Dual cytokine release from microsphere-containing decellularized extracellular matrix immune regulation promotes bone repair and regeneration

Most bone filler materials currently achieve bone regeneration by mimicking the natural bone extracellular matrix. However, it is difficult for these materials to replicate the structural functions and bioactivities, including immunomodulation, of natural bone perfectly to reduce inflammation and promote bone regeneration synergistically. Repairing bone defects with scaffolds using a decellularised extracellular matrix (dECM) as a matrix material is an important clinical application and research direction. Here, we processed bovine cancellous bone via an optimised combination of decellularisation methods and preferred dECM, which has the shortest processing time and lowest immunogenicity. Hexagonal mesoporous silica (HMS)/poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with bone morphogenetic protein-2 (BMP-2) were prepared using the complex emulsion method. The HMS/PLGA microspheres had longer cytokine release periods than did the separate HMS and PLGA microspheres. Composite BMP-2/HMS/PLGA microspheres were used to prepare dual-loaded cytokine scaffolds with bone immunomodulatory capacity, which were prepared from composite BMP-2/HMS/PLGA microspheres to increase the osteogenic activity of the dECM and to adsorb interleukin-4 (IL-4) on the surface of the scaffolds. The results showed that the dECM had good cytocompatibility and mechanical strength, and the composite microspheres and IL-4 further endowed the dECM with an ordered spatiotemporally controlled release function, which could release BMP-2 for more than 4 months in the long term and release IL-4 for approximately 10 days in the short term. The composite scaffold not only effectively promoted the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) but also immunomodulated the M1 to M2 polarisation of macrophages (MPs) and mediated the M2 polarisation of MPs, which in turn promoted the osteogenic differentiation of BMSCs, creating a favourable immune microenvironment for bone regeneration. In vivo, the dual drug-loaded scaffolds also exhibited good biocompatibility and significantly superior immunomodulatory bone-enhancing properties compared with those of the other groups. In summary, the combination of dECM scaffolds with cytokine-carrying microspheres and immunomodulatory factors can promote the orderly spatiotemporal release of cytokines, which significantly enhances the bone regeneration and repair effects of dECM scaffolds and is a promising bone filler material for clinical application.