Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate

Abstract

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, Triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, versus Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-Mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies we have further investigated the effectiveness of MABP in experiments that examined: 1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; 2) Furthermore, recognizing the importance of alkaline phosphatase in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of alkaline phosphatase activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced alkaline phosphatase activity, and in vivo have no significant calcification in long term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.

Triglycidyl Amine Crosslinking Combined With Ethanol Inhibits Bioprosthetic Heart Valve Calcification

“…Moreover, intrinsic alkaline phosphatase activity is not diminished by glutaraldehyde fixation [5]. Therefore, hydrolysis of phospho-esters, such as the residual nucleic acids present in crosslinked cells, by either intrinsic or extrinsic alkaline phosphatase, can form inorganic phosphorus, leading to calcium-phosphate mineral[2, 6]. Glutaraldehyde cross-links are also unstable, and this can lead to both material failure and local inflammation due to the leaching of glutaraldehyde from treated tissue[7–8].…”

“…We previously reported the synthesis and mechanistic characterization of an epoxy-crosslinker, triglycidyl amine (TGA) [16], that was demonstrated to result both in stable cross-linking of porcine aortic valve and bovine pericardial tissue, and significant, but not complete calcification inhibition in rat subdermal implants [6, 16–17]. These prior studies indicate that TGA anticalcification mechanisms may be due to unique alternations in collagen structure and diminished accumulation of extracellular matrix proteins associated with calcification such as tenascin-C and MMP9.…”

The Pathogenesis and Treatment of the Valvulopathy of Aortic Stenosis: Beyond the SEAS

“…Thus, NCBPs may provide a unique and novel means to slow AVC in addition to their statin-like effects. Additionally, pretreatment of bioprosthetic aortic valve cups with synthesized thiol bisphosphonate, 2-mercaptoethylidene-1, 1-bisphosphonic acid inhibited degenerative calcification [43]. These data suggest that NCBPs may serve as an effective and well-tolerated way to slow AVC, and clinical data supporting this theory are beginning to emerge.…”

Decellularization, Stabilization and Functionalization of Collagenous Tissues Used as Cardiovascular Biomaterials

“…However, in another report the crosslinking of bovine perichardium and porcine aortic valves with triglycidylamine, a molecule of high polarity and solubility in water, resulted an improvement in biocompatibility (assessed using bovine aortic valve interstitial cells, human umbilical endothelial cells and rats artery smooth muscle cells) and resistance to calcification (subcutaneous implantation in rats) compared with glutaraldehyde-fixed tissues (Connolly et al, 2005). Furthermore, triglycidylamine-fixed tissues showed stable mechanical properties (Sacks et al, 2007) and optimal reduction of calcification when treatments included mercapto-aminobisphosphonate (Rapoport et al, 2007). It was hypothesized that the difference between these two results, which explored the chemistry of epoxy in the crosslinking of tissue, may be due to differences in water solubility, chemical heterogeneity and contamination with used epoxy residual reactants (Connolly et al, 2005).…”