The electrospinning of a thermo-responsive polymer with peptide conjugates for phenotype support and extracellular matrix production of therapeutically relevant mammalian cells.

Current cell expansion methods for tissue engineering and regenerative medicine applications rely on the use of enzymatic digestion passaging and 2D platforms. However, this enzymatic treatment significantly reduces cell quality, due to the destruction of important cell-surface proteins. In addition, culture in 2D results in undesired de-differentiation of the cells caused by the lack of 3D similarity to the natural extracellular matrix (ECM) environment. Research has led to the development of thermo-responsive surfaces for the continuous culture of cells. These thermo-responsive materials properties can be used to passage cells from the surface when the cell culture temperature is reduced. Here we report the development of a PLA/thermo-responsive (PDEGMA) blend 3D electrospun fibre-based scaffold to create an enzymatic-free 3D cell culture platform for the expansion of mammalian cells with the desired phenotype for clinical use. Human corneal stromal cells (hCSCs) were used as an exemplar as they have been observed to de-differentiate to an undesirable myo-fibroblastic phenotype when cultured by conventional 2D cell culture methods. Scaffolds were functionalised with a cell adherence peptide sequence GGG-YIGSR by thiol-ene chemistry to improve cell adherence and phenotype support. This was obtained by functionalising the thermo-responsive polymer with a thiol (PDEGMA/PDEGSH) by co-polymerisation. These incorporated thiols react with the norbornene acid functionalised peptide (Nor-GGG-YIGSR) under UV exposure. Presence of the thiol in the scaffold and subsequent peptide attachment on the scaffolds were confirmed by fluorescence labelling, ToF-SIMS and XPS analysis. The biocompatibility of the peptide containing scaffolds was assessed by the adhesion, proliferation and immuno-staining of hCSCs. Significant increase in hCSC adherence and proliferation was observed on the peptide containing scaffolds. Immuno-staining showed maintained expression of the desired phenotypic markers ALDH, CD34 and CD105, while showing no or low expression of the undesired phenotype marker α-SMA. This desired expression was observed to be maintained after thermo-responsive passaging and higher when cells were cultured on PLA scaffolds with 10 wt% PDEGMA/4 mol% PDEGS-Nor-GGG-YIGSR. This paper describes the fabrication and application of a first generation, biocompatible peptide conjugated thermo-responsive fibrous scaffold. The ease of fabrication, successful adherence and expansion of a therapeutically relevant cell type makes these scaffolds a promising new class of materials for the application of cell culture expansion platforms in the biomaterials and tissue engineering field.

A design of experiments approach to identify the influencing parameters that determine poly-D,L-lactic acid (PDLLA) electrospun scaffold morphologies.

Electrospun fibrous materials have increasing applications in regenerative medicine due to the similarity of fibre constructs to the morphology of certain extracellular matrices. Although experimentally the electrospinning method is relatively simple, at the theoretical level the interactions between process parameters and their influence on the fibre morphology is not yet fully understood. Here, we hypothesised that a design of experiments (DoE) model could determine combinations of process parameters that result in significant effects on poly-D,L-lactic acid (PDLLA) fibre morphology. The process parameters used in this study were applied voltage, needle-to-collector distance, flow rate and polymer concentration. Data obtained for mean fibre diameter, standard deviation (SD) of the fibre diameter (measure of fibre morphology) and presence of 'beading' on the fibres (beads per µm2) were evaluated as a measure of PDLLA fibre morphology. Uniform fibres occurred at SDs of ≤500 nm, 'beads-on-string' morphologies were apparent between ±500 and 1300 nm and large beads were observed at ±1300-1800 nm respectively. Mean fibre diameter was significantly influenced by the applied voltage and interaction between flow rate and polymer concentration. Fibre morphology was mainly influenced by the polymer concentration, while bead distribution was significantly influenced by the polymer concentration as well as the flow rate. The resultant DoE model regression equations were tested and considered suitable for the prediction of parameters combinations needed for desired PDLLA fibre diameter and additionally provided information regarding the expected fibre morphology.