Influences of topography and composition on hepatocytes within electrospun polycaprolactone scaffolds for liver tissue engineering

Severe liver disease is one of the most common causes of death globally and its mortality has been rising since the 1970s. A quarter of the global population is predicted to have non-alcoholic fatty liver disease, which increases their risk of developing chronic conditions like fibrosis and hepatocellular carcinoma (HCC). Currently, whole organ transplantation is the only definitive cure for end-stage liver disease, however, the need for donor organs far outweighs demand. Recently liver tissue engineering is starting to show promise for alleviating the burden on liver disease patients and healthcare providers. It is essential to find effective pharmaceutical and regenerative therapies that can slow or halt disease progression. Achieving this goal involves using in vitro methods to produce liver models for testing target and drug molecules, expanding cells with regenerative capacity, and creating bioartificial liver devices. In recent decades, in vitro hepatocyte culture methods have evolved from 2D culture to 3D organoids and scaffold cultures that offer a biomimetic environment and elicit phenotypic responses from hepatocytes. Electrospinning is a well-known method to fabricate a nanofibre scaffold which mimics the natural extracellular matrix (ECM) which can support cell growth. This thesis has sought to optimise electrospun polycaprolactone (PCL) scaffolds for the culture of hepatocytes, with a focus on topography and composition. Three methods were utilized: 1) investigation of surface topography of electrospun PCL scaffolds on hepatocytes; 2) incorporation of decellularized rat liver extracellular matrix (dECM) into topographical featured PCL scaffolds and assess its impact on hepatocytes; 3) incorporation of human dECM into topographical featured PCL scaffolds and assess its impact on hepatocytes. All fabricated scaffolds were conducted for physical and chemical analyses and cultured with immortalised hepatic cell line HepG2 or mouse primary hepatocytes MPHs. The biological influence of the scaffolds on cellular behaviours was assessed through proliferation assays, immunohistochemistry, osmium staining and RT-qPCR gene expression analysis. Our results show that fibre surface topography have an influence on cell attachment, proliferation, morphology and functionality. Small surface nanotopographies at around 0.37 μm show to significantly increase the attachment and proliferative activities of HepG2 compared to large microtopographies (2 μm). The incorporation of rat and human dECM into topographically modified fibres both kept the morphological consistency (all randomly oriented and have similar fibre diameters in each study). Both rat and human dECM-containing scaffolds have promoted the HepG2 to grow as a densely packed monolayer-like structure, and showed an increasing trend of albumin gene expression. All topographically modified scaffolds or dECM-contained hybrid scaffolds exhibit good compatibility and have the ability to maintain HepG2 steady growth compared to smooth PCL fibres. Primary cells displayed sustained bioactivity after three days of culture in all scaffolds, with the results showing the trends of higher DNA content and higher expression of fibronectin observed in these cultured on the hybrid scaffolds. These studies show that the topography and composition can be utilized to modulate the physiological activity of electrospun PCL scaffolds and have a measurable impact on hepatocyte cultures. Our method provides a reproducible approach and emphasizes the synergism of topographical stimuli and biochemical cues on electrospun scaffolds, which have wide-ranging application prospects in in vitro hepatic microenvironments.