Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO((R)), an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 mu m in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 mu m PTS remain unclear. Here, we use HALO((R)) to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 mu m PTS compared to 100 mu m PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 mu m PTS. When subcutaneously implanted in MyD88KO mice, 40 mu m PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 mu m PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.