Immune response to the long-term grafting of cryopreserved small-diameter arterial allografts

Introduction. The viability and immunological response induced by cryopreserved arterial allografts remain unclear. This study examines the post-graft behaviour of this type of vessel substitute. Materials and methods. Both iliac arteries were extracted from Lewis rats (donors) and used to establish groups of allogeneic fresh (group I) or cryopreserved (group II) grafts in Fisher-344 rats (recipients). Cryopreserved segments for grafting were prepared by automated controlled freezing at a cooling rate of 1°C/min followed by storage in liquid nitrogen vapour at -145°C for 30 days. Before grafting, the vessels were slowly thawed. Animals were sacrificed at 14, 30, 90 and 180 days post-surgery when graft specimens were obtained for light and electron microscopy and immunohistochemical detection of inflammatory cells (CD45, ED1, CD4, CD8). Results. After surgery, 85.71% of the grafts in group I and 82.14% in group II were patent. Following long-term implant, both the fresh and cryopreserved allografts showed complete loss of the muscle compartment of the media. Inflammatory or CD45-positive cells (mainly macrophages and CD8 T-lymphocytes) were detected at earlier time points in suture zones and adventitia. In the fresh allografts, the number of immunolabelled cells steadily increased until they were seen to occupy the entire adventitia at 90 days, with high numbers persisting at 6 months. In the cryopreserved allografts, this adventitial inflammatory infiltrate was significantly reduced. Conclusions. The cryopreservation/slow thawing protocol used diminished the immune response induced by fresh arterial allografts improving their behaviour after grafting

Isolation of intact aortic valve scaffolds for heart-valve bioprostheses: Extracellular matrix structure, prevention from calcification, and cell repopulation features

Extracellular matrix (ECM) scaffolds isolated from valvulated conduits can be useful in developing durable bioprostheses by tissue engineering provided that anatomical shape, architecture, and mechanical properties are preserved. As evidenced by SEM, intact scaffolds were derived from porcine aortic valves by the combined use of Triton X-100 and cholate (TRI-COL) or N-cetylpyridinium (CPC) and subsequent nucleic acid removal by nuclease. Both treatments were effective in removing most cells and all the cytomembranes, with preservation of (1) endothelium basal membranes, (2) ECM texture, including the D-periodical interaction of small proteoglycans with normally D-banded collagen fibrils, and (3) mechanical properties of the treated valves. Ultrastructural features agreed with DNA, hexosamine, and uronic acid biochemical estimations. Calcification potential, assessed by a 6-week rat subdermal model, was significantly reduced by TRI-COL/nuclease treatment. This was not true for CPC only, despite better proteoglycan preservation, suggesting that nucleic acids also are involved in calcification onset. Human fibroblasts, used to repopulate TRI-COL samples, formed mono- or multilayers on surfaces, and groups of cells also were scattered within the valve leaflet framework. A biocompatible scaffolds of this kind holds promise for production of durable valve bioprostheses that will be able to undergo probable turnover and/or remodeling by repopulating recipient cells