[Show abstract][Hide abstract]ABSTRACT: We determined the possible effects of age, antimineralization treatments, circulatory implant conditions, prosthesis design, and valve-related structural aspects on valve calcification in adolescent sheep.
Calcium content was measured by means of atomic absorption spectrometry in bioprostheses implanted in 120 sheep (age <1 year) for a period of 3 or 6 months.
Bioprostheses calcified significantly in adolescent sheep, but the extent of calcification was multifactorial. Multivariate analysis of the calcium content reveals that age, mitral or pulmonary implant position, prosthesis design (stented or stentless), structure (porcine or pericardial, wall portion or cusp), and antimineralization treatment are independent factors influencing calcification; implant duration beyond 3 months was not. In juvenile sheep (age 5 months) the wall portion, as well as the cusps of the prosthesis, calcified significantly more than in adolescent sheep (age 11 months). Irrespective of age, the cusps of valves implanted in the mitral position calcified more than those in the pulmonary position. The wall portion of stentless valves calcified more than that of stented valves, and pericardial valves calcified less than porcine valves. The surfactant (Tween 80, No-React, and alpha-amino-oleic acid) and alcohol (ethanol and octanediol) treatment significantly reduced cusp calcification; sodium dodecylsulfate did not. None of the anticalcification treatments was able to prevent wall calcification in stentless porcine valves.
These findings suggest that tissue valve calcification is determined by many independent factors, which can be identified by using adolescent sheep as a preclinical in vivo model.
Full-text · Article · Aug 2006 · The Journal of thoracic and cardiovascular surgery
[Show abstract][Hide abstract]ABSTRACT: In gene expression studies, endogenous controls that are constitutively expressed (housekeeping genes) are commonly used to normalize for variations in cDNA synthesis efficiency. In the present study, a frequently used control gene, beta-actin, was examined in ovine heart valves to evaluate its applicability as a housekeeping gene for this tissue.
Interstitial cells (IC) of the four heart valves were isolated using the outgrowth explant method. Cells were cultured under different serum conditions (10% or 20% fetal bovine serum or 20% sheep serum) up to passage (P) 5. mRNA from fresh tissue and from cells at P0 and P5 was isolated, and expression of beta-actin determined using reverse transcription-polymerase chain reaction (RT-PCR). An identical control sample was used for each PCR and each gel electrophoresis. Data were expressed as a relative value of this control sample.
beta-Actin expression in the aortic valve was significantly lower than in other valves. The mRNA level of beta-actin was four-fold lower in freshly isolated IC than in cultured IC. Once up-regulated by in-vitro culturing conditions, beta-actin expression did not change from P0 to P5. An important increase in the variation of beta-actin expression was observed in cultured cells as compared to fresh cells. Different serum conditions did not lead to different beta-actin levels.
Due to the variation in expression, beta-actin cannot be used as a reference for gene expression of ovine-derived heart valve IC in culture.
Full-text · Article · Oct 2004 · The Journal of heart valve disease
[Show abstract][Hide abstract]ABSTRACT: At present the involvement of cardiac valve interstitial cells (VICs) in growth, repair, and tissue engineering is understudied. Therefore, this study aims at characterizing ovine VICs in order to provide a solid base for tissue engineering of heart valves. Ovine ICs of the four heart valves were isolated by the explant outgrowth method and expanded in vitro up to passage 5. Vimentin and collagen I gene expression from freshly isolated or cultured ICs was measured by reverse transcriptase-polymerase chain reaction. Immunocytochemical stainings of vimentin, alpha-smooth muscle actin (ASMA), smooth muscle myosin, and procollagen I were performed on aortic VICs. In addition, migration and extracellular matrix deposition were studied in vitro in aortic VICs. ICs show stable vimentin and collagen I expression in culture. Expression is approximately doubled in cultured ICs compared with fresh isolates. More than 95% of ICs in each passage stain for vimentin and procollagen I. Freshly isolated ICs are ASMA and myosin negative, but ICs in culture partially stain for these contractile markers. ICs have stable matrix production for up to five passages, associated with stable migration of the cells. We conclude that ovine valve interstitial cells undergo phenotypic modulation to activated myofibroblasts under culture conditions but retain stable matrix production.
No preview · Article · Sep 2004 · Tissue Engineering
[Show abstract][Hide abstract]ABSTRACT: The tissue-engineered (TE) heart valve was developed to improve the durability of tissue heart valves. The aim of this study was to evaluate morphological and histological changes in a TE heart valve consisting of decellularized porcine matrices seeded with viable autologous vascular endothelial cells (AVEC).
TE valves were implanted into the right ventricular outflow tract of eight juvenile sheep and explanted after 7 days, 3 and 6 months. The valves were evaluated visually, by invasive pressure measurements, X-ray, light microscopy, scanning, and transmission electron microscopy. The calcium content of the cusps was determined quantitatively by atomic absorption spectrometry.
After valve implantation, all animals showed fast recovery with no complications during the observation period. Invasive pressure measurements presented a mean TE valve pressure gradient of 1.5+/-0.5 mm Hg at 3 and the same at 6 months. Light microscopy showed a monolayer of AVEC on all explanted heart valves, confirmed by scanning electron microscopy and immunohistochemical staining. X-ray examination of explanted TE heart valves showed no cusp calcification, confirmed by atomic absorption spectrometry.
All explanted TE heart valves showed AVEC at the inner surface and ingrowth of fibroblast into the decellularized matrix, increasing during the observation period. The calcium contents were very low at explantation in these viable new heart valves.
No preview · Article · May 2003 · Medical science monitor: international medical journal of experimental and clinical research
[Show abstract][Hide abstract]ABSTRACT: Evidence has been gathered that biomechanical factors have a significant impact on cell differentiation and behavior in in vitro cell cultures. The aim of this bioreactor is to create a physiological environment in which tissue engineered (TE) aortic valves seeded with human cells can be cultivated during a period of several days. The bioreactor consists of 2 major parts: the left ventricle (LV) and the afterload consisting of a compliance, representing the elastic function of the large arteries, and in series a resistance, mimicking the arterioles and capillaries. The TE aortic valve is placed between the LV and the compliance. With controllable resistance, compliance, stroke volume and frequency, and hydrodynamic conditions can be changed over a wide physiological range. This study resulted in a prototype of a compact pulsatile flow system for the creation of TE aortic valves. In addition a biocompatibility study of the used materials is performed.
No preview · Article · Sep 2002 · Artificial Organs
[Show abstract][Hide abstract]ABSTRACT: To improve the durability of stentless valves without losing their excellent hemodynamic function, a new-generation auto-xenograft was developed and evaluated. A piece of vein was harvested from 3 juvenile sheep 6 weeks before implantation of the valve. Endothelial cells from the vein material were cultivated and used to reendothelialize a decellularized porcine pulmonary valve. The tissue-engineered valve was implanted into the right ventricular outflow tract of the juvenile sheep. It was explanted after 100 days and assessed macroscopically as well as by x-ray, light microscopy (hematoxylin and eosin staining and von Kossa staining), and scanning electron microscopy. Calcium content of the cusps was determined quantitatively by atomic absorption spectrometry. The sheep implanted with the valve recovered quickly without any problems during the observation period. X-ray examination of the 3 explanted valves showed no cusp calcification, which was confirmed by histological study. Atomic absorption spectrometry showed low tissue calcium content. A clinical safety and feasibility trial with an allograft valve prepared the same way showed excellent short-term results in 6 patients.
[Show abstract][Hide abstract]ABSTRACT: The presence of viable endothelial cells may support longer durability and the absence of calcification in valve prostheses. This study shows the development of a tissue engineered heart valve, constructed from viable autologous endothelial cells on an acellular matrix and its evaluation in juvenile sheep. In 3-month-old sheep (n = 8) a piece of vein was harvested to culture autologous endothelial cells. A porcine acellular matrix was reendothelialized and implanted in juvenile sheep as pulmonary interposition. The valves were explanted after 1 week, 3 and 6 months. Examination was performed by X-ray, light microscopy, and atomic absorption spectrometry. Reendothelialization mean rate was 10.3 x 10(5) cells/cm(2) with a mean endothelial cell viability of 95.5% (0.98 x 10(5) cells/cm(2)). X-ray examination showed no cusp calcification at 1 week, 3 and 6 months, which was confirmed by light microscopy. Immunostaining for factor VIII demonstrated colonization of viable mature autologous endothelial cells on the heart valve after the seeding process. The atomic absorption spectrometry showed no significant increase of the calcium content after 3 (P value >.1) and 6 months (P value >.1) compared with nonimplanted tissue engineered heart valves. The tissue engineered valve showed no cusp calcification in the juvenile sheep after 3 and 6 months.
No preview · Article · Nov 2001 · Seminars in Thoracic and Cardiovascular Surgery
[Show abstract][Hide abstract]ABSTRACT: 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).
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.
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).
Photo-oxidation of a porcine stentless valve prevents calcification not only in the cusps, but also in the aortic wall portion.
No preview · Article · Aug 2001 · The Journal of heart valve disease
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
SDS is inefficient as an antimineralization treatment, in contrast to ethanol or AOA. Cusp hematoma after ethanol treatment needs further investigation.
No preview · Article · May 2001 · The Annals of Thoracic Surgery