Endothelialization of a New Dacron Graft in an Experimental Model: Light Microscopy, Electron Microscopy and Immunocytochemistry

Two types of synthetic vascular grafts, Dacron Triaxial and Dacron Gelseal Triaxial, were implanted into both the common carotids of sheep. The animals were sacrificed 1, 2, 8, and 16 weeks after surgery. Multiple specimens, obtained from grafts and anastomoses, were studied by light microscopy, transmission and scanning electron microscopy. A parallel immunocytochemical analysis was performed on some specimens. Dacron Triaxial grafts failed to develop a complete neointimal coverage. Myofibroblasts and fibroblasts were the dominant cells in such synthetic graft. Moreover, focal areas of stripping, platelet deposition, and thrombosis were observed at 8 and 16 weeks. In contrast, a stable endothelial coverage developed on the Gelseal Triaxial grafts after 16 weeks

Electron Microscopy of Lipid Deposits in Human Atherosclerosis

The filipin probe associated with tannic acid stain was used to study intra-and extracellular lipids in surgically removed human atherosclerotic lesions (n = 20). In particular, intimal thickenings, fatty streaks and fibrolipidic plaques have been investigated by using mainly transmission and scanning electron microscopy. In the intimal thickenings, the lipid deposits were mainly localized in the subendothelial space as homogeneously sized particles (40-140 nm) and more heterogeneous uni-multilamellar vesicles (35-700 nm). Intermediate lipid forms were also observed. In the fatty streaks, the lipid deposits were intracellular and mainly observed in cells with a monocyte/macrophagic phenotype. Lipid inclusions, lipid lysosomal bodies and intracellular cholesterol crystals very similar to those observed in experimentally induced atherosclerosis were documented. In the fibrolipidic plaque the lipid deposits were found both in the intracellular and in the extracellular compartments. Lipids accumulated within arterial macrophages and smooth muscle cells, usually as lipid droplets. Clusters of lipoprotein-like particles (50 nm in diameter) as well as larger uni-multilamellar lipids (700 nm) with an occasional compound appearance were particularly observed bound to elastic tissue and collagen fibers. These morphological observations outline the complexity of lipid metabolism in the various histological aspects of human atherosclerosis

Development of a Rotation Device for Microvascular Endothelial Cell Seeding

A rotation device (RD) specifically designed to achieve sterile endothelial cell (EC) seeding of vascular grafts has been developed. The basic characteristics of the RD include: small dimensions, fully autoclavable components, and perfectly sealed graft holders. These features make it possible to maintain sterility during all the steps of EC seeding. This was documented by negativity of all bacteriological assays performed . Moreover, the RD can simultaneously support three vascular grafts with different lengths (20, 40, and 60 cm) and diameters (4-8 mm). EC seeding is performed in the climatized chamber (37 °C; 5 % C02) with constant rotation (0.1 -3 rpm). The rotation cycle can be completed automatically. The practical efficacy of the RD was investigated by seeding 2 x 105/cm2 of human microvascular EC on 20 cm length, 4 mm internal diameter (ID) fibronectin- coated polytetrafluoroethylene (ePTFE) grafts for 24 and 48 hours respectively . Further, the effect of a highly viscous plasma expander, i.e., haemagel, on cell retention was also evaluated. Results were not as favorable as expected. However, it should be emphasized that after 48 hours of eel! incubation by using the RD, 42 % of the initially seeded EC were still present and approximately 15 % were fully spread over the graft surface. Moreover, the 10 minute perfusion with haemagel did not decrease the number of adherent microvascular EC

Healing of Prosthetic Arterial Grafts

Numerous synthetic biomaterials have been developed as vascular substitutes. In vitro, ex vivo and in vivo studies have demonstrated that in animals, selected materials, i.e., Dacron and ePTFE (expanded polytetrafluoroethylene) grafts, are successfully incorporated in both the large and the small caliber host arteries through a process which is generally referred to as graft healing. Morphologically, this process consists of a series of complex events including fibrin deposition and degradation, monocyte-macrophage recruitment and flow-oriented cell-layer generation, this last event being the complete endothelialization of the arterial substitute. In contrast to experimental animals, the flow surface of synthetic vascular grafts remains unhealed in humans, particularly in the small caliber conduits. Healing in man consists of graft incorporation by the perigraft fibrous tissue response with a surface covered by more or less compacted, cross-linked fibrin. It is therefore obvious that: i) marked differences in graft healing exist between animals and man; and ii) the usual mechanisms of graft endothelialization are partially ineffective in man. In order to guarantee the patency of synthetic vascular grafts for human small artery bypass, new strategies and approaches have recently been attempted. In particular, the endothelial cell seeding approach has been successfully accomplished in animals and is being experimented in human clinical studies. The problems and results of this biological approach are outlined in this paper

Endothelial Injury in Human Atherosclerosis

A light and electron microscopic investigation (scanning and transmission electron microscopy) was performed on 51 human atherosclerotic carotid lesions. The purpose of this study was to establish whether features of endothelial injury such as those described in animals occur in man and whether these features can be related to specific stages of human atherosclerosis. Irrespective of their histological appearance the atherosclerotic lesions were covered with endothelium which showed non-specific changes in cell shape and size. However, all complicated lesions appeared denuded. Moreover, a peculiar interaction of endothelium with monocytes and lymphocytes as well as blood components (e.g., fibrin and lipoproteins) was observed in intimal thickenings, fatty streaks and uncomplicated plaques. The surface exposure of macrophage-derived foam cells was seen on florid fatty lesions. Large areas of the arterial surface lacking any endothelial coverage Were characteristic of complicated plaques. They appeared to be a consequence of the arterial wall degeneration with an associated failure in endothelial repair

Are Long Term Cryopreservation and Patency of Vein Allograft Truly Achievable?

Despite extensive experimental work, neither the effect of long term cryopreservation on vein graft architecture nor the failure of alloveins due to graft rejection have yet been investigated. Herein, we investigated ultrastructurally: a) the integrity of rabbit jugular veins following 1, 2 and 3 months of cryopreservation; b) the outcome of the three-month cryopreserved vein auto- and allografts after 1 month of implantation in the rabbit carotid artery; and c) the immunologic response to cryopreserved vein allografts with and without seeded autologous endothelium. Prior to implantation, the cryopreserved rabbit veins were well-maintained except for endothelial cell damage. Following implantation, the cryopreserved vein autografts were comparable to fresh veins with a complete endothelial lining. Conversely, only one of the allograft was still patent with features of acute rejection. After seeding with autologous endothelium , these explants failed shortly after surgery. We found absence of endothelium and necrosis of the media components with neutrophil infiltration. Although three months of cryopreservation does not affect vein graft architecture significantly, endothelial cells are damaged irrespective of the time of cryopreservation. Vein autografts promptly healed after one month of implantation at which time a viable endothelial cell lining was restored from the host artery. Conversely, vein allografts, with and without seeded autologous endothelium, failed due to graft rejection. This study highlights that current methods of cryopreservation do not reduce antigenicity of venous allografts significantly