Post-operative echocardiographic evaluation of bioprosthetic mitral valve implantation in sheep.

The ovine model is generally considered to be the best for testing bioprosthetic heart valve durability. Although echocardiography is the method of choice for the interim evaluation of the valve, literature on sheep echocardiography is scarce. Within the context of a study on treatment of pericardial heart valve prostheses, 19 adolescent sheep underwent transthoracic echocardiography six days after mitral implantation of bioprosthetic valves. Echocardiographic examination was performed under mild anesthesia and animals were put in a right lateral decubitus position. Four images were obtained: right parasternal long axis four and five chamber views, right parasternal long axis view with left ventricular outflow, and right parasternal short axis view through the mitral valve. We measured aortic annulus and velocity time integral over the aortic valve to determine stroke volume, cardiac output and cardiac index. The mitral valve was evaluated through color Doppler imaging for valvular and paravalvular leakages. Pulsed wave spectral Doppler was used for the measurement of velocities, pressures and velocity time integrals. For the evaluation of valve stenosis deceleration time and pressure half-time were determined. Effective orifice area of the mitral valve was derived. And, although not measured, other structures could clearly be visualized: right and left ventricle and atrium, wall thicknesses, tricuspid valve. This study shows that echocardiography in sheep is feasible, and that right parasternal images, obtained in animals in a right lateral decubitus position, are well qualified for the interim evaluation of bioprosthetic valves implanted in the mitral position. Besides the implanted valve, other cardiac structures like atria and ventricles can be visualized and evaluated.

Evolving Bioprosthetic Tissue Calcification Can Be Quantified Using Serial Multislice CT Scanning.

Background. We investigated the value of serial multislice CT scanning for in vivo determination of evolving tissue calcification in three separate experimental settings. Materials and Methods. Bioprosthetic valve tissue was implanted in three different conditions: (1) glutaraldehyde-fixed porcine stentless conduits in pulmonary position (n = 6); (2) glutaraldehyde-fixed stented pericardial valves in mitral position (n = 3); and (3) glutaraldehyde-fixed pericardial tissue as patch in the jugular vein and carotid artery (n = 16). Multislice CT scanning was performed at various time intervals. Results. In stentless conduits, the distribution of wall calcification can be reliably quantified with CT. After 20 weeks, the CT-determined mean calcium volume was 1831 ± 581 mm³, with a mean wall calcium content of 89.8 ± 44.4 µ g/mg (r (2) = 0.68). In stented pericardial valves implanted in mitral position, reliable determination of tissue mineralization is disturbed by scattering caused by the (continuously moving) alloy of the stent material. Pericardial patches in the neck vessels revealed progressive mineralization, with a significant increase in mean HU and calcium volume at 8 weeks after implantation, rising up to a level of 131.1 ± 39.6 mm³ (mean calcium volume score) and a mean calcium content of 19.1 ± 12.3 µ g/mg. Conclusion. The process of bioprosthetic tissue mineralization can be visualized and quantified in vivo using multislice CT scanning. This allows determination of the kinetics of tissue mineralization with intermediate in vivo evaluations.

Improved endothelialization and reduced thrombosis by coating a synthetic vascular graft with fibronectin and stem cell homing factor SDF-1α.

Failure of synthetic small-diameter vascular grafts is determined mainly by the lack of endothelial cells, as these cells inhibit thrombosis and intimal hyperplasia. Coating of graft material with homing factors for circulating stem cells has the potential to improve endogenous endothelialization of these grafts and to reduce graft failure. Synthetic knitted polyester grafts (6mm diameter) were coated with FN and SDF-1α before surgical interposition in the carotid artery of sheep. Similar uncoated vascular grafts were implanted in the contralateral side as internal controls. To study the early attraction of stem cells, grafts were implanted in a first series of nine sheep and explanted after 1 or 3 days. In coated grafts, four times higher fractions of CD34(+) and three to four times higher fractions of CD117(+) cells adhering to the vessel walls were found than in control grafts (P<0.05). When such coated and non-coated grafts were implanted in 12 other sheep and explanted after 3 months, all coated grafts were patent, while one control graft was occluded. EcNOS staining revealed that FN-SDF-1α coating significantly increased coverage with endothelial cells from 27 ± 4% of the graft to 48 ± 4% compared with the controls (P=0.001). This was associated with a significant reduction of intimal hyperplasia (average thickness 1.03 ± 0.09 mm in controls vs. 0.69 ± 0.04 mm in coated grafts; P=0.009) and significantly less adhesion of thrombotic material in the middle part of the graft (P=0.029). FN-SDF-1α coating of synthetic small-caliber vascular grafts stimulated the attraction of stem cells and was associated with improved endothelialization and reduced intimal hyperplasia and thrombosis.

Cyclically stretching developing tissue in vivo enhances mechanical strength and organization of vascular grafts.

Tissue-engineered vascular grafts must have qualities that rival native vasculature, specifically the ability to remodel, the expression of functional endothelial components and a dynamic and functional extracellular matrix (ECM) that resists the forces of the arterial circulation. We have developed a device that when inserted into the peritoneal cavity, attracts cells around a tubular scaffold to generate autologous arterial grafts. The device is capable of cyclically stretching (by means of a pulsatile pump) developing tissue to increase the mechanical strength of the graft. Pulsed (n=8) and unpulsed (n=8) devices were implanted for 10 days in Lovenaar sheep (n=8). Pulsation occurred for a period of 5-8 days before harvest. Thick unadhered autologous tissue with cells residing in a collagen ECM was produced in all devices. Collagen organization was greater in the circumferential direction of pulsed tissue. Immunohistochemical labelling revealed the hematopoietic origin of >90% cells and a significantly higher coexpression with vimentin in pulsed tissue. F-actin expression, mechanical failure strength and strain were also significantly increased by pulsation. Moreover, tissue could be grafted as carotid artery patches. This paper shows that unadhered tissue tubes with increased mechanical strength and differentiation in response to pulsation can be produced with every implant after a period of 10 days. However, these tissue tubes require a more fine-tuned exposure to pulsation to be suitable for use as vascular grafts.

Dehiscence of a valved conduit in the ascending aorta following low-velocity blunt chest trauma: case report.

We report a case of a 56-year old man presenting with dehiscence of a valved conduit in the ascending aorta following low-velocity blunt thoracic trauma. The patient had a history of a Bentall procedure in 1994. Two weeks before referral to our hospital, the patient fell during a bicycle ride and hit the handlebars of the bicycle with his chest. During the days following the accident, the patient developed progressively worsening fatigue, shortness of breath, and intolerance for even minor physical effort. The presence of an enlarged ascending aorta surrounding the implanted valved graft was confirmed, and the patient was referred to our department for surgical repair, after which the patient had an uneventful recovery and was discharged home on postoperative day 12.

Research on biological and mechanical heart valves: experimental studies in chronic animal models.

In the total pre-clinical evaluation of new heart valve substitutes, there is an absolute need for chronic experimental valve testing in different animal models. Also fundamental research towards mechanisms of calcification, tissue degeneration and valve thrombosis requires standardized and well-controlled animal models. Possible clinical use of a new experimental valve type and/or future developments and improvements in prosthetic heart valves, all depend on such research activities. Our recent studies concerning prosthetic heart valves resulted in following conclusions. 1. Photo-oxidation, a new tissue treatment, seems to have many possible advantages over currently existing valve fixation techniques (glutaraldehyde-fixation). The stentless porcine photo-fixed valve shows, in contrast to standard stentless valves, no aortic wall mineralization together with a good preservation of cuspal function. 2. For clinical right-sided valve implantations, the presence of a stent, together with the fixation pressure of the valve, can have its influence on the long-term behaviour of the valves in this low-pressure environment. Both stented valves, as valves fixed under pressure, seem to suffer more from fibrous tissue overgrowth 3. Mechanical stress is an important factor in the degeneration and calcification of biological valve tissue, mainly when an unfavourable stress pattern is present. Not all currently used animal models are equally reliable for valve testing and evaluation. 4. Implantation of aortic wall samples in the jugular vein of juvenile sheep is a simple, reliable and cost-effective model of aortic wall calcification. Calcification of glutaraldehyde-fixed aortic wall tissue is initiated at the level of cellular remnants, with little or no contribution from elastic fibers. Acellularization can avoid this cell-mediated calcification, but an additional treatment will be necessary to avoid the inflammation leading to elastolysis and consequent calcification of elastic fibers. 5. Mechanical valve implantation in pulmonary position delivers a reliable and reproducible test of mechanical valve thrombosis. The model allows us to compare the thrombogenic potential of different mechanical valve types, while it can also serve as a test for new therapeutic or diagnostic tools for mechanical valve thrombosis.

Dissociation of cardiomyocyte apoptosis and dedifferentiation in infarct border zones.

AIMS: Cardiomyocyte apoptosis is known to occur in infarct border zones, where cardiomyocyte dedifferentiation, as seen in hibernating myocardium, can also be observed. The aim of the study is to determine whether dedifferentiated cardiomyocytes represent a population of cells stably surviving or undergoing apoptosis. METHODS AND RESULTS: Microinfarctions were induced in sheep (n=8) by intracoronary injection of polymer macrobeads. The sheep were killed when cardiac function was gradually decreased (ejection fraction 37+/-6%, mean+/-SEM), but not earlier than 6 weeks after embolization. Transmural biopsies were taken from embolized and remote areas, based on flow measurements with positron emission tomography. Cells were classified as dedifferentiated when sarcomere content was depleted by >10% and glycogen content increased. Apoptosis was detected using the Tdt-mediated nick-end labelling (TUNEL) method and activated caspase-3 immunolabelling. Dedifferentiated cardiomyocytes were identified by morphology and by immunohistochemical evaluation of dedifferentiation related expression patterns of desmin, titin, cardiotin and alpha-smooth muscle actin. Cardiomyocyte apoptosis was detected in both the infarction border zones and remote areas. Dedifferentiated cardiomyocytes accounted for up to 30% of the cells in embolized areas and were almost exclusively non-apoptotic. CONCLUSION: In embolization induced microinfarcted tissue, dedifferentiated cardiomyocytes are preferentially spared to undergo apoptosis. It is hypothesized that dedifferentiated cardiomyocytes and apoptotic cardiomyocytes represent two different cell populations. The dedifferentiated cells can be considered as stable surviving cells.

Antimineralization treatments in stentless porcine bioprostheses: an experimental study.

BACKGROUND AND AIM OF THE STUDY: Photo-oxidation treatment of porcine stentless bioprostheses (Photofix) was compared with glutaraldehyde fixation, with either AOA (Freestyle valve) or Tween-80 (Edwards Prima Plus valve). METHODS: Six valves of each type were implanted in juvenile sheep, in the pulmonary position. Valves were explanted after three or six months and examined macroscopically, by X-radiography, and by light and transmission electron microscopy. Calcium content was determined by atomic absorption spectrometry. RESULTS: The cusps of all valves were free of calcification, and had normal histology and function. Calcium contents (median +/- IQR) were 0.63+/-0.45, 0.73+/-1.46 and 0.46+/-1.42 microg/mg for the Photofix, Freestyle and Prima Plus valves, respectively (p = NS). Calcium contents of the aortic wall portions were 0.71+/-1.27 (Photofix), 10.78+/-77.22 (Freestyle) and 28.70+/-66.53 (Prima Plus) (p <0.05 for Photofix versus Freestyle or Prima Plus). CONCLUSION: Photo-oxidation of a porcine stentless valve prevents calcification not only in the cusps, but also in the aortic wall portion.

Calcification characteristics of porcine stented valves in a juvenile sheep model.

BACKGROUND: Different antimineralization treatments of stented porcine bioprostheses were evaluated: ethanol (Epic), alpha-amino-oleic acid (AOA) (Mosaic), and sodium dodecyl sulfate (SDS) (Hancock II). A nontreated, glutaraldehyde-fixed valve (Labcor) served as control. METHODS: For each treatment, six valves were implanted in juvenile sheep in the pulmonary position. Valves were explanted after 3 and 6 months and examined macroscopically, by roentgenogram and light and transmission electron microscopy. Calcium content (microg/mg) was determined by atomic absorption spectrometry. RESULTS: The Labcor valves revealed small calcium deposits in the cusps, although calcium content remained low (median value 0.4+/-0.8 microg/mg). SDS did not prevent cusp calcification as assessed by histology and calcium content measurement, which was higher than in all other valves: 1.9+/-4.6 microg/mg (p < 0.05). Cusp retraction and rupture were occasionally found in the Hancock. The Mosaic and Epic valves showed no cusp calcification and had low calcium contents (0.3+/-2.4 microg/mg and 0.7+/-0.6 microg/mg, respectively). Epic showed less pannus formation, but had hematoma or iron staining in the cusps. CONCLUSIONS: SDS is inefficient as an antimineralization treatment, in contrast to ethanol or AOA. Cusp hematoma after ethanol treatment needs further investigation.

Influence of ischemic time and temperature on endothelial cell growth after transport.

BACKGROUND: The preparation of tissue-engineered material is a complex procedure. The possibility to transport tissue between laboratories without losing endothelial cell (EC) function was examined. METHODS: In 3 month old juvenile sheep (n = 6) a piece of vein (n = 14) was harvested and transported over 900 km to the tissue laboratory in Dulbecco's Modified Eagle's Medium (= DMEM). Vein material of each animal was transported at 4 degrees C (Group I, n = 6) and 25 degrees C (Group II, n = 8). EC growth potential was evaluated in function of the medium temperature and the ischemic time (between 8-24 hours). At the end of the first passage the EC of Group I and II were put together to save autologous serum of the sheep. After the 2nd passage the EC were cryopreserved at -80 degrees C to evaluate if EC viability would change. RESULTS: The growth potential of hypothermic Group I was equal in 16.7% (n = 1), higher in 33.3% (n = 2) and lower in 50% (n = 3) than Group II which had the same ischemic time during transport. Increase in ischemic time up to 24 hours showed no decrease of growth potential. Cryopreservation had no significant influence on EC viability. Viability at the end of the second passage, after recultivation and at the end of the third passage was 97.4% +/- 1.52, 95.5% +/- 1.34 and 94.5% +/- 1.08 respectively. CONCLUSIONS: In sheep there is no need to transport the EC at a temperature of 4 degrees C. Up to 24 hours growth potential and viability are maintained also at 25 degrees C.

Influence of design and fixation pressure on fibrous sheathing in right-sided porcine aortic bioprostheses.

Valve dysfunction in right side implanted bioprostheses can be caused by fibrous sheathing and cusp retraction. A scoring system was employed to grade the macroscopic appearance of the cusp retraction. Stentless porcine aortic valves exhibit less cusp retraction when implanted in right-sided position than stented porcine aortic valves.

Influence of species, environmental factors, and tissue cellularity on calcification of porcine aortic wall tissue.

Valve tissue calcification has complex host, implant, and mechanical determinants. We studied the influence of species (rat v sheep), environmental factors (presence v absence of blood contact and arterial stress), and tissue cellularity (normal v acellularized tissue) on porcine aortic wall mineralization. Porcine aortic wall samples underwent standard glutaraldehyde-fixation or combined enzyme-detergent acellularization. Samples were implanted subcutaneously in rats (n = 8) and in juvenile sheep (n = 8). Furthermore, in juvenile sheep, similar samples were implanted into the jugular vein (blood contact) and into the carotid artery (blood contact and arterial stress). After 8 and 12 weeks, tissue was explanted and evaluated by X-ray, light- and electron-microscopy, and calcium content measurement (atomic absorption spectrometry). On the Von Kossa staining, auto-fluorescence of elastic fibers was used to identify the relation between calcific deposits and elastin. Subcutaneously implanted, glutaraldehyde-fixed tissue calcified severely in rat, but much less in sheep (calcium content: 56.2 +/- 13.6 v 9.9 +/- 9.0 microg/mg, respectively; P <.001). In sheep, the presence of blood contact (venous implants) increased wall calcification significantly (36.9 +/- 15.8; P <.001), but hemodynamic stress (arterial implants) had no additional mineralizing effect on the aortic wall (P >.05 v venous implants). Calcification of glutaraldehyde-fixed tissue occurred predominantly at the level of cells and cellular remnants, as confirmed by electron- and fluorescence-microscopy, locating calcific deposits in between elastic fibers. Acellularized tissue calcified significantly less, but an inflammatory response towards the tissue led to fragmentation, lysis, and subsequent calcification of elastic fibers. Results from subcutaneous implantations show large inconsistencies in calcification between the species. In sheep, blood contact increases aortic wall calcification significantly, while arterial stress has no additional effect. The sheep-jugular implantation model can be used as a simplified model for further study of aortic wall calcification and new antimineralization treatments. Calcification of glutaraldehyde-fixed aortic wall tissue is initiated at the level of cellular remnants, with little or no contribution from elastic fibers. Acellularization can avoid this cell-mediated calcification, but an additional treatment (glutaraldehyde, cryopreservation, photo-fixation,.) will be necessary to avoid the inflammation leading to elastolysis and consequent calcification of elastic fibers.

Observational Study of Travelers' Diarrhea.

Background: European air travelers returning from Algeria, Egypt, Mexico, Morocco, and Tunisia were interviewed about their experience of travelers' diseases upon arrival in Brussels. Diarrhea was mentioned by 37% of the adults and 27% of the children. These subjects were questioned about the types of measures taken, type and duration of drug treatment (if any), and about duration of diarrhea and side effects experienced. Methods: Final analysis was performed based on 2160 interviews. The largest proportion of diarrhea was reported in the age group 15-24 years (46%). Results: The majority of the 2160 subjects had opted for drug treatment (81%): 927 subjects for loperamide alone, 235 for loperamide in combination with nifuroxazide, and 178 for nifuroxazide alone. Other drugs had been used less frequently. The median time to recovery was 2.4 days with loperamide compared to 3.2 days with nifuroxazide and to 3.4 days for the no-treatment group. Conclusions: A stratification of the results by severity of the diarrhea suggests a rank of antidiarrheal potency as follows: loperamide > nifuroxazide > no-drug treatment. The side effect with the highest incidence was constipation (2.4% with loperamide). (J Travel Med 2:11-15, 1995) Travelers' diarrhea is usually defined as the passage of at least three unformed stools per day or any number of such stools when accompanied by fever, abdominal cramping, or vomiting. The definition may be broadened to include more trivial bowel disturbance.1,2 The duration of this self-limited disease generally is 3 to 5 days. Medical intervention aims at shortening the duration of disease, thus allowing the sufferer to resume his or her usual activities at an early stage. A shortened period of recovery to physical well-being has obvious favorable economic implications if the traveler is on business and may help the maintenance of a desired level of quality of life while a traveler is on holiday. An observational study of various medical complaints made by European travelers about their stay in areas outside Europe (Algeria, Egypt, Mexico, Morocco, and Tunisia) was conducted. Air travelers returning from these areas between July 15 and August 16, 1992, were interviewed upon arrival at Brussels airport by means of a standardized questionnaire written up in lay language. As shown in Table 1, the total number of complaints in the adult group (>= 15 years of age, n = 5373) was 4919 and 446 in the pediatric group (n = 818). With fever as an exception, there were fewer complaints in children. Only approximately 50% of the travelers did not suffer