Bioengineered growth factor delivery molecules for vascularisation and wound healing

Chronic wounds, such as those exhibited by diabetic ulcers are a global health issue affecting approximately 3.75 million people worldwide. Chronic wounds are slower to heal, or fail to heal, due to decreased vascularisation among other factors. Growth factors are key signalling molecules which promote wound healing, however, they experience rapid degradation by proteolytic enzymes present in the chronic wound environment. Proteoglycans (PGs) are the natural binding, protective and signalling partners for many growth factors. However, the yield of PGs from natural sources is limited due to their low abundance in tissues and cell cultures. Recombinant DNA technology and metabolic engineering offer alternative PG production methods to increase the yield of PGs and to alter the structure of their glycosaminoglycan (GAG) chains. Serglycin is an intracellular PG, which has eight GAG attachment sites. Unlike other PGs, serglycin can be decorated with heparin, heparan sulphate (HS), chondroitin sulphate (CS) and/or dermatan sulphate (DS). Through these various GAG chains, serglycin present in intracellular granules can bind and release cytokines, chemokines and growth factors, which are ideal properties for growth factor delivery and signalling applications. Thus, this thesis examined the influence of various culture microenvironments on the yield and GAG structure of recombinant serglycin produced by both adherent and suspension mammalian cells. In addition, this thesis explored the ability of recombinant serglycin to support angiogenic growth factor binding and signalling. Adherent human embryonic kidney cells expressing recombinant serglycin (HEK-SGN) cultured in shaker flasks and continuously stirred tank reactors (CSTR) produced more protein decorated with GAGs compared to culture flasks. The cells cultured in CSTR produced more HS/heparin and CS/DS chains compared to the other culture flasks. HEK-SGN cells maintained in medium containing 25 mM glucose achieved the hi