Extraction of keratin macrofilament bundles from human hair Mariana Cabral, Atara Israel, Dr. Roche C. de Guzman Fred DeMatteis School of Engineering and Applied Science, Hofstra University, Hempstead, NY 11549 INTRODUCTION
METHODS
Keratins are classified as intermediate filaments (IFs) of animal cytoskeletal proteins: type I and type II, acidic and basic keratins, respectively. IFs are unique to each cell and their main function is to maintain cell integrity by conferring organization and support. IF extracts can self-assemble into supramolecular structures and form hydrogels in right conditions which can be used in drug delivery and tissue engineering.
Different batches of human hair clippings were treated (Fig. 3) with: 200 mM dithiothreitol (DTT) and 5001000 mM thioglycolic acid (TGA) reducers, 0% or 25% ethanol (EtOH), 1-8 M urea denaturant, 25-100 mM Tris buffer, and sodium hydroxide (NaOH) for basic pH adjustment, then incubated for 1-13 days at 37-50 °C. The supernatant (soluble) and residual hairs (insoluble bulk gels) were separated using a sieve. Supernatants were dialyzed (to remove chemicals) (Fig. 4) and visualized for proteins in SDS-PAGE. Residual hairs were extensively washed with water (to remove chemicals) (Fig. 5), seeded and cultured with mesenchymal stem cells (MSCs) for biocompatibility, pulled using a mechanical tester for tensile strength (Fig. 6), and dehydrated and air-dried for SEM imaging.
crosslinked KAPs
Figure 3. Treated hair.
Figure 5. Residual hair during and after washing with water. Figure 4. Dialysis bag.
Figure 6. Tensile testing.
RESULTS Treatments lead to exposure of the cortical components of hair including keratin macrofibril bundles (Fig. 7), which are more pronounced in longer incubation (Fig. 8) and shown to support MSCs attachment and survival for a week in vitro (Fig. 9). Residual hair’s (resembling hydrogels) tensile strengths exponentially decreased over time (Fig. 10), suggesting increasing KAPs solubilization (since KAPs are responsible for hair strength).
OBJECTIVE Test the hypothesis. ▪ Residual hair (keratin macrofilaments): scanning electron microscopy (SEM) (Fig. 2) ▪ Supernatant (KAPs): sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Figure 11. Electrophoresis of supernatant at various conditions.
CONCLUSIONS
Hair keratins (Fig. 1) are formed via keratinization and cornification, assemble into exclusive type I/II heterodimers, build to macrofilament bundles, and embedded in keratin associated proteins (KAPs), held together by disulfide crosslinks. Hypothesis: To obtain keratins, KAPs can initially be removed via reduction of disulfides in the presence of alcohol which renders keratin IFs insoluble.
Figure 1. Hair showing locations of keratin IFs and KAPs.
KAPs
Low molecular weights (MWs) in the soluble materials (Fig. 11) indicate the extraction of KAPs that contain active free thiols. Dialysis crosslinks them to high MWs and addition of DTT reduced KAPs back to low MWs. With each extraction cycle (or treatment time), more KAPs were removed from the residual hair. It was also demonstrated that the process was repeatable using various hair types.
Keratin macrofilaments
The hypothesis was conditionally accepted since the TGA reducing agent with EtOH alcohol hair treatment showed evidence of keratin macrofilament preservation in the residual hair and dissolution of low MW KAPs. Process improvements are suggested, including minimizing NaOH quantities to prevent protein degradation and increased washing and equilibration steps. Future work will involve hydrophobic interaction liquid chromatography keratin separation of solubilized residual hair and in vivo mouse implantation of keratin-based gels for biocompatibility testing and tissue engineering potential evaluation.
REFERENCES 1. de Guzman RC, Tsuda SM, Ton MN, Zhang X, Esker AR, Van Dyke ME. “Binding interactions of keratin-based hair fiber extract to gold, keratin, and BMP-2.” PLOS ONE (2015) 10:e0137233. PMID: 26317522. 2. Fujii T, Takayama S, Ito Y. “A novel purification procedure for keratin-associated proteins and keratin from human hair.” J. Biol. Macromol (2013) vol. 13(3):92-106. 3. Moll R, Divo M, Langbein L. “The human keratins: biology and pathology.” Histochem Cell Biol (2008) vol. 129:705– 733. 4. Plowman JE, Harland DP. “Fibre Ultrastructure.” Springer Nature Singapore Pte Ltd (2018) vol.1054.
ACKNOWLEDGMENTS Figure 7. (Left) Normal hair and (Right) chemically-treated residual hair.
Figure 2. (Left) SDS-PAGE procedure. (Right) SEM system.
Figure 10. Weakening of residual hair gels over time.
Figure 8. (Left to Right) Increasing treatment time leads to release of more macrofilaments.
Figure 9. MSCs on keratin bundles.
This work was made possible through Dean Sina Rabbany and the generous donors of the ASPiRe 2021 Program. Hair samples were provided by Lori Castoria, Hazel Consunji, Shannya Niveyro, and Atara Israel. We also like to thank the Biology Department and Dr. Jason Williams for the SEM facility access and use. Hair protein extraction experiments were conducted in 208 Weed Hall, Department of Engineering.