Generating an ex vivo vascular model

Realistic ex vivo anthropometric vascular environments are required for endovascular device optimization and for preclinical evaluation of interventional procedures. The objective of this research is to build an anthropomorphic model of the human carotid artery. The combination of magnetic resonance angiography image processing and computer-aided design and manufacturing techniques allowed fabrication of multicomponent morphologically precise casts of the carotid artery. The lost core technique was used to produce a hollow vessel prototype incorporating polyvinyl alcohol cryogel (PVA-C) as a tissue-mimicking vessel wall material. PVA-C was mechanically characterized by uniaxial tensile testing after different numbers of freeze/thaw cycles. The novel model construction approach outlined in this study accounts for the morphologic complexities of the human vasculature, and proved successful for the production of realistic compliant ex vivo arterial model.     

Antiatherogenic effect of grape flavonoids in an ex vivo model

The effects of grape phytoestrogens on cholesterol accumulation were studied in primary culture of human blood monocytes incubated with blood serum from postmenopausal women obtained before and 2, 4, and 6 h after single intake of plant components of grapes. Phytoestrogens from grape seeds, pressed out grapes, and fermented grape ridges prevent cholesterol accumulation in cells and can be regarded as prospective components for the development of natural preparations for the prevention of atherosclerosis in postmenopausal women. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 6, pp. 660–663, June, 2006     

Hematologic and biochemical effects of the Gradiflow in an ex vivo ovine model

The Gradiflow (Life Therapeutics, Frenchs Forest, Australia) system is a novel electrophoretic technique that uses the dual characteristics of size and charge to separate target macromolecules from complex biological solutions. The system has the potential to selectively remove a range of moieties from blood and plasma in an in vivo system such as hemodialysis or, alternatively, in an in vitro setting such as a blood bank. In this study, the safety of a scaled down Gradiflow prototype device with a membrane surface area of 16 cm2 was investigated using an ex vivo ovine model. Physiologic, hematologic, and biochemical parameters were assessed in 12 sheep: 6 animals treated using the Gradiflow and 6 controls. The effects of the Gradiflow procedure on both whole blood and plasma were analyzed. The Gradiflow procedure was well tolerated, and the application of an electrical potential or exposure to the Gradiflow membrane did not cause any significant changes in the parameters measured. Hemoglobin levels remained stable in all groups during the 4-hour experiment. An early neutropenia was observed in all groups, although this appeared to be more pronounced with exposure to a plasma filter; the presence of the Gradiflow component had no separate influence. One sheep in the plasma group experienced septic shock, caused by Staphylococcus contamination of the separation membrane. Overall, the results indicate that there were no gross physiologic, hematologic, or biochemical adverse reactions to the ex vivo Gradiflow procedure.     

Cervico-vaginal tissue ex vivo as a model to study early events in HIV-1 infection

Vaginal intercourse remains the most prevalent route of infection of women. In spite of many efforts, the detailed mechanisms of HIV-1 transmission in the female lower genital tract remain largely unknown. With all the obvious restrictions on studying these mechanisms in humans, their understanding depends on the development of adequate experimental models. Isolated cell cultures do not faithfully reproduce important aspects of cell-cell interactions in living tissues and tissue responses to pathogens. Explants and other types of ex vivo tissue models serve as a bridge between cell culture and tissues in vivo. Herein, we discuss various cervico-vaginal tissue models and their use in studying HIV vaginal transmission and consider future directions of such studies.     

HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model

Infection and dissemination of human immunodeficiency virus (HIV)-1 through the female body after vaginal intercourse depends on the activation/differentiation status of mucosal CD4 T cells. In this study, we investigated this status and the susceptibility to HIV-1 infection of human cervico-vaginal tissue ex vivo. We found that virtually all T cells are of the effector memory phenotype with broad CC chemokine receptor 5 (CCR5) expression. As it does in vivo, human cervico-vaginal tissue ex vivo preferentially supports the productive infection of R5 HIV-1 rather than that of X4 HIV-1 in spite of the broad expression of CXC chemokine receptor 4 (CXCR4). X4 HIV-1 replicated only in the few tissues that were enriched in CD27⁺CD28⁺ effector memory CD4 T cells. Productive infection of R5 HIV-1 occurred preferentially in activated CD38⁺CD4 T cells and was followed by a similar activation of HIV-1-uninfected (bystander) CD4 T cells that may amplify viral infection. These results provide new insights into the dependence of HIV-1 infection and dissemination on the activation/differentiation of cervico-vaginal lymphocytes.     

Porphyromonas gingivalis-induced inflammatory mediator profile in an ex vivo human whole blood model

Periodontitis is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. Leukocytes play a major role in the host response to Porphyromonas gingivalis, a major aetiological agent of chronic periodontitis. Secretion of high levels of inflammatory mediators, including cytokines and prostaglandins, by leucocytes is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of an ex vivo whole blood model to P. gingivalis stimulation. The production of interleukin-1 beta (IL-1beta), IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), IFN-gamma-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), Regulated on Activation Normal T cell Expressed and Secreted (RANTES) and prostaglandin E2 (PGE2) were quantified by enzyme-linked immunosorbent assays. P. gingivalis induced the secretion of the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and IFN-gamma, the chemokines IL-8, RANTES and MCP-1 and the inflammatory mediator PGE2 in an ex vivo human whole blood model. The secretion levels were dependent on the strain and the infectious dose used. While the mediator profiles were comparable between six healthy subjects, a high interindividual variability in the levels of secreted mediators was observed. This study supports the view that P. gingivalis, by inducing high levels of inflammatory mediators from a mixed leucocyte population, can contribute to the progression of periodontitis.     

Evaluation of the in vivo and ex vivo optical properties in a mouse ear model

Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, mu(a), scattering coefficients, mu(s), and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 degrees C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 degrees C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.     

3D thoracoscopic ultrasound volume measurement validation in an ex vivo and in vivo porcine model of lung tumours

The purpose of this study was to validate the accuracy and reliability of volume measurements obtained using three-dimensional (3D) thoracoscopic ultrasound (US) imaging. Artificial "tumours" were created by injecting a liquid agar mixture into spherical moulds of known volume. Once solidified, the "tumours" were implanted into the lung tissue in both a porcine lung sample ex vivo and a surgical porcine model in vivo. 3D US images were created by mechanically rotating the thoracoscopic ultrasound probe about its long axis while the transducer was maintained in close contact with the tissue. Volume measurements were made by one observer using the ultrasound images and a manual-radial segmentation technique and these were compared with the known volumes of the agar. In vitro measurements had average accuracy and precision of 4.76% and 1.77%, respectively; in vivo measurements had average accuracy and precision of 8.18% and 1.75%, respectively. The 3D thoracoscopic ultrasound can be used to accurately and reproducibly measure "tumour" volumes both in vivo and ex vivo.     

Effect of C1-INH on ischemia/reperfusion injury in a porcine limb ex vivo perfusion model

Revascularization of an amputated limb within 4–6 h is essential to avoid extensive ischemia/reperfusion (I/R) injury leading to vascular leakage, edema and tissue necrosis. I/R injury is a pathological inflammatory condition that occurs during reperfusion of an organ or tissue after prolonged ischemia. It is characterized by a complex crosstalk between endothelial cell activation and the activation of plasma cascades. Vasculoprotective pharmacological intervention to prevent I/R injury might be an option to prolong the time window between limb amputation and successful replantation. We used C1-easterase inhibitor (C1-INH) in this study because of its known inhibitory effects on the activation of the complement, coagulation and kinin cascades. Forelimbs of 8 large white pigs were amputated, subjected to ischemia, and then reperfused with autologous whole blood. All limbs were exposed to 9 h of cold ischemia at 4 °C. After 2 h of cold ischemia the limbs were either perfused with of C1-INH (1U/ml in hydroxyethyl starch, n = 8) or hydroxyethyl starch alone (n = 7). After completion of the 9-h ischemia period, all limbs were ex vivo perfused with heparinized autologous whole blood for 12 h using a pediatric heart lung machine to simulate in vivo revascularization. Our results show that I/R injury in the control group led to a significant elevation of tissue deposition of IgG and IgM, complement C3b/c, C5b-9 and MBL. Also, activation of the kinin system was significantly increased, namely bradykinin in plasma, and expression of bradykinin receptors 1 and 2 in tissue. In addition, markers for endothelial integrity like expression of CD31, VE-cadherin and heparan sulfate proteoglycans were decreased in reperfused tissue. Limb I/R injury also led to activation of the coagulation cascade with a significant elevation of fibrin and thrombin deposition and increased fibrinogen-like protein-2 expression. C1-INH treated limbs showed much less activation of plasma cascades and better protection of endothelial integrity compared to the reperfused control limbs. In conclusion, the use of the cytoprotective drug C1-INH significantly reduced I/R injury by protecting the vascular endothelium as well as the muscle tissue from deposition of immunoglobulins, complement and fibrin.     

Nitinol versus stainless steel stents: acute thrombogenicity study in an ex vivo porcine model

Acute and subacute stents thrombosis along with thrombus mediating neointimal proliferation within the stent struts remain major concerns in coronary stenting. Up to date, there is an obvious lack of data on the thrombogenicity of stent materials in physiological conditions. This study was performed to compare the relative thrombogenicity of nitinol versus stainless steel stents. Nitinol stents were laser cut to reproduce the exact geometry of the stainless steel Palmaz stents and tested in an ex vivo AV shunt porcine model under controlled conditions. Nitinol stents presented only small amounts of white and/or red thrombus principally located at the strut intersections while Palmaz stents clearly exhibited more thrombus. As a result, 125I-fibrin(ogen) adsorption and (111)I-platelets adhesion were significantly lower on nitinol than on stainless steel devices (36%, p = 0.03 for fibrin(ogen) and 63%, p = 0.01 for platelet). These results were confirmed by scanning electron observations showing different thrombus morphologies for nitinol and stainless steel. Along with the unique mechanical properties of nitinol, its promising haemocompatibility demonstrated in our study may promote their increasing use for both peripheral and coronary revascularization procedures.     

An ex vivo model for functional studies of myofibroblasts

Migration, proliferation and invasive growth of myofibroblasts are key cellular events during formation of granulation tissue in situations of wound healing, arteriosclerosis and tumor growth. To study the invasive phenotype of myofibroblasts, we established an assay where arterial tissue from chicken embryos was embedded in fibrin gels and stimulated with growth factors. Addition of serum, PDGF-BB and FGF-2, but not VEGF-A, resulted in an outgrowth of cellular sprouts with a pattern that was similar to the organization of cells invading a provisional matrix in an in vivo model of wound healing using the chicken chorioallantoic membrane. Sprouting cells were defined as myofibroblasts based on being alpha-smooth muscle actin-positive but desmin-negative. There was no contribution of endothelial cells in outgrowing sprouts. The acquired myofibroblastic phenotype was stable since sprout-derived cells resumed sprouting in a growth factor-independent manner when re-embedded as spheroids in a fibrin matrix. Invasive growth and sprouting of vascular smooth muscle cells was not limited to chicken cells since a similar response was seen when spheroids composed of purified primary human aortic smooth muscle cells were embedded in fibrin. Finally, a technique for flat visualization of the three-dimensional sprouting and a quantification method is described. This ex vivo model allows quantitative analysis of invasive growth and differentiation of vascular smooth muscle cells and fibroblasts into myofibroblasts.     

Atherosclerotic aneurysm in aortocoronary vein graft.

An atherosclerotic aneurysm was found in aortocoronary vein grafts of two patients who had a second revascularization because of reappearance of anginal symptoms five and six years, respectively, after the initial bypass operation.     

Comparison of lung preservation solutions in human lungs using an ex vivo lung perfusion experimental model

OBJECTIVE:

Experimental studies on lung preservation have always been performed using animal models. We present ex vivo lung perfusion as a new model for the study of lung preservation. Using human lungs instead of animal models may bring the results of experimental studies closer to what could be expected in clinical practice.

METHOD:

Brain-dead donors whose lungs had been declined by transplantation teams were used. The cases were randomized into two groups. In Group 1, Perfadex® was used for pulmonary preservation, and in Group 2, LPDnac, a solution manufactured in Brazil, was used. An ex vivo lung perfusion system was used, and the lungs were ventilated and perfused after 10 hours of cold ischemia. The extent of ischemic-reperfusion injury was measured using functional and histological parameters.

RESULTS:

After reperfusion, the mean oxygenation capacity was 405.3 mmHg in Group 1 and 406.0 mmHg in Group 2 (p = 0.98). The mean pulmonary vascular resistance values were 697.6 and 378.3 dyn·s·cm^-5, respectively (p = 0.035). The mean pulmonary compliance was 46.8 cm H₂O in Group 1 and 49.3 ml/cm H₂O in Group 2 (p = 0.816). The mean wet/dry weight ratios were 2.06 and 2.02, respectively (p = 0.87). The mean Lung Injury Scores for the biopsy performed after reperfusion were 4.37 and 4.37 in Groups 1 and 2, respectively (p = 1.0), and the apoptotic cell counts were 118.75/mm² and 137.50/mm², respectively (p = 0.71).

CONCLUSION:

The locally produced preservation solution proved to be as good as Perfadex®. The clinical use of LPDnac may reduce costs in our centers. Therefore, it is important to develop new models to study lung preservation.     

采用猪颈椎标本对滑动型与限制型颈椎前路固定板的生物力学试验研究

目的采用18 具猪颈椎骨标本进行生物力学测试,对比研究滑动型和限制型颈椎前路固定系统的临床生物力学特性的优劣。方法实验选用三种固定板,其中两种滑动型前路固定板(Swift 板,Premier板),一种限制型前路固定板(Peak 板)。使用 18 具猪颈椎骨(C2 - C7)模拟两阶段椎体切除,采用高度可调节的支撑植入椎体切除位置,模拟植骨(内置微型压力传感器)。每一种固定板均采用六具,分三种工况进行颈椎标本实验测试。施加的载荷控制在生理载荷范围内。结果在各标本运动极限内,限制型和滑动型固定板在全支撑时固定板上载荷无显著区别;在支撑缩短时,所有三种固定板上载荷均明显减少,而该现象在滑动型固定板表现的更明显。在缩短支撑情况下,滑动型固定板在缩短支撑状态下板上的载荷减少。就滑动型固定板而言,在支撑缩短的状态下,支撑上的载荷减少较少而板上的载荷增加也不大。在全长支撑的载荷极限状态下,Swift型固定板的载荷分配比Premier型的固定板更好一些。在载荷极限状态下,Premier型的固定板的载荷分配优于Swift型固定板。结论实验证明滑动型固定板在2阶段椎体次全切除术中的生物力学特性更好。     

Assessment of early docetaxel response in an experimental model of human breast cancer using DCE‐MRI,ex vivo HR MAS,and in vivo 1H MRS

The purpose of this study was to evaluate the use of dynamic contrast‐enhanced (DCE) MRI, in vivo ¹H MRS and ex vivo high resolution magic angle spinning (HR MAS) MRS of tissue samples as methods to detect early treatment effects of docetaxel in a breast cancer xenograft model (MCF‐7) in mice. MCF‐7 cells were implanted subcutaneously in athymic mice and treated with docetaxel (20, 30, and 40 mg/kg) or saline six weeks later. DCE‐MRI and in vivo ¹H MRS were performed on a 7 T MR system three days after treatment. The dynamic images were used as input for a two‐compartment model, yielding the vascular parameters K^trans and v_e. HR MAS MRS, histology, and immunohistochemical staining for proliferation (Ki‐67), apoptosis (M30 cytodeath), and vascular/endothelial cells (CD31) were performed on excised tumor tissue. Both in vivo spectra and HR MAS spectra were used as input for multivariate analysis (principal component analysis (PCA) and partial least squares regression analysis (PLS)) to compare controls to treated tumors. Tumor growth was suppressed in docetaxel‐treated mice compared to the controls. The anti‐tumor effect led to an increase in K^trans and v_e values in all the treated groups. Furthermore, in vivo MRS and HR MAS MRS revealed a significant decrease in choline metabolite levels for the treated groups, in accordance with reduced proliferative index as seen on Ki‐67 stained sections. In this study DCE‐MRI, in vivo MRS and ex vivo HR MAS MRS have been used to demonstrate that docetaxel treatment of a human breast cancer xenograft model results in changes in the vascular dynamics and metabolic profile of the tumors. This indicates that these MR methods could be used to monitor intra‐tumoral treatment effects. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of an ex vivo model to investigate the effects of altered haemodynamics on human bypass grafts

The insertion of vein grafts into the arterial circulation may contribute to vessel wall thickening and accelerated atherosclerosis, a common feature of late vein graft failure. We aimed to develop a model suitable for investigation of the effects of altered haemodynamics on human saphenous vein following its implantation into the arterial circulation. Segments of human saphenous vein obtained from patients undergoing coronary artery bypass surgery were sutured at each end to PTFE and placed into a flowsystem. Pressure and flowrates to stimulatethe arterial and venous systems were achieved. A theoretical model of the flowchamber was created and computational fluid dynamics software (FLOTRAN, Swanson Analysis Systems) was used to determine the flowprofile within the model. In summary, a flow model has been developed to investigate the effect of altered haemodynamics on the molecular and pathological changes that occur in vein grafts incorpor-ated into the arterial circulation.