Biomedical subjects

L Visai

Publications and source records attributed to L Visai.

16 recordsLinked to original sources

PubMed2006

Antibacterial activity of zinc modified titanium oxide surface.

Titanium-based implants are successfully used for various biomedical applications. However, in some cases, e.g. in dental implants, failures due to bacterial colonization are reported. Surface modification is a commonly proposed strategy to prevent infections. In this work, titanium oxide, naturally occurring on the surface of titanium, was modified by promoting the formation of a mixed titanium and zinc oxide, on the basis of the idea that zinc oxide on titanium surface may act as the zinc oxide used in pharmaceutical formulation for its lenitive and antibacterial effects. The present work shows that it is possible to form a mixed titanium and zinc oxide on titanium surfaces, as shown by Scanning Electron Microscopy and XPS analysis. To this end titanium was preactivated by UV on crystalline titanium oxide, both in the anatase form or in the co-presence of anatase and rutile. By performing antibacterial assays, we provide evidence of a significant reduction in the viability of five streptococcal oral strains on titanium oxide surfaces modified with zinc. In conclusion, this type of chemical modification of titanium oxide surfaces with zinc might be considered a new way to reduce the risk of bacterial colonization, increasing the lifetime of dental system applications.

Anti-Bacterial Agents↗

PubMed2005

Decreased bacterial adhesion to surface-treated titanium.

Osteointegrative dental implants are widely used in implantology for their well-known excellent performance once implanted in the host. Remarkable bacterial colonization along the transgingival region may result in a progressive loss of adhesion at gum-implant interface and an increase of the bone area exposed to pathogens. This phenomenon may negatively effect the osteointegration process and cause, in the most severe cases, implant failure. The presence of bacteria at implant site affect the growth of new bone tissue and consequently, the achievement of a mechanically stable bone-implant interface, key parameters for a suitable implant osteointegration. In the present work, a novel surface treatment has been developed and optimized in order to convert the amorphous titanium oxide in a crystalline layer enriched in anatase capable of providing not only antibacterial properties but also of stimulating the precipitation of apatite when placed in simulated body fluid. The collected data have shown that the tested treatment results in a crystalline anatase-type titanium oxide layer able to provide a remarkable decrease in bacterial attachment without negatively effecting cell metabolic activity. In conclusion, the surface modification treatment analyzed in the present study might be an elegant way to reduce the risk of bacterial adhesion and increase the lifetime of the transgingival component in the osteointegrated dental implant.

Animals↗

PubMed2000

Monoclonal antibodies to CNA, a collagen-binding microbial surface component recognizing adhesive matrix molecules, detach Staphylococcus aureus from a collagen substrate.

Previous studies showed that Staphylococcus aureus expresses a collagen-binding MSCRAMM (Microbial Surface Component Recognizing Adhesive Matrix Molecules), CNA, that is necessary and sufficient for S. aureus cells to adhere to cartilage and is a virulence factor in experimental septic arthritis. We have now used a monoclonal antibody (mAb) approach to further analyze the structure and function of CNA. 22 mAbs raised against the minimal ligand binding domain, CNA-(151-318), were shown to bind to the MSCRAMM with similar affinity. All mAbs appear to recognize conformation-dependent epitopes that were mapped throughout the CNA-(151-318) domain using a chimeric strategy where segments of CNA are grafted on ACE, a structurally related MSCRAMM from Enterococcus faecalis. These mAbs were able to inhibit (125)I-collagen binding to CNA-(151-318) as well as to intact S. aureus cells. They also interfered with the attachment of bacteria to collagen substrates. Furthermore, some of the mAbs could effectively displace (125)I-collagen bound to the bacteria. These displacing mAbs were also able to detach bacteria that had adhered to a collagen substrate in a preincubation, raising the possibility that some of the mAbs may be used as therapeutic agents.

Amino Acid Sequence↗

PubMed2000

Quantification of Staphylococcus aureus cell surface adhesins using flow cytometry.

The initiation of many infectious diseases involves specific adhesion of bacteria to host tissue proteins and carbohydrates. Staphylococcus aureus is known to bind specifically to several proteins in the extracellular matrix (ECM). We report the quantification of the collagen and fibronectin adhesin densities on the staphylococcal surface using flow cytometry. Our results are in agreement with previous reports on the transcription of the respective genes and demonstrate different patterns of temporal expression for the two adhesins in the strains studied. We demonstrate a convenient technique for quantification of bacterial adhesins that can be used in studies aimed at characterization of bacterial adhesion to ECM components and understanding expression of adhesins during the course of an infection.

Adhesins, Bacterial↗

PubMed2000

Novel poly(urethane-aminoamides): an in vitro study of the interaction with heparin.

In order to obtain heparin-binding polyurethanes, tertiary amino-groups have been introduced in the polymer backbone by attributing a key-role to the chain extender, i.e. substituting butanediol, commonly used in polyurethane synthesis, with a tailor-made diamino-diamide-diol. In this work a poly(ether-urethane-aminoamide) (PEU/PIME/al) was obtained with poly(oxytetramethylene) glycol 2000, 1,6-hexamethylene-diisocyanate and the new chain extender, in the molar ratio 1:2:1. The heparin binding capacity of PEU/PIME/al was evaluated with 125I labelled heparin, using for comparison the analogous polymer obtained with a diamide-diol (i.e. the poly(ether-urethane-amide) PEU/PIBLO/al), and two commercially available biomedical polyurethanes (Pellethane 2363 and Corethane). pH and ionic strength dependence of the heparin uptake were investigated by treating all the polyurethanes with solutions of 125I heparin into buffers from pH 4 to 9 or NaCl molarity from 0.0 to 1.0. The stability of the interaction with bound heparin was investigated by sequential washing treatments (PBS, 1 N NaOH, 2% SDS solution), then analysing the residual radioactivity on the materials. Results indicated that the heparin binding of PEU/PIME/al is significantly higher and more stable than that of the other polyurethanes, with a time-dependent kinetic. The interaction with heparin appears to be prevalently ionic, with the contribution of other electrostatic and hydrophobic interactions. Activated partial thromboplastin time (APTT), performed on human plasma with polyurethane-coated, heparinized test tubes, indicated that bound heparin maintains its biological activity after the adsorption.

Animals↗

PubMed1998

Heparin-binding domain of human fibronectin binds HIV-1 gp120/160 and reduces virus infectivity.

In vitro experiments indicate that components of the host present in body fluids may prevent the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells. Fibronectin (Fn), a dimeric 440-kDa extracellular matrix adhesion protein, is secreted by mesenchymal cells and assembled into insoluble matrices. Fn exerts important effects on cell growth and differentiation through a number of discrete functional domains. Several microorganisms are known to bind Fn. We show that, under physiological conditions, HIV-1 gp120 and gp160 are capable of binding plasma and cellular Fn as well as laminin and vitronectin. Experiments were set up to analyze in detail the binding of HIV gp120 and gp160 to Fn. The gp120 and gp160 specifically recognize the C-terminal heparin-binding domain of Fn (Fn-CTHBD) with a calculated KD of 2.8 x 10(-7) M for gp160. Binding of gp160 to Fn-CTHBD is a saturable and specific process that is blocked by antibodies to Fn-CTHBD and by heparin and is inhibited to a minor extent by heparan sulfate and dextran sulfate. These observations suggest that gp120/160 specifically recognize the III15 repeat within Fn-CTHBD. Intact Fn and Fn-CTHBD strongly inhibit the interaction of gp120/160 with soluble CD4 and, under low serum conditions, are capable of neutralizing the infectivity of HIV-1 for CD4-positive T cells. Thus, Fn that is present in plasma and mucinous secretions may well affect HIV infectivity and virus distribution in vivo.

Antibodies↗

PubMed1998

Antibody response to fibronectin-binding adhesin FnbpA in patients with Staphylococcus aureus infections.

We have analyzed antibody reactivity to a fibronectin-binding microbial surface component that recognizes adhesive matrix molecules (MSCRAMM) in blood plasma collected from patients with staphylococcal infections. All patients had elevated levels of anti-MSCRAMM antibodies compared to those of young children who, presumably, had not been exposed to staphylococcal infections. The anti-MSCRAMM antibodies preferentially reacted with the ligand-binding repeat domain of the adhesin. However, these antibodies did not inhibit fibronectin binding. Essentially, all patients had antibodies which specifically recognized the fibronectin-MSCRAMM complex but not the isolated components. Epitopes recognized by these anti-ligand-induced binding sites antibodies were found in each repeat unit of the MSCRAMM. These results demonstrate that staphylococci have bound fibronectin some time during infection and that each repeat unit in the MSCRAMM can engage in ligand binding. Furthermore, our previously proposed model, suggesting that an unordered structure in the MSCRAMM undergoes a conformational change upon ligand binding (K. House-Pompeo, Y. Xu, D. Joh, P. Speziale, and M. Höök, J. Biol. Chem. 271:1379-1384, 1996), is presumably operational in patients during infections.

Adhesins, Bacterial↗

PubMed1996

A monoclonal antibody enhances ligand binding of fibronectin MSCRAMM (adhesin) from Streptococcus dysgalactiae.

A monoclonal antibody 3A10, generated from a mouse immunized with the Streptococcus dysgalactiae fibronectin (Fn) binding protein FnbA, was isolated, and its effect on ligand binding by the antigen was examined. The epitope for 3A10 was localized to a previously unidentified Fn binding motif (designated An) just N-terminal of the repeat domain which represents the primary ligand binding site on FnbA. Fn binding to Au was enhanced by 3A10 rather than inhibited. This effect was demonstrated in two different assays. First, in the presence of 3A10 the Au-containing proteins and synthetic peptide more effectively competed with bacterial cells for binding to Fn. Second, 3A10 dramatically increased the binding of biotin-labeled forms of the Au-containing proteins to Fn immobilized on a blotting membrane. Pure 3A10 IgG did not recognize the antigen by itself, and Fn was required for the immunological interaction between the antibody and the epitope. This induction effect of Fn was shown in both Western blot and enzyme-linked immunosorbent assay in which immobilized Au-containing molecules were probed with 3A10 in the presence of varying concentrations of Fn. Specificity analyses of 3A10 revealed that the monoclonal also recognized a ligand binding motif in a Streptococcus pyogenes Fn binding MSCRAMM but not the corresponding motifs in two related adhesins from Staphylococcus aureus and S. dysgalactiae. Furthermore, 3A10 stimulated Fn binding by S. pyogenes cells. These results together with subsequent biophysical studies presented in the accompanying paper (House-Pomepeo, K., Xu, Y., Joh, D., Speziale, P., and Höök, M. (1996) J. Biol. Chem. 271, 1379-1384) indicate that the ligand binding sites of Fn binding MSCRAMMs have little or no secondary structure. However, on binding to Fn, they appear to undergo a structural rearrangement resulting in a defined structure rich in beta sheet and expressing a ligand-induced binding site for antibodies such as 3A10.

Adhesins, Bacterial↗

PubMed1995

Isolation and characterization of a novel collagen-binding protein from Streptococcus pyogenes strain 6414.

In this report we have analyzed the binding of collagen to Streptococcus pyogenes strain 6414. This binding was rapid, specific, and involved a limited number of receptor molecules (11,600 copies per cell). When the proteins in a streptococcal lysate were blotted onto a nitrocellulose filter and probed with 125I-labeled collagen, a prominent collagen-binding protein of 57 kDa was identified as well as minor 130-150-kDa components. The major 57-kDa protein was isolated by affinity chromatography on collagen-Sepharose followed by gel filtration chromatography. The 57-kDa protein purified from S. pyogenes was used to raise a monospecific antibody which also reacted with a collagen-binding protein of similar molecular size isolated from Streptococcus zooepidemicus. The two collagen-binding proteins from streptococci have a similar amino acid composition and isoelectric points. Isolated collagen-binding protein was specifically recognized by 125I-collagen in a solid-phase binding assay and displayed an affinity for the ligand quite similar to that exhibited by intact bacteria (Kd = 3.1 versus 3.5 x 10(-9) M, respectively). Surface-labeled bacteria attached to microtiter wells coated with different collagen types and the 57-kDa protein blocked the adhesion to collagen substrate. We propose that the 57-kDa protein is an adhesin involved in the attachment of streptococci to host tissues.

Amino Acid Sequence↗

PubMed1992

Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.

The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.

Binding Sites↗

PubMed1992

Cloning and expression of two different genes from Streptococcus dysgalactiae encoding fibronectin receptors.

Binding of bacteria to fibronectin has been implicated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcus dysgalactiae to fibronectin. The cells bind to a site in the NH2-terminal domain of the protein via trypsin-sensitive cell surface components. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding proteins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with 125I-labeled fibronectin, a 140-kDa fibronectin-binding protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibronectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction mapping and cross-hybridization experiments. The two encoded proteins were also immunologically distinct although both bound to the same region of the fibronectin molecule, and both effectively inhibited the binding of 125I-fibronectin to bacterial cells. Immunological analyses showed that only one of the two proteins tentatively identified as fibronectin receptors was expressed in detectable quantities in the Streptococcus dysgalactiae strain under the culture conditions employed.

Bacterial Adhesion↗

PubMed1991

Binding sites in fibronectin for an enterotoxigenic strain of E. coli B342289c.

The binding of fibronectin and fibronectin fragments to the enterotoxigenic strain E. coli B34289c was studied. E. coli cells bound to two distinct sites of fibronectin, one being the N-terminal domain, which also contains the binding sites for staphylococci and streptococci, and the other located within the central heparin binding region. In addition, the N-terminal and the heparin binding domain mediated the attachment of bacteria in a solid phase binding assay. E. coli cells expressed two classes of receptors, the first, a 17 kDa protein, recognized by the N-terminal fragment and the second, having a mol. mass of 55 kDa, which interacts with the internal heparin binding domain. Bacterial receptors, which bind the N-terminal end of fibronectin, may be structurally related.

Bacterial Adhesion↗

PubMed1990

Binding of collagens to an enterotoxigenic strain of Escherichia coli.

An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity (G. Fröman, L. M. Switalski, A. Faris, T. Wadström, and M. Höök, J. Biol. Chem. 259:14899-14905, 1984). We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides [alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4] were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure.

Binding Sites↗

PubMed1986

Binding of collagen to Staphylococcus aureus Cowan 1.

Collagen binds to a receptor protein present on the surfaces of Staphylococcus aureus cells. Binding of 125I-labeled type II collagen to its bacterial receptor is reversible, and Scatchard plot analysis indicates the presence of one class of receptor that occurs on an average of 3 X 10(4) copies per cell and binds type II collagen with a Kd of 10(-7) M. Studies on the specificity of collagen cell binding indicate that the receptor does not recognize noncollagenous proteins but binds all of the different collagen types tested (types I to VI). Furthermore, isolated collagen alpha chains and peptides generated by cyanogen bromide cleavage of type I collagen alpha chains are recognized by the receptor as indicated by the ability of these polypeptides to inhibit binding of 125I-labeled type II collagen to staphylococcal cells. Synthetic collagen analogs were tested as inhibitors of type II collagen binding to bacterial cells. The peptides (Pro-Gly-Pro)n, (Pro-Pro-Gly)10, and (Pro-OH-Pro-Gly)10 were recognized by the receptor, whereas the peptides (Pro-Ala-Gly)n and polyproline showed no inhibitory activity.

Collagen↗

PubMedDate not supplied

Calcified matrix production by SAOS-2 cells inside a polyurethane porous scaffold, using a perfusion bioreactor.

The repair and regeneration of damaged or resected bone are problematic. Bone autografts show optimal skeletal incorporation, but often bring about complications. Hence, there is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic cross-linked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells within a porous scaffold. The polyurethane porous scaffold had an average pore diameter of 624 microm. Using a perfusion bioreactor, we investigated the effect of shear stress on SAOS-2 human osteoblast proliferation and calcified matrix production. The physical, morphological, and compressive properties of the polyurethane foam were characterized. At a scaffold perfusion rate of 3 mL/min, in comparison with static conditions without perfusion, we observed 33% higher cell proliferation; higher secretion of osteopontin, osteocalcin, decorin, and type I collagen (9.16-fold, 71.9-fold, 30.6-fold, and 18.12-fold, respectively); and 10-fold increased calcium deposition. The design of the bioreactor and the design of the polyurethane foam aimed at obtaining cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.

Biocompatible Materials↗

PubMedDate not supplied

Stimulation of osteoblast growth by an electromagnetic field in a model of bone-like construct.

The histogenesis of bone tissue is strongly influenced by physical forces, including magnetic fields. Recent advances in tissue engineering has permitted the generation of three dimensional bone-like constructs. We have investigated the effects of electromagnetic stimulation on human osteoblast cells grown in a hydrophobic polyurethane scaffold. Bone-like constructs were stimulated by pulsed electromagnetic fields in a bioreactor. Proliferation, bone protein expression and calcified matrix production by osteoblasts were measured using histochemical methods. In stimulated cultures, the number of cells was significantly higher compared to static (control) cultures. In both stimulated and control cultures, cells were immunoreactive to osteoblast markers, including type-I collagen, osteocalcin and osteopontin, thus suggesting that the expression of bone-related markers was maintained throughout the in vitro experiments. Morphometric analysis of von Kossa-stained sections revealed that stimulation with electromagnetic field significantly increased matrix calcification. The data lend support to the view that the application of a magnetic field can be used to stimulate cell growth in bone-like constructs in vitro. This finding may be of interest for the production of biomaterials designed for clinical applications.

Animals↗