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|Title:||Understanding the biochemical properties of human hair keratins : self-assembly potential and cell response||Authors:||Lai, Hui Ying||Keywords:||Engineering::Materials::Biomaterials||Issue Date:||2020||Publisher:||Nanyang Technological University||Source:||Lai, H. Y. (2020). Understanding the biochemical properties of human hair keratins : self-assembly potential and cell response. Doctoral thesis, Nanyang Technological University, Singapore.||Abstract:||Keratin is a class of cysteine rich intermediate filament (IF) proteins, existing in abundance and readily available in bio-wastes such as human hair. A total of 17 keratin types are present in human hair, which can be further classified as Type I and II keratin subtypes. They associate in 1:1 ratio to form strongly bonded heterodimers and further assemble into microfibrils (7 – 10 nm). Mature hair fiber is formed through a highly regulated morphogenesis process. In recent decades, keratin as a novel natural biomaterial has shown excellent bioactivity, biocompatibility, and angiogenic properties in a wide range of biomedical applications. Much work has been done on extracting and understanding the profile of keratins from hair since the 20th century. Much efforts were also made to elucidate their detailed structure and to understand their molecular assembly kinetics. Although the conditions for assembly of soluble IF proteins into characteristic 10 nm wide filaments vary, no co-factors are required, this thereby makes biochemical studies of IF practical. However, the chemistry and biology of hair keratin subtypes expression and their potential interaction mechanisms are still yet to be understood. In fact, among all the past studies, limited effort has been invested in understanding the molecular self-assembly of crude hair keratin and no study has been performed to fractionate hair keratins, which would be a critical step to unravel the current knowledge gaps of interaction and function of these proteins. This Ph.D. project, therefore, aims to understand and evaluate the self-assembling potential of the hair keratin extracts and further perform separation of the different keratin subtypes within the extracts for behavior study. It is hypothesized that the enriched specific hair keratin subtypes would present unique cell-material behaviors and characteristics. In order to validate this hypothesis, a number of objectives were identified, and the scope of the experiments was formulated. Specifically, the self-assembly potential of hair keratins was evaluated and cellular response to the crude and purified keratins solution were explored. The antioxidant properties of total hair proteins, keratins proteins and keratin associated proteins were compared. Potential applications of such materials could range from biomedical to water remediation. The protocol to reconstruct self-assembled intermediate filaments from crude keratin extracts was established via step-down dialysis in acidic buffer conditions. Continuous self-assembled fibers were achieved, which demonstrated average diameters ranging from 6 to 10 nm when assembled in buffer below pH 3.3. Furthermore, coating casted with the assembled protein fibers were able to remain stable up to five days in fully supplemented culture media and were cell compatible. Surface morphology and integrity of the assembled coating were characterized using Atomic Force Microscopy (AFM), optical microscope and immunohistochemistry. As type I and II keratin subtypes are strongly bound as dimers and have similar molecular weights and isoelectric points (pI), it is extremely challenging to isolate the individual subtypes. Among the different chromatography approaches evaluated Gel Permeation Chromatography (GPC), Asymmetrical Field Flow Fractionation (AFFF) and High-Performance Liquid chromatography (HPLC), HPLC utilizing a weak anion-exchange (WAX) column revealed six distinct peaks detected by the UV detector, indicating the highest possibility in fractioning the keratin extracts based on pI differences. A two-step salt elution method was ascertained to achieve type II enriched fractions. Lastly, antioxidant properties of the purified fractions were found to be retained and comparable to the crude keratin extracts. Interestingly, the crude keratin extracts showed similar DPPH scavenging ability (IC50) compared to KAPs, although the latter possesses greater cysteine content. Additionally, within the same concentration range, KAPs induced cell toxicity in the in vitro study as it formed precipitates. Keratins exhibited antioxidant properties as a media supplement, which was comparable to the well-established antioxidant compound, Acetylcysteine (NAC) at 1 mM. Human dermal fibroblasts (HDFs) were protected from acute hydrogen peroxide-induced oxidative stress and showed increased proliferation rate in the presence of 40 µM keratin. In conclusion, the above findings provide new perspectives into the self-assembly and antioxidant ability of crude and purified human hair keratins, which can be further exploited as an advanced biomaterial across a wide discipline of applications.||URI:||https://hdl.handle.net/10356/146707||DOI:||10.32657/10356/146707||Rights:||This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).||Fulltext Permission:||open||Fulltext Availability:||With Fulltext|
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Updated on Sep 22, 2021
Updated on Sep 22, 2021
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