F Dijk

VACUUM CELL SEEDING - A NEW METHOD FOR THE FAST APPLICATION OF AN EVENLY DISTRIBUTED CELL LAYER ON POROUS VASCULAR GRAFTS

The study was to develop a method to induce rapid endothelial coverage of vascular prostheses by cell seeding. The method uses vacuum pressure and is therefore called vacuum cell seeding. A special seeding device was constructed, in which grafts of different length and/or inner diameter could be positioned. Microporosity of the grafts was a prerequisite for this method. Two types of commercially available microporous grafts were tested. The ePTFE graft routinely used clinically needed pretreatment to enable the method, whilst a polyurethane-based graft could be seeded as received. Vacuum cell seeding applied cells from a suspension in culture medium within 10 min in an evenly distributed cell layer on to the luminal graft surface. The adhering cells immediately started flattening, thereby completely covering the luminal surface. It was concluded that the vacuum cell seeding method rapidly introduced a confluent layer of seeded cells on porous vascular grafts in a simple way, which in the clinical setting could easily be performed on the operating table.

Regeneration of the arterial wall in microporous, compliant, biodegradable vascular grafts after implantation into the rat abdominal aorta

SummaryThe ultrastructure of a new type of vascular graft, prepared from a mixture of polyurethane (95 weight %) and poly-L-lactic acid (5 weight %), was examined six weeks after implantation into the abdominal aorta of rats. These microporous, compliant, biodegradable, vascular grafts function as temporary scaffolds for the regeneration of the arterial wall.Smooth muscle cells, covering the grafts, regenerated a neo-media underneath an almost completely regenerated endothelial layer (neo-intima). These smooth muscle cells varied in morphology from normal smooth muscle cells to myofibroblasts. They were surrounded by elastic laminae and collagen fibers.Macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries were present in the disintegrating graft lattices. The epithelioid cells and multinucleated giant cells engulfed polymer particles of the disintegrating grafts.The regeneration of the endothelial and smooth muscle cells is similar to the natural response of arterial tissue upon injury. The presence of macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries in the graft lattices resembles the natural response of tissue against foreign body implants. Both of these responses result in the formation of a neo-artery that possesses sufficient strength, compliance and thromboresistance to function as a small caliber arterial substitute.

Biofilm formation on voice prostheses: influence of dairy products in vitro.

Laryngectomized patients use silicone rubber voice prostheses to regain their speech; however, the lifetime of these devices is limited due to biofilm formation. Following anecdotal evidence, the influence of various dairy products on biofilm formation on voice prostheses was studied, using the artificial throat-model. Biofilms were grown on Groningen and Provox®2 voice prostheses by inoculating two artificial throats with the total microflora isolated from an explanted Groningen voice prosthesis. After 3 days, one throat was perfused three times daily with 650 ml dairy product; the other was perfused with phosphate buffered saline, used as a control. After 12 days the microflora on each voice prosthesis was determined. Perfusion of the artificial throat with buttermilk three times daily for 9 days reduced the amount of bacteria and yeasts in the biofilm on Groningen voice prostheses to 3% and 15% of the control, respectively. These effects were not observed with a pasteurized conservable buttermilk product. Yakult fermented milk drink, Mona mild yoghurt, Mona vifit yoghurt, semi-skimmed milk and low-fat yoghurt reduced the amount of bacteria by various degrees, ranging from 12% (Yakult) to 88% (Mona mild) of the control, but these products did not inhibit, and sometimes even stimulated, yeast growth. A combination of buttermilk and Yakult did not show a synergistic effect, as expected. Effects for the Provox®2 voice prosthesis were less pronounced. These in vitro experiments in the artificial throat demonstrated that the formation of the biofilm on voice prostheses can be lessened by the daily use of certain dairy products, of which buttermilk had the strongest inhibitory effect, followed by Yakult.Laryngectomized patients use silicone rubber voice prostheses to regain their speech, however, the lifetime of these devices is limited due to biofilm formation. Following anecdotal evidence, the influence of various dairy products on biofilm formation on voice prostheses was studied, using the artificial throat-model. Biofilms were grown on Groningen and Provox2 voice prostheses by inoculating two artificial throats with the total microflora isolated from an explanted Groningen voice prosthesis. After 3 days, one throat was perfused three times daily with 650 ml dairy product; the other was perfused with phosphate buffered saline, used as a control. After 12 days the microflora on each voice prosthesis was determined. Perfusion of the artificial throat with buttermilk three times daily for 9 days reduced the amount of bacteria and yeasts in the biofilm on Groningen voice prostheses to 3% and 15% of the control, respectively. These effects were not observed with a pasteurized conservable buttermilk product. Yakult fermented milk drink, Mona mild yoghurt, Mona vifit yoghurt, semi-skimmed milk and low-fat yoghurt reduced the amount of bacteria by various degrees, ranging from 12% (Yakult) to 88% (Mona mild) of the control, but these products did not inhibit, and sometimes even stimulated, yeast growth. A combination of buttermilk and Yakult did not show a synergistic effect, as expected. Effects for the Provox2 voice prosthesis were less pronounced. These in vitro experiments in the artificial throat demonstrated that the formation of the biofilm on voice prostheses can be lessened by the daily use of certain dairy products, of which buttermilk had the strongest inhibitory effect, followed by Yakult.

(Electron) microscopic observations on tissue integration of collagen-immobilized polyurethane.

The foreign body reactions to collagen-immobilized polyurethane (PU-CI) films during subcutaneous implantation in rats were characterized. The underlying concept is that collagen-immobilization will improve the tissue integration. Since the method of collagen-immobilization involves the covalent coupling of collagen to an acrylic acid (AA) based surface graft, both non-modified PU and PU-AA were used as controls. Bare PU has a flat surface, whereas both PU-AA and PU-CI displayed a slightly roughened surface. Implantation showed that PU-CI induced early after implantation a far more intense foreign body reaction than PU and PU-AA. This reaction consisted of increased presence of fibrin, granulocytes and macrophages. Roughening of the surface as with PU-AA induced only a small increase in fibrin formation and cellular migration. At day 5 the reaction to PU-CI had slowed down; giant cell formation now slowly started but was decreased compared to PU and PU-AA. At day 10 capsules around each type of material looked similar, but in contrast to PU. PU-CI films could no longer be dissected from their capsules. Only at week 3 this also occurred with PU, at which time point again similar capsules with the three materials were observed. At week 6, of the three materials PU-CI showed the thinnest capsule with most immediate adherence of connective tissue. These results show that collagen-immobilization of PU increased the early tissue reaction and therefore the tissue integration. The thin capsule observed at 6 weeks may be beneficial in e.g. infectious circumstances, when easy access for immune reactions is needed. This, and the long-term performance of PU-CI will be a matter of future investigations.

Early hypergravity exposure effects calbindin-D28k and inositol-3-phosphate expression in Purkinje cells

In this study the effects of hypergravity were analyzed on cerebellar Purkinje cells during early development in rats. The cerebellum is a key structure in the control and the adaptation of posture and anti-gravity activities. This holds particularly when external conditions are modified. Three groups of rats were conceived, born and reared in hypergravity (2g). At postnatal day 5 (P5), P10 or P15, they were exposed to normal gravity and at P40, the cerebella were investigated on the expression of calbindin-D28k and inositol-3-phosphate (IP3) in Purkinje cells. Control animals were bred in the same conditions but at 1g. Immunoreactivity of Purkinje cells was studied in lobules III and IX of the vermis. Lobule IX of the vermis is one of the targets of primary otolithic vestibular projections, and lobule III served as a control, being much less related with vestibular inputs. The results show that hypergravity induces a decrease in calbindin and IP3 labeling in 20% of Purkinje cells of lobule IX without any change in lobule III. Animals transferred from 2g to 1g at P5 or P10 showed the most pronounced effects and much less at P15. This study demonstrates that early development of the cerebellum is highly sensitive to changes in gravity. Ages until P10 are critical for the development of vestibulo-cerebellar connections, and in particularly the calcium signaling in Purkinje cells.

Scanning electron microscopic analysis of endothelial cell coverage and quality in large vessels from multi-organ donors: effects of preservation on endothelial cell integrity.

Endothelial cell integrity (coverage and quality) of large donor vessels is important because these vessels are used for vascular reconstructions in solid‐organ transplantation. Disruption of the endothelial cell monolayer will initiate blood coagulation and may lead to thrombosis of large vessels, often resulting in the loss of the transplanted organ. Iliac arteries and veins, removed from 10 heart‐beating multi‐organ donors at the end of the donor procedure, were analyzed using scanning electron microscopy at three different time points of preservation. Endothelial cell coverage and quality were determined immediately after removal from the donor, after 10 h (time of transplantation) and 7 d storage in ‘University of Wisconsin’ cold preservation solution (UW). Endothelial cell coverage decreased during the preservation of arteries, but was maintained in veins. Storage of the veins for 7 d in plastic bags showed a decreased endothelial cell coverage compared to storage in glass vials. Early removal of the blood vessels and proper storage, free floating and in clean UW, may improve maintenance of the endothelial cell integrity. These findings may be important in order to reduce the risk of thrombosis and, consequently, organ failure after transplantation. Furthermore, vessels with maintained endothelial cell integrity after 7 d may be used for in vitro research.

Morphology of donor lens-capsule material studied by SEM

The lens capsule of a 70-year-old male donor with a cataractous lens was carefully prepared for SEM by first washing the capsule with buffer solution to remove lens-fibres and subsequently attaching it to silicon rubber. During the fixation and drying stages of the preparation procedure the capsule stayed attached to the rubber substratum. In the equatorial zone germinating cells were found with knob-shaped microvilli, closely connected to lensfibres. Large units of pathological capsule epithelial cells were found, only slightly inter connected by a few pseudoposia. In addition, single pathological epithelial cells with pseudopodia, arranged on top of the cell in a rosette-like configuration, were found at certain locations. Both types are probably related to the original lens-cataract.

Secondary cataract material collected with a glass cannula

Secondary cataract material from three patients, collected with a glass cannula approx. 18, 24 and 30 months respectively after operation, was prepared for SEM examination. For the soft samples this was done by filtration through a millipore filter followed by fixation and drying. The more solid material was suspended in a fixation solution, followed by centrifuging, suspension in 70% ethanol and drying on a specimen-holder. The short residence samples (18 months) showed mainly erythrocytes, some (inflammatory) cells and degenerated lens-fibre material. Most of the more solid material, which was collected more than 20 months after operation, showed fragments of (regenerated) capsule epithelium and pieces of solid lens-fibre material with fragments of capsule epithelium attached.