Ultrastructural pathology of bioprosthetic heart valves with infectious endocarditis

Aim. To study the ultrastructure of mitral bioprosthetic heart valves (BHVs) which failed due to infective endocarditis.Materials and Methods. Here we examined 7 ethylene glycol diglycidyl ether-treated xenopericardial BHVs excised during repeated BHV replacement because of prosthetic endocarditis. After being fixed in formalin and postfixed in osmium tetroxide, BHVs were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the BHV microanatomy by means of backscattered scanning electron microscopy at 15 kV voltage.Results. The extracellular matrix underwent degradation and disintegration resulting in loosening, fragmentation, and reduction in the electron density of collagen and elastin fibers. We observed a number of recipient cells (macrophages, multinucleated giant cells, neutrophils, endothelial cells and smooth muscle cells) within the BHVs. The highest number of cells was localized on the valve surfaces. The localization of the recipient cells on the ventricular and atrial surfaces was different. The central part of the valves was abundantly populated by macrophages.Conclusion. Prosthetic endocarditis is accompanied by the migration of recipient cells into the BHV structure, which is the consequence of surface and extracellular matrix disintegration.

Elemental analysis of valvular and atherosclerotic calcification

Aim. To analyze the topographic patterns of valvular and atherosclerotic calcification growth.Methods. Dysfunctional aortic valves (n = 18) and atherosclerotic plaques (n = 20) were fixed in formalin, postfixed in 1% osmium tetroxide, consecutively stained by 2% osmium tetroxide and 2% uranyl acetate, and embedded into epoxy resin (Epon) with the further grinding and polishing ofthe samples. Upon the counterstaining by lead citrate and sputter coating with carbon, samples were visualized by backscattered scanning electron microscopy. Elemental analysis was conducted via energy-dispersive X-ray spectroscopy. Measurement of Ca/P ratio within the mineral deposits was carried out employing a pool table principle (i.e., in the center of the deposit, in the near and far circumferences (clockwise), and in control regions around the mineral deposit). Topographic patterns of calcifications were modeled using the correlation analysis. Results. Significant correlation was revealed between the Ca/P ratio in the deposit center and in the near and far circumferences of deposit in both in valvular (r = 0,35-0,78 - near circumference; r = 0,63-0,69 - far circumference) and atherosclerotic mineral deposits (r = 0,37-0,56 - near circumference; r = 0,48-0,63 - far circumference), suggesting the hierarchical growth of cardiovascular calcification around the initial nucleation sites.Conclusion. Valvular and atherosclerotic calcifications development is concentric.

Ultrastructural mitral valve abnormalities in infective endocarditis

Aim. Using an original method based on backscattered scanning electron microscopy, to study the structural features of the mitral valve leaflets in infective endocarditis.Material and methods. We examined 9 mitral valves extracted during surgical interventions due to structural malfunction in patients with infective endocarditis (IE). The samples were fixed in buffered paraformaldehyde with osmium tetraoxide postfixation. After dehydration by increasing alcohol concentration and acetone, the samples were placed in epoxy resin. After the resin has polymerized, the samples were ground and then polished to the desired depth. To increase the electronic contrast, the samples were treated with a uranyl acetate alcohol solution during dehydration and with Reynolds' lead citrate after polishing the epoxy blocks. The samples were visualized by backscattered scanning electron microscopy at an accelerating 15-kV voltage.Results. Structural leaflet injuries caused by IE were most pronounced in the central part and the base. Necrotic areas were extensive electron-dense formations located in the central leaflet layers, or displaced towards the ventricular surface. The electron-dense material in the necrotic area was poorly structured and contained individual cells and bacteria. Bacteria were also present outside the necrotic area. Necrotic areas were surrounded by a layer of a modified extracellular matrix, usually covered with a fibrin layer. Among the extracellular matrix fibers, the macrophages, smooth myocytes and fibroblasts was noted. The fibrin layer, in addition to these cells, contained a large number of blood vessels and was often covered with endothelium.Conclusion. Infection of the mitral valve leaflets causes a simultaneous inflammatory response and regeneration activation. Without adequate regulatory factors, the processes of inflammation and connective tissue creation lead to structural and functional leaflet failure. Specific causes may be overgrowth of necrotic and inflammatory areas, edema and fiber orientation disorder, as well as leaflet rupture.

Histomorphometric Analysis of Osseointegrated Grade V Titanium Mini Transitional Implants in Edentulous Mandible by Backscattered Scanning Electron Microscopy (BS-SEM)

The purpose of this study is to assess the use of grade V titanium mini transitional implants (MTIs) immediately loaded by a temporary overdenture. For this, a histomorphometric analysis of the bone area fraction occupancy (BAFO) was performed by backscattered scanning electron microscopy (BS-SEM). Four female patients were submitted to surgery in which two MTIs were installed and immediately loaded with a temporary acrylic prosthesis. During the same surgery, two regular diameter implants were placed inside the bone and maintained without mechanical load. After 8 months, the MTIs were extracted using a trephine and processed for ultrastructural bone analysis by BS-SEM, and the regular-diameter implants were loaded with an overdenture device. A total of 243 BAFOs of MTIs were analyzed, of which 94 were mainly filled with cortical bone, while 149 were mainly filled with trabecular bone. Bone tissue analysis considering the total BAFOs with calcified tissues showed 72.13% lamellar bone, 26.04% woven bone, and 1.82% chondroid bone without significant differences between the samples. This study revealed that grade V titanium used in immediately loaded MTI was successfully osseointegrated by a mature and vascularized bone tissue as assessed from the BAFO.