In vivo proof of persisting biofunctionalization of biological implants by fibronectin coating in a small animal model of aortic valve implantation

Objectives: Biological cardiovascular implants are proposed as biomimetic materials that may trigger recipient derived repopulation and regeneration. However, an unfavorable in vivo remodeling may also develop, e.g. triggered by adverse events like early thrombus formation at the blood-implant interface. Therefore, strategies for a rapid in vivo endothelialization and interstitial repopulation are warranted to increase the biofunctional properties of biological implants, e.g. by protein coating.

Methods: Aortic rat conduits (n=18) were decellularized according to a detergent-based protocol and subsequently coated with covalently Alexa488-labelled (green emission) fibronectin (FN; 50µg/ml, 24 hours). Coated conduits were implanted into the infrarenal aorta in an end-to-side manner in wildtype Wistar rats (n=9). Uncoated implants served as controls (n=9). Fluorescence-based detection of FN coating on aortic conduits was performed before implantation and at explantation after 1, 7 and 28 days. Histological and immunohistochemical (IHC) analyses were performed to detect cellular repopulation.

Results: All rats survived without clinical signs of lower body malperfusion. Before implantation and after 1 day in vivo, confocal microscopic evaluation revealed bright green FN fluorescence on the luminal as well as on the adventitial surface of the coated aortic conduits, which was diminished on postoperative day 7 and absent after 28 days. IHC demonstrated remarkable adventitial, but sparse endothelial repopulation of all conduits with no significant difference between both groups on day 7. Twenty-eight days after implantation, all implants showed complete endothelialization and a myofibroblast hyperplasia with increased ratio of intima-to-media thickness in the perianastomotic regions, while the aortic arch was incompletely repopulated. In both groups staining against inflammatory cell markers (CD3 & CD68) was negative.

Conclusions: Using a small animal aortic valve implantation model in combination with a fluorescent fibronectin, we demonstrate for the first time the feasibility of persisting biofunctionalization of cardiovascular implants in the systemic circulation beyond 7 days. However, in our model FN coating led to no significant reduction of intimal hyperplasia of decellularized aortic conduits. Further exploration of alternative coating substances is warranted for an improved biofunctionalization of cardiovascular implants.