The development of a tissue engineered vascular graft using a poly(glycerol sebacate) methacrylate scaffold

Cardiovascular disease is the number one cause of death worldwide. In the treatment of such disease, vascular surgery commonly utilises grafts to replace or bypass damaged regions of the circulatory system. Currently, autograft vessels represent the gold standard for vascular bypass; however, these are of limited availability and quality. Synthetic conduits are also available, but these are of little use as small diameter vessels (<5 mm) due to high incidence of failure through infection or thrombosis. A need exists for a better vascular graft with tissue engineering offering an attractive solution. Although the development of tissue engineered vascular grafts (TEVGs) is being explored by a number of research groups using a wide range of methods, to date, a TEVG has not yet been produced that closely matches the mechanical performance of the autograft vessels currently favoured in vascular surgery. This research aimed to develop a method of manufacturing TEVGs with mechanical properties more similar to the current gold standard vessels. These TEVGs would be biocompatible, non-immunogenic and able to grow and remodel in vivo. Additionally, the TEVG manufacturing process would also be readily adaptable to producing more complex geometric shapes than simple tubes. It was hypothesised that this may result in an improvement in the performance of the TEVGs and may also expand the possible clinical applications. Following a review of the literature, presented in Chapter 1, a synthetic polymer scaffold based tissue engineering approach was selected as the method for producing the TEVGs. This approach has been widely adopted in the field of vascular graft tissue engineering. Synthetic polymer scaffold based methods are compatible with a range of manufacturing processes; have shown success in a variety of in vivo studies of TEVGs, including human trials; and had the potential to be adapted to produce a TEVG in a variety of predefined geometries. A synthetic polymer scaffold would be developed with properties, such as elasticity, degradation rate and porosity, specifically optimised towards the development of a TEVG with mechanical properties similar to the current gold standard autografts. TEVGs would be produced in vitro by seeding cells onto the synthetic polymer scaffold and then culturing them in a bioreactor under physiologically relevant flow to encourage cell proliferation and appropriate ECM deposition. A novel photocurable form of poly(glycerol sebacate), poly(glycerol sebacate) methacrylate (PGS-M) was proposed as the material for the synthetic polymer scaffold. This material had the potential to provide the mechanical performance and degradation properties identified as requirements for the scaffold, along with being biocompatible and easy to process into various scaffold geometries. In Chapter 2, different variants of PGS-M were produced which varied in molecular weight and degree of methacrylation (DM). These were characterised using various analytical chemistry techniques. It was determined that both the molecular weight and DM could be controlled by altering the reaction conditions used in the synthesis of the polymer. The degradation of the different variants of PGS-M was examined and this revealed that the polymer was susceptible to enzymatic degradation. Increasing the DM appeared to have an inverse effect on the degradation rate. The mechanical properties of PGS-M were also assessed and found to largely depend on the DM of the polymer and not the molecular weight. A 30% DM, low molecular weight (30% Low Mw) PGS-M was selected as the most suitable variant of PGS-M for producing a scaffold for use in culturing a TEVG. In Chapter 3, the biocompatibility of 30% Low Mw PGS-M is presented. Flat surfaces of the polymer were able to support the growth and proliferation of human dermal fibroblasts, human adipose derived stem cells and human coronary artery smooth muscle cells (SMCs) for several days in culture. Additionally, growth on the PGS-M surfaces does not appear to alter the phenotype of the SMCs. It was determined that culture on PGS-M surfaces may have effects on the metabolic activities of the three cell types investigated and that these effects may be subtle and cell specific. In Chapter 4, porous scaffold structures, suitable for use in tissue engineering, were produced from 30% Low Mw PGS-M using a porogen leaching method with sucrose particles. Combining PGS-M with sucrose particles of different sizes, at different ratios, allowed variation of the scaffolds’ handling properties, pore sizes, porosities and wettability. SMCs seeded onto the porous PGS-M scaffolds remained viable for 7 days in static culture and partially infiltrated the scaffold interiors. A method was then developed to produce the porous scaffolds as tubes of suitable geometry and porosity for use in the generation of TEVGs. Additionally, a method for producing porous PGS-M scaffolds in a variety of geometries was also demonstrated as a proof-of-concept. This method used a novel hybrid additive manufacturing and porogen leaching approach. A bioreactor was required for the culture of the tubular PGS-M scaffolds, once seeded with cells, to produce TEVGs. In Chapter 5, a design brief was proposed for a bioreactor capable of culturing the TEVGs under dynamic conditions and applying mechanical stimulation. This had been identified as advantageous in previous studies in TEVGs. A design process was implemented, with a number of initial ideas evaluated to determine an integrated final solution. The final bioreactor design utilised a pulsatile flow to provide mechanical stimulation to the developing TEVGs. The bioreactor was manufactured and assessed for sterility and its ability to provide mechanical stimulation to developing TEVGs. The pulsatile flow could be modulated to produce pressures and flow rates within the range of physiological blood flow which were appropriate for the culture of TEVGs. Modifications to the design were implemented, as required, to improve performance. The knowledge gained from the previous chapters was combined in Chapter 6. Porous tubular scaffolds, produced from 30% Low Mw PGS-M were seeded with human coronary artery SMCs and cultured in the bioreactor as TEVGs. The TEVGs were cultured for 7 days under dynamic and static conditions. TEVGs cultured under dynamic conditions, with mechanical stimulation produced by the pulsatile flow in the bioreactor, displayed highly variable results, but demonstrated the partial formation of blood vessel-like tissue in a small instance. TEVGs cultured under static conditions produced repeatable results, although with reduced vascular tissue formation compared to the grafts cultured under dynamic conditions. Both culture regimes produced TEVGs containing collagen and elastin and both also appeared to cause a change in the phenotype of the attached SMCs, from contractile to proliferative. Finally, Chapter 7 suggests the possible further work that may be conducted to explore the PGS-M scaffold based TEVGs, as the original aims of the research were not fully realised. Suggestions of how the bioreactor culture may be modulated and optimised are made along with ideas for generating TEVGs of varied geometries. Publications Pashneh-Tala, S. , MacNeil, S. & Claeyssens, F. The Tissue-Engineered Vascular Graft— Past, Present, and Future. Tissue Engineering Part B: Reviews 22, 68–100 (2015). List of Abbreviations ADSCs Adipose-derived stem cells PDI Polydispersity index AVFs Arteriovenous fistulas PEG Polyethylene glycol α-SMA Alpha-smooth muscle actin PEUU Poly(ester urethane)urea BM-MNCs Bone marrow-derived mononuclear cells PGA Polyglycolide BSA Bovine serum albumin PGS Poly(glycerol sebacate) CABG Coronary artery bypass grafting PGS-A Poly(glycerol sebacate) acrylate CDl3 Deuterated chloroform PGS-M Poly(glycerol sebacate) methacrylate DA Degree of acrylation PLGA Poly(lactide-co-glycolide) DM Degree of methacrylation PLLA Poly-L-lactide DMEM Dulbecco’s modified eagle’s medium PTFE Polytetrafluoroethylene ECs Endothelial cells PU Polyurethane ECM Extracellular matrix PVA Polyvinyl alcohol EDTA Ethylenediaminetetraacetic acid P4HB Poly-4-hydroxybutyrate EPCs Endothelial progenitor cells P(GA/CL) Poly(glycolide/caprolactone) Fem-pop Femoropopliteal P(LA/CL) Poly(lactide/caprolactone) GFP Green fluorescent protein P(L/D)LA Poly(L/D)lactide GPC Gel permeation chromatography RR Resazurin reduction HUVECs Human umbilical vein endothelial cells SEM Scanning electron microscopy H&E Haematoxylin and eosin SIS Small intestinal submucosa IMS Industrial methylated spirit SLS Selective laser sintering ITA Internal thoracic artery SMCs Smooth muscle cells MCP-1 Monocyte chemoattractant protein 1 SV Saphenous vein Mn Number average molecular weight TCP Tissue culture plastic MSC Mesenchymal stem cells TESA Tissue engineering by self-assembly MTT 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide TEVG Tissue engineered vascular graft Mw Weight average molecular weight TGF-β1 Transforming growth factor beta 1 NMR Nuclear magnetic resonance v/v Volume/volume OCT Optimum cutting temperature w/v Weight/volume PCL Poly-ε-caprolactone w/w Weight/weight