Introduction: : Small diameter vascular grafts (sdVGs) are used clinically in over 400,000 patients annually in the US. For patients experiencing severe atherosclerosis, bypass surgeries are critical for re-establishing blood flow, and, while the clinical gold standard for bypass grafting is autologous saphenous vein grafts, they have exhibited limited patency and availability. Synthetic sdVGs, even with the progress in the field, still suffer from post-implantation thrombogenicity, graft rupture, and infection. For athero-prone patients who receive arterial bypasses, conduits are plagued by insufficient elasticity and compliance, failure to form a functional endothelium, and neointimal hyperplasia. Consequently, it is imperative to develop effective anti-atherosclerotic synthetic sdVGs with mechanical properties similar to native vessels, demonstrating low thrombogenicity and immunogenicity for maintaining long-term patency in patients with severe atherosclerosis.
We previously developed sdVG composed of electrospun fibrin hydrogel layers surrounded by a polycaprolactone (PCL) sheath and showed its regenerative capacity in mice and porcine models. While this formulation was successful in promoting a stable, confluent endothelium, it was still plagued by tissue overgrowth contributing to stenosis in the porcine model. To address these shortcomings, we are introducing genipin, a biopolymer derived from gardenia fruits, as our crosslinker to replace the PCL sheath, improving suturability while incorporating genipin’s potential capacity to redirect macrophages towards the pro-regenerative lineage and reduce TNF--promoted smooth muscle cell (SMC) recruitment. Our novel genipin-crosslinked sdVG aims to (1) improve degradation and compliance, both during surgical intervention and for maintaining patency, and (2) reduce inflammation and SMC overgrowth for long-term integration in athero-prone environments.
Materials and
Methods: : Fibrin tubes are synthesized following a published protocol; in short, fibrin hydrogel microfiber sheets are electrospun aseptically, with longitudinal or circumferential alignment formed by rolling the sheets about mandrels arranged parallel or perpendicular to the fiber orientation. Following our prior design, PCL sheaths are prepared to fit around the fibrin microfiber tubes by heat treatment, forming the fibrin-PCL (F-PCL) scaffold. Alternatively, for our novel conduit, tubes will instead be crosslinked in a genipin solution following synthesis for 72 hours, followed by dehydration allowing for compaction of structure and stable shelf storage (Fig. 1A). Following synthesis of the genipin-crosslinked fibrin scaffold, grafts are: (1) analyzed for surface features by SEM, (2) seeded with endothelial colony-forming cells (ECFCs) as an assay for cytotoxicity and cellular adherence, and (3) analyzed for mechanical endurance and properties including stress-strain curves for tensile strength, suture retention strength, burst pressure strength, and degradation rate.
We are implanting this novel fibrin-based arterial scaffold as a carotid interposition graft in ApoE-/- mice to evaluate efficacy through measurements of endothelialization, integration, durability, and remodeling of the sdVGs. We anticipate to observe reduced intimal hyperplasia and immunogenicity of our scaffolds compared to isogenic vein grafts.
Results, Conclusions, and Discussions:: To evaluate the sdVGs in the carotid interposition athero model, we have produced sdVGs using the prior F-PCL formulation that remained patent 4wk after implantation in ApoE-/- mice (Fig. 1). Like the vein grafts, these sdVGs demonstrated endothelium lining their luminal surface and vaso vasora on their adventitial surface (Fig. 1A,C). SMCs appeared on the periphery and subintimal layers of the sdVGs, yet appeared only in the subintimal domain of the vein grafts (ACTA2, Fig 1C). The sdVGs also had smaller lumens, because of greater cellular infiltration, with many of these cells being leukocytes (CD45+, Fig 1E). Unfortunately, multiple occluded murine models demonstrated stiffening of the vessel and loss of patency due to thrombosis and hyperplasia, motivating removal of the noncompliant PCL sheath and the introduction of a crosslinker to reduce SMC over-infiltration.
Genipin-crosslinked fibrin synthesized in the aforementioned protocol maintained its fibrillar structure as confirmed using SEM, and the genipin-crosslinking did not affect cell viability (Fig. 2B,C). Degradation of the graft reached a mass fraction of 0.47 after 21 days, which suits the regenerative timeline compared to the 2-3 year degradation timeline for PCL (Fig. 2D). Tensile ring-pulling conducted on the genipin-crosslinked sdVGs compared to the previously developed F-PCL sdVG demonstrated a non-significant difference in their characteristic stress-strain curves (Fig. 2E,F). While the PCL sheath had technical limitations in suturability and compliance due to its mechanical mismatch with native tissue, it was able to support the fibrin layer in retaining patency in vivo after 4wks without tears or bursts. Matching the strength of the F-PCL sdVG even with removal of the PCL is important for ensuring mechanical stability once implanted in vivo. Although there were still mechanical shortcomings compared to the native aortas, the mechanics of the fibrin-only conduit appeared strong enough to withstand in vivo pressures and flow conditions. Ongoing studies are evaluating the mechanical endurance of these grafts in vivo compared to vein grafts.
In tackling common modalities of failure of synthetic sdVGs in patients with athero, we are engineering a scaffold to overcome the limitations of autologous bypasses, providing a prefabricated, tunable conduit for vascular grafting.
.jpg "Katherine Tang photo")