The characterisation of extracellular matrix materials for vascular tissue engineering: an in vitro evaluation of cell behaviour and genetic response

The prevalence of atherosclerosis and other degenerative cardiovascular pathologies continues to challenge the capabilities of modern medicine. Despite progressive treatment options, vessel replacement is the often the only solution to many cardiovascular pathologies, thus verifying the need for improved vascular graft substitutes. The assessment of potential vascular graft materials requires extensive, physiologically-realistic, in vitro analysis prior to clinical application. This study presented a comprehensive evaluation platform of cell-material interactions, including cell compatibility, viability, proliferation trends and gene response to the substrate materials in order to evaluate the potential of ECM materials in vascular applications. Cellular mechano-transductory pathways are known to be highly regulated by the mechanical and chemical properties of the scaffold on which they are grown, thus heightening the need for pre-clinical assessment of the cell/material interactions of each material. In order to obtain clinically relevant data, cell-seeded substrates were subjected to physiological flow conditions mimetic of the in vivo environment.

This study focused on cellular attachment and behaviour on naturally derived cell?seeded extracellular matrix (ECM) materials due to their unique natural structure and bioactive nature. The ECMs studied were porcine urinary bladder matrix (UBM) and small intestinal submucosa (SIS). Primary human aortic cell lines were chosen to assess the cell-material interactions – Human Aortic Endothelial Cells (HAEC) and Smooth Muscle Cells (SMC). Cell morphology, proliferation, metabolic activity and gene expression were analysed under both static and wall shear stress (WSS) flow conditions. WSS profiles, mimetic of physiological arterial fluid flow, were applied utilising a cone and plate bioreactor. The bioreactor system was modified and validated for the incorporation of 3D scaffold whilst ensuring a controlled level of accuracy within the flow environment. Cell culture conditions analysed included: HAEC culture, SMC culture, a direct HAEC/SMC co-culture, and HAEC and SMC seeded on to the ECM materials both in mono-culture and co-culture conditions. Metabolic activity levels were quantified for all conditions to evaluate cellular health and cell proliferation/apoptosis was monitored using the xCELLigence real time cell analyser system, in addition to LIVE/DEAD® assay which provided visual confirmation of cell viability. Genetic analysis of cell interactions of co-culture and ECM, in static and dynamic conditions was conducted utilizing real time RT-PCR techniques for the quantification of an array of inflammatory and remodelling biomarkers.

The analysis of genetic responses to physiological relevant conditions gave great insight into the differential inflammatory and remodelling gene expression for both ECM materials. A direct co-culture model was achieved and studied as a comparative control for the co-cultured ECM materials. It was found that the gene expression levels were more regulated by the different ECM substrate conditions in comparison to the variation from atheroprotective to atherogenic WSS flow conditions. In all, greater cellular metabolic activity was observed for UBM ECM when compared to SIS for HAEC and SMC culture conditions in addition to the HAEC/SMC co-culture conditions. UBM ECM showed greater promise of remodelling activity demonstrated by up regulation of MMP expression. In addition to this, HAEC/SMC co-cultured UBM ECM exhibited reduced expression of MCP-1 and IL-1β inflammatory biomarkers in comparison to SIS ECM when exposed in an atherogenic flow environment. This finding for UBM ECM may translate to more favourable integrative potential and adaptation to the in vivo host environment.

The evaluation of implantable graft materials at a pre-clinical genetic level remains understudied. This field of testing provides an indication of anticipated host response to physiological shear through gene expression analysis whilst also allowing the shear resistance of cell-substrate attachment to be analysed in appropriate mechanical environment simulative of the intended vascular application. In keeping with the imperative need to drive laboratory research towards clinical translation, this study presents a promising in vitro platform for pertinent analysis of potential vascular graft materials.