Triglycidylamine Crosslinking of Porcine Aortic Valve Cusps or Bovine Pericardium Results in Improved Biocompatibility, Biomechanics, and Calcification Resistance

Abstract

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.

Polyphenol-Stabilized Tubular Elastin Scaffolds for Tissue Engineered Vascular Grafts

“…Calcium content was then measured using atomic absorption spectrophotometry as described before. [45][46][47] For quantification of proteoglycans, samples were treated with papain overnight, and glycosaminoglycans (GAGs) released into solution were assayed with 1,9-dimethylmethylene blue reagent using a 0-to 1.25-ug chondroitin sulfate standard curve, as described by Hoemann et al 48 …”

“…Subdermal implantation has been extensively used for testing cardiovascular biomaterials as it anticipates bioreactor and intra-vascular graft implantation experiments. [48][49][50][51][52][53][54][55] The subdermal milieu is adequate for evaluation of basic implant properties such as host cell infiltration, degeneration and remodeling, toxicity and biocompatibility, inflammation, and calcification. 52,53 Moreover, FDA and ISO standards clearly mandate subdermal implantation as a required test for cardiovascular devices, before implantation in large animals.…”

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

“…Triglycidyl amine (TGA) was primarily synthesized as previously described [10], in brief reacting aqueous ammonia with excess epichlorohydrin. Following several additional reaction steps, a novel cold crystallization technique resulted in a coarse white water-soluble powder.…”

“…Our group has previously reported an alternative protein stabilization strategy based on the use of the polyepoxide crosslinker, Triglycidyl amine (TGA) [10]. Optimization experiments with TGA determined that a 7-day crosslinking protocol was necessary for maximal tissue stabilization and collagenase resistance [10].…”

“…Optimization experiments with TGA determined that a 7-day crosslinking protocol was necessary for maximal tissue stabilization and collagenase resistance [10]. Rat subcutaneous implant studies with TGAprepared materials demonstrated significant calcification resistance over Glut controls for porcine cusp and bovine pericardial implants, but not porcine aortic wall [11].…”