Cryopreserved Arteries Research Articles

The Viability, Structure, and Mechanical Properties of Cryopreserved Rabbit Carotid Artery

New techniques have been developed for cryopreservation of arteries for use in transplantations or bypass surgery. In our experiments, rabbit carotid arteries were cryopreserved in a cryoprotective medium containing 1.5 M 1,2- propanediol (PROH). After storage in liquid nitrogen for over 10 days, the frozen arteries were thawed slowly in an ice bag that had been pre-cooled in liquid nitrogen to prevent thermal stress and fracture. Fresh carotids were used as normal controls. The fresh and cryopreserved arteries were cultured. The growth of smooth muscle cells (SMCs), the development of endothelial cells (ECs), the integrity of carotid walls (the elastic lamellae or fibers), and the mechanical properties of arteries (elastic modulus and fracture strength) were investigated. The results showed that SMCs survived cryopreservation. It took approximately the same amount of time (∼24–36 h) for the SMCs of cryopreserved arteries to regenerate as those of the fresh arteries. After cryopreservation, only a small n...

New approach to improving endothelial preservation in cryopreserved arterial substitutes

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig ( n=20) were frozen at a temperature reduction rate of 1 °C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (−80, −145 or −196 °C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens ( p<0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at −145 °C), significant differences ( p<0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at −80 °C followed by rapid thawing led to greatest endothelial cell loss (61.36 ± 9.06% covered endothelial surface), while a temperature of −145 °C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38 ± 16.67% surface cover). In conclusion, storage at a temperature of −145 °C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.

Effect of the Thawing Process on Cryopreserved Arteries

This study was designed to explore the changes that occur in cryopreserved pig arteries following different thawing procedures, before and after being placed in an in vitro flow circuit. Segments of minipig iliac artery were cryopreserved in complete minimal essential medium plus 10% dimethylsulphoxide and stored in liquid nitrogen at -196 degrees C for 30 days. Three study groups were established according to whether the arterial specimens were fresh (control, n = 20), cryopreserved and rapidly thawed (RT) at 37 degrees C (n = 22) or cryopreserved and subjected to controlled, automated slow thawing (ST) (n = 22). Half of the specimens of each group were subsequently placed in the flow circuit for 72 hr. Evaluation was made of morphological and ultrastructural changes. Cell damage was established using the TUNEL method. All cryopreserved specimens showed endothelial denudation that was most extensive in those subjected to rapid thawing. Slowly thawed specimens showed improved cell viability and organization of the vessel wall, compared to those thawed rapidly. Under conditions of flow, the damage induced by the freezing/thawing process was enhanced. These findings suggest that (a) slow thawing of cryopreserved arteries results in improved preservation of the structure and viability of vessels, and (b) the damage induced by freezing/thawing is enhanced when vessels are subjected to flow in an in vitro circuit.

Gradual Thawing Improves the Preservation of Cryopreserved Arteries

This study was designed to test a slow, controlled, automated process for the thawing of cryopreserved arteries, whereby specimen warming is synchronized with the warming of its environment. Segments of minipig iliac artery, 4–5 cm in length, were subjected to controlled, automated cryopreservation in a biological freezer at a cooling rate of 1°C/min to −120°C, followed by storage in liquid nitrogen at −196°C for 30 days. Following storage, the arterial segments were subjected to rapid (warming rate of approximately 100°C/min) or gradual (1°C/min) thawing. Thawed specimens were processed for light microscopy and for scanning and transmission electron microscopy, Cell death was determined by the TUNEL method. Metalloproteinase (MMP) expression was estimated by immunohistochemical analysis. Most of the cryopreserved vessels subjected to rapid thawing showed spontaneous fractures, mainly microfractures, whereas these were absent in slowly thawed specimens. In rapidly thawed vessels, the proportion of damaged cells was double that observed in those thawed more gradually. Increased intensity and extent of MMP-2 expression was shown by rapidly thawed specimens. The slow-thawing protocol tested avoids the formation of spontaneous fractures and microfractures and the accumulation of fluid within the arterial wall tissue. This results in improved tissue preservation.

Rheological properties of fresh and cryopreserved human arteries tested in vitro

Cryopreservation is widely used to preserve blood vessels for a while but is controversially suspected to affect the mechanical behavior of these allografts. The aim of this study was to determine whether differences in the three-dimensional mechanical behavior exist or not between fresh and cryopreserved arteries. Using a previously developed experimental system, in vitro inflation tests were performed on twenty segments of human fresh and cryopreserved arteries, in static conditions. Opening angles were also measured from images of rings in zero-stress state. The initial reference state was chosen as the unloaded state and tests were performed on specimens stretched at natural “in vivo” length. Mechanical measures calculated are “natural” (Hencky) strains (finite deformations), “true” (Cauchy) stresses in radial, circumferential, and longitudinal directions as well as strain energy per unit volume. Tangent moduli are derived from radial and circumferential stress-strain characteristics using non-linear curve fitting. Values of incremental and pressure-strain elastic parameters, wall stiffness, and compliance per unit length are also calculated. Results are presented in terms of characteristics of stresses and strains in the three directions, axial force, tangent moduli vs strains or stresses, and energy per unit volume, for both types of artery, with reference to transmural pressure. Detailed numerical results are given at mean transmural pressure or in the physiological range. Significant differences are indicated by statistic Student T-tests. Results obtained show that significant differences exist between rheological properties of fresh and cryopreserved segments of human artery. Strains, stresses, axial force, strain energy, and wall stiffness values highlight those differences whereas elastic parameters, compliance, and opening angle do not. The usefulness of some parameters to compare the mechanical behavior existing between fresh and cryopreserved arteries is therefore underlined.

Changes in the cooling rate and medium improve the vascular function in cryopreserved porcine femoral arteries.

The purpose of this study was to design an adequate technique with which to cryopreserve pig femoral arteries and to assess the influence of storage times in vascular function. Fifty-two femoral arteries were distributed in seven groups. In group A (control), 10 arteries were studied after harvest; in groups B1 and B2, 19 arteries were suspended in RPMI 1640 plus fetal calf serum plus dimethylsulfoxide and were cryopreserved at 1 degrees C per minute or 0.3 degrees C per minute, respectively. In groups C1 to C4, 23 arteries were suspended in modified Krebs-Henseleit plus dimethylsulfoxide plus sucrose, cryopreserved at 0.7 degrees C per minute, and kept frozen for 1, 15, 60, or 180 days, respectively. After being thawed, arteries were examined for contraction and endothelial-dependent vasodilation (organ bath studies), antithrombotic properties of the endothelial layer(perfusion studies), and vessel structure (electron microscopy). Endothelial cells were present in both cryopreserved and control arteries. The control vessels showed a mean contraction to norepinephrine (10(-7) mol/L) of 13010 +/- 3181 mg. Arteries in groups B1 and B2 did not respond to norepinephrine. Contraction in groups C1 to C4 was as follows: C1, 5354 +/- 1222 mg; C2, 5187 +/- 2672 mg; C3, 6867 +/- 2292 mg; C4, 7000 +/- 2858 mg, which represent 50% of the control values (P <.001). Vasodilation was similar in control (99% +/- 3%) and cryopreserved arteries (C1, 90% +/- 13%; C2, 93% +/- 12%; C3, 89% +/- 15%; C4, 88% +/- 22%). Storage time did not influence vascular function. Platelet interaction was almost absent and similar in all groups. A modified cryopreservation technique preserves endothelial function independently of the storage time up to 6 months.

Effect of Cryopreservation on Endothelin-1 Productions of Human Mammary Artery

The aim of this study was to determine the ability of human mammary artery cells to maintain a metabolic activity by measuring the artery concentration of two vasoactive substances, endothelin-1 (ET-1) and cyclic guanosyl monophosphate (cGMP), and a neurohumoral substance—neuropeptide Y (NPY)—prior to and following cryostorage. Ten distal segments of internal mammary arteries were obtained at the time of surgery in patients who had undergone coronary artery bypass grafting. One ring of each vessel served as a fresh control for the other ring that was used in cryopreservation experiments. The arteries were frozen with liquid nitrogen at a controlled rate down to –130°C with an automatic freezing machine and were then stored in a liquid nitrogen vapor at −150°C. After mammary artery extraction, ET-1, cGMP, and NPY concentrations were studied before and after cryopreservation. Cryopreserved, compared to fresh arteries, exhibited an increase in ET-1 (11.11±1.61 vs. 3.09±0.06 pg/mg; p=0.004) and a decrease in cGMP (9.88±2.04 vs. 8.55±2.07 p moles/mg; p<0.02), whereas there was no significant NPY variation. An increase in ET-1 and decrease in cGMP was found in 10 out of 10 and 6 out of 10 of cryopreserved artery specimen, respectively. There was no significant correlation between ET-1 and cGMP production in fresh or in cryopreserved arteries. The present method of cryostorage is effective in preserving “hormonal” mammary artery activity. However, the particularly high ET-1 concentration without associated cGMP concentration may be deleterious by increasing smooth-muscle cell proliferation and vascular tone of cryopreserved arteries.

Banking and Distribution of Large Cryopreserved Arterial Homografts in Brussels: Assessment of 4 Years of Activity by the European Homograft Bank (EHB) with Reference to Implantation Results in Reconstruction of Infected Infrarenal Arterial Prostheses and Mycotic Aneurysms

In 1991 European Homograft Bank (EHB) initiated a program of cryopreservation and distribution of large arteries to meet a new demand for quality-controlled arterial homo grafts of various sizes. From May 1991 to June 1995, 308 arteries have been registered from 136 donors: 122 brain death cases and 14 cadavers (mean age 34 years, male/female ratio 1.52/1); 263 arteries were cryopreserved (113 aortas, 64 aortic bifur cations, and 86 femoral); 19 were discarded for atherosclerosis (6.7%); 10 batches of arteries were partially or totally discarded because of persistent contamination and further eight batches for positive or doubtful viral serology. One hundred patients were treated in nine European centers with one (N = 69) or more EHB homografts. Indications were: infected prosthesis 70 (17 with aortoenteric fistula); mycotic aneurysm 19 (four ascending aortas, two with bronchial fistula); neoplastic infiltration of subrenal aorta one; extracardiac reconstructions/shunts 10. (continued on next page) (Abstract continued) Results from homograft reconstructions in infected prosthesis or mycotic aneurysm were available in 90 patients. There were 19 early deaths and 24 early complications, three were directly graft-related and included a fatal case of homograft rupture. Sixty- seven vascular cases were followed up from 1 month onward (mean: 16 months): 50 were uneventful; there were nine late deaths, of which two resulted from graft-related digestive hemorrhage; there were eight cases of late complications; three arteries were partly explanted as a result of focal thrombosis. Four patients were lost to follow-up. In the cases of aortoenteric fistula, however, the results were disappointing with only five late survivors of the 16 treated patients. Finally, these results show that cryopreserved arteries seem to perform as well in the midterm as the fresh ones. Both the banking activity of cryopreserved homografts and the short- to mid-term performances of the implants in cases of prosthetic or native arterial infection are very satisfactory, provided no aortoenteric fistula is present. Cryopreserved arteries can also be used for extracardiac shunts and reconstructions.

Evaluation of Cryopreserved Arteries as Alternative Small Vessel Prostheses

Biologic or synthetic grafts have had limited success in small vessel applications. Studies were initiated to assess the potential use of cryopreserved (CP) arteries as coronary artery bypass conduits. Sheep carotid arteries (internal diameter: 4 mm; length: 10 cm) were cryopreserved in a nutrient media containing 10% DMSO and were stored in a nitrogen vapor at -150 degrees C. After thawing, histological, enzyme-histochemical and functional studies showed slight histological alterations, preservation of enzymal activities and an abolition of the contractile response. In a sheep model, arterial substitution of a 10 cm segment of carotid artery was realised by implantation of fresh autografts ( n = 4); fresh allografts (n = 9) and CP allografts (n = 9). After 3 months, all autografts were patent with slight histological alterations. Fresh and CP allografts showed similar modifications: patency rate was 7/9 in both groups. Intimal thickening with cell proliferation was seen in fresh (3/7) and CP (4/8) arteries; loss of smooth muscle medial cells was constant. Adventitia was always involved by a marked inflammatory reaction. One characteristic of CP allografts was the frequent presence of large dystrophic calcifications. In conclusion, morphologic and functional arterial changes occurred after freezing and thawing. In spite of vascular rejection, the patency rate of allografts after 3 months of implantation in arterial circulation remained high and does not seem influenced by cryopreservation.