Origin of cells that line damaged native blood vessels following endothelial cell transplantation.

Endothelial cell (EC) transplantation has been proposed as a method to reduce the thrombogenicity of both vascular grafts as well as injured native blood vessels. While techniques have been developed to establish EC monolayers on these surfaces, a major question that remains is whether the cells that exist on the blood flow surface are the same cells placed on the surface at the time of transplantation. We have developed an intravital fluorescent staining technique that permits isolated, autologous, fat-derived microvascular endothelial cells (MVEC) to be labeled and subsequently detected following their transplantation. In our study, rat abdominal aortas (AA) were injured with a 3F embolectomy catheter, and the injured surfaces were immediately treated with fluorescently labeled MVEC. Five days after transplantation, AA were evaluated by both scanning electron and fluorescence microscopy. Results of scanning electron microscopy showed the existence of nonthrombogenic regions in the areas of injury, and fluorescence microscopy of the identical areas established that these cells contained fluorescent dye. Our results indicate that the cells that line these injured areas of native vessels are the same cells that were originally transplanted. Our intravital fluorescence technique provides a method to trace the origin and disposition of transplanted cells on the vascular surfaces.

Microvascular endothelial cell sodding of ePTFE vascular grafts: improved patency and stability of the cellular lining.

Small diameter (< 6 mm) synthetic vascular grafts fail at a clinically unacceptable rate due in large part to their inherent thrombogenicity. The development of a new cellular lining on synthetic vascular grafts would most likely improve the patency rates observed for these grafts in small diameter positions. We have evaluated the use of endothelial cell transplantation to accelerate the formation of a cell lining using microvascular endothelial cells derived from canine falciform ligament fat. This source of fat is histologically similar to human liposuction fat and was isolated using a collagenase digestion technique identical to methods used for human liposuction fat microvessel endothelial cell isolation. The isolated fat endothelial cells were sodded onto 4 mm ePTFE grafts using pressure to force the cells onto the luminal surface. This pressure sodding method permitted cell deposition in less then 3 min. Sodded and control (non-cell-treated) grafts were implanted as interpositional paired grafts using end-to-end anastomoses in the carotid arteries of mixed breed dogs. Each dog therefore received a sodded graft on one side and a control graft on the contralateral side. After 12 weeks of implantation all control grafts were occluded while 86% of the cell-sodded grafts remained patent. Statistical evaluation of the data revealed a significant improvement in patency of cell sodded grafts (McNemar's chi 2 P = .02). Morphological evaluation of grafts explanted at 5, 12, 26, and 52 weeks following implantation revealed the presence of a cell lining on sodded grafts which remained stable for a period of at least one year. This new cell lining exhibited morphologic characteristics of a nonthrombogenic endothelial cell lining. The development of this new intima, evaluated 5 weeks-1 year after implantation, was not associated with a progressive intimal hyperplasia. From these data we conclude that microvessel endothelial cells derived from canine falciform ligament fat can be rapidly isolated using an operating room compatible method. Cell deposition on synthetic grafts is subsequently accelerated using a pressure sodding technique. A cellular lining forms on the inner surface and is associated with a statistically significant improvement in the function of sodded grafts in a canine carotid artery model.

Formation of a multilayer cellular lining on a polyurethane vascular graft following endothelial cell sodding.

Small-diameter (less than 6 mm) clinically available vascular grafts often fail due in part to the inherent thrombogenicity of artificial polymers. Transplantation of endothelial cells onto the lumen of these vascular grafts has been suggested as one method to overcome this thrombogenicity. We have developed a compliant polyurethaneurea (PEUU) 4-mm graft with a luminal surface modified by a glow discharge gas plasma. Autologous microvessel endothelial cells were isolated from canine falciform ligament fat, were transplanted onto the luminal surface of the grafts using an intraoperative isolation and sodding technique, and both endothelial-cell-treated and non-cell-treated grafts were placed as bilateral carotid interposition grafts in a canine model. After 5 weeks of implantation, explanted control (non-cell-treated) grafts exhibited a deposition of platelets, white cells and fibrin characteristic of a thrombogenic surface. MVEC sodded grafts exhibited a multicellular lining within but distinct from the lumen of the PEUU graft. The blood-contacting surface of this lining exhibited an antithrombogenic endothelial cell monolayer. We suggest that the PEUU graft supported the initial deposition of MVEC and development of and endothelial cell lining. During the 5 weeks of implantation this lining continued to proliferate and detached from the PEUU graft substratum. The final neocellular lining exhibited a luminal diameter and histological features similar to a native artery.

Thrombus-free, human endothelial surface in the midregion of a Dacron vascular graft in the splanchnic venous circuit--observations after nine months of implantation.

The addition of an endothelial cell lining to a prosthetic vascular graft may reduce the thrombogenicity of the blood-contacting surface. An endothelialized mesoatrial graft was implanted in a patient with Budd-Chiari syndrome caused by a primary inferior vena caval leiomyosarcoma. During the initial surgery a Dacron vascular graft was preclotted with plasma and then lined with microvascular endothelial cells derived from the patient's subcutaneous adipose tissue. The patient did well initially but 9 months later required resection of a mechanical stricture of the graft that occurred as it passed beneath the costochondral junction. Grossly, the luminal surface of the resected graft was free of thrombus, with a smooth, glistening, white surface. Light microscopy demonstrated a surface layer of cells morphologically consistent with an endothelial cell monolayer, a subendothelial layer composed of extracellular matrix and spindle-shaped cells, and granulation tissue around the Dacron fabric. Immunohistochemistry and electron microscopy confirmed the presence of vascular endothelium on the luminal surface. This report documents the successful achievement of a human endothelial cell monolayer that persisted for 9 months in the midportion of a Dacron vascular graft.

Human microvessel endothelial cell isolation and vascular graft sodding in the operating room.

We have evaluated multiple factors inherent to an operating room-compatible endothelial cell procurement and sodding procedure. Microvessel endothelial cell isolations have been performed on fat tissue obtained from over 140 patients with a 100% success rate. Liposuction-derived fat was optimal with respect to cell yield, and isolation time. The devices and equipment used were acceptable to the operating room and the complete cell procurement procedure was successful even in the hands of personnel with minimal training. Fat digestion was achieved using crude clostridial collagenase, with an average cell yield of 1 x 10(6) microvessel endothelial cells/gm of fat. Evaluation of this procedure with canine fat using an operating room acceptable procedure resulted in a 100% procurement success rate requiring 1.5 hours (+/- .5 hrs) for completion of the fat isolation, and cell isolation procedure. Microvessel EC could subsequently be used in graft seeding or sodding techniques to establish endothelial cell monolayers on vascular grafts. Our results indicate that one person with minimal cell isolation background can reproducibly isolate large quantities of sterile autologous endothelial cells in the operating room for immediate use in endothelial cell seeding/sodding procedures.