Effect of fluid dynamics on decellularization efficacy and mechanical properties of blood vessels.

Decellularization of blood vessels is a promising approach to generate native biomaterials for replacement of diseased vessels. The decellularization process affects the mechanical properties of the vascular graft and thus can have a negative impact for in vivo functionality. The aim of this study was to determine how detergents under different fluid dynamics affects decellularization efficacy and mechanical properties of the vascular graft. We applied a protocol utilizing 1% TritonX, 1% Tributyl phosphate (TnBP) and DNase on porcine vena cava. The detergents were applied to the vessels under different conditions; static, agitation and perfusion with 3 different perfusion rates (25, 100 and 400 mL/min). The decellularized grafts were analyzed with histological, immunohistochemical and mechanical tests. We found that decellularization efficacy was equal in all groups, however the luminal ultrastructure of the static group showed remnant cell debris and the 400 mL/min perfusion group showed local damage and tearing of the luminal surface. The mechanical stiffness and maximum tensile strength were not influenced by the detergent application method. In conclusion, our results indicate that agitation or low-velocity perfusion with detergents are preferable methods for blood vessel decellularization.

Ultrastructural analysis and residual DNA evaluation of rabbit vein scaffold.

PURPOSE: To investigate the ultrastructural characteristics and analysis of residual DNA in scaffold models, produced with decellularized vena cava in an experimental model with rabbits. METHODS: Three groups were created for ultrastructural and residual DNA analysis: group 1 - control, consisting of samples of vena cava in natura; group 2 - SD, consisting of vein fragments submitted to 2% sodium deoxycholate decellularization by shaking (160rpm - Shaker News Brunswick Scientific®) for 1 hour at controlled temperature shaker at 37°C; group 3 - SDS, consisting of vein fragments submitted to 1% sodium dodecyl sulfate decellularization under the same previous condition, for 2 hours. RESULTS: The ultrastructural matrix of the blood vessel maintained its vintegrity after either decellularization models. The results of the two quantification methods demonstrated a significant decrease in the DNA content of the decellularized vena cava samples as compared to the control samples and, differed statistically from each other, p <0.05. CONCLUSION: The 2% DS protocol for vein decellularization, in this experimental model, was considered the best protocol because it presented less amount of residual DNA without causing substantial destruction of the extracellular matrix.

Ultrastructural analysis and residual DNA evaluation of rabbit vein scaffold

Abstract Purpose: To investigate the ultrastructural characteristics and analysis of residual DNA in scaffold models, produced with decellularized vena cava in an experimental model with rabbits. Methods: Three groups were created for ultrastructural and residual DNA analysis: group 1 - control, consisting of samples of vena cava in natura; group 2 - SD, consisting of vein fragments submitted to 2% sodium deoxycholate decellularization by shaking (160rpm - Shaker News Brunswick Scientific®) for 1 hour at controlled temperature shaker at 37°C; group 3 - SDS, consisting of vein fragments submitted to 1% sodium dodecyl sulfate decellularization under the same previous condition, for 2 hours. Results: The ultrastructural matrix of the blood vessel maintained its vintegrity after either decellularization models. The results of the two quantification methods demonstrated a significant decrease in the DNA content of the decellularized vena cava samples as compared to the control samples and, differed statistically from each other, p <0.05. Conclusion: The 2% DS protocol for vein decellularization, in this experimental model, was considered the best protocol because it presented less amount of residual DNA without causing substantial destruction of the extracellular matrix.

[HISTOGENESIS AND PECULIARITIES OF STRUCTURAL ORGANIZATION OF THE CARDIAC MUSCLE TISSUE IN THE WALLS OF HUMAN CAVAL AND PULMONARY VEINS].

To study the structural organization and histogenesis of the cardiac muscle tissue in the walls of human caval and pulmonary veins, the heart was examined in 3 human embryos (at weeks 6-7 of development) and 20 fetuses (at weeks 9-10, 16, 19, 22 and 24 of development), as well as segments of caval and pulmonary veins of adult men and women (n = 50) located at various distances from the heart. The methods of light and electron microscopy were used in this work. To obtain the isolated cells from the walls of caval and pulmonary veins, the method of tissue alkaline dissociation was used. An immunohistochemical study with the monoclonal antibodies against cardiac troponin T was performed. It was found that the cardiomyocytes in humans were located in the middle and outer tunics of caval and pulmonary veins, where they formed thick layers. In the pulmonary veins of the adult humans, cardiac muscle fibers did not reach the intrapulmonary areas, in the inferior vena cava their layer did not extend beyond the pericardium, in the superior vena cava, its length was 2.5-3.0 cm. The formation of the pulmonary vein orifices occured by sequential inclusion of the wall of the common pulmonary vein, and later--of the right and left pulmonary veins into the wall of the left atrium. During the formation of the orifices of the caval veins, the gradual inclusion of the wall of the venous sinus in the wall of the right atrium was observed, resulting in caval veins opening directly into the cavity of the right atrium. The veins studied were referred to the veins of the muscular type with the strong development of muscular elements containing the myocardial component.

Inhibitory effects of ethanol extract from Radix Ophiopogon japonicus on venous thrombosis linked with its endothelium-protective and anti-adhesive activities.

The in-vivo inhibitory effects of the ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) on venous thrombosis were studied in mouse and rat models and in-vitro endothelial cell-protective and anti-adhesive activities were observed in ECV304 cells injured by sodium dithionite and HL-60 adhesion to ECV304 cells injured by TNF-alpha. The in-vivo results showed that ROJ-ext significantly inhibited venous thrombosis induced by tight ligation of the inferior vena cava for 6 h in mice and for 24 h in rats by once oral administration at doses of 12.5 and 25 mg/kg. Meanwhile, ROJ-ext had no obvious effect on some coagulation parameters, which was different from warfarin, which remarkably prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) in rats at the same time. Histological analysis under light microscope and scanning electron microscope (SEM) of inferior vena cava indicated that ROJ-ext could protect endothelial cells from anoxic injury and alleviate inflammatory changes in the vein wall. On the other hand, the in-vitro studies approved that ROJ-ext significantly enhanced viability of ECV304 cells injured by sodium dithionite at the concentrations of 0.1, 1.0 and 10 mug/ml when given before and after the anoxic induction. Meanwhile, ROJ-ext remarkably inhibited adhesion of HL-60 cells to ECV304 cells injured by rh TNF-alpha at above concentrations in a dose-dependent manner. The findings of this study showed that ethanol extract of Radix Ophiopogon japonicus (ROJ-ext) inhibited venous thrombosis, which linked with its endothelial cell-protective and anti-adhesive activities. This lends scientific support to the therapeutic use of the plant for thrombotic diseases.

Relationship in the chick of the developing pulmonary vein to the embryonic systemic venous sinus.

Previous studies have shown that the relationship of the systemic venous sinus (sinus venosus) to the developing pulmonary vein are very similar in mice, rats, and man, with the pulmonary vein gaining access to the heart through a persisting segment of the dorsal mesocardium. It has been suggested that this process differs in avian development, with the pulmonary vein being connected to the systemic venous sinus with subsequent transfer to the left atrium. Here we have investigated the anatomical sequence of events in the chick, using serial histological sections and microdissection followed by scanning electron microscopy. We examined a temporal series of chick embryos, ranging from Hamburger and Hamilton stage 15 to stage 30. Although there are some differences in detail, the development of the pulmonary venous connections in the chick was found to be directly comparable to that already described in eutherian mammals. In both mammals and the chick, the dorsal mesocardial connection, which connects the primitive atrium to the posterior thoracic wall, forms a fixed point through which the pulmonary vein gains access to the atrial compartment of the heart, only varying if the connection itself is anomalous. The tributaries of the systemic venous sinus and the primary atrial septal structures develop around the dorsal connection.

Anatomy and development of the sinoatrial valves in the dogfish (Scyliorhinus canicula).

BACKGROUND: We describe the adult anatomy and the development of the cardiac sinoatrial valves in the dogfish (Scyliorhinus canicula). METHODS: We use scanning electron microscopy, histological and histochemical techniques in 39 hearts from embryos and adult specimens. RESULTS: The sinoatrial valvular set of the adult dogfish is composed of two transverse valves laterally attached to the sinoatrial junction at their bases. Both valves are composed of two muscular layers, the sinusal and the atrial, whose histological features are similar to the cardiac wall which they face. Collagen bundles, elastic fibers and fibroblasts are present between the muscular layers. The extracellular matrix between the valvular layers also contains sulphated and non-sulphated glycosaminoglycans. The sinoatrial valves develop from two lateral infoldings of the cardiac wall. The left fold is deeper than the right, causing a shift of the sinoatrial communication to the right. The epicardium progressively covers the outer sinoatrial groove and the space between the folds becomes populated by mesenchymal cells. The posterior atrioventricular endocardial cushion is in contact with the base of the left fold until the embryo has about 40 mm TL. CONCLUSIONS: The sinoatrial valves, in the dogfish, develop from lateral infoldings of the cardiac wall. This origin results in histological and histochemical differences between the two muscular layers which constitute the valves of the adult. The comparison of the sinoatrial valve morphogenesis between the dogfish and some higher vertebrates suggests that the right sinoatrial valve, but not the left, is homologous throughout the vertebrate phylogeny.

Cardiac musculature of the cranial vena cava in the common tree shrew (Tupaia glis).

Cardiac musculature of the cranial vena cava in the common tree shrew (Tupaia glis) was examined by light and transmission electron microscopy. The common tree shrew has well developed cardiac myocyte layers in the tunica media of the cranial vena cava, extending from the right atrium to the root of the subclavian vein. Because the common tree shrew belongs to a primitive group of mammals, the occurrence of cardiac musculature in the cranial vena cava may be a common feature in lower mammals. The development of this musculature indicates that active contraction of the cranial vena cava wall occurs in this species. Electron micrographs showed the typical ultrastructure of myocytes and nerve endings. These observations suggest that this musculature may serve as a regulatory pump for the return of venous blood to the right atrium and as a blood reservoir system under conditions of rapid heart rate. Additionally, the presence of atrial natriuretic polypeptide (ANP) was also demonstrated in the myocytes of the vena cava immunohistochemically. These findings show that the cardiac endocrine organ for ANP develops even in the principal veins including the cranial vena cava.

Cardiac musculature of the cranial vena cava in the rat.

The distribution and morphological features of cardiac musculature in rat cranial venae cavae were examined by light and transmission electron microscopy. Cardiac myocytes are encountered from the right atrium to the root of the subclavian vein. The musculature consists of several well-developed circular or spiral myocyte layers. The ultrastructure of myocytes in the cranial venae cavae exhibits a similar structure to that of atrial myocytes. Abundant myofibrils and mitochondria are detected within the cytoplasm of these myocytes, suggesting an active contraction of the musculature. Characteristic caveolae are accumulated near the sarcolemma of cardiac myocytes in the cranial venae cavae showing their high pinocytotic activity. Immunohistochemical analysis reveals the presence of an atrial natriuretic polypeptide-like substance in the cranial vena cava and the proximal portion of the subclavian vein. Ultrastructural studies also demonstrate the distribution of atrial granules within the musculature. This musculature in the vena cava may be considered part of the endocrine atrium.

Contributions of K+, Na+, and Cl- to the membrane potential of intact hamster vascular endothelial cells.

The transmembrane potential (Vm) of vascular endothelial cells (EC) is an important property that may be involved in intra- and intercellular signal transduction for various vascular functions. In this study, Vm of intact aortic and vena caval EC from hamsters were measured using conventional microelectrodes. Vascular strips with the luminal surface upwards were suffused in a tissue chamber with Krebs solution in physiological conditions. The resting Vm of aortic and vena caval EC was found to be -40 +/- 1 mV (n = 55) and -43 +/- 1 mV (n = 15), respectively. The Vm recordings were confirmed to have originated from EC by scanning and transmission electron microscopy combined with the comparison of electrical recordings between normal and endothelium-denuded aortic strips. The input resistance varied from 10-240 M omega, which implied the presence of electrical coupling between vascular EC. Elevating the K+ level in the suffusate from 4.7 mM to 50 and 100 mM depolarized aortic EC by 19% and 29% and vena caval EC by 18% and 29%, respectively. These low percentages indicated a relatively small contribution of [K+] to the resting Vm of vascular EC. A positive correlation (r > 0.69) between the resting Vm and the magnitude of depolarization by the high [K+]o may be related to the involvement of voltage-dependent K+ channels. The hyperpolarization caused by lowering both [Na+]o and [Cl-]o suggested the disengagement of some electrogenic transport systems in the membrane, such as a Na(+)-K(+)-Cl- cotransporter. The transference number (t(ion)), as an index of membrane conductance for specific ions, was calculated for K+ (15-20%), Na+ (16%), and Cl- (9-15%), demonstrating that both Na+ and Cl- as well as K+ contribute to the overall resting Vm. Our study documented some basic electrophysiology of the vascular EC when both structural and functional properties of the cell were maintained, thus furthering the understanding of the essential role of endothelial cells in mediating vascular functions.

Intermediate filaments and ATPase activity in the vascular wall of vertebrates.

The vascular wall of aorta and vena cava was examined for adenosine triphosphatase (ATPase) activity and cytoskeletal intermediate filaments (IF) in different representatives of vertebrates. Enzyme activity was studied by the modified method of Padykula and Herman. A streptavididin-biotin immunohistochemical method was applied to reveal desmin (D) and vimentin (V) IF. Endothelial cells of all vessels were V-positive and D-negative and exhibit high ATPase activity. Vascular smooth muscle cells (SMC) in lower vertebrates (pisces and amphibia) were also V-positive and D-negative, but showed low ATPase activity. SMC were D-positive and V-negative and possessed high enzyme activity in aves and mammals, similar to that of the endothelium. In cow vascular wall D-reactivity and high ATPase activity were mostly expressed in bundles of mosaically arranged thick SMC fibres of the outer aortic media as well as in the longitudinal fibres in the inferior vena cava. In higher vertebrates SMC of vasa vasorum were both V- and D-positive and showed high enzyme activity. The results demonstrate that D-immunoreactivity is mostly expressed in SMC of layers of high functional activity, which correlates with the intense ATPase reaction in these cells.

Atherosclerosis- and age-related multinucleated variant endothelial cells in primary culture from human aorta.

Endothelial cells were cultured from human aortas and inferior venae cavae of autopsied subjects ranging in age from infancy to 85 years. Endothelial cells in 32 of more than 100 attempted cultures were pure enough for evaluation. Emerged endothelial cells in primary culture were classified into two types: typical endothelium and variant endothelium. Typical endothelial cells were small, round to polygonal shaped, and were arranged uniformly. Their diameter ranged from 50 to 70 microns. Variant endothelial cells were larger, ranging from 100 to 200 microns in diameter, and giant endothelial cells measuring more than 250 microns in diameter were scattered among them. Variant endothelial cells were usually multinucleated and possessed endothelium-specific markers of vWF and Weibel-Palade bodies. No incorporation of [3H]thymidine was found in the nuclei of cultured variant endothelial cells. Although most cultured endothelial cells were of the typical type, variant endothelial cells were interspersed throughout the culture. The ratio of variant endothelial cells to typical cells correlated well with the severity of atherosclerosis, but less so with aging. The number of variant endothelial cells in cultures from inferior venae cavae was slight and constant throughout all age groups. The presence of multinucleated endothelial cells in in vivo aortas was confirmed by both scanning and transmission electron microscopy. They sometimes existed in colonies in the aortas from elderly subjects with intimal-thickened or advanced atherosclerotic lesions. These results indicate that variant endothelial cells were present in vivo and their ratio in primary culture reflected the in vivo population. It is likely that these cells were formed by adhesion of adjacent typical endothelial cells and that this process was affected more by atherosclerosis than by aging. Although it is not clear if the multinucleated variant cells were formed before the formation of atherosclerotic plaque or after the plaque formation, they will contribute to further development of atherosclerotic lesions, which in turn cause malfunction of the cell membrane. We suggest that there is a cyclic effect of these processes for multiplication of the variant endothelial cells and advancement of atherosclerotic lesions.

Cardiovascular complications of chronic catheterization of the jugular vein in the dog.

Cardiovascular changes associated with indwelling catheters were evaluated in 51 adult beagle dogs catheterized for 4 to 9 weeks. Pathologic changes consistent with traumatic injury were in the vena cava and endocardium of the right atrium of 88% of cannulated dogs. Lesions were characterized by surface denudation and diffuse intimal thickening due to myointimal hyperplasia and deposition of extracellular matrix. Affected intima was lined by hyperplastic, poorly differentiated endothelial cells and contained round to oval cells with characteristics of smooth muscle cells. After 9 weeks, thickened intima was vascularized and composed of spindle-shaped cells and fibrillar stroma. Intimal sclerosis and localized proliferative papillary projections in the vena cava cranial to areas of myointimal hyperplasia occurred infrequently. Traumatic lesions, regardless of location or severity, did not extend below the internal elastic membrane. Inflammatory cellular responses, when present, were minimal. The location, distribution, and morphogenesis of catheter-related cardiovascular lesions distinguishes them from those induced by chemical toxicity or pharmacotoxicity.

Vascular anastomoses with laser energy.

Laser-assisted arterial and venous anastomoses are now feasible. A microscope-guided CO2 laser was used to deliver 60 to 100 mW to anastomose end to end 44 rabbit carotid arteries (1.5 to 2.0 mm) and 27 rabbit vena cavae (4 to 6 mm). These were compared with control arteries repaired with interrupted suture technique. Anastomoses were examined from between 24 hours and 19 weeks. Laser carotid anastomoses yielded 93% patency (41 of 44) and 9% aneurysms (4 of 44), whereas hand-sewn carotid anastomoses produced 91% patency (40 of 44) and no aneurysms. In the vena cava, 26 of 27 laser anastomoses were patent (96%) compared with 19 of 20 (95%) sutured controls. Venous aneurysmal dilatation was seen in 2 of 27 laser (7%) and in 3 of 20 (15%) hand-sewn anastomoses. Histologic examination of laser-assisted anastomoses showed local full-thickness thermal injury. Repair was by fibroblast and myofibroblast proliferation, and luminal cell coverage was complete by 14 days in both laser and sutured repairs. Laser arterial and venous anastomoses are attractive because of their simplicity and rapidity of performance. Their patency is comparable to sutured anastomoses, but arterial aneurysms remain a hazard despite use of extremely low laser energy.

Experimental replacement of vena cava with expanded polytetrafluoroethylene graft.

Expanded polytetrafluoroethylene (Gore Tex) grafts were used for the replacement of the superior or inferior vena cava in dogs. When examined 2 weeks to 18 months after the replacement, the overall patency rate was 82%. If neither deformation nor narrowing of the graft on examination by cavagram had occurred within 14 days after implantation, the patency of the grafts remained unchanged. Ninety days after implantation, the formation of pseudointima was almost complete. However, since the grafts were not firmly anchored, superficial desquamation in their mid-portion continued for long periods. A composite graft made up of Gore Tex and noncrimped Dacron was studied in order to know the reason why high patency of the Gore Tex graft was obtained. The pseudointima of the Gore Tex graft gradually increased in thickness for 60 min, after which no further increase was observed. The pseudointima in the Dacron graft thickened markedly as time progressed and additional layers of blood cells accumulated on it. Although the ultimate fate of Gore Tex graft has not yet been determined, experimental trials indicate its suitability for patients requiring replacement of the vena cava.

Negative staining of whole cells: transmission electron microscopy of peripheral organelles in rat venous endothelial cells.

The study of whole negatively stained cells has revealed details of cellular organelles in rat venous endothelial cells. In particular, details of surface membrane organelles and small tubular structures were demonstrated. The surface membrane organelles which appeared "vesicular-like" were found to be connected with small tubular attachments. These findings were correlated with those described by other techniques. It is significant that this simple technique appears to permit the demonstration of fine details of three-dimensional cytoplasmic structures.