Hydroxyapatite Particle Shape and Size Influence MSC Osteogenesis by Directing the Macrophage Phenotype in Collagen-Hydroxyapatite Scaffolds

The field of bone tissue engineering has seen the advancement of a variety of biomaterials with a diverse range of material properties. Biomaterial properties such as particle shape and size, stiffness, and pore size all influence the osteogenic capacity of biomaterials, typically evaluated in vitro by analyzing their potential to promote osteogenesis in mesenchymal stem cells (MSCs). There is now accumulating evidence highlighting the role of macrophages in driving bone regeneration responses. In this study, we evaluated the osteogenic capacity of collagen scaffolds functionalized with hydroxyapatite particles of varying shapes (needle vs spherical) and sizes (5 μm vs 100 μm) using an in vitro culture system of MSCs alone and in coculture with macrophages. We show that macrophage response to HA particles was elevated in the presence of a scaffold with 5 μm needle-shaped particles (Coll N5), with an increase in the expression and secretion of both pro-inflammatory (TNFα, IL6, and MIP1α) and anti-inflammatory (IL10 and IL1Ra) factors. When MSCs alone were cultured on the scaffolds, we show that scaffolds with HA particles were highly osteogenic, with superior osteogenesis observed in scaffolds with large 30 μm spherical particles (Coll S30) compared to small 5 μm needle-shaped particles (Coll N5). A coculture of MSCs with macrophages increased osteogenesis in all groups, with the most dramatic increase on Coll N5 scaffolds, leading to an elimination of the differences observed during monoculture. Through gene expression analysis, we showed that this correlated with an enhanced pro-osteogenic macrophage phenotype on Coll N5 scaffolds. These results highlight the potential of modulating material properties such as particle shape and size to develop osteoimmunomodulatory materials that direct osteogenic responses by influencing macrophage response.