1,721,216 research outputs found
Humanization of porcine pulmonary valvulated conduits after decellularization and repopulation with human endothelial cells
No full textIntroduction. In heart valve surgical replacement, the most suitable hemodynamic properties and mechanical performances depend on the preservation of cusp anatomical shape and stromal structure. In addition, post-operatory valve calcification can be avoided only after removing all native cells from bioprostheses. Previously, complete cell extraction was achieved from porcine valve leaflets with concurrent preservation of their extracellular matrix (ECM) (1). These acellular scaffolds allowed "in vitro" repopulation with homologous valve interstitial cells, which also re-differentiated into all four cell phenotypes existing in heart valves (2). Methods. Porcine pulmonary valvulated segments (PVCs) were decellularized using combined non -denaturating neutral detergents Triton X-100 and Cholate, followed by Benzonase® digestion. Acellular PVCs were (i) orthotopically implanted in recipient pigs for 1-2 months, or (ii) "in vitro" seeded with endothelial cells derived from human umbilical cord (HUVEC), and incubated for 1-2 weeks. Histological and TEM-SEM ultrastructural analysis was performed, also after histochemical reactions for glycosaminoglycan (GAG) localization and laminin immuno-localization. Results. The treated PVCs exhibited complete cell remotion, good ECM preservation and surface reactivity for laminin. (i) After 2-month implantation, "in vivo" cell colonization spontaneously occurred by two distinct cell populations: endothelial-like cells, adhering to PVC luminal areas, and mesenchimal-like cells, migrating through PVC interstitium. (ii) After cell seeding and 1-week incubation, monolayers of human endothelial cells completely covered PVC luminal surfaces. Cell adhesion to the retained basal "lamina" and cell junction formation were also observed. In addition, valve interstitium was enriched by newly secreted GAGs at the subendothelial aspects. After cell seeding and 2-week incubation, micropinocytotic activity by endothelium and increased GAG-reactivity were observed. Conclusions. The decellularized PVCs are propensive for both (i) "in vivo" homologous cell repopulation, and (ii) "in vitro" heterologous endothelization with HUVEC. In addition, PVC stroma acquired more and more hybrid character because human-endothelium-generated GAGs were added to the native ECM macromolecules retained within the treated porcine PVCs. Thus these engineered PVCs appear as promising autologous-like, glutaraldehyde-free, and anti-thrombogenic bioprostheses. 1. Spina M., Ortolani F., El Messlemani A., Gandaglia A., Bujan J., Garcia-Honduvilla N., Vesely I., Gerosa G., Casarotto D., Petrelli L., Marchini M.: Isolation of intact aortic valve scaffolds for heart valve bioprostheses: extracellular matrix structure, prevention from calcification and cell repopulation features. J. Biomed. Mater. Res., 67, 1338-1350, 2003. 2. Bertipaglia B., Ortolani F., Petrelli L., Gerosa G., Spina M.,, Pauletto P., Casarotto D., Marchini M., Sartore S.: Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells. Ann. Thorac. Surg., 75, 1274-1282, 2003
"GA-banding": a new terminology and a study of the glutaraldehyde-induced band pattern of type I collagen fibrils
No full textThe negative staining D-band patterns of glutaraldehyde-reacted collagen fibrils were compared to those of fresh collagen fibrils. Negative staining was obtained by using 1% phosphotungstic acid (PTA) diluted in phosphate buffer 0.1 M, pH 7.4. The stain was dripped onto grids where native type I collagen fibrils, isolated from bovine dermis, were collected. Ultrastructural pictures were digitized to form microdensitometric traces. The glutaraldehyde-induced patterns showed fifteen light bands (micrographs) or negative peaks (microdensitograms), whose D-locations were constant and characteristic. In order to make this ultrastructural feature a precise reference parameter, these bands were called "GA-bands" and numbered. When comparing this averaged microdensitogram with that of negatively stained fresh fibrils, peak "GA1" and peak "GA7" were observed to correspond to peak "X2" (known as N-terminal telopeptide region) and peak "X3" (known as C-terminal telopeptide region) respectively, while there was no correspondence between the other peaks of the two traces. It means that the regions where preexistent crosslinks exist are unaffected by interaction with glutaraldehyde, while in the other regions, where new glutaraldehyde-crosslinks occur, the band pattern modifies. The unchanged D-location of peaks "GA1" and "GA7" leads to the conclusion that the D-shortening induced by glutaraldehyde is not due to shifting of tropocollagen molecules but to changes in their orientation with respect to fibril long axis or in secondary-tertiary structure of collagen
Elastin and collagen interact with cell-derived acidic phospholipid membranes in the progression of mineralization in calcifying aortic valves
No full textINTRODUCTION Elastin fibers have been reported to be prone to mineralization in ageing and in a number of cardiovascular diseases including bioprosthetic valve calcification. Different mechanisms seem to exist which direct these mineralization processes such as in aorta atherosclerotic plaques, where calcification is preceded by accumulation of cholesterol esters and neutral lipids inside altered elastin fibers [1], or that in subdermally implanted aorta wall segments, where calcification occurs at the outer aspect of elastin fibers interacting with undefined amorphous material [2]. Recently, we found that fixation/reactions with glutaraldehyde and cationic copper-phthalocyanine Cuprolinic blue (GACB) at low salt-critical-electrolyte-concentration and pH were suitable for studying calcification in subdermally implanted aortic valves, because of tissue unmasking form mineral and simultaneous visualization at the ultrastructural level of unique, electrondense layers outlining calcifying cells and matrix-vesicle-like structures [3,4]. The observed reactivity and susceptibility to extraction with chloroform-methanol suggested these pericellular layers to be formed by acidic phospholipids which will cluster at cell surfaces replacing cell plasma membranes. Here, a modified glutaraldehyde-malachite-green method (GA-MG) was used to support the concept of lipid involvement in valve calcification and to assess how elastin fibers and collagen fibrils are involved in the mineralization progression from the cells into the extracellular matrix. MATERIAL AND METHODS Animal model inducing calcification: 6-week long implantation of porcine aortic valve leaflets in rat subcutis [5]. Processing of explanted samples: (A) immersion in 25mM sodium acetate buffer, containing 0.05% Cuprolinic blue + 0.05M MgCl2 + 2,5% glutaraldehyde, pH 4.8, at room temperature for 4 days (GACB); (B) immersion in 0.067 mol/L cacodylate buffer solution, containing 3% glutaraldehyde and 0.1% Malachite green, pH 4.8, at 4°C for 18h (GAMG). Post-fixation in 2% OsO4 in phosphate buffer, pH 7.2; dehydration in graded ethanols; embedding in Araldite/Epon. (A-C) thin section staining with uranyl acetate and lead citrate. RESULTS On thin sections, GAMG-subjected samples showed reactivity patterns superimposable to those observed for GACB-treated samples. Namely, decalcifying effect occurred with associated unmasking of cells and matrix vesicles, which appeared to be outlined by MG-reactive, electrondense borders (Fig.1). In addition, mineralization spreading from cells to adjacent extracellular matrix was characterized by outward growing of the MG-reactive material, which enveloped electron-lucent elastin fibers and collagen fibrils, and the additional presence of amorphous material embedding all these components (Fig.2). Despite demineralization, calcium precipitates were observed to be retained by most elastin fibers interacting with MG-reactive material , whereas no mineral deposits was found either on iuxta-cellular fibers still not involved in such a relationship, or those lying in uncalcified extracellular matrix. DISCUSSION GA-MG method reveals acidic phospholipid distribution because it allows lipid retention in the sample, with subsequent formation of electron-dense GA-MG-OsO4-lipid complexes during standard post-fixation with osmium tetroxyde [6]. The lower pH here used allowed to obtain tissue demineralization with preservation of proper reactivness. In the experimental conditions adopted, acidic phospholipids appeared to be accumulated at cell and matrix vesicle surfaces, i.e. where primary calcification occurs, and to be also involved in subsequent mineralization progression into the ECM. Here, elastic fibers and collagen fibrils seem to require prior surface interaction with cell-derived acidic phospholipids before undergoing mineralization, in agreement with the concept that their calcification need interaction with additional molecules [1,3] and/or cell degradation products [3,7]. The concept is supported that elastin fibers can undergo calcification according to different mechanisms which include intrinsic alterations as well as interaction with polyanionic molecules such as proteoglycans, as suggested for pseudoxanthoma elasticum [8], or acidic phospholipids, as suggested by the present data. ACKNOWLEDGEMENTS This work was supported by a special grant by Cassa di Risparmio di Padova e Rovigo Foundation. REFERENCES [1] Bobryshev YV, Lord RS. Atherosclerosis 42, 197-198 (1999). [2] Paule WJ, Bernick S, Strates B, Nimni ME. J. Biomed. Mater. Res. 26, 1169-1177 (1992). [3] Ortolani F, Petrelli L, Tubaro F, Spina M, Marchini M. Connect. Tiss. Res. 43, 44-55 (2002). [4] Ortolani F, Tubaro F, Petrelli L, Gandaglia A, Spina M, Marchini M. Histochem. J. 34, 41-50 (2002). [5] Schoen FJ, Tsao JW, Levy RJ. Am. J. Pathol. 123, 134-145(1986). [6] Bonucci E, Silvestrini G. Bone 15, 153-160 (1994). [7] Kim KM. Scan. Electron. Microsc. 9, 1137-1175 (1995). [8] Pasquali Ronchetti I, Baccarani-Contri M, Fornieri C., Mori G., Quaglino D. jr. Micron 24, 75-89 (1993)
Critical effects of inorganic phosphate at threshold concentrations on cultured aortic valve interstitial cells. Macroautophagocytosis versus procalcific cell degeneration
Full text linkThe conventional threshold values ascribed to inorganic phosphate concentration ([Pi]) in diagnosing normophosphatemia range between 0.8mM and 1.45mM to 2.0mM [Pi].
In cultures mimicking metastatic calcification ([Pi]=3.0mM) a major role was found to be played by [Pi] (Pi-cultures) in priming a procalcific cell degeneration of bovine aortic valve interstitial cells (bAVICs), with mineralization enhancing subsequent to superstimulation with bacterial lipopolysaccharide (LPS) plus conditioned medium from cultured LPS-stimulated macrophages (Pi-LPS-CM-cultures) [1]. Here, bAVIC primary cultures were carried out which contained different [Pi] (0.4, 0.6, and 1.3mM in added solutions, i.e. 0.8, 1.3, and 2.0mM in final cultures), so including border concentrations on respect to hypophosphatemic- and hyperphosphatemic-like conditions. At 0.8mM and 1.3 [Pi] and for each incubation time (3, 9, 15, 21, and 28 days), bAVICs from Pi-cultures and Pi-LPS-CM-cultures shared common ultrastructural features showing prominent macroautophagocytosis to occur, consistently with the immunohistochemical detection of the specific marker of mature autophagosomes MAP1-LC3A. Neither cell death signs nor appearance of calcific nodules were observed. At 2.0 [Pi], most bAVICs were affected by degenerative fragmentation as described for severe metastatic-like calcifcation, i.e. the appearence of phthalocianin-positive material outcropping at cell surface, acting as hydroxyapatite nucleator and being source of real calcospherulae. Quantitative spectrophotometric estimation of calcium amounts and alkaline phosphatase activity were consistent with the ultrastructural data, with (i) similar values for Pi-LPS-CM-cultures versus Pi-cultures and control cultures, at 0.8 and 1.3mM [Pi], and (ii) significantly higher values for Pi-LPS-CM-cultures versus Pi-cultures and these latter versus controls, at 2.0mM [Pi]. Restriction of immunopositivity to caspase-8 to very few cells and complete immunonegativity to annexin-V suggested apoptosis to be a negligible epiphenomenon. In conclusion, the propensity of bAVICs to undergo procalcific degeneration resulted to correlate with [Pi] in such a manner that a differential discrimination of this parameter within the conventional normophosphatemic range is suggested for a proper evaluation of the risk for dystrophic valve calcification. Moreover, bacterial-derived inflammation seems to be regarded as an effective trigger for the higher normophosphatemic [Pi].
References
[1] Bonetti A, Della Mora A, Contin M, Tubaro F, Marchini M, Ortolani F. (2012). Anat Rec 295: 1117-1127
Immunolocalization of interleukin-1 receptor antagonist in healthy and infarcted myocardium
Full text linkIschemic heart disease is a widespread cause of death. During infarction, myocardial injury is mediated by release of several pro-inflammatory cytokines including multifunctional interleukin-1 (IL-1). In various tissues, IL-1-mediated deleterious effects are known to be attenuated via the over-expression of natural anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra). In the present investigation, IL-1ra distribution in healthy and infarcted myocardium was studied by light and electron microscopy. After immunostaining, weak positivity resulted for cardiomyocytes in normal myocardium and, at higher degrees, in infarction border areas and ischemic ones. In ischemic areas, additional reactivity was displayed by the extracellular matrix and intravascular plasma. Immunogold labelling provided further details on intracytoplasmatic and extracellular distribution; in particular, noticeable gold particle distribution appeared on intercalated discs in normal and hypertrophic cardiomyocytes, as well as on thickened Z-lines for these latter. The present results suggest that cardiomyocytes represent a major source of IL-1ra in vivo, even though additional contribution by blood derived IL-1ra is to be taken in account in ischemic areas. In addition, ischemia-associated intracytoplasmic IL-1ra increase and its additional presence in the extracellular matrix is consistent with the concept that this cytokine plays a cardioprotective role at different levels and by distinct mechanisms
STAIN EXCLUSION PATTERNS OF FIXED COLLAGEN FIBRILS AND MECHANISM OF GLUTARALDEHYDE FIXATION
No full text
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Playing with Black and Yellow: the Evolvability of a Batesian Mimicry
No full textIrrespective of the selective advantage deriving from similar color pattern, the evolution of Batesian (and
Mu ̈llerian) mimicry between distantly related insects
groups has been perhaps facilitated by the availability to both models and mimics of similar pattern units more likely to be expressed, and to be modified in parallel ways, due to shared developmental constraints. We explore this
hypothesis in a comparison of units of black-and-yellow
color patterns between wasps (Vespidae) and those syrphids
(Syrphidae) that are considered to be their Batesian mimics. As a proxy for evolvability we analyzed the cooccurrence of multiple color pattern within species (either as serial homologues or as expression of intraspecific variation) in 203 species of syrphids and 127 species of wasps. In both the wasps and the syrphids, the most frequent black-and-yellow patterns on the abdomen—all shared between the two insect groups—are also the most extensively linked in the networks of intraspecific co-occurrence,
but are not the same in the two insect groups: in accordance with our hypothesis, this suggests positively biased evolvability
- …
