Introduction: Scaffolds are used to turn mono-layers of stem cells into highly organized 3-dimensional tissues. In the native form keratin IF have structural integrities, mechanical properties and others like biocompatibility, biodegradability, conductivity and immunotolerance that make them possible candidates for improved growth of stem cells in tissue engineering.
Methods: Living cell specimens obtained from bovine hoof and sheep skin were extracted in urea buffers under reducing conditions and extracts purified by a series of ultracentrifugations. Supernatants were collected and dialysed in buffer solutions (10mM Tris-HCl, pH 8.0, 2.5 mM EDTA and 5mM BME. Dialysates were examined by using transmission electron microscopy (TEM) of negatively stained grids. Preparations were dried as films in Petri dishes for testing of physical properties. Isolated native IF were used as a control.
Results: Reconstituted keratin IF of 7-8 nm diameter and lengths exceeding 1 micrometre were observed in TEM preparations. Cryo-prepared control IF demonstrated a central core. Films of mass per unit area (n=3) 55.28 ± 1.45 g/m2, and thickness (n=34) of 133.02 ± 9.02 µm were obtained. A calculated Young’s Modulus (n=2) of 2.28 ± 1.18 MPa was determined. (For comparison, Rubber; 10-100MPa, Teflon; 500MPa, Nylon; 3000-7000MPa)* Results expressed as average ± sem.
Conclusion: The extracted polypeptides of bovine epidermal living cells forming the various sub- units of keratin IF reassemble into long filaments in vitro using dilute salt solutions and the absence of denaturing agents and additional co-factors. The reconstituted filaments have dimensions and morphological properties similar to IF observed in situ.