Erschienen:
[Erscheinungsort nicht ermittelbar]: University of New South Wales. Graduate School of Biomedical Engineering, 2021
Sprache:
Englisch
Entstehung:
Hochschulschrift:
Dissertation, University of New South Wales. Graduate School of Biomedical Engineering, 2021
Anmerkungen:
Beschreibung:
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 highest yields of protein (1.5 mg/L) and GAGs (1.9 mg/L). The cells maintained in medium containing 5.5 mM glucose produced less GAG chains compared to when these cells were grown in medium containing 25 mM glucose, however, heparin was produced.Suspension HEK-293 cells expressing recombinant serglycin (HEK-S-SGN) were established in this thesis. The presence of serum in the culture medium promoted the suspension cells to adhere during culture. HEK-S-SGN cells maintained in serum free medium produced a 4.6-fold higher yield of protein decorated with HS/heparin compared to the cells maintained in serum containing medium. Recombinant serglycin supported fibroblast growth factor 2 (FGF2) binding in vitro via its HS/heparin chains, while vascular endothelial growth factor 165 (VEGF165) binding was mediated via HS/heparin and CS/DS chains as well as the protein core of serglycin. In addition, recombinant serglycin potentiated FGF2 signalling via its GAG chains and FGF receptor 1c in vitro. Furthermore, recombinant serglycin supported angiogenesis in vivo by potentiating FGF2 and VEGF165 through its HS/heparin chains in the chicken chorioallantoic membrane assay. Thus, this thesis demonstrated that the culture conditions influenced the protein yield and type of GAG chains that decorated recombinant serglycin. In addition, recombinant serglycin was found to support angiogenesis via binding and signalling angiogenic growth factors via its GAG chains. These results suggest the potential of incorporating recombinant serglycin into tissue engineering or regenerative medicine strategies for growth factor-mediated tissue repair applications.