[Recellularization of tissue engineered vascular grafts in perfusion bioreactor].

Small diameter tissue engineered vascular grafts could be a potential solution to the shortage of vascular substitutes in reconstructive cardiovascular surgery. Previously, we have developed a decellularization method for human umbilical arteries, which could be used as a scaffold in vascular tissue engineering. Objective of the study was to optimize the recellelularization of decellularized scaffolds with mesenchymal stem cells. In the study, the possibility of cell growth on decellularized vessel has been shown. We also has proved that the use of perfusion-bioreactor improves the results of recellularization.

Endothelial nitric oxide synthase immunreactivity and the ultrastructure of endothelial cells of umbilical artery in normal and preeclamptic pregnancies.

Our objective was to investigate the endothelial nitric oxide synthase (eNOS) immuno-reactivity and the ultrastructure of endothelial cells of a human umbilical artery in both normal and preeclamptic pregnancies. The umbilical cords from normal and preeclamptic pregnancies were collected immediately after vaginal and abdominal deliveries. Umbilical arteries were isolated and fixed in 10% neutral formaline solution, embedded in paraffin, and then stained with hematoxylin and eosin (H&E) for the histologic investigation, and eNOS activation were examined in samples by streptavidine-biotine immunohistochemical methods. The arterial sections were also fixed in phosphate-buffered 2.5% glutaraldehyde solution (pH 7.2) for 3 h and post-fixed with 1% osmium tetroxide at 4°C for 2 h for the investigation of the ultrastructural examination. In the umbilical artery of preeclamptic pregnancies, endothelial cells were oval, triangular, or polygonal, and were disorganized. Some endothelial cells were separated by enlarged intercellular spaces. A dilated endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some endothelial cells displayed deep invaginations and irregular outlines. Most endothelial cells had a high number of cytoplasmic vacuoles. In preeclampsia, eNOS immunoreactivity increased considerably in endothelial cells when compared to normal pregnancies. We believe that preeclampsia plays an important role in the pathogenesis of endothelial cell dysfunction and activation in the umbilical artery. However, the disturbance mechanism of endothelial cells is not known, and further studies are necessary to clarify the exact mechanism.

Recellularization potential of small diameter vascular grafts derived from human umbilical artery.

BACKGROUND: The primary therapeutic strategy in cardiovascular disease is the coronary artery bypass surgery, which in- volves the use of small diameter vascular grafts (<6 mm). Human umbilical arteries could be used as a source for the development of these grafts. OBJECTIVE: The aim of this study was the decellularization of human umbilical arteries and the evaluation of their re- cellularization potential. METHODS: Decellularization of human umbilical arteries was performed with a detergent based protocol. Histological analysis was performed in order to determine the effect of decellularization. Then, recellularization was performed by using two different approaches. The first approach was the dynamic seeding of human umbilical arteries with Mesenchymal Stromal Cells and the second approach involved the recellularization by using a bioreactor system. RESULTS: Histological analysis showed the successful removal of cellular and nuclear materials from the umbilical arteries. In addition, successful recellularization of the vessels was observed with both approaches. CONCLUSION: The results of this study indicated that human umbilical arteries could serve as an alternative material for the proper development of small diameter vascular grafts.

Stem cells cardiac patch from decellularized umbilical artery improved heart function after myocardium infarction.

The construction of the high biocompatible biomaterials pretreated with MSC offers a promising strategy to improve the effects of stem cell therapy for the myocardial infarction (MI). However, assembling vascularized three-dimensional (3-D) myocardial tissues remains an enormous challenge. In this study, we optimized the decellularization protocol with the umbilical artery to construct microporous 3-D scaffold which is suitable for the stem cells (SC) proliferation. The SD rats underwent proximal left coronary ligation and a 5-mm diameter microporous SC patch was implanted directly on the infarct area (SC patch group). The LV contractile function, regional myocardial wall compliance, and tissue histology were assessed 4 weeks after patch implantation. The MSC patch integrated to the local heart tissue and the neo-vessels have been observed in the MSC patch. The vessels in the MSC patch were positive for the CD31 (marker for the mature endothelial cells). The left ventricle wall was thicker in the MSC patch group than the control group (p<0.05 vs. empty patch group). And the LVEF has been improved in the MSC patch group than empty patch group (59±6.7% vs. 31±4.5%, p<0.05). CONCLUSIONS: Our results showed that the implantation of the MSC patch improved cardiac contractile function in heart infarction rat model. The construction of artificial tissue from the decellularized umbilical artery and the MSC may open a promising perspective for the tissue therapy for MI.

Ultrastructural features of smooth muscle and endothelial cells of isolated isobaric human placental and maternal arteries.

The ability of a blood vessel to develop tone is dependent upon morphological parameters of the smooth muscle cells (SMC), including density, relationship with the endothelium and subcellular distribution of myofilaments and intracellular organelles. Consequently, wall ultrastructure of isolated human placental chorionic plate arteries (n=12), fixed when pressurised to mimic their in vivo geometry, was examined qualitatively using electron microscopy, and compared with maternal arteries (omental, n=10, myometrial, n=6). Arteries from women with uncomplicated pregnancy were tested for contractile viability before fixing, with some vessels post-fixed in osmium-ferricyanide for sarcoplasmic reticulum (SR) identification. In contrast to maternal arteries, placental arteries had no internal elastic lamina but exhibited considerable extracellular matrix separating circularly orientated SMC. Human SMC contained tightly packed arrays of myofilaments running parallel to the plasma membrane, enveloping cellular organelles. Synthetic SMC, with few myofilaments and much rough SR, were observed in placental arteries only. SR in SMC from maternal arteries was located centrally, often encircling mitochondria, and also near the plasma membrane associated with caveolae. Positive SR staining was rarely observed in SMC of placental arteries. This study highlights ultrastructural differences between placental and maternal arteries that may underlie specialised mechanisms of regulating vascular tone in the placenta.

Propagation and morphologic phenotypes of human umbilical cord artery endothelial cells.

Human umbilical cord artery endothelial cells can be propagated on fibronectin-coated dishes for approximately 50 cumulative population doublings in the presence of crude preparations of endothelial cell growth factor (ECGF) and serum. The cells were characterized by immunofluorescent staining and their ultrastructure. Different morphologic phenotypes could be demonstrated: closely attached cell monolayers, atypical cells, giant cells, tube-like structures. The formation of tube-like structures can be induced by proteolytic modification of fibronectin. Our data demonstrate that umbilical arteries may provide an excellent source for the routine serial cultivation of human arterial endothelial cells.

Binding of plasma von Willebrand factor by arterial microfibrils.

We developed an ELISA test to measure the binding of plasma von Willebrand factor (vWF) to arterial microfibrils and compared this interaction to the binding of vWF to collagen under the same conditions. We found that vWF binds to microfibrils in a similar manner as it binds to collagen: the binding was independent of the presence of cations, and temperature of incubation and was displaced by 1 M NaCl. Using purified 125I-vWF we showed that the binding was saturable and could be displaced by cold vWF in excess. Using immunoblotting we showed that vWF binds to a 97 kDa protein present in the microfibrils different from the 128 kDa thrombospondin-like structure (GP 128) which in microfibrils is known to interact with blood platelets. These results indicate that in the subendothelium, microfibrils bind plasma vWF and this reinforces the thrombogenic role of these structures.

Kinetics and some characteristics of uptake of noradrenaline by the human umbilical artery.

The uptake of exogenously added noradrenaline (NA) (0.5-2.5 mug/ml) by the human umbilical artery was linear with time up to 10 minutes. The uptake was saturable and could be described by the Michaelis-Menten equation. The uptake was cocaine-resistant, normetanephrine-sensitive, was considerably inhibited in the cold and was partially inhibited by Na+- deficiency. Of NA accumulated in the artery 31% could be washed out by NA-free medium. It is concluded that the mechanism of uptake of NA by the human umbilical artery is similar to the uptake2 mechanism.

Ultrastructure of human umbilical vessels: a possible role in amniotic fluid formation?

Human umbilical vessels obtained from neonates delivered at term after uneventful pregnancies were examined by light and transmission electron microscopy, with the aim of determining whether or not their structure is compatible with possible fluid exchange between the circulating blood and Wharton's jelly. A comparison of arteries and veins showed that although these vessels have common characteristics, they differ in some elements of their fine structure. The endothelium of both vessels appeared to be highly active metabolically. In the artery, the endothelial cells often protruded into the lumen. This aspect was related to the fine filaments concentrated in the basal part of the cells. This zone, free of organelles, was absent in the venous endothelium, but here pinocytotic vesicles and Weibel-Palade bodies were more abundant. The media included the same elements but was much thicker in the arteries than in the veins. There were two cellular types: typical myocytes and myofibroblasts rich in organelles. Their cytoplasmic processes extended into the interstitial space which was occupied by a material with a loose structure, that is, material containing a well-developed ground substance at the expense of the elastic and collagen fibres. The ultrastructural features of the umbilical vessels suggest an increased endothelial permeability, and it is suggested that transfer across the umbilical vessels may play a role in the formation of amniotic fluid.

Morphological changes in the umbilical arteries of babies born to pre-eclamptic mothers: an ultrastructural study.

The umbilical arteries of babies from ten normal pregnancies and from ten babies born to pre-eclamptic mothers were studied. Ultrastructurally, there were marked abnormalities of umbilical arterial morphology in the pre-eclamptic group. A loss of intercellular junctions, distension of the endoplasmic reticulum and an increase in cell organelles in the endothelial cells leading to a partial de-endothelialization was noted. Smooth muscle cells showed the morphological features of metabolic activation. These ultrastructural findings can be attributed to fetal hypoxia and to disturbances in cellular lipid metabolism; they suggest that there may be an abnormal fetal arterial blood flow in maternal pre-eclampsia.

Magnesium supplement in pregnancy-induced hypertension. A clinicopathological study.

The placenta and the umbilical cord obtained from 18 women with pregnancy-induced hypertension were investigated by light microscopy. The umbilical artery was studied by electron microscopy. 10 placentae and umbilical cords from normal pregnancies served as controls. The study was performed as a double-blind randomized controlled study in which 11 women were allocated to magnesium and 7 to placebo treatment. The treatment comprised a 48-hour intravenous magnesium/placebo infusion followed by daily oral magnesium/placebo intake until one day after delivery. Magnesium supplement increased birth weight and placental weight significantly. Light microscopic study of the placentae and the umbilical cord arteries showed no difference between the three groups concerning the occurrence of infarctions, cytotrophoblastic hyperplasia, vasculo-syncytial membranes, basement membrane thickening, stromal fibrosis or intervillous fibrin. Ultrastructurally, the endothelial cells of the umbilical arteries from women with pregnancy-induced hypertension showed a significant increase in the amount of dilated endoplasmic reticulum and basal laminae thickness when all 18 cases were compared with the controls. There was no significant difference when the magnesium group, the placebo group and the control group were compared separately. The present study suggests that magnesium supplement has a beneficial effect on fetal growth in pregnancy-induced hypertension. With regard to the light and electron microscopic changes we were unable to demonstrate any significant difference between the magnesium, placebo and control groups.

Organ culture of arteries for experimental studies of vascular endothelium in situ.

The objective of this study was to determine whether organ culture of arteries could be used as a more physiological model than endothelial cell culture for the study of vascular endothelium in vitro. Small pieces of artery from rat, pig, piglet and man were cultured in 24-well plates for up to seven or eight days to study the characteristics of the vascular endothelial cell layer during the first week of culture, in particular its integrity, viability and propensity for cell division. Using conventional and confocal microscopy, silver-stained endothelial cell boundaries were shown to be intact at all time points, up to and including day 7. However, occasional very small gaps between endothelial cells were seen with the scanning electron microscope under high power at day 7. Using the bromodeoxyuridine technique, no endothelial cell division was seen at day 4 in any species, except for the occasional endothelial cell in rat aorta. At day 7, pig, piglet and human arteries showed only very occasional dividing endothelial cells, but many endothelial cells had divided by day 7 in rat aorta. Viability of the endothelium was assessed using fluorochromes and examination of the endothelial layer en face using confocal microscopy. Viability was always excellent (> 95%) up to day 4. By day 7, occasional patches of dead cells could be seen, which were most obvious in rat aorta. This study demonstrates that endothelial cells can be studied in situ in organ culture with intact morphology, lack of cell division and excellent viability for a minimum of four days. For many research questions involving vascular endothelium--for example the pathophysiology of hyperacute rejection--short-term organ culture of vessels is likely to represent a more physiological model than endothelial cell culture.

Prostacyclin producing activity of human umbilical blood vessels in adrenergic innervated and non-innervated portions.

The present experiment was performed in order to clarify the significance of prostacyclin (PGI2) in the regulation of human umbilical blood flow. Distribution of adrenergic nerve fibers in umbilical cord was examined by means of a modification of the glyoxylic acid fluorescence histochemical technique. PGI2 producing activity in various portions of umbilical blood vessels was measured by platelet bioassay. Adrenergic nerve fibers were observed only in the region surrounding umbilical arteries at the fetal end of the cord. PGI2 producing activity of umbilical arteries was significantly lower in the innervated region than in the non-innervated region. There were no significant regional differences in umbilical vein which has no adrenergic innervation. The relationship between vascular PGI2 producing activity and adrenergic innervation, and the significance of PGI2 in the regulation of human umbilical blood flow are discussed.

Further studies on the presence of functional tissue factor activity on the subendothelium of normal human and rabbit arteries.

Although tissue factor (TF) activity has been observed on the subendothelial surface of rabbit aorta and human umbilical cord, immunofluorescent and in situ hybridization methods have failed repeatedly to demonstrate TF in the intima of human blood vessels. In the present study, TF activity on everted, de-endothelialized arteries was studied by two methods. One utilized a flow system and measured fibrin deposition and fibrinopeptide A formation. The other utilized a newly developed rotating probe system and measured the conversion of factor X to factor Xa in the presence of factor VIIa and Ca+2. The study attempted to control, or assess, the possibility that functional TF could have been exposed on the vessel surface by the procedures used to prepare the arterial segments. By both methods, TF activity was detected on the subendothelium of rabbit aortae and human umbilical arteries, and was unaffected by the length of storage or by inclusion of actinomycin D in the storage buffer. TF activity was also observed in the subendothelium of adult human ileo-colic, internal mammary, and renal arteries, studied by the rotating probe method. The latter may underestimate TF activity, as some of the factor Xa formed appears to bind to the subendothelial surface. TF activity (Xa formation) was detected on the luminal surface (subendothelium) of non-everted arteries, but increased activity was observed after eversion of the vessel. The source of the subendothelial TF, and its presence in normal subendothelium in vivo, requires further study. In addition, if any of the TF activity observed in this study was derived from injured endothelial or myointimal cells during preparation of the everted vessel segments, the techniques described could serve as a useful model for studying TF-induced thrombosis and factor Xa formation on injured blood vessels, and for evaluating the anti-thrombotic properties of TF-inhibitors.

Human umbilical artery for microvascular grafting. Experimental study in the rat.

As microvascular surgery has developed, the necessity for smaller arterial prostheses of less than 2 mm in internal diameter (ID) has increased markedly. Glutaraldehyde-stabilized human umbilical arteries with 1.5 to 2.0 mm ID are smaller than any other graft material currently available. This study was designed to determine whether this graft material has potential clinical application for microanastomosis. Twenty male albino rats, each weighing 400 to 500 gm, were used in this study. The abdominal aorta was exposed and a 3- to 4-mm segment was resected. A 10-mm interposition graft of glutaraldehyde-stabilized human umbilical artery was implanted by end-to-end anastomosis. The patency of the grafts was determined by repeated operative exploration at intervals of 1 week, and 1, 3, 9, and 12 months. Two grafts were found to be occluded each time at the 1-, 3-, and 9-month explorations. At each exploration time, five rats were sacrificed and histopathological studies conducted. All five remaining grafts were patent at 12 months. There was no evidence of endothelial proliferation on the luminal surface of the patent grafts as determined by scanning electron microscopy. The luminal surface and underlying region consisted of an amorphous proteinaceous-like material. Significant degeneration of the vessel wall was noted in all grafts surviving over 9 months. Central necrosis surrounded by a chronic inflammatory process that extended to and included the adventitia was observed in the occluded grafts. While the patency demonstrated was good, the tissue changes noted in the walls of the grafts indicate that further study is necessary before this material can be used as a graft in humans.

Umbilical vessels of spontaneously hypertensive rats.

Umbilical vessels of spontaneously hypertensive rats, control strain Wistar-Kyoto rats and rats treated with alpha-methyldopa were compared using the scanning electron microscope and light microscope. Observations with the scanning electron microscope revealed that the venous endothelial cells were relatively flat, giving the luminal surface of the vein a smooth appearance. The nuclear region of the fusiform arterial endothelial cells was responsible for the bumpy appearance of the luminal surface of the artery. Microvilli were a consistent feature of the endothelium in both umbilical vessels. There was no consistent pattern of distribution or density of microvilli within either vessel, but microvilli were more abundant on the luminal surface of the artery than in the vein. The luminal surface of some endothelial cells of the artery had long straight processes which crossed several cells before terminating. Light microscopic observations revealed that the endothelial cells and cells of the tunica intima and media contained an abundance of glycogen. The same layers stained sparsely for acid glycosaminoglycans. Maternal hypertension and treatment of spontaneously hypertensive rats with the antihypertensive drug, alpha-methyldopa, did not result in significant morphological alterations of either the endothelium or tunica media of the umbilical blood vessels.

Human umbilical cord cells for cardiovascular tissue engineering: a comparative study.

OBJECTIVE: Tissue engineering of viable, autologous cardiovascular replacements with the potential to grow, repair and remodel represents an attractive approach to overcome the shortcomings of available replacements for the repair of congenital cardiac defects. Currently, vascular myofibroblast cells represent an established cell source for cardiovascular tissue engineering. Cell isolation requires the invasive harvesting of venous or arterial vessel segments prior to scaffold seeding, a technique which may not be preferable, especially in pediatric patients. This study evaluates cells isolated from human umbilical cord artery, umbilical cord vein and whole cord as alternative autologous cell sources for cardiovascular tissue engineering. METHODS: Cells were isolated from human umbilical cord artery (UCA), umbilical cord vein (UCV), whole umbilical cord (UCC) and saphenous vein segments (VC), and were expanded in culture. All three expanded cell groups were seeded on bioabsorbable copolymer strips and grown in vitro for 28 days. Isolated cells were characterized by flow cytometry, histology, immunohistochemistry, proliferation assays and compared to VC. Morphological analysis of the seeded polymer strips included histology, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and uniaxial stress testing. RESULTS: UCA, UCV and UCC demonstrated excellent cell growth properties comparable to VC. Following isolation, all three cell groups showed myofibroblast-like morphology and characteristics by staining positive for alpha-smooth muscle actin (ASMA) and vimentin. Histology and immunohistochemistry of seeded polymers showed good tissue and extracellular matrix formation containing collagen I, III and elastin. TEM showed viable myofibroblasts and the deposition of collagen fibrils and progressive growing tissue formation, with a confluent surface, was observed in SEM. No difference was found among the mechanical properties of UCA, UCV, UCC and VC tissue engineered constructs. CONCLUSIONS: Tissue engineering of cardiovascular constructs by using UCA, UCV and UCC is feasible in an in vitro environment. Cell growth, morphology, characteristics and tissue formation were comparable between UCA, UCV, UCC and VC. UCC represent an attractive, readily available autologous cell source for cardiovascular tissue engineering offering the additional benefits of utilizing juvenile cells and avoiding the invasive harvesting of intact vascular structures.

Fetal Doppler shift waveforms in intrauterine growth retardation: laplace transform analysis technique.

An approach to the analysis of fetal blood flow velocity/time waveforms is described using a Doppler shift flowmeter. The waveform shape is described in terms of its Laplace transform. Variations in the value of the dominant coefficient in the Laplace transform in the descending thoracic aorta appear to distinguish growth retarded from normally grown fetuses early in pregnancy. Growth retardation was defined by an index of size specifically aimed at detecting the disproportionately grown fetus. Simpler methods of waveform shape description fail to detect the growth retarded fetus early in the second trimester.

Chromatin changes of endothelial cells in umbilical arteries in smokers.

Fine granular spots within the nucleus of endothelial cells have been demonstrated in umbilical arteries from newborns. These nuclear changes were predominantly found in vessels from newborns delivered by heavy smoking mothers (greater than or equal to 10 cigarettes per day) chi 2 = 8.28, p less than 0.005. Similar nuclear changes have until now only been reported in man in relation to tumors or other pathological conditions. The vessels showed signs of increased cellular turnover compatible with the findings in experimental atherosclerosis. The nuclear changes thus could be interpreted as an alteration of nuclear activity.

Fetal cardiovascular system as influenced by maternal smoking.

Primary prevention of atherosclerosis should be initiated early in life, preferably in childhood. Nevertheless, it tends to be forgotten that a child has existed for nine months before birth, and that the newborn child might already have been exposed to agents capable of causing vessel damage. Studies of the umbilical artery, umbilical vein, and vessels of the placental villi (2, 3, 4, 5, 6) revealed that, in this portion of the fetal cardiovascular system, severe damage to the vessel wall is associated with maternal tobacco smoking during pregnancy. These alterations within the fetal cardiovascular system were never found in the children of non-smoking mothers. If similar changes occur in the other vessels of the newborn child, which it seems reasonable to expect, they might give rise to sequelae later in life. It should therefore strongly be advocated that pregnant women abandon tobacco smoking.