Vitrification of tissue engineered pancreatic substitute.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

In vivo evaluation of the effects of a new ice-free cryopreservation process on autologous vascular grafts.

Conventionally cryopreserved vascular grafts have performed poorly as arterial grafts. One possible mechanism that causes the poor function is the extracellular ice damage in tissue. We used a novel new ice-free cryopreservation (namely, vitrification) method for prevention of ice formation in cryopreserved venous grafts. This study was designed to evaluate the in vivo effects of the vitrification process on autologous vascular grafts using a short-term transplantation model and to examine the morphology and patency of vitrified grafts in correlation with control grafts. New Zealand White rabbits underwent a right common carotid interposition bypass graft. Fresh and vitrified reversed ipsilateral external jugular veins were used as autologous grafts. Animals were sacrificed at either 2 or 4 weeks after implantation, and fresh and vitrified vein grafts were harvested for histology studies. The results, comparing the patency of fresh and vitrified grafts, demonstrated similar short-term patency rates (approximately 90%). There were no signs of media disruption, aneurysm, or graft stenosis in vitrified vein grafts. Vitrification had not altered the pathophysiological cascade of events that occur when a vein graft is inserted into the arterial system. The vitrification process had no adverse effects locally or systemically in vivo. In addition, vitrification has preserved endothelial cell and smooth muscle cell integrity posttransplantation. In conclusion, this study, using an autologous animal model, clearly demonstrated a significant benefit of vitrification for preservation of graft function, and vitrification may be an acceptable approach for preservation of blood vessels or engineered tissue constructs.

Interstitial ice formation in cryopreserved homografts: a possible cause of tissue deterioration and calcification in vivo.

BACKGROUND AND AIM OF THE STUDY: Cryopreserved valve homografts often fail in infants. Controversies are ongoing concerning the relative contributions of cryopreservation variables, immune responses, cellular viability, and durability of the extracellular matrix to the mode of tissue failure. METHODS: Tissues to be examined for patterns of ice crystal distribution were cryopreserved. Tissue water was substituted with methanol and the tissues cryopreserved conventionally using dimethylsulfoxide, after which they were warmed and processed for light or electron microscopy. Selected specimens were vitrified to prevent ice crystal formation, cryopreserved, and subsequently warmed and processed for light and electron microscopy. RESULTS: Cryosubstitution of conventionally cryopreserved heart valves, while still frozen, demonstrated extensive extracellular ice formation, with smaller crystals in the ventricularis than in either the spongiosa or fibrosa. Extracellular ice formation was prevented by vitrification, a process in which the biological system is stabilized as an amorphous solid in the absence of crystalline ice. CONCLUSION: It is proposed that the extensive ice formation observed in conventionally cryopreserved heart valves may cause extracellular matrix damage that predisposes the valves to calcification. Future studies will assess the above hypothesis by comparison of conventional and ice-free (vitrification) cryopreservation methods in animal models of calcification.

Cardiac tissue iron: effects on post-ischemic function and free radical production, and its possible role during preconditioning.

We determined whether prior treatment of rats (study 1) with subthreshold doses of iron (no evidence of cardiac tissue overload), or in vitro ischemic pre-conditioning (IP: 5 min. Ischemia (I)/5 min. Reperfusion (R) x 2 cycles) of hearts from untreated rats (study 2), can modulate redox-active cardiac tissue iron levels or distribution, leading to alterations in post-ischemic lipid peroxidation-derived free radical (FR) production and severity of reperfusion injury. In study 1, rats received biweekly i.p. injections of 0 (saline=S), 3, 6, or 12 mg FeCl3/ml for 3-wks prior to imposing 30 min. I/15 min. R in vitro. The highest dose caused no elevations in plasma or heart tissue Fe levels, but did further reduce post-ischemic recoveries of left ventricular developed pressure (17% lower), cardiac work (57%) and output (54%), and increased effluent lipid hydroperoxides (2.1-fold) compared to the S-group. Post-ischemic FR production was assessed in toluene-extracted effluent by ESR spectroscopy and alpha-phenyl-N-tert butylnitrone (PBN=2.5 mM perfusate) spin trapping. PBN/alkoxyl (alphaH=1.90 G, alphaN=13.63 G) was the dominant signal detected in all groups; however, Fe-treated groups displayed significant dose-dependent increases in total alkoxyl content (3, 6, 12 mg/ml: 1.8-, 2.3-, 2.7-fold higher) compared to the S-group. These data suggest that even mild, non-overloading doses of iron can be functionally and oxidatively detrimental to hearts when an I/R stress is imposed. In study 2, isolated hearts from untreated rats were exposed to two-IP cycles: during IP, total effluent iron content (atomic absorption) increased 11.4-fold compared to control and analysis of cardiovascular tissue iron distribution (X-ray microanalysis) suggested that iron loss from capillary endothelium was far greater than from tissue myocytes. Moreover, iron-catalyzed production of alkoxyl radicals following severe I/R stress (40 min. I/15 min. R) was substantially lower (73%) in IP hearts compared to the non-IP counterparts. These preliminary findings suggest that cardioprotection resulting from IP may, in part, be related to IP-induced release of cardiovascular endothelial iron (redox-active) prior to imposing severe I/R stress.

1-Deamino-8-D-arginine vasopressin (DDAVP) increases platelet membrane expression of glycoprotein Ib in patients with disorders of platelet function and after cardiopulmonary bypass.

1-deamino-8-D-Arginine vasopressin (DDAVP) shortens the bleeding time in some patients with platelet dysfunction and decreases blood loss in some cardiopulmonary bypass patients. We studied platelet membrane glycoproteins in patients with von Willebrand disease (vWD), disorders of platelet function, and in cardiopulmonary bypass patients after infusion of 0.3 microgram/kg of DDAVP. Platelets from 8 cardiopulmonary bypass patients, receiving DDAVP immediately after surgery, were compared to those of 14 patients not receiving DDAVP. We also studied 12 patients with vWD, and 8 patients with platelet dysfunction receiving DDAVP. Fixed platelets, stained with monoclonal fluorescein (FITC)-labeled antibodies directed against GPIb (CD42b antigen), GPIb/IX, GPIIb/IIIa (CD41a antigen), CD63 antigen (a platelet activation protein), and P-selectin (CD62 antigen) were studied by flow cytometry. Binding of CD42b monoclonal antibody (MoAb) and anti-GPIb/IX to platelets from both groups of bypass patients increased during the 18-20 hr after surgery, but the group receiving DDAVP showed the greater increase (P = 0.032). Platelets from patients receiving DDAVP for vWD or for platelet dysfunction, had increases in CD42b MoAb and anti-GPIb/IX binding (P < 0.01) that coincided with shortening of their bleeding time. No changes were seen in binding of other antibodies. When platelets from normal donors were incubated with DDAVP for 20 hr, there were increases in platelet surface CD42b MoAb binding, while immunogold-stained transmission electron micrographs of permeabilized platelets demonstrated decreases in cytoplasmic CD42b MoAb binding. DDAVP increases platelet membrane GPIb expression in a variety of patients and may account for improvement in hemostasis seen in some studies. Redistribution of GPIb from the cytoplasm to the membrane may account for this increased expression.

Dietary fiber and intestinal adaptation: effects on lipid absorption and lymphatic transport in the rat.

Adult male rats fed defined diets containing various fiber supplements or cholestyramine for 4 wk were surgically provided with lymphatic drainage catheters and starved overnight. After duodenal administration of a standard lipid test emulsion, absorption rates and lipoprotein distributions of cholesterol and oleic acid were determined. Prefeeding diets containing cellulose or alfalfa had no significant effect on oleic acid absorption. Diets containing pectin, guar gum, metamucil, mixed fibers (Fibyrax), or cholestyramine caused decreased lymphatic recovery in the initial period; except for the metamucil diet, no decrease was caused in the 24-h recovery, suggesting delayed but not impaired absorption. Fatty acid distribution among lipoproteins and chylomicron size were not altered by diet. All supplements caused a significant reduction in cholesterol absorption during the initial period, and cholesterol absorption remained depressed in animals prefed pectin, guar gum, mixed fibers, metamucil, and cholestyramine.

Lipid accumulation in jejunal and colonic mucosa following chronic cholestyramine (Questran) feeding.

The hypolipidemic agent, cholestyramine (Questran), when fed to rats inhibits intestinal absorption of cholesterol and triglycerides and causes significant epithelial cell damage in both small and large intestine. In this study, we report significant accumulation of lipids in the mucosal layer of both jejunum and colon in rats administered 2% cholestyramine for a four-week period, when compared to a control group maintained on regular chow. The total lipid increment with cholestyramine was 4.7-fold in the jejunum and 3.7-fold in the colon. The triglyceride fraction increased substantially in the small but not the large intestine. Relative phospholipid levels decreased in the treated jejunum but not in the colon. The biochemical data were reflected in morphological evidence of lipid-laden enterocytes obtained by light and transmission electron microscopy. Since cholestyramine has been shown to sequester 99.8% of micellar phospholipid in vitro, it is concluded that the presence of cholestyramine in the intestinal lumen may interfere with phospholipid availability for chylomicron synthesis and serosal lipid exit from the epithelium. This unusual deposition of lipid within the mucosal layer may also be correlated with the known cocarcinogenic effect of this resin in experimentally induced intestinal cancer.

Interactions of alfalfa plant and sprout saponins with cholesterol in vitro and in cholesterol-fed rats.

The in vitro interactions of saponins from alfalfa plant and alfalfa sprouts with cholesterol and the effects of alfalfa plant and sprout and saponin-free alfalfa plant on diet-induced liver cholesterol accumulation, bile acid excretion, and jejunal and colonic morphology were examined. Cholesterol-saponin interactions have been suggested as mechanisms for the observed hypocholesterolemic effects of alfalfa as well as the changes in intestinal morphology. Alfalfa plant saponins bound significant quantities of cholesterol both from ethanol solution and from micellar suspension. Alfalfa sprout saponins interacted with cholesterol to a lesser but significant extent. Sprout saponins also inhibited growth of Trichoderma viride significantly, another measure of saponin-cholesterol interaction. Bile acid adsorption was greatest for alfalfa plant and was not reduced by removal of saponins from the plant material. The ability of alfalfa to reduce liver cholesterol accumulation in cholesterol-fed rats was enhanced by removal of saponins and alfalfa sprouts did not prevent accumulation. Removal of saponins from alfalfa reduced the changes in intestinal morphology previously reported, but interaction with membrane cholesterol did not appear to be the cause of this effect of saponins. Saponin-cholesterol interaction is an important part of the hypocholesterolemic action of alfalfa but interaction of bile acids with other components of alfalfa may be of equal importance.

Effect of chronic intake of dietary fibers on the ultrastructural topography of rat jejunum and colon: a scanning electron microscopy study.

This report is an attempt to quantitate the observable topographical characteristics of small and large intestine after a specific dietary regimen under well-defined states of lipid absorption and metabolism. Alfalfa, white wheat bran, cellulose, and pectin were fed for 6 wk at a level of 15 g/100 g diet to four groups of rats (12 rats per dietary group). A 5th control group was maintained on Purina Rat Chow. Three animals from each group were blind-coded for morphological assessment. After anesthesia, the jejunum and mid-colon were removed and processed for scanning electron microscopy. Beginning with the mildest mucosal surface changes, the observed order in terms of increasing severity is bran less than cellulose less than pectin less than alfalfa. Our observations suggest that altered rates of cell loss in intestinal tract cytokinetics may be occurring with particular feeding patterns and should be considered as a possible mechanism in the nutritional consequences of dietary fiber intake.

Structural-functional modulation of mucin secretory patterns in the gastrointestinal tract.

1. Mucus-producing cells are clearly identifiable sub-populations of cells in many of the epithelial transport model systems studied, i.e., gastric, jejunal, and colonic epithelia of the rat. 2. The distribution of these cells varies from a predominant location on the mucosal surface of the stomach to a regular degree of interspersion among the dominant columnar epithelial cell population in the small and large intestine. 3. The functional state of these cells can be assessed by a variety of morphological techniques and their biochemical synthetic and secretory capacity estimated by isotopic tracer procedures. 4. In the gastric or jejunal acute ulceration model, sulfomucin production is decreased by aspirin and markedly enhanced by Prostaglandin E1, providing additional evidence for the cytoprotective role of the mucin blanket. 5. With chronic feeding of the bile salt sequestrants which provoke colonic mucosal irritation and injury, there is also a stimulated mucin output. 6. Disruption of the cytoskeletal framework of the mucosal cells by Cytochalasin B or colchicine depresses mucin production. 7. Many of the compounds currently known to phenomenologically perturb mucus synthesis and/or secretion (Table 7) are utilized as probes in the delineation of electrolyte and water transport processes in these epithelial models. It is of interest to not in this volume that renal epithelial tissue do not appear to possess a significant mucin cell population. The developing concept of an association between mucus secretion and antigenic or immune responses may account for this fact. In several epithelial systems, however, modulation of mucin cell function should be taken into consideration in the construction of transport models.

Effect of bile salt-binding resins on the morphology of rat jejunum and colon. A scanning electron microscopy study.

One mechanism suggested to account for the hypocholesteremic effect of dietary fibers is their ability to sequester bile salts. Since bile salts have been found to alter intestinal structure, the morphological effects of several commonly used, xenobiotic, bile salt-binding agents was investigated. Wistar rats were fed a purified stock diet, ad libitum, for 6 weeks containing either 2% cholestyramine, 2% colestipol, or 2% DEAE-Sephadex. The bile salt-binding capacity of these substances was tested in vitro using taurocholate and glycocholate. The effect of in vivo feeding of the resins was to evoke ultrastructural topographical deviations from control appearance in both jejunal and colonic mucosae. Colonic cell injury was more severe than that observed in the jejunum of both colestipol- and DEAE-Sephadex-fed animals while the reverse was true for the rats fed cholestyramine. The degree of distortion in each condition was positively correlated with the extent of bile salt-binding capability in vitro. The rank order of both effects in terms of increasing severity was DEAE-Sephadex less than colestipol less than cholestyramine.