Human hair keratin gradient metallogel and their applications for wound healing

• Main Author: Yee, Zhen Lin
• Other Authors: Ng Kee Woei
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2024
• Subjects: Engineering; Human hair keratin; Hydrogels; Wound healing
• Online Access: https://hdl.handle.net/10356/175545
• Institution: Nanyang Technological University

Recapitulating the microstructure of our human skin is a challenge due to the presence of heterogeneous cellular microenvironments and this challenge is accentuated when developing hydrogel scaffolds to facilitate the restoration of normal tissue function during wound healing. To alleviate these difficulties, research on developing gradient hydrogels has mushroomed. Among the fabrication strategies reported, the use of layering to develop biomimetic skin substitutes is common. One limitation is the presence of discontinuity between the layers, creating interfacial stress that can lead to delamination. This compromises the structural and functional integrity of the substitute. Another common challenge amongst other techniques is the limited scalability of the engineered constructs due to the multi-step approach with numerous equipment requirements. Moreover, synthetic materials lack inherent bioactivity and natural biomaterials create the possibility of interspecies pathogen transfers. Looking no further but to what is already present, human hair keratins (HHKs) extracted from hair waste are a sustainable source of biomaterial with scalable potential to be valorised into templates for biomedical applications. Among these templates, the use of metal ions as a hydrogelator for HHKs through the metal-thiolate complexation process has yet to be explored. This PhD aims to exploit the high cysteine content of HHKs and their high affinity to metal ions to establish a novel facile methodology to create a gradient HHK metallogel. In one step, the gradient gelation is facilitated by the diffusion of silver ions (Ag+) into the solubilized HHKs through a thin membrane first formed upon a dropwise addition of HHK into the ion bath. By varying the thiol:Ag ratios from 0.50 to 1.50, hydrogels with different gradient profiles were formed and their Ag+ concentration-dependent gelation kinetics were elucidated with the Turbiscan analyser. Beyond the free thiols, the saturation and inhibition studies identified the presence of additional interactions between HHKs and other protein side groups. These interactions alone can form an irregular hydrogel but without the free thiols, a gradient HHK-Ag metallogel cannot be formed. The material performance of these HHK-Ag gradient metallogels was correlated to their microstructure in which varying the thiol:Ag ratio during the fabrication can lead to a tunable porosity gradient profile of dissimilar surface topographies with a non-uniform pore wall thickness. Moreover, the microarchitecture of the gradient construct mimics that of native skin. A higher fraction of the large flat pores with walls of greater thickness present in the structure improved the hydrogels’ mechanical properties and endowed these scaffolds with a higher water uptake capacity and a slower rate of enzymatic and hydrolytic degradation. To establish the HHK-Ag gradient metallogels as a dermal template, a series of in vitro studies were conducted with human dermal fibroblasts (HDFs). Through the metabolic activity assay and cell viability analysis, the 1.25 hydrogel was found to be the best substrate for HDF culture amongst the other gradient metallogels. Also, this substrate could support HDFs to adhere and spread, as well as promote proliferation characterized by cell nuclei counts in a manner comparable to a collagen type 1 hydrogel control. Although the metabolic activity of HDFs seeded on the 1.25 hydrogel was lower than the control, this HHK-Ag substrate did not impede the production of ECM proteins such as collagen III and fibronectin and the expression of alpha-smooth muscle actin. Moreover, their expressions were higher than the HDFs cultured on the control, which could be beneficial in the phases of wound healing. Also, the concentration of the Ag-based hydrogel leachates did not elicit a cytotoxic response to human epidermal keratinocytes and the 1.25 hydrogels were found to be antibacterial against Gram-positive Staphylococcus aureus. Through these results, this thesis has met the overarching objective, which is to develop a novel HHK-Ag gradient hydrogel as a potential dermal template for wound healing application. Not limited to using HHKs only to form a gradient metallogel, the versatility of this system is explored with Gelatin Methacrylate for the formation of a composite hydrogel with HHKs, first through the thiol-methacrylate Michael addition and then the photocrosslinking of the methacrylate groups with a photoinitiator. Also, with Ag+ added, a porosity gradient was imparted. Preliminary material characterization was conducted to investigate the protein interactions and to evaluate the material properties of the composite system.