Poly(ethylene Oxide)/β-lactoglobulin Electrospun Nanofibers: Chemical Crossliking Assessment and Thymosin-β4 Functionalization

Poly(ethylene Oxide)/β-lactoglobulin Electrospun Nanofibers: Chemical Crossliking Assessment and Thymosin-β4 Functionalization By Daniel E. Vargas November, 2016 Director of Thesis: Barbara Muller-Borer, PhD Major Department: Department of Engineering Introduction: Using electrospun nanofiber scaffolds have emerged as a technique for tissue engineering (TE) applications. In 2011, Sullivan et al. reported on the process to effectively electrospin and crosslink nanofibers from poly(ethylene oxide) (PEO) and β-lactoglobulin (BLG) aqueous solutions. PEO and BLG are both biodegradable and biocompatible materials. Crosslinking PEO/BLG nanofibers is necessary to improve their aqueous stability for TE applications. However, the heat treatment process suggested by Sullivan et al. is time intensive. The purpose of this study was to a) investigate an alternative crosslinking method for electrospun nanofibers made from an aqueous protein solution b) assess the resulting nanofibers for their potential use as scaffolds for TE applications, and c) evaluate the effect of biologically treated nanofiber scaffolds on stem cell proliferation. Chemical crosslinking techniques using Sodium Trimetaphosphate (STMP) combined with sodium hydroxide (NaOH) were evaluated. STMP has been shown to effectively crosslink polysaccharide nanofibers in situ during electrospinning. Methods: STMP, at various concentrations, was added to PEO/BLG electrospinning solutions. The effects of STMP were characterized by measuring the solution’s viscosity, pH and conductivity. Confocal laser scanning microscopy (CLSM) images were acquired to qualitatively assess electrospun nanofiber morphology and scaffold topography. Human mesenchymal stem cells (hMSC) were grown on PEO/BLG scaffolds under control conditions and when treated with the protein Thymosin-β4 (Tβ4). HMSC proliferation was assessed to evaluate the effects of PEO/BLG nanofiber scaffolds and different Tβ4 treatments at day 2, 4 and 8. Results: Using STMP to chemically crosslink PEO/BLG electrospun scaffolds affected solution properties, nanofiber morphology and scaffold topography. PEO/BLG/STMP nanofibers were highly beaded and wavy with little structure relative to PEO/BLG nanofibers. Fibers were not stable in an aqueous solution. Using Tβ4 to treat the PEO/BLG nanofiber scaffolds and/or cell culture media improved hMSC proliferation with increased time in culture. HMSCs remained viable throughout the growth period for all treatments. However, hMSCs did not integrate into PEO/BLG nanofiber scaffolds, but attached to the scaffold surface. Conclusion: Using STMP, at the tested concentrations, as an alternative crosslinker for PEO/BLG nanofibers was ineffective and did not result in usable electrospun scaffolds. Chemically crosslinking PEO/BLG nanofibers requires further research in polymer chemistry to identify an alternative in situ crosslinking mechanism. Treating the scaffolds and/or media with Tβ4 did result in improved hMSC proliferation. However, while hMSC cultures remained viable and proliferation increased with Tβ4 treatments, further research is necessary to develop protocols that will enable hMSC integration with PEO/BLG nanofiber scaffolds. Poly(ethylene Oxide)/β-lactoglobulin Electrospun Nanofibers: Chemical Crossliking Assessment and Thymosin-β4 Functionalization Thesis Presented to The Faculty of the Department of Engineering East Carolina University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biomedical Engineering By Daniel E. Vargas November, 2016 © Daniel E. Vargas, 2016 Poly(ethylene Oxide)/β-lactoglobulin Electrospun Nanofibers: Chemical Crossliking Assessment and Thymosin-β4 Functionalization By Daniel E. Vargas