Understanding Fibroblast Behaviour in an Electrospun Microfibrous Scaffold

Fibroblasts are connective tissue cells with multiple functions in health and disease. They are responsible for maintenance of the extracellular matrix and become activated during the wound healing response. Persistently active fibroblast populations are implicated in the development of fibrotic tissue and have come under increasing scrutiny as key constituents of the tumour stroma. Given their complex roles in health, regeneration and disease and accounting for the fibrous matrix in which fibroblasts reside in vivo, these cells have been extensively studied in three-dimensional tissue culture models. The aim of this thesis was to explore the behaviour of fibroblast cells cultured on 3D electrospun polycaprolactone scaffolds. The hypothesis that scaffolds mimic the microarchitecture of the extracellular matrix and are sufficient to maintain a population of low activity in-vivo like fibroblasts was tested with scaffolds fabricated at the University of Sheffield, School of Clinical Dentistry. These scaffolds were characterised using SEM image analysis, water contact angle and uniaxial mechanical testing and mercury intrusion porosimetry. Fibre widths and orientation resembled the arrangement of collagen in the extracellular matrix. Fibroblasts were seeded onto electrospun scaffolds and 2D tissue culture plastic controls. On scaffolds fewer normal oral fibroblasts (NOF) expressed the proliferation marker Ki67 and qPCR revealed that this was due to cellular quiescence, not senescence. On scaffolds qPCR showed that the expression of activation marker α-SMA and ECM protein coding genes collagen 1, fibronectin and versican was reduced compared to 2D controls. Next generation RNA sequencing revealed that the MAPK pathway was suppressed in scaffold grown fibroblasts. Finally, cancer associated fibroblasts (CAF) cultured on electrospun scaffolds showed reduced expression of CAF markers ACTA2, PDGFR, TNC and VCAN. These results indicate that electrospun polycaprolactone scaffolds can trigger a reduction in the activation behaviour of NOF and CAF. In the future this model can be used to develop our understanding of fibroblast diseases and to seek novel therapeutic targets.