The effect of decellularisation on the real time mechanical fatigue of porcine aortic heart valve roots.

Decellularised heart valve roots offer a promising option for heart valve replacement in young patients, having the potential to remodel and repair. Replacement heart valves have to undergo billions of opening and closing cycles throughout the patient's lifetime. Therefore, understanding the effect of cyclic loading on decellularised heart valve roots is important prior to human implantation. The aim of this preliminary study was to investigate the influence of low concentration sodium dodecyl sulphate (SDS) decellularisation treatment on the in vitro real time mechanical fatigue of porcine aortic heart valve roots under physiological real time cyclic loading conditions. This required a specific real time in vitro method to be developed, since previous methods relied on accelerated testing, which is non-physiological, and not appropriate for valve replacement materials that exhibit time dependent characteristics. The effects of the real time fatigue on hydrodynamic function and mechanical properties of the heart valve roots were assessed. The mechanical fatigue of decellularised porcine aortic heart valve roots (n = 6) was assessed and compared to cellular porcine aortic heart valve roots (n = 6) in a modified Real time Wear Tester (RWT) at a physiological frequency and under cyclic pressure conditions for a maximum of 1.2 million cycles. Periodically, the heart valve roots were removed from the RWT to assess the influence of cyclic loading on valve competency (static leaflet closure). At the end of testing further hydrodynamic performance parameters were ascertained, along with determination of leaflet material properties. A real time mechanical fatigue assessment method was developed and applied; with two cellular and two decellularised porcine aortic leaflets in different heart valve roots showing tears in the belly region. The decellularised aortic heart valve roots exhibited comparative functionality to the cellular heart valve roots under in vitro static and pulsatile hydrodynamic conditions. However, the material properties of the decellularised aortic leaflets were significantly altered following cyclic fatigue assessment and showed increases in elastin and collagen phase slopes and ultimate tensile strength compared to the cellular porcine aortic leaflets in the circumferential direction. This preliminary study demonstrated that low concentration SDS decellularised porcine aortic heart valve roots can withstand physiological cyclic deformations up to 1.2 million cycles in a RWT whilst maintaining their overall hydrodynamic function and leaflet mechanical properties. This is the first full report of preclinical mechanical fatigue assessment of decellularised porcine aortic heart valve roots under physiological real time conditions.

Repopulation of decellularised porcine pulmonary valves in the right ventricular outflow tract of sheep: Role of macrophages.

The primary objective was to evaluate performance of low concentration SDS decellularised porcine pulmonary roots in the right ventricular outflow tract of juvenile sheep. Secondary objectives were to explore the cellular population of the roots over time. Animals were monitored by echocardiography and roots explanted at 1, 3, 6 (n = 4) and 12 months (n = 8) for gross analysis. Explanted roots were subject to histological, immunohistochemical and quantitative calcium analysis (n = 4 at 1, 3 and 12 months) and determination of material properties (n = 4; 12 months). Cryopreserved ovine pulmonary root allografts (n = 4) implanted for 12 months, and non-implanted cellular ovine roots were analysed for comparative purposes. Decellularised porcine pulmonary roots functioned well and were in very good condition with soft, thin and pliable leaflets. Morphometric analysis showed cellular population by 1 month. However, by 12 months the total number of cells was less than 50% of the total cells in non-implanted native ovine roots. Repopulation of the decellularised porcine tissues with stromal (α-SMA+; vimentin+) and progenitor cells (CD34+; CD271+) appeared to be orchestrated by macrophages (MAC 387+/ CD163low and CD163+/MAC 387-). The calcium content of the decellularised porcine pulmonary root tissues increased over the 12-month period but remained low (except suture points) at 401 ppm (wet weight) or below. The material properties of the decellularised porcine pulmonary root wall were unchanged compared to pre-implantation. There were some changes in the leaflets but importantly, the porcine tissues did not become stiffer. The decellularised porcine pulmonary roots showed good functional performance in vivo and were repopulated with ovine cells of the appropriate phenotype in a process orchestrated by M2 macrophages, highlighting the importance of these cells in the constructive tissue remodelling of cardiac root tissues.

Effects of Chemical and Radiation Sterilisation on the Biological and Biomechanical Properties of Decellularised Porcine Peripheral Nerves.

There is a clinical need for novel graft materials for the repair of peripheral nerve defects. A decellularisation process has been developed for porcine peripheral nerves, yielding a material with potentially significant advantages over other devices currently being used clinically (such as autografts and nerve guidance conduits). Grafts derived from xenogeneic tissues should undergo sterilisation prior to clinical use. It has been reported that sterilisation methods may adversely affect the properties of decellularised tissues, and therefore potentially negatively impact on the ability to promote tissue regeneration. In this study, decellularised nerves were produced and sterilised by treatment with 0.1% (v/v) PAA, gamma radiation (25-28 kGy) or E Beam (33-37 kGy). The effect of sterilisation on the decellularised nerves was determined by cytotoxicity testing, histological staining, hydroxyproline assays, uniaxial tensile testing, antibody labelling for collagen type IV, laminin and fibronectin in the basal lamina, and differential scanning calorimetry. This study concluded that decellularised nerves retained biocompatibility following sterilisation. However, sterilisation affected the mechanical properties (PAA, gamma radiation), endoneurial structure and basement membrane composition (PAA) of decellularised nerves. No such alterations were observed following E Beam treatment, suggesting that this method may be preferable for the sterilisation of decellularised porcine peripheral nerves.