Fulvia Ortolani

Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells: the VESALIO project (vitalitate exornatum succedaneum aorticum labore ingenioso obtenibitur)

BACKGROUND Heart valve bioprostheses for cardiac valve replacement are fabricated by xeno- or allograft tissues. Decellularization techniques and tissue engineering technologies applied to these tissues might contribute to the reduction in risk of calcification and immune response. Surprisingly, there are few data on the cell phenotypes obtained after cellularizing these naturally-derived biomaterials in comparison to those expressed in the intact valve. METHODS Aortic valve interstitial cells (VIC) were used to repopulate the corresponding valve leaflets after a novel decellularization procedure based on the use of ionic and nonionic detergents. VIC from leaflet microexplants at the third passage were utilized to repopulate the decellularized leaflets. Intact, decellularized and repopulated valve leaflets and cultured VIC were examined by immunocytochemical procedures with a panel of antibodies to smooth muscle and nonmuscle differentiation antigens. Intact and cellularized leaflets were also investigated with Western blotting and transmission electron microscopy, respectively. RESULTS Myofibroblasts and smooth muscle cells (SMC) were mostly localized to the ventricularis of the leaflet whereas fibroblasts were dispersed unevenly. Cultured VIC were comprised of myofibroblasts and fibroblasts with no evidence of endothelial cells and SMC. Two weeks after VIC seeding into decellularized leaflets, grafted cells were found penetrating the bioscaffold. The immunophenotypic and ultrastructural properties of the grafted cells indicated that a VIC heterogeneous mesenchymal cell population was present: fibroblasts, myofibroblasts, SMC, and endothelial cells. CONCLUSIONS VIC seeding on detergent-treated valve bioscaffolds has the cellular potential to reconstruct a viable aortic valve.

The influence of heart valve leaflet matrix characteristics on the interaction between human mesenchymal stem cells and decellularized scaffolds

The potential for in vitro colonization of decellularized valves by human bone marrow mesenchymal stem cells (hBM-MSCs) towards the anisotropic layers ventricularis and fibrosa and in homo- vs. heterotypic cell-ECM interactions has never been investigated. hBM-MSCs were expanded and characterized by immunofluorescence and FACS analysis. Porcine and human pulmonary valve leaflets (p- and hPVLs, respectively) underwent decellularization with Triton X100-sodium cholate treatment (TRICOL), followed by nuclear fragment removal. hBM-MSCs (2x10(6) cells/cm(2)) were seeded onto fibrosa (FS) or ventricularis (VS) of decellularized PVLs, precoated with FBS and fibronectin, and statically cultured for 30 days. Bioengineered PVLs revealed no histopathological features but a reconstructed endothelium lining and the presence of fibroblasts, myofibroblasts and SMCs, as in the corresponding native leaflet. The two valve layers behaved differently as regards hBM-MSC repopulation potential, however, with a higher degree of 3D spreading and differentiation in VS than in FS samples, and with enhanced cell survival and colonization effects in the homotypic ventricularis matrix, suggesting that hBM-MSC phenotypic conversion is strongly influenced in vitro by the anisotropic valve microstructure and species-specific matching between extracellular matrix and donor cells. These findings are of particular relevance to in vivo future applications of valve tissue engineering.

Ultrastructural evaluation of human metaphase II oocytes after vitrification: closed versus open devices.

OBJECTIVE To compare the ultrastructural appearance of oocytes after vitrification and warming with two different devices. DESIGN Oocytes were examined by ultrastructural analysis after vitrification and warming with use of closed (CryoTip; Irvine Scientific, Santa Ana, CA) or open (Cryotop; Kitazato BioPharma Co., Ltd., Shizuoka, Japan) devices. SETTING Pordenone Hospital IVF Unit and Medical Morphological Research Department, University of Udine. PATIENT(S) Surplus oocytes from 10 patients (aged 31-39 years) undergoing assisted reproductive technologies at the Pathophysiology Unit of Human Reproduction and Sperm Bank between 2006 and 2008. INTERVENTION(S) Oocytes with normal invertoscopic appearance underwent vitrification and warming with closed (CryoTip) or open (Cryotop) devices and were processed for transmission electron microscopy. MAIN OUTCOME MEASURE(S) Cryodamage extent and cell alterations in oocytes after open or closed vitrification and warming procedures and their rehydration rate. RESULT(S) A higher rate of complete oocyte rehydration and less-severe ultrastructural alterations were observed after vitrification and warming with the open Cryotop device. CONCLUSION(S) These preliminary data suggest that oocyte ultrastructure is better preserved with an open rather than closed vitrification and warming protocol.

Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts.

H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.

Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

BackgroundNitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time.ResultsWith this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle.ConclusionsThe dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.

Olfactory mucosa histological findings in laryngectomees.

After total laryngectomy, the patients often report immediate and marked olfactory deficit. The aim of this study was to determine whether hyposmia in laryngectomees reflects olfactory epithelial damage. Ten laryngectomized patients and ten rhinologically normal subjects were subjected to olfactory testing, after which histological examination of biopsied olfactory mucosa was performed. Olfactory testing in laryngectomees revealed a marked reduction in odor perception. Histological examination of olfactory mucosa specimens showed that in laryngectomees some neuroepithelial structural features were comparable with those found in normal subjects. However, additional signs of damage were also observed, consisting mainly of various degrees of epithelial degeneration, above and beyond those that are characteristic of physiological epithelium turnover. These different degenerative features consisted of severe damage to the neuroepithelium, culminating in complete topical loss. Bowmans glands were also observed to be involved in the degenerative process. Laryngectomy-induced hyposmia seems to be correlated with the almost complete loss of nasal airflow due to the disconnection between the upper and lower airways, which prevents odor molecules from reaching the olfactory area, together with degenerative phenomena, which affect the neuroepithelium, and consequent failure in neurosensorial performance.

Galectin-1 in cartilage: expression, influence on chondrocyte growth and interaction with ECM components.

Galectin-1 is a 14 kDa beta-galactoside binding protein, capable of forming lattice-like structures with glycans of cellular glycoconjugates and inducing intracellular signaling. The expression of Galectin-1 in porcine cartilage is described in this work for the first time. Immunocytochemical methods revealed distinct distribution patterns for both articular and growth plate cartilage. In articular cartilage, the highest reactivity for Galectin-1 was found in all chondrocytes at the superficial zone and in most of those at the lower layer of the middle zone. In the growth plate, marked reactivity was seen in chondrocytes at the proliferative zone and reached a maximum level for the column-forming cells at the hypertrophic zone. In addition, different Galectin-1 distribution patterns were observed at the subcellular level. With regards to the metabolic effects of Galectin-1, the results in vitro seem to indicate an inhibitory effect of Galectin-1 on articular chondrocyte anabolism (i.e. inhibition of cell proliferation and anabolic gene expression) and a stimulation of catabolic processes (i.e. induction of matrix degradation and hypertrophy marker expression). These data represent a starting point for the understanding the molecular mechanisms underlining ECM-Galectin-1 interaction and the subsequent signaling-cell transduction processes involving cartilage formation and maturation.

Carotenoids co-localize with hydroxyapatite, cholesterol, and other lipids in calcified stenotic aortic valves. Ex vivo Raman maps compared to histological patterns

Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.

Texture analysis of TEM micrographs of alginate gels for cell microencapsulation

In this work, the morphological characteristics of a calcium alginate gel and a binary (gel) mixture composed of (calcium) alginate and lactose‐modified chitosan (chitlac) are evaluated and compared to quantify the differences between the two three‐dimensional (3D) structures. A set of textural descriptors based on histogram analysis as well as on gray level co‐occurrence matrix and on fractal dimension is extracted from transmission electron microscopy micrographs to describe the morphological differences that the images present. The obtained results reveal significant quantitative morphological differences between the calcium alginate gel and the binary gel mixture that were already inferred from rheological experiments, so as to provide a structural basis for developing new encapsulation systems based on such mixed polymer gels. Microsc. Res. Tech. 74:58‐66, 2011.