The Development of a Fibrin-Collagen-Glycosaminoglycan Scaffold for Heart Valve Tissue Engineering

Valvular heart disease is predicted to be the next cardiac epidemic (d’Arcy et al., 2011) and valve replacement, using bioprosthetic or mechanical valves, is the only therapy currently available for treating dysfunctional valves. While these valve substitutes undoubtedly save lives, they also have well documented limitations and for paediatric patients, their most debilitating limitation is an inability to grow concurrently with a growing body. Tissue engineered heart valves (HV) offer the potential of a valve replacement that can integrate fully with the native vasculature, facilitating growth and remodelling of the valve over time. However, many tissue engineering approaches have failed due to cell-mediated contraction, resulting in valves with leaflets that cannot maintain a tight seal over time. Therefore, the overall goal of the research presented in this PhD project was to develop a natural biomaterial scaffold for HV tissue engineering, capable of resisting cell-mediated contraction.

In the study presented in Chapter 2, a natural biomaterial composite was developed by reinforcing a fibrin gel with a freeze-dried collagen-glycosaminoglycan (CG) matrix. The resultant fibrin-collagen-glycosaminoglycan (FCG) scaffold demonstrated unique mechanical and biological properties and a controlled porous microstructure. Crucially, the ability of this FCG scaffold to resist cell-mediated contraction was established, with no change in scaffold dimensions when cultured with contractile cells. These results showed the potential of this biomaterial for use in applications where dimensional stability is crucial to the functionality of the construct.

A biofabrication process was developed in Chapter 3 to scale up this FCG scaffold into an anatomically accurate HV shape. Freeze-drying has not been previously used to successfully create a matrix with a large height to width ratio, thus creating a HV shaped CG matrix was a major challenge. Through control of parameters such as the mould material, thermal transfer through the mould and final freezing temperature, a repeatable process to create a HV shaped CG matrix was established. The collagen concentration of the HV shaped CG matrix was optimised for HV applications. Fibrin was successfully infiltrated through the porous microstructure of the CG matrix to create a HV shaped version of the FCG scaffold which demonstrated excellent integration of all components and consistent mechanical properties between the valve leaflets and wall.

In the final chapter (Chapter 4), a pulmonary valve substitute was developed through dynamic conditioning of the FCG construct. The application of this dynamic regime encouraged synthesis of appropriate, aligned, cell-mediated extra cellular matrix (ECM). Furthermore, when endothelial cells were cultured on the construct, it enabled the development of an intact endothelial cell layer. In addition, the leaflets of the FCG construct remained dimensionally stable and functionality testing demonstrated full coaptation of valve leaflets and unobstructed valve opening. The burst strength of the FCG construct increased two-fold from its initial strength, and crucially when tested at pulmonary flow conditions, the FCG construct passed the performance criteria outlined in ISO 5840-2 for pulmonary valve substitutes.

In conclusion, this project has led to the development of a FCG scaffold that can resist cell-mediated contraction, an attribute with many potential applications. The development of a biofabrication process to create a HV shaped version of this scaffold has demonstrated for the first time how freeze-drying can be used to create porous scaffolds with a complex geometry. Finally, the development of a tissue engineered pulmonary valve substitute through dynamic conditioning of this FCG scaffold brings us one step closer to the realisation of a clinically relevant tissue engineered HV and also holds potential as a 3D in vitro model for drug screening, device testing, and investigation of valvular cell interactions and disease states.