In vitro recellularization of decellularized bovine carotid arteries using human endothelial colony forming cells

Nicolai Seiffert,Anja Reutzel-Selke,Dag Wulsten,Karl H Hillebrandt,Hendrik Napierala,Peter Tang,Igor M Sauer,Hannah Everwien,Simon Moosburner,Johann Pratschke,Eriselda Keshi,Benjamin Struecker

doi:10.1186/s13036-021-00266-5

Abstract

BackgroundMany patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. However, in some patients no suitable replacements (i.e. autologous or prosthetic bypass grafts) are available. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials. Clinical translation of this technique, however, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients.Bovine carotid arteries (BCA) were decellularized using a combined SDS (sodium dodecyl sulfate) -free mechanical-osmotic-enzymatic-detergent approach to show the feasibility of xenogenous vessel decellularization. Decellularized BCA chips were seeded with human ECFC, isolated from a high cardiovascular risk patient group, suffering from diabetes, hypertension and/or chronic renal failure. ECFC were cultured alone or in coculture with rat or human mesenchymal stromal cells (rMSC/hMSC). Decellularized BCA chips were evaluated for biochemical, histological and mechanical properties. Successful isolation of ECFC and recellularization capabilities were analyzed by histology.ResultsDecellularized BCA showed retained extracellular matrix (ECM) composition and mechanical properties upon cell removal. Isolation of ECFC from the intended target group was successfully performed (80% isolation efficiency). Isolated cells showed a typical ECFC-phenotype. Upon recellularization, co-seeding of patient-isolated ECFC with rMSC/hMSC and further incubation was successful for 14 (n = 9) and 23 (n = 5) days. Reendothelialization (rMSC) and partial reendothelialization (hMSC) was achieved. Seeded cells were CD31 and vWF positive, however, human cells were detectable for up to 14 days in xenogenic cell-culture only. Seeding of ECFC without rMSC was not successful.ConclusionUsing our refined decellularization process we generated easily obtainable TEVG with retained ECM- and mechanical quality, serving as a platform to develop small-diameter (< 6 mm) TEVG. ECFC isolation from the cardiovascular risk target group is possible and sufficient. Survival of diabetic ECFC appears to be highly dependent on perivascular support by rMSC/hMSC under static conditions. ECFC survival was limited to 14 days post seeding.

Highlights

Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery
Microscopic evaluation of extracellular matrix (ECM) showed no loss of abundant ECM components in respective areas of typical appearance, the compressed configuration following cell removal resulted in an intensified appearance of ECM-elements, especially in the area of the tunica media (Fig. 1e-l)
In this study we evaluate the capability of endothelial colony forming cells (ECFC) acquired through peripheral blood draw from hospitalized patients to reendothelialize decellularized Bovine carotid arteries (BCA) chips when applied in co-culture with rat mesenchymal stromal cells (rMSC) and Human mesenchymal stromal cells (hMSC)

Summary

Introduction

Many patients suffering from peripheral arterial disease (PAD) are dependent on bypass surgery. Advances have been made to develop autologous tissue engineered vascular grafts (TEVG) using endothelial colony forming cells (ECFC) obtained by peripheral blood draw in large animal trials Clinical translation of this technique, still requires additional data for usability of isolated ECFC from high cardiovascular risk patients. Synthetic prosthetic grafts may be used as an alternative for large diameter arteries (> 6 mm), mechanical mismatch, adverse host response, reduced patency rates and increased susceptibility to infections have impeded clinical applicability, especially for medium or small diameter arteries (inner diameter ≤ 6 mm) [8,9,10,11,12] To address these problems and create viable alternatives, tissue engineered vascular grafts (TEVG) have been developed using self-assembly and biodegradable scaffold techniques of both synthetic and natural origin [13]. While the results seem promising and are pursued up to clinical trials, no widespread clinical application has been established so far [13, 14]

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