Injectable system for spatio-temporally controlled delivery of hypoxia-induced angiogenic signalling

Abstract

While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo, namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O2). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5°C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37°C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol–gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.