Waste-to-wealth : American bullfrog skin-derived collagen for wound dressing applications

• Main Author: Cimenoglu, Cigdem
• Other Authors: Dalton Tay Chor Yong
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Engineering::Materials::Biomaterials
• Online Access: https://hdl.handle.net/10356/152474
• Institution: Nanyang Technological University

Wound healing is a complex process in which four successive steps involve along with various cell types, extracellular matrix proteins and growth factors. Successful wound healing process is highly dependent on the completion of each step, and many complications could be developed due to the complexity. Therefore, all-inclusive interactive wound dressings produced using collagen as an active compound have been extensively studied and applied in clinical applications. The majority of collagen-based wound dressings are fabricated from mammalian-derived collagen, which could potentially possess various risks such as disease transmission and allergic reaction as well as religious restrictions. In addition, the high production cost of mammalian-derived collagen-based products discourages the use in wound healing applications. Therefore, in the recent years, marine-derived collagen as an alternative source of collagen in wound healing application has been explored albeit its low extraction yield and low thermal stability which hinders the scale-up production and the applicability. In this regard, there is a necessity to investigate an alternative non-mammalian source of collagen with higher thermal stability and a more cost-effective production process. In this thesis, collagen was extracted from bullfrog skin via an optimized mechano-chemical extraction method with high yield (70.3%) and short time (2 weeks). Physicochemical analysis revealed that the extracted acid solubilised bullfrog skin-derived collagen (ABFC) is a Type I collagen, and the triple helical structure was conserved. Promising applicability was observed due to the high thermal stability and unique nano-featured fibre thickness. Chemical crosslinking with 1,4-Butanediol diglycidyl ether (BDE) improved the structural stability of the ABFC wound dressing construct. BDE-crosslinked ABFC (ABFC-BDE) wound patch displayed nano-scale topographical features which could maximize cell-materials interaction. In vitro immunogenic response studies proved the reliability of ABFC-BDE with its low immunogenic profile. ABFC-BDE was shown to be able to enhance the re-epithelisation process. In vitro cellular studies on human keratinocytes interestingly demonstrated that ABFC-BDE stimulated cellular motility and showed moderate cell adhesion. The nanofiber structure of ABFC-BDE supports the proliferation of keratinocytes, promoting keratinocytes migration with the help of moderate cell adhesion indicating the advantageous utility of the material for the use in wound healing applications. In addition, high tissue remodelling ability of ABFC-BDE was exhibited by proliferation and ECM secretion profiles of fibroblasts. The effectiveness of ABFC-BDE as a wound dressing material was partially proven by in vitro cellular studies, and further confirmation was carried out by in vivo full-thickness excisional wound healing model together with immunohistological analysis. Better integration of ABFC-BDE construct with neighbouring native tissue due to the nano-feature of the fibres led to swift epithelial tongue formation (i.e. re-epithelisation) which in turn reduced the risk of infection. ABFC-BDE showed balanced collagen synthesis resulted in the healthy collagen content at the wound bed upon the closure, which improved the tissue remodelling ability. Validation of the applicability of bullfrog skin-derived collagen as wound dressing may display the potential of ABFC and widen its applications as a therapeutic agent.