Comparison of a closed system to a standard open technique for preparing tissue-engineered vascular grafts

Abstract

We developed a prototype for a closed apparatus for assembling tissue-engineered vascular grafts (TEVGs) with the goal of creating a simple operator-independent method for making TEVGs to optimize safety and enable widespread application of this technology. The TEVG is made by seeding autologous bone marrow-derived mononuclear cells onto a biodegradable tubular scaffold and is the first man-made vascular graft to be successfully used in humans. A critical barrier, which has prevented the widespread clinical adoption of the TEVG, is that cell isolation, scaffold seeding, and incubation are performed using an open method. To reduce the risk of contamination, the TEVG is assembled in a clean room. Clean rooms are expensive to build, complex to operate, and are not available in most hospitals. In this investigation, we used an ovine model to compare the safety and efficacy of TEVGs created using either a standard density centrifugation-based open method or the new filter-based closed system. We demonstrated no graft-related complications and maintenance of growth capacity in TEVGs created using the closed apparatus. In addition, the use of the closed system reduced the amount of time needed to assemble the TEVG by ∼ 50%. Adaptation of similar methodologies may facilitate the safe translation and the widespread use of other tissue engineering technologies.

Figures

FIG. 1.

Schematic representation of the closed disposable seeding system for assembling tissue-engineered vascular grafts (TEVGs). Heparinized bone marrow is added to the bone marrow bag. The bone marrow bag is then suspended at a head-height of 18 inches and the bone marrow is transferred into the drip chamber, which acts to filter out large particles (1). The bone marrow then flows out through the drip chamber filter and through the cell harvest filter (2), which entraps bone marrow-derived mononuclear cells (BM-MNC) (2a). The effluent is collected in the effluent bag. Sixty milliliters of harvest solution (10% Dextran 40) is then back-flushed through the filter (3), releasing entrapped BM-MNC (3a) and collecting them in the seeding chamber. The BM-MNCs are then seeded onto the scaffold using vacuum seeding (4, 4a). The effluent is then transferred to the seeding chamber thus bathing the seeded scaffold (5). At that time, the tubing is heat sealed and the TEVG is placed in the incubation chamber for 2 h, at which point it is ready for surgical implantation. Color images available online at www.liebertpub.com/tec

FIG. 2.

Summary of morbidity and mortality data for TEVG implanted in the lamb model. TEVG were implanted as intrathoracic IVC interposition grafts and monitored over a 6-month time course. A total of 12 TEVG were implanted and divided into two equal groups (n=6/group). Group 1consisted of TEVG seeded using a closed filter-based system and group 2 consisted of TEVG seeded using an open density centrifugation-based method. (A) Survival curves: There was one surgical complication, which occurred in the open method group. It consisted of a neurological injury that resulted from a prolonged clamp time. Due to the severity of the neurological deficit, the animal required euthanasia. (B) Graft-related complications: There were two graft-related complications (critical stenosis), which developed in the open method group and also required euthanasia. No deaths or graft-related complications occurred in the TEVG assembled using the closed system. IVC, inferior vena cava.

FIG. 3.

Computed tomography (CT) analysis of TEVG implanted in lamb model. Serial CT imaging was used to assess the change in size and shape of the TEVG implanted in juvenile lambs over a 6-month time course. All surviving animals were serially imaged at early (2 months) and late (6 months) time points. At the early time point, the TEVG showed thickening of the graft wall accompanied in some instances by graft luminal narrowing (n=11). There was no radiographic evidence of thrombosis in any graft. The graft luminal volume increased over the course of the experiment in all (n=9) animals that survived the 6-month time course. There was no radiographic evidence of aneurismal dilation in any grafts. Arrows show implanted graft by horizontal CT slice. “J” shows parts of implanted grafts by long-axis CT slice. Color images available online at www.liebertpub.com/tec

FIG. 4.

Histological analysis of TEVG: TEVGs in lambs explanted at 6 months after implantation resemble native blood vessels. (A) Scaffold. Before surgical implantation, all grafts were measured and cut to 20 mm in length. (B) At time of explantation, seeded scaffolds had transformed into neovessels resembling native inferior vena cava. (C–E) H&E staining revealed cellular architecture in the grafts resembling that of native vessels. Masson's trichrome staining showed robust collagen formation in both the groups. There was no evidence of ectopic calcification (Von Kossa). Elastin staining was present in both the groups but not as well developed as seen in the native IVC (Elastica Van Gieson [EVG] and Hart's stain). Glycosaminoglycan staining was seen in both the groups (Alcian blue stain). H&E, hematoxylin and eosin. Color images available online at www.liebertpub.com/tec