Development of an acellular annuloplasty ring for atrioventricular valve repair

Annuloplasty is a common technique used for repair of the mitral valve. It is hypothesised that an acellular biological ring may provide a superior solution by encouraging tissue integration and improved annular dynamics whilst offering similar structure and composition to the native annulus.
Human mitral valve annuli (n=3) were characterised using histological methods to reveal the histoarchitecture of the native tissue. The histological features of porcine mitral valve annuli (n=5) were compared to the human annuli, as an alternative source of tissue. Haematoxylin and eosin, alcian blue and Sirius red Miller’s stained sections illustrated the morphology of both species revealing a highly fibrous region at the anterior of the annulus compared to the posterior region. The continuum of the fibrosa layer in the mitral leaflet into the fibrous region around the annulus was also visualised. Differences between species were observed at the trigones where cartilaginous tissue was present in the porcine annuli compared to fibrous tissue in the human tissue. The human mitral valve annuli were heavily calcified and showed signs of ossification, hence porcine annuli were used as the starting material for developing a biological acellular annuloplasty ring.
Porcine mitral valve annuli (n = 36) were decellularised using a series of eight iterative protocols. The annuli were successfully decellularised using a protocol comprising two freeze thaw cycles (one cycle in hypotonic buffer), followed by four alternate cycles of washing in hypotonic buffer plus proteinase inhibitors and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus proteinase inhibitors followed by treatment with nuclease solution, washing in hypertonic buffer and final disinfection wash in 0.1% (v/v) peracetic acid. Histological analysis of the treated tissue revealed removal of nuclei apart from apparent “ghost nuclei” in the trigones. Immunohistochemical analyses revealed loss of collagen IV and laminin associated with the basement membrane but retention of collagens I, II and III responsible for structural integrity. Total DNA content of the processed tissue was less than 50 ng.mg-1 wet weight and in-vitro biocompatibility assays showed decellularised porcine mitral valve annulus tissue was not cytotoxic to BHK and L929 cells. Biomechanical tensile tests using low strain rate to failure of the decellularised porcine mitral annuli showed increased tensile strain and transition strains and stresses compared to the native porcine annuli. A method for functional testing revealed that the acellular rings performed as well as synthetic annuloplasty rings in static tests of regurgitant porcine hearts.