Design and fabrication of microstructured and mechanically-controlled electrospun corneal membranes

Corneal blindness is the third leading cause of blindness worldwide. Current treatments require the use of donor corneas or amniotic membrane as a cell carrier. While these treatments are successful to a degree, they present downsides including accessibility of tissue and the risk of disease transmission. The use of synthetic scaffolds is a potential solution; current research has exhibited the importance of controlling mechanical properties when designing new approaches to corneal regeneration. The aim of this project is to create synthetic electrospun scaffolds with mechanically tailored stiffness to explore corneal cell behaviour in vitro, as well as to explore the inclusion of topographical cues in these membranes. Mechanically tailored membranes were fabricated using electrospinning and blending PLGA (poly(lactic-co-glycolic acid)) and PCL (Polycaprolactone) in different concentrations. Membranes were characterised in dry (storage) and wet conditions (submerged in PBS at 37°C). Characterisation of the membranes was done using Scanning Electron Microscopy (SEM) to analyse microstructure and fibre diameter. Uniaxial tensile testing was used to obtain stiffness, Ultimate Tensile Strength (UTS), and strain at UTS from the membranes. Gas chromatography was performed to measure the remnant solvents in them. Differential Scanning Calorimetry (DSC) was executed to analyse the thermal properties in the membranes. Biological testing was accomplished using rabbit and porcine limbal explants on the membranes and growing them for 2 and 3 weeks. Cell outgrowth in the membranes was analysed using different microscopy techniques (SEM, Light Sheet microscopy, and epifluorescence microscopy). Topographical cues were introduced in electrospun membranes by casting metal collectors with particular designs after the input of specialist of L. V. Prasad Eye Institute. Fibre alignment and fibre orientation were examined in the membranes by analysing SEM images and processing them with the ImageJ software with a protocol developed by us introduced in this thesis.
Electrospun scaffolds with different mechanical properties were successfully manufactured blending PLGA and PCL. DCM and DMF as solvents produced a lower amount of remnant solvents in the electrospun scaffolds made of PLGA and PCL than the maximum permissible by EMA and FDA. Adding PCL showed statistical differences in the stiffness of the membrane for blends with 10% or more PCL on it, an effect that is increased when the membranes are analysed in dry and wet conditions. From all the conditions analysed, 30%PCL - 70%PLGA is able to maintain its mechanical properties in wet/warm and dry conditions which we envisage is a very important point for the material to be used in theatre. PLGA and PLGA-PCL membranes showed outgrowth from rabbit and porcine limbal explants at 2 and 3 weeks no differences were observed in the amount of cell outgrowth between them. All mechanically tailored membranes developed in this research showed good capacity as cell carriers of limbal corneal cells. 30%PCL – 70%PLGA displayed cell outgrowth in both sides of the membrane, suggesting that the cells penetrated through the membrane and populated the other side. As well, Topographical cues in the membranes were successfully introduced and characterised analysing fibre orientation and fibre alignment in different zones on them. With all this in mind, 30%PCL – 70%PLGA showed the best results in its mechanical properties and its cell outgrowth when seeded with porcine limbal explants for two weeks and we suggest moving forward the research using this electrospun membrane with longer culture times.