Imuno – Engenharia Baseada nos Neutrófilos para Regeneração de Tecidos

The N_TARGET project AIMS to develop an innovative biomaterial-based approach to tackle the escalating problem of non-healing CW. CW represent a “silent epidemic” amongst the world population, affecting the quality of life of millions of people globally [26]. Our overarching goal is to investigate the role of neutrophils as pivotal immunomodulatory agents in biomaterial-facilitated tissue repair. Through N_TARGET, we aim to initiate a paradigm shift in biomaterial-driven immunomodulation, transitioning from a macrophage-centric approach to a novel focus centered on neutrophils. It is now widely acknowledged that neutrophils play diverse roles beyond their traditional function as "suicidal killer" cells, serving as essential effectors of the innate immune system and orchestrators of inflammation resolution and tissue regeneration processes [27]. Building upon this we postulate that the design of immunomodulatory biomaterials should prioritize neutrophils as the primary cell type. By effectively modulating neutrophil activation and response, we anticipate eliciting appropriate reactions from other effector cells, including monocytes and macrophages, among others. Our objective is to pioneer an innovative biomaterial-based approach that establishes a conducive 3D microenvironment for tissue repair by capitalizing on the pivotal role of neutrophils in mediating immunomodulatory and pro-angiogenic processes. To achieve this, the following specific objectives are considered: (i) To develop an immunomodulatory and pro-angiogenic hydrogel. We will utilize a hydrophilic alginate (ALG) backbone chemically modified with hydrophobic cyclooctyne groups (K), resulting in amphiphilic derivatives (ALG-K). These derivatives will be combined with either LxA4 or RvE1. Upon mixing with gel-precursor solutions, these compounds will become incorporated into ALG-K hydrogels upon ionic crosslinking (ALG-K-Lx, ALG-K-Rv). Their retention inside the 3D network will be promoted by reversible hydrophobic interactions with the K domains. (ii) To evaluate the immunomodulatory properties of ALG-K-Lx and ALG-K-Rv through a) in vitro studies with primary human neutrophils, primary human macrophages, and co-cultures of both cell types; b) in vivo in a rodent air-pouch model of inflammation. ALG-K-Lx and ALG-K-Rv are expected to modulate the polarization of neutrophils and macrophages towards an anti-inflammatory and pro-regenerative N2 and M2 phenotype, respectively. (iii) To evaluate the pro-angiogenic potential of ALG-K-Lx and ALG-K-Rv in vitro using human endothelial cells and in vivo using the chick-embryo Chorioallantoic Membrane Assay (CAM-assay). This will be an important goal, since angiogenesis is fundamental for proper wound healing as new vessels will supply the growing tissue with oxygen and nutrients. (iv) To evaluate the wound healing capacity of the ALG-K-Lx or ALG-K-Rv in a rodent skin incision wound model. With the developed hydrogels, we aim to create a pro-regenerative microenvironment and thus accelerate the wound healing process. (v) To assess the presence of neutrophils and their different polarization states in human samples of CW retrieved from patients. This important step will allow a better understanding of the prevalence and role of neutrophils in CW. The samples retrieved from CW will be submitted to histological and immunohistochemical studies to evaluate the neutrophil inflammatory microenvironment in these wounds.