Introducing a Model to Investigate Neutrophil-Mediated Biomaterials Immunomodulation in Osteogenesis

Immune regulation is essential during the regeneration process, influencing the resolution of inflammation following tissue injury or infection. Neutrophils are the first immune cells recruited to inflammation sites, acting as primary responders that coordinate subsequent inflammatory events after implantation. This early activation of the inflammatory response is considered crucial for determining future outcomes, although it has not been extensively studied. The purpose of this study was: 1) to introduce a model for studying how neutrophils influence the differentiation of mesenchymal stromal cells (MSCs) through their secretome, and 2) to assess the effects of surface coatings and topographies on neutrophil behavior. Polycaprolactone (PCL, MW: 45,000 Da) scaffolds were fabricated using an extrusion-based bioprinting technique and through creating 3D molds with diverse surface topographies (smooth, pillar-like, and microgroove) using the Original Prusa SL1S SPEED 3D printer. These scaffolds were then coated with solutions of FBS and collagen type I. Human primary neutrophils, isolated from peripheral blood, were seeded onto the biomaterials, and their responses were assessed using various techniques, including CTB, LDH, Olink®, and ELISA assays. Additionally, MSCs were differentiated to the bone lineage in presence of neutrophil-conditioned medium. Neutrophils cultured in FBS-containing conditions exhibited reduced metabolic activity and increased LDH release. ELISA assays revealed elevated levels of neutrophil elastase and MMP-9 in the culture media of neutrophils exposed to FBS(Figure1-B). Olink® immunoassay showed that neutrophils treated with FBS produced higher levels of inflammatory proteins compared to those without FBS (Figure1-C). In contrast, PCL scaffolds coated with FBS displayed lower ROS production than their FBS-free counterparts. The collagen coating did not significantly affect ROS production, metabolic activity, or LDH release (Data not shown). Alizarin Red S staining demonstrated increased mineralization in MSCs treated with conditioned medium from FBS-coated PCL and PMA groups, and osteogenic medium, relative to other groups (Figure1-D). Furthermore, qRT-PCR analysis indicated that the NC-treated groups exhibited higher expression of osteogenic markers ALPL, RUNX2/SOX9, and COL1A1 on day 7 compared to those induced with osteogenic medium (Figure1-E). In this study, a model has been developed to investigate neutrophil-mediated immunomodulation. Early findings indicate that neutrophil responses can influence MSC osteogenic differentiation. Leveraging the osteoimmunomodulatory functions of neutrophils may improve strategies for bone repair and regeneration. For any figures or tables, please contact the authors directly.