Extracellular matrix scaffolds derived from different musculoskeletal tissues drive distinct macrophage phenotypes and direct tissue-specific cellular differentiation

The host immune response, specifically macrophage function, is a critical determinant of biomaterial success or failure post-implantation. Extracellular matrix (ECM) derived scaffolds have been shown to promote a pro-regenerative macrophage phenotype and a more constructive remodelling outcome. Here we demonstrate that macrophages adopt distinct phenotypes when exposed to articular cartilage (AC), ligament (LIG) and growth plate (GP) derived ECM scaffolds. Macrophages were generally unresponsive to LIG-derived ECM, adopted an M2-like phenotype when exposed to AC-derived ECM, and a hybrid M1-M2 phenotype when exposed to GP-ECM. Furthermore, macrophages expressed higher levels of pro-chondrogenic factors, such as FGF2, when exposed to AC-ECM, and higher levels of angiogenic and pro-osteogenic factors, such as VEGF, IL-6 and TNF, when exposed to GP-ECM. In addition, we observed that they can differentially direct the differentiation of skeletal stem cells, whereby AC-ECM promotes the chondrogenic differentiation and GP-ECM the osteogenic differentiation of multipotent stem/stromal cells (MSCs). In vivo characterisation of immune cell subsets following scaffold implantation into a large bone defect demonstrated that AC-ECM drives an M2 macrophage phenotype, while GP-ECM containing scaffolds promoted a hybrid M1-M2 phenotype and enhanced vascularisation and vessel maturation. This distinct response to the implantation of GP-ECM containing scaffolds was associated with increased CD45⁺ leukocyte and CD3⁺ T cell infiltration, accompanied by elevated concentrations of IFN-γ and IL-17. Taken together this work demonstrates that the source tissue of ECM scaffolds plays a key role in regulating the phenotype of both macrophages and skeletal stem cells. Furthermore, these ECMs can direct the cellular differentiation and production of growth factors essential for the regeneration of their source tissue. This work highlights the need for a more thorough characterisation of innate immune cell subsets post-biomaterial implantation.