Human hair keratin electrospun matrices for tissue regeneration
Sow, Wan Ting
Date of Issue2016-05-26
School of Materials Science and Engineering
Keratins present themselves as potential autologous alternatives to current natural proteins used as matrices in regenerative medicine applications, because they are abundant, bioactive and easily extracted from human hair. The aim of this work is to study the feasibility of electrospinning human hair keratin and evaluate the potential of electrospun keratin fibers as random and aligned templates for tissue regeneration. Keratins extracted from human hair using sodium sulfide were blended with poly (ethylene oxide) (PEO) at various concentrations and electrospun. The fiber morphology and diameter distribution of the optimised fibrous platforms were analysed by scanning electron microscopy (SEM). Material properties of the resulting keratin fibrous platforms were characterized using fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA) to understand their chemical, thermal and mechanical properties respectively. Biodegradation profiles of keratin fibers were also studied under hydrolytic and enzymatic conditions. Cytotoxicity of the keratin fibers were examined by assessing cell viability and morphology of L929 murine fibroblasts cultured on the fibers through Live/Dead staining assay and SEM. In addition, the potential of keratin fibers for wound healing was evaluated by culturing primary human dermal fibroblasts (HDFs) on them. Cell viability of cultured HDFs was quantified using PicoGreen and AlamarBlue assays while the cell morphology and distribution was observed using Live/Dead and F-actin staining. Immunostaining was also done to assess the ability of cultured HDFs in secreting extracelluar matrix (ECM) proteins such as fibronectin and collagen III. The interaction between HDFs and keratin was then elucidated by cell-material interaction study through integrin inhibition assay and Western blotting. Results showed that keratin fibers were successfully electrospun using a solution of 30 wt% keratin and 0.5 wt% PEO. The resulting fibrous platforms consisted of well-formed sub-micron fibers with mean fiber diameter of 0.70 ± 0.09 µm and 0.74 ± 0.10 µm for random and aligned keratin fibers respectively. Chemical analysis indicated that keratin retained its chemical conformation after electrospinning. L929 murine fibroblasts cultured on keratin fibers showed healthy cell growth, demonstrating no cytotoxicity issues. Results from HDFs studies showed that proliferation and metabolic activity of HDFs on keratin fibers were comparable to the positive control and significantly higher on aligned keratin fibers compared to random keratin fibers on day 3 and day 5 after cell seeding. Live/dead staining indicated that HDFs cultured on the keratin fibers could form cellular networks according to the architecture of the keratin fibrous platforms. Besides, F-actin staining revealed that the HDFs exhibited their typical spindle morphology and responded well to the topography of the keratin fibers. It was also shown that keratin fibers could stimulate the HDFs to secrete and deposit ECM proteins essential for early stage of wound healing. Cell-material interaction study demonstrated that α4β1 integrin played a significantly role in HDFs attachment to keratin. In this work, it is revealed that keratin fibrous platforms provide a conducive environment to facilitate the growth of primary HDFs and promote ECM production. From these results, it is concluded that electrospun human hair keratin fibers have the potential to be developed into templates for tissue regeneration.