Bi-layered living cellular constructs promote recruitment and activation of an M2 macrophage phenotype

Background Skin substitutes, also known as Cellular and/or Tissue-based Products (CTPs), and wound dressings often are designed to support the innate wound healing response of the patient. The mechanism of action for these products varies, with some products strictly acting as bacterial barriers while others can alter the wound microenvironment by providing additional extracellular matrix components or modulating the inflammatory response. Bi-layered living cellular constructs (BLCCs) are CTPs that mimic human skin and can heal itself in response to wounding in vitro . How could signals from BLCC impact a wound microenvironment? Methods Biopsy punches of BLCC were placed atop dermal equivalent layers and cultured for up to 9 days. Outgrowth area was assessed and biopsy + outgrowth was evaluated for genetic regulation shifts. Proteins generated by BLCC and secreted into the media were confirmed and characterized by ELISAs, and this growth media was used as conditioned media to assess the potential recruitment and activation of monocytes/macrophages using THP-1 cells. Results Biopsies of BLCC had outgrowth for up to 9 days with histological characterization confirming presence of a mature epithelium. Genetic assessment identified several genes involved in activation and migration of cells, as well as several genes involved in the recruitment and activation of monocyte/macrophages, including CCL7, G-CSF, and GM-CSF. Assessment of spent/conditioned media confirmed presence of these proteins and use of spent/conditioned media as a chemoattractant resulted in monocyte recruitment. Cells activated with PMA and exposed to conditioned media were also found to express an M2-like phenotype, as assessed by flow cytometry. Conclusion BLCC wounding promotes monocyte recruitment and subsequent polarization into an M2-like population, a critical immune cell effector for the promotion of a wound healing phenotype.