A Macrophage‐Based Cell Therapy Approach Promotes Collagen Deposition in Diabetic Wounds

ABSTRACT Macrophages play a critical role in wound healing. Modulating macrophage activity has thus been identified as a potential strategy to stimulate immune‐mediated tissue regeneration. This study utilised endotoxin tolerization as a strategy to dampen cytokine production upon the ex vivo activation of M2 monocytic cells before collecting their secretome for therapeutic application. The M2 derived secretome was harvested from GM‐CSF differentiated THP‐1 cells followed by IL4‐induced M2 polarisation with or without prior endotoxin tolerization. The protein constituents of the secretome were determined and quantified using label free LC–MS/MS analysis and cytokine levels using enzyme‐linked immunosorbent assays. The efficacy of the M2 derived secretome (with and without prior tolerization) to stimulate fibroblast activity was assessed in vitro (scratch assay) and in vivo (murine full thickness wound model). In culture, the secretome (regardless of tolerization) stimulated fibroblast migration and increased the release of hydroxyproline, which is an essential requirement for collagen synthesis. Similarly, in full thickness excisional wounds, a single application of the M2 monocytic cell secretory products post wounding significantly increased collagen deposition within the wounded area compared to controls. There was however no difference evident in the healing outcomes between the wounds treated with M2 monocytic cell derived secretome without tolerization and those treated with secretome derived from tolerized M2 monocytic cells. Despite its impact on ECM deposition in the wound bed, the secretome showed no benefit for superficial wound closure and did not improve the overall histology score. Taken together, the data suggest that M2 secretory products pose a risk for excessive scar formation.