Scanning Electron Microscopy Methodology for Study of the Pathophysiology of Calcification in Bioprosthetic Heart Valves

Scanning electron microscope (SEM) morphologic analysis combined with energy dispersive characteristic X-ray (EDX) microprobe analysis provides insight into the mechanisms associated with disease-related crystal formation in biological materials. SEM and EDX were employed in analyzing specimens which were embedded in standard fashion in glycolmethacrylate (JB-4). The specimen surfaces under electron microscope investigation resulted from microtomy used in the preparation of reference light microscope histological sections; thus histology served as a direct reference for the SEM and EDX analyses. The particular application of these methods was in the study of bioprosthetic heart valve calcification, largely responsible for clinical failure of these heart valve substitutes. To simulate the clinically observed mineralization processes, glutaraldehyde-pretreated porcine heart valve leaflets were implanted subcutaneously in rats and subsequently removed at various time intervals from 1 to 56 days. Also, to address the hypothesis that the calcification process generates crystalline materials analogous to those in bone, EDX data obtained from pure hydroxyapatite were compared with the embedded tissue results. Further, EDX results were compared with data obtained by chemical analysis of the bulk specimens to assess the validity of the electron microscope technique

A Computational Model of Aging and Calcification in the Aortic Heart Valve

The aortic heart valve undergoes geometric and mechanical changes over time. The cusps of a normal, healthy valve thicken and become less extensible over time. In the disease calcific aortic stenosis (CAS), calcified nodules progressively stiffen the cusps. The local mechanical changes in the cusps, due to either normal aging or pathological processes, affect overall function of the valve. In this paper, we propose a computational model for the aging aortic valve that connects local changes to overall valve function. We extend a previous model for the healthy valve to describe aging. To model normal/uncomplicated aging, leaflet thickness and extensibility are varied versus age according to experimental data. To model calcification, initial sites are defined and a simple growth law is assumed. The nodules then grow over time, so that the area of calcification increases from one model to the next model representing greater age. Overall valve function is recorded for each individual model to yield a single simulation of valve function over time. This simulation is the first theoretical tool to describe the temporal behavior of aortic valve calcification. The ability to better understand and predict disease progression will aid in design and timing of patient treatments for CAS

Covalent binding of aminopropanehydroxydiphosphonate to glutaraldehyde residues in pericardial bioprosthetic tissue: Stability and calcification inhibition studies

Calcification has limited the clinical utility of bioprosthetic heart valves fabricated from either glutaraldehyde-pretreated bovine pericardium or porcine aortic valves. Aminopropanehydroxydiphosphonate (APDP), covalently bound to residual aldehyde groups in glutaraldehyde-treated pericardial bioprosthetic tissue, has been shown to inhibit cardiovascular calcification in the rat subdermal model. Using 3H-labeled glutaraldehyde (GLUT) at a concentration of 0.02 M and 0.14 M 14C-labeled APDP, we assessed the effects of GLUT incubation temperature (4[deg] or 25[deg]C) and pH of the GLUT incubation solution (pH 4.0, 7.4, or 10.0) on the GLUT incorporation step and subsequent APDP binding (24 hr 25[deg]C) into bioprosthetic valve (BPV) tissue (bovine pericardium). Increased incorporation of GLUT and APDP occurred at lower GLUT incubation temperature (GLUT, 346.05 +/- 1.9 nM/mg, 4[deg]C vs 259.76 +/- 1.39 nM/mg, 25[deg]C; APDP, 57.56 +/- 4.43 nM/mg, 4[deg]C vs 36.36 +/- 0.46 nM/mg, mean +/- standard error, at 25[deg]C). There also was a greater incorporation of GLUT but not APDP at the higher glutaraldehyde pretreatment pH (GLUT, pH 10.0, 213.73 +/- 73 nM/mg vs pH 4, 132.08 +/- 43 nM/mg; APDP, pH 10.0, 51.41 +/- 12 nM/mg vs pH 4.0, 49.97 +/- 6 nM/mg). In vivo studies revealed that all groups with treated BPV implanted for 21 days in male 3-week-old CD rats demonstrated a loss of both GLUT (12%-50%) and APDP (48%-64%) compared to preimplant content. BPV implant calcification was significantly inhibited in all groups treated with APDP compared to control Ca2+ (5.54 +/- 2.1-9.64 + 1.2 [mu]g/mg, APDP pretreated, vs 93.64 +/- 11.65 [mu]g/mg, control; P [les] 0.001) despite the progressive loss of both GLUT and APDP with time. It is concluded that preincubation of BPV tissue in GLUT at lower temperature (4[deg]C) and higher pH (10.0) enhanced BPV GLUT uptake and subsequent APDP covalent binding. In addition, in the rat subdermal model, BPV tissue calcification was markedly inhibited by APDP, despite a significant loss of bound drug.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27918/1/0000341.pd

Controlled release of ethanehydroxy diphosphonate from polyurethane reservoirs to inhibit calcification of bovine pericardium used in bioprosthetic heart valves

Calcification (CALC) of bioprosthetic heart valves (BHVs) fabricated from either glutaraldehyde-pretreated bovine pericardial tissue or porcine aortic valves is the most frequent cause of clinical failure of these devices. Previous studies have demonstrated that calcification is inhibited by diphosphonate compounds released into the vicinity of bioprosthetic tissue implanted subcutaneously in rats. Controlled release of the anticalcification agent ethanehydroxy diphosphonate (EHDP), as a 1:1 mixture of Na2 EHDP and CaEHDP from cylindrical polyurethane (PU) reservoirs (o.d. = 0.36 cm i.d. = 0.33 cm, length = 4 cm) fabricated by solvent casting was assessed in vitro and in vivo. The diffusivity (D), determined independently using standard diffusion cells, for ionic EHDP diffusion across the PU membrane was 1.2 x 10 cm2/s. Volume influx of buffer into the reservoirs in vitro was observed experimentally to reach a maximum at 7.8 days (288 +/- 44 [mu]l) with a biexponential decline to 147 +/- 6 [mu]l at 70 days. The cumulative EHDP released in vitro after 70 days was 4.2 +/- 0.6% (4.8 +/- 0.7 mg) compared to 15.7 +/- 3.2% (18.1 +/- 3.7 mg) in vivo (subcutaneously in 3 week-old, male, CD rats) over 21 days. The release rate of EHDP from the reservoirs was not a zero-order process. Reservoir administration of EHDP effectively inhibited pericardial BHV-CALC in 21-day subdermal explants (Ca2+ = 4.5 +/- 1.4 [mu]g Ca2+/mg tissue; control, Ca2+ = 120 +/- 13 [mu]g Ca2+/mg tissue) without diphosphonate-related untoward effects at a dose of approx. 3 mg/kg per day.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28671/1/0000488.pd

After 50 Years of Heart Transplants: What Does the Next 50 Years Hold for Cardiovascular Medicine? A Perspective From the International Society for Applied Cardiovascular Biology

The first successful heart transplant 50 years ago by Dr.Christiaan Barnard in Cape Town, South Africa revolutionized cardiovascular medicine and research. Following this procedure, numerous other advances have reduced many contributors to cardiovascular morbidity and mortality; yet, cardiovascular disease remains the leading cause of death globally. Various unmet needs in cardiovascular medicine affect developing and underserved communities, where access to state-of-the-art advances remain out of reach. Addressing the remaining challenges in cardiovascular medicine in both developed and developing nations will require collaborative efforts from basic science researchers, engineers, industry, and clinicians. In this perspective, we discuss the advancements made in cardiovascular medicine since Dr. Barnard's groundbreaking procedure and ongoing research efforts to address these medical issues. Particular focus is given to the mission of the International Society for Applied Cardiovascular Biology (ISACB), which was founded in Cape Town during the 20th celebration of the first heart transplant in order to promote collaborative and translational research in the field of cardiovascular medicine

Tissue-engineered valved conduits in the pulmonary circulation

AbstractObjective: Bioprosthetic and mechanical valves and valved conduits are unable to grow, repair, or remodel. In an attempt to overcome these shortcomings, we have evaluated the feasibility of creating 3-leaflet, valved, pulmonary conduits from autologous ovine vascular cells and biodegradable polymers with tissue-engineering techniques. Methods: Endothelial cells and vascular medial cells were harvested from ovine carotid arteries. Composite scaffolds of polyglycolic acid and polyhydroxyoctanoates were formed into a conduit, and 3 leaflets (polyhydroxyoctanoates) were sewn into the conduit. These constructs were seeded with autologous medial cells on 4 consecutive days and coated once with autologous endothelial cells. Thirty-one days (±3 days) after cell harvesting, 8 seeded and 1 unseeded control constructs were implanted to replace the pulmonary valve and main pulmonary artery on cardiopulmonary bypass. No postoperative anticoagulation was given. Valve function was assessed by means of echocardiography. The constructs were explanted after 1, 2, 4, 6, 8, 12, 16, and 24 weeks and evaluated macroscopically, histologically, and biochemically. Results: Postoperative echocardiography of the seeded constructs demonstrated no thrombus formation with mild, nonprogressive, valvular regurgitation up to 24 weeks after implantation. Histologic examination showed organized and viable tissue without thrombus. Biochemical assays revealed increasing cellular and extracellular matrix contents. The unseeded construct developed thrombus formation on all 3 leaflets after 4 weeks. Conclusion: This experimental study showed that valved conduits constructed from autologous cells and biodegradable matrix can function in the pulmonary circulation. The progressive cellular and extracellular matrix formation indicates that the remodeling of the tissue-engineered structure continues for at least 6 months. (J Thorac Cardiovasc Surg 2000;119:732-40

Controlled release of 1-hydroxyethylidene diphosphonate: in vitro assessment and effects on bioprosthetic calcification in sheep tricuspid valve replacements

Calcification (CALC) is the most frequent cause of the clinical failure of bioprosthetic valves (BHV's). Controlled-release (paravalvar) administration of the anticalcification agent ethanehydroxydiphosphonate (EHDP), as either Na2EHDP or in combination (1:1) with the less soluble CaEHDP, from a silicone rubber matrix (20% w/w EHDP) was studied both in vitro and in vivo for the prevention of BHV CALC. Seventeen sheep (6-7 months old, male, Suffolk) underwent tricuspid valve replacement using Hancock I, 25 mm porcine aortic bioprostheses. BHV explant evaluation after 16-20 weeks revealed that two of the 7 control BHV were calcified (139 +/- 20.8 [mu]gCa2+/mg of tissue), while none of the 9 BHV retrieved from animals receiving controlled release EHDP demonstrated CALC (4.41 +/- 1.09 [mu]g Ca2+/mg of tissue). No adverse effects of EHDP on bone or calcium metabolism were noted. The cumulative percent of EHDP released per electron microprobe analysis was 40.4% +/- 9.68 (Na, CaEHDP) to 79.0% +/- 4.82 (Na2EHDP) in vivo compared to 35.7% +/- 7.72 and 78.6 +/- 11.1 in vitro, respectively. Assessment of the Young's modulus (Y) using thermomechanical analysis (TMA) revealed a 1.5-fold (Silastic Q7-4840) to 9.5-fold (Silastic 382) increase in Y following drug loading. The Y for explanted, Silastic Q7-4840 polymer matrices ranged from 2.84 x 104 to 5.57 x 105 dyne/cm2. In vitro osmotic related matrix swelling of the Na2EHDP loaded, unsealed matrices (20% w/w) after 75 days was minimized to a 35.8% increase in weight due to coincorporation of CaEHDP with Na2EHDP in a 1:1 ratio and was further reduced (22.2% increase in weight) by sealing 76% of the releasing surface, compared to Na2EHDP matrices which demonstrated a 414% and 141% increase in weight, respectively.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27902/1/0000322.pd

Cardiovascular implant calcification: a survey and update

Calcification of cardiovascular prosthetic implants is a common and important problem. This review provides an update based upon the Conference on Cardiovascular Implant Calcification held as part of the 13th World Congress of the International Society for Heart Research, 1989. A variety of cardiovascular prostheses are affected clinically by calcification, including bioprosthetic heart valves, aortic homografts and trileaflet polymeric valve prostheses. In addition, experimental studies have demonstrated calcification of artificial heart devices in ventricular assist systems in long-term calf studies. The pathophysiology of this disease process is incompletely understood. A common element between the various types of cardiovascular implant calcification is the localization of calcific deposits to devitalized cells and membranous debris. Prevention of cardiovascular implant calcification by either biomaterial modifications or regional drug therapy (controlled release) is being investigated.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29115/1/0000154.pd

Hemodynamic Environments from Opposing Sides of Human Aortic Valve Leaflets Evoke Distinct Endothelial Phenotypes In Vitro

The regulation of valvular endothelial phenotypes by the hemodynamic environments of the human aortic valve is poorly understood. The nodular lesions of calcific aortic stenosis (CAS) develop predominantly beneath the aortic surface of the valve leaflets in the valvular fibrosa layer. However, the mechanisms of this regional localization remain poorly characterized. In this study, we combine numerical simulation with in vitro experimentation to investigate the hypothesis that the previously documented differences between valve endothelial phenotypes are linked to distinct hemodynamic environments characteristic of these individual anatomical locations. A finite-element model of the aortic valve was created, describing the dynamic motion of the valve cusps and blood in the valve throughout the cardiac cycle. A fluid mesh with high resolution on the fluid boundary was used to allow accurate computation of the wall shear stresses. This model was used to compute two distinct shear stress waveforms, one for the ventricular surface and one for the aortic surface. These waveforms were then applied experimentally to cultured human endothelial cells and the expression of several pathophysiological relevant genes was assessed. Compared to endothelial cells subjected to shear stress waveforms representative of the aortic face, the endothelial cells subjected to the ventricular waveform showed significantly increased expression of the “atheroprotective” transcription factor Kruppel-like factor 2 (KLF2) and the matricellular protein Nephroblastoma overexpressed (NOV), and suppressed expression of chemokine Monocyte-chemotactic protein-1 (MCP-1). Our observations suggest that the difference in shear stress waveforms between the two sides of the aortic valve leaflet may contribute to the documented differential side-specific gene expression, and may be relevant for the development and progression of CAS and the potential role of endothelial mechanotransduction in this disease.National Institutes of Health (U.S.) (Molecular, Cellular, and Tissue Biomechanics training grant (T32 EB006348))National Institutes of Health (U.S.) (NHLBI RO1-HL7066686)Charles Stark Draper Laboratory (Fellowship

Transcriptome, Methylome and Genomic Variations Analysis of Ectopic Thyroid Glands

Congenital hypothyroidism from thyroid dysgenesis (CHTD) is predominantly a sporadic disease characterized by defects in the differentiation, migration or growth of thyroid tissue. Of these defects, incomplete migration resulting in ectopic thyroid tissue is the most common (up to 80%). Germinal mutations in the thyroid-related transcription factors NKX2.1, FOXE1, PAX-8, and NKX2.5 have been identified in only 3% of patients with sporadic CHTD. Moreover, a survey of monozygotic twins yielded a discordance rate of 92%, suggesting that somatic events, genetic or epigenetic, probably play an important role in the etiology of CHTD.Journal ArticleResearch Support, Non-U.S. Gov'tValidation StudiesSCOPUS: ar.jinfo:eu-repo/semantics/publishe