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Maria Antonietta CROCE

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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Pubblicazioni

2017 - Surface engineering of Solid Lipid Nanoparticle assemblies by methyl α-d-mannopyranoside for the active targeting to macrophages in anti-tuberculosis inhalation therapy [Articolo su rivista]
Maretti, Eleonora; Costantino, Luca; Rustichelli, Cecilia; Leo, Eliana Grazia; Croce, Maria Antonietta; Francesca, Buttini; Truzzi, Eleonora; Iannuccelli, Valentina
abstract

This study describes the development of new mannosylated Solid Lipid Nanoparticle assemblies (SLNas) delivering rifampicin for an inhaled treatment of tuberculosis. SLNas were surface engineered with mannose residues to recognize mannose receptors located on infected alveolar macrophages and facilitate cell internalization. Two sets of SLNas were produced by the melt emulsifying technique using biocompatible lipid components, i.e. cholesteryl myristate combined with palmitic acid (PA set) or tripalmitin (TP set), in the presence of the targeting moiety, methyl α-d-mannopyranoside. Mannosylated SLNas were examined for their physical properties, drug payloads and release, as well as respirability in terms of emitted dose and respirable fraction determined by Next Generation Impactor. The most appropriate formulations were assessed for mannosylation using FTIR, XPS, SEM coupled with EDX analysis, and wettability assay, in comparison with the respective non-functionalized SLNas. Besides, cytotoxicity and cell internalization ability were established on J774 murine macrophage cell line. Mannosylated SLNas exhibited physical properties suitable for alveolar macrophage passive targeting, adequate rifampicin payloads (10-15%), and feasible drug maintenance within SLNas along the respiratory tract before macrophage internalization. Despite respirability impaired by powder cohesiveness, surface mannosylation provided quicker macrophage phagocytosis, giving evidence of an active targeting promotion.

2014 - Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis [Articolo su rivista]
Maretti, Eleonora; Rossi, Tiziana; Bondi, Moreno; Croce, Maria Antonietta; Hanuskova, Miriam; Leo, Eliana Grazia; Sacchetti, Francesca; Iannuccelli, Valentina
abstract

The goal of the work was to evaluate an anti-tubercular strategy based on breathable Solid Lipid Microparticles (SLM) to target alveolar macrophages and to increase the effectiveness of the conventional tuberculosis (TB) therapy. Rifampicin loaded SLM composed of stearic acid and sodium taurocholate were characterized for aerodynamic diameter, surface charge, physical state of the components, drug loading and release as well as drug biological activity on Bacillus subtilis strain. Moreover, SLM cytotoxicity and cell internalization ability were evaluated on murine macrophages J774 cell lines by MTT test, cytofluorimetry and confocal laser microscopy. SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium, negative charged surface able to promote uptake by the macrophages and preserved drug antimicrobial activity. The negligible in vitro release of rifampicin indicated the capacity of the microparticle matrix to entrap the drug preventing its spreading over the lung fluid. In vitro studies on J774 cell lines demonstrated SLM non-cytotoxicity and ability to be taken up by cell cytoplasm. The microparticulate carrier, showing features suitable for the inhaled therapy and for inducing endocytosis by alveolar macrophages, could be considered promising in a perspective of an efficacious TB inhaled therapy by means of a Dry Powder Inhaler device.

2013 - Inhalated drug delivery systems to target alveolar macrophages for tuberculosis therapy: design of safe SLM loaded with rifampicin [Abstract in Atti di Convegno]
Maretti, Eleonora; Iannuccelli, Valentina; Leo, Eliana Grazia; Bondi, Moreno; Croce, Maria Antonietta; Sacchetti, Francesca; Rossi, Tiziana
abstract

The present research aimed to improve the effectiveness of TB treatment by a non conventional therapy and using teh respiratory tract as a novel adminictration route for rifampicin. The study dealt with the design of Solid Lipid Microparticles (SLM) to be delivered by a Dry Powder Inhaler (DPI) device and to target the alveolar macrophages. The negligible in vitro drug release indicated the capacity of the matrix to firmly entrap the drug. Rifampicin maintained its biological activity during the preparation steps. Moreover, SLM were suitable to be taken up by murine J774 cells.

2013 - Unravelling genomic diversity of Zygosaccharomyces rouxii complex with a link to its life cycle [Articolo su rivista]
Solieri, Lisa; T., Chand Dakal; Croce, Maria Antonietta; Giudici, Paolo
abstract

Zygosaccharomyces rouxii and the related species Zygosaccharomyces sapae (hereafter referred to as Z. rouxii complex) are protoploid hemiascomycete yeasts relevant in the elaboration and spoilage of foodstuff. Divergence of Z. rouxii complex before whole genome duplication, leading to the genus Saccharomyces, makes these yeasts very attractive for genome evolution study. Relatively little is known, however, about the diversity in this branch at the genetic and physiological levels. In this work, we investigated Z. rouxii complex, encompassing strains that in other works have been studied separately and comparing them in a comprehensive way. We showed that the majority of strains are unusually heterogeneous in their ribosomal DNA, a signal of relaxation of concerted evolution. Further analysis showed that they have hypervariable karyotypes, different levels of ploidy, and that housekeeping markers vary both in copy number and sequence. Overall, the results provide compelling evidence that the strains considered in this study are a complex of haploid, aneuploid and diploid mosaic lineages. The reproductive mode and life cycle of Zygosaccharomyces could lead to this unsuspected diversity

2009 - BIOCOMPATIBILITY OF COLLAGEN MEMBRANES ASSESSED BY CULTURING HUMAN J111 MACROPHAGES CELLS. [Articolo su rivista]
Aruta, CLAUDIA GAETANA; Croce, Maria Antonietta; Quaglino, Daniela; Guerra, Deanna; Tiozzo, Roberta
abstract

We have carried out an in vitro study on the interactions of human macrophages (J111) with two different membranes made of collagen type I and II, isolated from horse tendon and from horse articular and trachea cartilagene in order to obtain data on their biocompatibility. We have described the morphology of cell seeded on collagen films, and we have evaluated their proliferation as well as cytokine production as indicator of macrophage activation. The inflammatory response may in fact induce the destruction of collagen membranes and may interfere with cell and tissue behaviour. Results might be relevant for in vivo application such ad “tissue engeneering” and /or specialized cells implantation.

2009 - Collagen modified based membranes for tissue engineering: influence of type and molecular weight of GAGs on cell proliferation [Articolo su rivista]
Ruozi, Barbara; B., Parma; Croce, Maria Antonietta; Tosi, Giovanni; Bondioli, Lucia; S., Vismara; Forni, Flavio; Vandelli, Maria Angela
abstract

This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes.Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to growth and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.

2008 - Biocompatibility of various root canal filling materilas ex vivo [Articolo su rivista]
R., Scotti; Tiozzo, Roberta; C., Parisi; Croce, Maria Antonietta; P., Baldissara
abstract

AIM: To evaluate the biocompatibility of a resin-based endodontic filler (RealSeal) using the indirect cytotoxicity test. METHODOLOGY: Human gingival fibroblasts were cultured ex vivo. Pellets of the materials to be tested were incubated for 24, 48, and 72 h at 37 degrees C under sterile conditions to obtain their eluates. The fibroblasts were exposed to either diluted (50%) or undiluted eluates for 24 h. A culture medium with foetal calf serum was added to the control wells. Cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The data concerning cell viability were statistically analyzed using one-way anova test and Bonferroni multiple comparisons test. Results: Eluates obtained after 24 h of incubation with the resin filler did not reduce cellular viability. An increase in cellular viability, as compared with control cells, was observed in the gutta-percha group. The undiluted eluate from the polyether material was cytotoxic, causing an 82 +/- 4% decrease in cellular viability. Eluates obtained after 48 h of incubation with the resin filler increased cellular viability, whereas the polyether significantly reduced viability. Gutta-percha did not cause any detectable change. After 72 h of incubation the eluate of the resin filler caused an increase in cellular viability, as did gutta-percha, whereas polyether caused a significant decrease. CONCLUSIONS: RealSeal resin filler was nontoxic in this laboratory model. Further investigations are necessary to verify its usefulness in clinical applications.

2008 - Genome size and ploidy level: New insights for elucidating relationships in Zygosaccharomyces species [Articolo su rivista]
Solieri, Lisa; Cassanelli, Stefano; Croce, Ma; Giudici, Paolo
abstract

Ploidy is a fundamental genetic trait with important physiological and genomic implications. We applied complementary molecular tools to highlight differences in genome size and ploidy between Zygosaccharomyces rouxii strain CBS 732T and other related wild strains (ATCC 42981, ABT 301, and ABT 601). The cell cycle analysis by flow cytometry revealed a genome size of 12.7 ± 0.2 Mb for strain CBS 732T, 21.9 ± 0.2 Mb for ATCC 42981, 28.1 ± 1.3 Mb for ABT 301, and 39.00 ± 0.3 Mb for ABT 601. Moreover, karyotyping analysis showed a high variability, with wild strains having a higher number of chromosomal bands than CBS 732T. The ploidy level was assessed comparing genome size from flow cytometry with the average haploid size from electrophoretic karyotyping. Strain CBS 732T showed an haploid DNA content, whereas the wild strains a diploid DNA content. In addition gene probe-chromosome hybridization targeted to ZSOD genes showed that wild strains with a diploid DNA content have two ZSOD copies located on different chromosomes.

2007 - Tropoelastin deposition is affected by addition of heparan sulphate. [Abstract in Rivista]
Guidetti, Rita; Croce, Maria Antonietta; Gheduzzi, Dealba; B., Parma; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela
abstract

The interactions between tropoelastin and heparan sulphate are described and their role in elastic fiber formation are discussed

2006 - 17 Beta-estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell cultures [Articolo su rivista]
M., Ricchi; Bertolotti, Marco; C., Anzivino; Carulli, Lucia; I., Canedi; Ml, Bormioli; Tiozzo, Roberta; Croce, Maria Antonietta; A., Lonardo; N., Carulli; P., Loria
abstract

Background: Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17 beta-estradiol (E-2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. Methods:HepG2 cells were exposed for 24 h to DCA (350 mu mol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 mu mol/L), or E-2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 mu mol/L) or mixtures of E-2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. Results:High concentrations of E-2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E-2 (P < 0.01) in physiological (1-10 nmol/L) doses. The cytoprotective effects of E-2 and UDCA was also observed in the WRL-68 cell line. Conclusions: 17 beta-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E-2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification.

2006 - Monitoring cell-cycle-related viscoelasticity by a quartz crystal microbalance [Articolo su rivista]
Alessandrini, Andrea; Croce, Maria Antonietta; Tiozzo, Roberta; Facci, P.
abstract

We have monitored viscoelasticity variation of a cell population during the cell cycle by a Quartz Crystal Microbalance (QCM). Balb 3T3 fibroblasts were synchronized in the G0/G1 phase and seeded in a QCM chamber placed in a cell incubator. After cell sedimentation, the frequency signal was characterized by an amplitude modulation attributed to the viscoelasticity variation of the cells proliferating in phase. A control experiment with nonsynchronized cells showed a similar signal trend, but without significant modulation. Interestingly, the system resulted also to perform as a device sensitive to the effect of drugs affecting the cell cycle, such as colchicine.

2005 - Dermal fibroblasts from pseudoxanthoma elasticum patients have raised MMP-2 degradative potential [Articolo su rivista]
Quaglino, Daniela; Sartor, L; Garbisa, S; Boraldi, Federica; Croce, Maria Antonietta; Passi, A; De Luca, G; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Cultured fibroblasts from the dermis of normal subjects and of Pseudoxanthoma elasticum (PXE) patients were analysed for enzyme activity, protein and mRNA expression of metalloproteases (MMP-2, MMP-3, MMP-9, MT1-MMP) and of their specific inhibitors (TIMP-1, TIMP-2 and TIMP-3). MMP-3, MMP-9 and TIMP-3 mRNAs and proteins failed to be detected in both the medium and the cell layer of both controls and PXE patients. MMP-2 mRNA was significantly more expressed in PXE than in control cell lines, whereas MT1-MMP, TIMP-1 and TIMP-2 mRNAs appeared unchanged. MMP-2 was significantly higher in the cell extracts from PXE fibroblasts than in control cells, whereas differences were negligible in the cell medium. Data suggest that PXE fibroblasts have an increased proteolytic potential, and that MMP-2 may actively contribute to connective tissue alterations in this genetic disorder.

2005 - On the pathogenesis of PXE-like clinical manifestations in Beta-thalassemic patients [Abstract in Rivista]
Boraldi, Federica; PAOLINELLI DEVINCENZI, Chiara; M. I., Garcia Fernandez; Quaglino, Daniela; Tiozzo, Roberta; Croce, Maria Antonietta; P., Cianciulli; F., Sorrentino; G. L., Forni; Ronchetti, Ivonne
abstract

Altered fibroblast phenotype and membrane transport properties are described in patients affected by beta-thalassemia with elastic fiber mineralization and PXE-like clinical manifestations.

2004 - Adhesion and proliferation of human dermal fibroblasts on collagen matrix [Articolo su rivista]
Croce, Maria Antonietta; Silvestri, C; Guerra, Deanna; Carnevali, E; Boraldi, Federica; Tiozzo, Roberta; Parma, B.
abstract

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.

2004 - Functional genomics: the PXE model [Abstract in Atti di Convegno]
Ronchetti, Ivonne; Quaglino, Daniela; Gheduzzi, Dealba; Croce, Maria Antonietta; Boraldi, Federica; Tiozzo, Roberta
abstract

Pseudoxanthoma elasticum represent a disease model for investigating the relationships bettween altered fibroblast phenotype and elastic fiber degeneration/calcification.

2004 - The skin equivalent model: developments and perspectives. [Articolo su rivista]
Croce, Maria Antonietta; Sammarco, Rita; PAOLINELLI DEVINCENZI, Chiara; Boraldi, Federica; Gheduzzi, Dealba; Damour, O.; Sommer, P.; Ronchetti, Ivonne; Tiozzo, Roberta; Quaglino, Daniela
abstract

Three-dimensional culture systems have been developed to mimic natural interactions among cells and between cells and the extracellular matrix. The “skin equivalent ” is a 3-D co-culture system of fibroblasts and keratinocytes used as a covering surface in extended wounds, and as a model for studying the influence of each cell type on the synthesis of the extracellular matrix. In the present study, a three-dimensional scaffold made of chitosan, glycosaminoglycans and collagen has been colonised by keratinocytes from newborn foreskin an by fibroblasts isolated from donors of different ages. In the skin equivalent, the neo-synthesized connective tissue is characterized by the presence of amorphous elastic fibers, strengthening the usefulness of this three-dimensional model as a powerful tool for investigating connective tissue metabolism in different physiological (i.e. aging) and pathological conditions. Interestingly, when fibroblasts isolated from aged donors were cultured in vitro, even though in the presence of abundant nutrients and growth factors, their old phenotype appeared not to be completely overcome, indicating that aging is the result of a continuous remodelling controlled by epigenetic as well as genetic factors. Finally, the aged phenotype of fibroblasts seems to exert a stronger influence on keratinocytes compared to that of keratinocytes on fibroblasts.

2003 - Cell-matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan [Articolo su rivista]
Boraldi, Federica; Croce, Maria Antonietta; Quaglino, Daniela; Sammarco, Rita; Carnevali, Elena; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell-matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5 x 10(5) to 2.7 x 10(6) Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell-substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type 1-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast-collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling. (C) 2003 Elsevier Science Ltd. All rights reserved.

2003 - Hyaluronan uptake by adult human skin fibroblasts in vitro [Articolo su rivista]
Croce, M. A.; Boraldi, F.; Quaglino, D.; Tiozzo, R.; Pasquali-Ronchetti, I.
abstract

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

2003 - Multidrug resistance protein-6 (MRP6) in human dermal fibroblasts. Comparison between cells from normal subjects and from Pseudoxanthoma elasticum patients [Articolo su rivista]
Boraldi, Federica; Quaglino, Daniela; Croce, Maria Antonietta; M. I. G., Fernandez; Tiozzo, Roberta; Gheduzzi, Dealba; Bacchelli, Barbara; Ronchetti, Ivonne
abstract

Multidrug resistance protein-6 (MRP6) is a membrane transporter whose deficiency leads to the connective tissue disorder Pseudoxanthoma elasticum (PXE). In vitro dermal fibroblasts from normal and PXE subjects, homozygous for the R1141X mutation, were compared for their ability to accumulate and to release fluorescent calcein, in the absence and in the presence of inhibitors and competitors of the MDR-multidrug resistance protein (MRP) systems, such as 3-(3-(2-(7-choro-2 quinolinyl) ethenyl)phenyl ((3-dimethyl amino-3-oxo-propyl)thio) methyl) propanoic acid (MK571), verapamil (VPL), vinblastine (VBL), chlorambucil (CHB), benzbromarone (BNZ) and indomethacin (IDM). In the absence of chemicals, calcein accumulation was significantly higher and the release significantly slower in PXE cells compared to controls. VBL and CHB reduced calcein release in both cell strains, without affecting the differences between PXE and control fibroblasts. VPL, BNZ and IDM consistently delayed calcein release from both control and PXE cells; moreover, they abolished the differences between normal and MRP6-deficient fibroblasts observed in the absence of chemicals. These findings suggest that VPL, BNZ and IDM interfere with MRP6-dependent calcein extrusion in in vitro human normal fibroblasts. Interestingly, MK571 almost completely abolished calcein release from PXE cells, whereas it induced a strong but less complete inhibition in control fibroblasts, suggesting that MRP6 is not inhibited by MK571. Data show that MRP6 is active in human fibroblasts, and that its sensitivity to inhibitors and competitors of MDR-MRPs' membrane transporters is different from that of other translocators, namely, MRP1. It could be suggested that MRP1 and MRP6 transport different physiological substances and that MRP6 deficiency cannot be overcome by other membrane transporters, at least in fibroblasts. These data further support the hypothesis that MRP6 deficiency may be relevant for fibroblast metabolism and responsible for the metabolic alterations of these cells at the basis of connective tissue clinical manifestations of PXE. (C) 2003 Elsevier B.V./Intemational Society of Matrix Biology. All rights reserved.

2003 - Study of the potential citotoxicity of dental impression materials. [Relazione in Atti di Convegno]
Tiozzo, Roberta; F., Magagna; Boraldi, Federica; Croce, Maria Antonietta; Bortolini, Sergio; Consolo, Ugo
abstract

The aim of this study was to assess the cytotoxicity of tow types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods.

2001 - Hyaluronan affects protein and collagen synthesis by in vitro human skin fibroblasts [Articolo su rivista]
Croce, Maria Antonietta; K., Dyne; Boraldi, Federica; Quaglino, Daniela; G., Cetta; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 muM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (- 40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.

2000 - Abnormal phenotype of in vitro dermal fibroblasts from patients with pseudoxanthoma elasticum (PXE) [Articolo su rivista]
Quaglino, Daniela; Boraldi, Federica; Barbieri, D; Croce, Maria Antonietta; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

Pseudoxanthoma elasticum (PXE) is a genetic connective tissue disease, whose gene and pathogenesis are still unknown. Dermal fibroblasts from patients affected by PXE have been compared in vitro with fibroblasts taken from sex and age-matched normal individuals. Cells were grown and investigated in monolayer, into three-dimensional collagen gels and in suspension. Compared with normal cells, PXE fibroblasts cultured in monolayer entered more rapidly within the S phase and exhibited an increased proliferation index; on the contrary, similarly to normal fibroblasts, PXE cells did not grow in suspension. Furthermore, compared with normal fibroblasts, PXE cells exhibited lower efficiency in retracting collagen type I lattices and lower adhesion properties to collagen type I and to plasma fibronectin. This behavior was associated with higher expression of integrin subunits alpha 2, alpha 5, alpha v, whereas beta 1 subunit as well as alpha 2 beta 1 and alpha 5 beta 1 integrin expression was lower than in controls. Compared to controls, PXE fibroblasts had higher CAM protein expression in accordance with their high tendency to form cellular aggregates, when kept in suspension. The demonstration that PXE fibroblasts have altered cell-cell and cell-matrix interactions, associated with modified proliferation capabilities, is consistent with the hypothesis that the gene responsible for PXE might have a broad regulatory role on the cellular machinery. (C) 2000 Elsevier Science B.V. All rights reserved.

1997 - Cell behaviour and cell-matrix interactions of human palmar aponeurotic cells in vitro [Articolo su rivista]
Quaglino, Daniela; G., Bergamini; Croce, Maria Antonietta; Boraldi, Federica; D., Barbieri; Caroli, Alessandro; A., Marcuzzi; Tiozzo, Roberta; Ronchetti, Ivonne
abstract

The present investigation has been performed to better characterize, in vitro, normal aponeurotic cells in comparison with dermal fibroblasts and with cells derived from Dupuytren´s affected aponeuroses. Cells were cultured in monolayer and/or into three-dimensional collagen gels. Cell structure, adhesion, and spreading capability on different substrates, as well as integrin expression were investigated by light and electron microscopy and by flow cytometry. Cell-matrix interactions were also analyzed by gel retraction experiments in the presence, or absence, of RGD peptides and anti-integrin antibodies. Normal aponeurotic cells, compared with dermal fibroblasts, exhibited in vitro peculiar structural features, which were substantially maintained in Dupuytren´s aponeurotic cells, irrespective of the substrate they were grown on. By contrast, the aponeurotic cell behavior was different in normal and diseased cells, these latter approaching that of dermal fibroblasts. Normal aponeurotic cells, in fact, were characterized by low efficiency in retracting the collagen gel, low alpha(2), alpha(1) and alpha(5) integrin subunit expression and low adhesion properties onto collagen and fibronectin, whereas cells isolated from the aponeuroses of Dupuytren´s patients exhibited higher capability of retracting the collagen gel, increased adhesion properties toward collagen and fibronectin, and higher levels of integrin expression. No differences were observed between dermal fibroblasts from Dupuytren´s patients or from normal subjects. These in vitro results are consistent with those previously obtained in situ, suggesting that palmar aponeurotic cells have a peculiar phenotype and that changes in cell-matrix interactions occur in Dupuytren´s contracture. Moreover, by comparing data obtained from the retracted fibrotic cords and the still clinically unaffected aponeuroses of the same patients, it may be noted that Dupuytren´s disease is not only confined to the clinically involved branches, but includes the whole aponeurosis of the affected hand.