-2 could promote the osteogenic differentiation of bone marrow mesenchymal stem cells. Micro-computed tomography analysis showed that, among all the experimental groups, implantation of VEGF//BMP-2 spheres into rat cranial critical-sized bone defects enhanced new bone formation to the greatest extent, resulting in the largest amount of new bone volume and the largest isolated bone islands. Histological examination showed that VEGF//BMP-2 spheres also significantly increased in-growth of blood vessels with positive CD31 staining. All these findings suggest that the submicron-scale core–shell VEGF//BMP-2 spheres developed in this study are capable of yielding sequentially coupled angiogenesis and osteogenesis, implying their extensive application in bone tissue regeneration.