INTRODUCTION: The use of fibrous proteins as keratin has been recently explored to improve the biological properties of scaffolds in tissue regeneration [1]. Electrospinning is one of the most recognized technologies able to confine - at the level of single fiber - a self-adapting pattern of morphological, chemical or physical signals to reproduce a functional ECM-like micro-environment. Herein, fibers made of Poly ?-caprolactone (PCL) and wool keratin will be investigated to evaluate the contribution of chemical and morphological features at in vitro epithelial cell behavior. METHODS: Bicomponent fibers were processed via electrospinning by combining keratin -extracted from wool by sulphitolysis- and PCL into a unique solution. Electrospinning conditions were optimized in order to fabricate keratin added fibers without beads and/or defects. Morphological features i.e., average diameters and fibre distribution was investigated via SEM supported by image analysis. In order to in vitro validate the scaffolds, hMSC and HaCat cells were seeded onto PCL and keratin/PCL fibers to compare the viability until 14 days. Cell morphology was also evaluated by SEM microscopy, while new collagen formation was estimated by Sirius red dye at 14 and 21 days. The presence of cytokeratin markers was finally evaluated by immunofluorescence. RESULTS & DISCUSSION: Electrospinning was optimized to form wool keratin and PCL composite fibres. The feasibility of the process was basically proved by ATR-FTIR analysis. Clear peaks for amide groups into the spectra confirm the presence of keratin into the fibers, without any alteration due to the electrostatic forces interactions during the process. The presence of keratin also induced an increase of the average diameter -144.1 ± 43.9 nm- respect to those of PCL fibers -81.7 ± 26.7 nm-. The increase in fibre wettability due to the presence of the hydrophilic protein, explained the improvement in cell adhesion in comparison to PCL fibres, used as control. SEM images on cellseeded samples underlined a higher tendency of hMSC to spread in the presence of keratin along the fibers corroborated by roliferation progressively increased until 14 days. The bioactive effect of keratin was also confirmed by the higher formation of ex novo collagen in vitro after 14 and 21 days while the presence of cytokeratins was confirmed by fluorescence microscopy. CONCLUSIONS: In this work, fabrication of PCL/keratin nanofibers was optimized to explore the potential use as cell-instructive scaffolds, with improved biological properties, due to the mutual effect of physical and biochemical cues. They promise to be a good candidate to regenerate epithelial tissues such as cornea. ACKNOWLEDGEMENTS: H2020, Marie Sk?odowska-Curie Actions (Grant n. 665403) and DGAPA-UNAM: PAPIIT IT203618. REFERENCES: [1] Cruz-Maya I et al. AIMS Mater Sci. 2018; 5(2):156-170
In vitro characterization of keratin added fibers for cornea regeneration
C Vineis;V Guarino
2019
Abstract
INTRODUCTION: The use of fibrous proteins as keratin has been recently explored to improve the biological properties of scaffolds in tissue regeneration [1]. Electrospinning is one of the most recognized technologies able to confine - at the level of single fiber - a self-adapting pattern of morphological, chemical or physical signals to reproduce a functional ECM-like micro-environment. Herein, fibers made of Poly ?-caprolactone (PCL) and wool keratin will be investigated to evaluate the contribution of chemical and morphological features at in vitro epithelial cell behavior. METHODS: Bicomponent fibers were processed via electrospinning by combining keratin -extracted from wool by sulphitolysis- and PCL into a unique solution. Electrospinning conditions were optimized in order to fabricate keratin added fibers without beads and/or defects. Morphological features i.e., average diameters and fibre distribution was investigated via SEM supported by image analysis. In order to in vitro validate the scaffolds, hMSC and HaCat cells were seeded onto PCL and keratin/PCL fibers to compare the viability until 14 days. Cell morphology was also evaluated by SEM microscopy, while new collagen formation was estimated by Sirius red dye at 14 and 21 days. The presence of cytokeratin markers was finally evaluated by immunofluorescence. RESULTS & DISCUSSION: Electrospinning was optimized to form wool keratin and PCL composite fibres. The feasibility of the process was basically proved by ATR-FTIR analysis. Clear peaks for amide groups into the spectra confirm the presence of keratin into the fibers, without any alteration due to the electrostatic forces interactions during the process. The presence of keratin also induced an increase of the average diameter -144.1 ± 43.9 nm- respect to those of PCL fibers -81.7 ± 26.7 nm-. The increase in fibre wettability due to the presence of the hydrophilic protein, explained the improvement in cell adhesion in comparison to PCL fibres, used as control. SEM images on cellseeded samples underlined a higher tendency of hMSC to spread in the presence of keratin along the fibers corroborated by roliferation progressively increased until 14 days. The bioactive effect of keratin was also confirmed by the higher formation of ex novo collagen in vitro after 14 and 21 days while the presence of cytokeratins was confirmed by fluorescence microscopy. CONCLUSIONS: In this work, fabrication of PCL/keratin nanofibers was optimized to explore the potential use as cell-instructive scaffolds, with improved biological properties, due to the mutual effect of physical and biochemical cues. They promise to be a good candidate to regenerate epithelial tissues such as cornea. ACKNOWLEDGEMENTS: H2020, Marie Sk?odowska-Curie Actions (Grant n. 665403) and DGAPA-UNAM: PAPIIT IT203618. REFERENCES: [1] Cruz-Maya I et al. AIMS Mater Sci. 2018; 5(2):156-170I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.