Self-assembling peptide-enriched electrospun polycaprolactone scaffolds promote the h-osteoblast adhesion and modulate differentiation-associated gene expression

Electrospun polycaprolactone (PCL) is able to support the adhesion and growth of h-osteoblasts and to delay their degradation rate to a greater extent with respect to other polyesters. The drawbacks linked to its employment in regenerative medicine arise fromits hydrophobic nature and the lack of biochemical signals linked to it. This work reports on the attempt to add five different self-assembling (SA) peptides to PCL solutions before electrospinning. The hybrid scaffolds obtained had regular fibers (SEM analysis) whose diameters were similar to those of the extracellularmatrix, more stable hydrophilic (contact angle measurement) surfaces, and anamorphous phase constrained by peptides (DSC analysis). They appeared to have a notable capacity to promote the h-osteoblast adhesion and differentiation process by increasing the gene expression of alkaline phosphatase, bone sialoprotein, and osteopontin. Adding an Arg-Gly-Asp (RGD) motif to a self-assembling sequence was found to enhance cell adhesion, while the same motif condensed with a scrambled sequence did not, indicating that there is a cooperative effect between RGD and 3D architecture created by the self-assembling peptides. The study demonstrates that self-assembling peptide scaffolds are still able to promote beneficial effects on h-osteoblasts even after they have been included in electrospun polycaprolactone. The possibility of linking biochemical messages to self-assembling peptides could lead the way to a 3D decoration of fibrous scaffolds

Hydraulic zonation of the lagoons of Marano and Grado, Italy. A modelling approach

The hydraulic regime-based zonation scheme of the Lagoons of Marano and Grado (Italy) has been derived by means of numerical models. A finite element modelling system has been used to describe the water circulation taking in account different forces such as tide, wind and rivers. The model has been validated by comparing the simulation results against measured water levels, salinity and water temperature data collected in several stations inside the lagoons. The analysis of water circulation, salinity and spatial distribution of passive tracers released at the inlets, led to a physically-based division of the lagoons system into six subbasins. The derived classification scheme is of crucial value for understanding the renewal capacity and pollutants distribution patterns in the lagoon

Specific Involvement of Pilus Type 2a in Biofilm Formation in Group B Streptococcus

Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells

Connectivity between coastal lagoons and sea: asymmetrical effects on assemblages' and populations' structure

Connectivity among marine populations plays a fundamental role in the dynamic of metapopulations and communities. Moreover, genetic connectivity is important for the evolutionary history and adaptive capability of species while demographic connectivity is essential to maintain ecological processes. In coastal lagoons, isolation degree or confinement is considered the main factor structuring biological assemblages. These environments also function as nursery areas for many marine species that colonize the lagoons as larvae or juveniles, returning to the sea for reproduction. It is therefore essential to know the connectivity between lagoons and sea for the management of biodiversity and the exploitation of coastal living resources. This work anases the role that connectivity between coastal lagoons and sea plays in the assemblages and subpopulations structure of the first. To this purpose, a finite element hydrodynamic model was used coupled with a lagrangian module to simulate the potential exchange of organisms between Mar Menor lagoon (Western Mediterranean) and the adjacent sea. Connectivity parameters from 40 stations, located inside and outside the Mar Menor, have been estimated. The outcomes of the eight simulations carried out were compared with field data, including ichthyoplankton species composition and genetic fluxes in 6 species with different life stories. The results suggest that pelagic larval phases are longer than expected or the species can extend PLD within certain limits if the conditions for the settlement are not adequate. Repetition can improve the chance of self-recruitment, coupling larval duration, competency period and finding the adequate location for settlement. The results also show that connectivity between all lagoon and Mediterranean stations is very low and is independent of geographical distance. There is a strong asymmetry in the probability of receiving particles, being lower the probability of colonization of the lagoon stations from the sea than vice versa. Despite its low values, connectivity can explain up to 65% of the similarities in species composition of the ichthyoplankton and between 30 and 96% of the variance in genetic differentiation of the studied species. The low value of connectivity and colonization rates is enough, however, to maintain the genetic fluxes between populations and, at the same time, restricted connectivity can play an important role in maintaining high diversity and heterogeneous assemblage structure

Peptidi derivati da fattori endogeni ad attività pro- ed anti-angiogenica: sintesi, caratterizzazione conformazionale e saggi biologici in vitro e in vivo

Angiogenesis, the formation of new capillaries, is among the key events in some physiological processes, like organ growth and development, wound healing and reproduction. However, an impairment of this process is also a hallmark of different pathologies, such as chronic inflammation, ischemic diseases, tumor growth and metastasis. Angiogenesis is a complex process involving extensive interplay among cells, soluble factors and extracellular matrix. Physiologically, the body controls angiogenesis through a series of "on" and "off" regulatory switches, maintaining a perfect balance of positive and negative angiogenesis modulators. Normally, endothelium exists in a quiescent state ("turned off"); angiogenic stimuli, including hypoxia, inflammation, and mechanical factors, activate endothelial cells (ECs), by initiating the autocrine or paracrine production and release of growth factors or cytokine. Uncontrolled growth of vessels, due to a balance breakdown, may lead to angiogenesis-dependent diseases. So, the regulation of both angiogenesis stimulators and inhibitors, can represent a promising target for therapy and drug development. Among endogenous modulators, the chemokine Stromal Derived-Factor 1 (SDF-1), and the small polypeptide Thymosin ß4 (Tß4), were found to play a crucial role in vascular biology, stimulating proliferation, migration and differentiation of ECs. The above mentioned events are also mediated by cell adhesion molecule. Compound containing adhesive sequences, such as RGD motif and heparin-binding domain, can be used either in tissue engineering as adhesion factors, or as anti-angiogenic agents, through a competition with extracellular matrix components for the binding to cell surface. This work focused on i) design and solid phase synthesis of peptide sequences contained in SDF-1, Tß4 and ECM protein; ii) conformational studies and iii) evaluation in vitro and in vivo of their angiogenic effects on human umbilical vein endothelial cells (HUVECs) culture and chorioallantoic membrane (CAM). Tß4 is a member of the ß-thymosins, molecules with hormone-like properties, and is the most abundant G-actin sequestering peptide in human blood platelets and white cells. Philp et al (2003), suggested that Tß4 exerts its pro-angiogenic activity in vitro e in vivo through the sequence motif 17LKKTET22, involved in actin binding. Starting from these considerations it was studied the behavior of three bio-active peptides representing the central part, the N and C terminus of Tß4, and characterized of containing the binding actin site. The structural analysis (NMR and Circular Dichroism), demonstrates that each peptide reproduces the conformation adopted in native protein and exerts both in vitro and in vivo pro-angiogenic effects, comparable to that induced by Fibroblast Growth Factor 2 (FGF-2). These results confirm the significant role of central binding actin site in the induction of neo-vessel formation and suggest in vitro a positively modulation by the N terminus part of Tß4. Stromal Derived Factor-1 (SDF-1) is an ?-chemokine that binds to G-protein-coupled CXCR4 receptor. The SDF-1/CXCR-4 axis plays an important role in the regulation of stem/progenitor cell trafficking, in normal development of the nervous system, and in hematopoietic system. Moreover, SDF-1 favours the formation of metastasis by increasing tumor cell motility (Libura et al. 2002), and plays an important role in the development of the vascular system by exerting a strong pro-angiogenic effect both in vitro and in vivo. SDF-1 is constitutively expressed in stromal cells, ECs, dendritic cells, and other cells. It is present mainly in two splice forms alpha and beta, which have identical amino acid sequences except for the presence of four additional amino acids at the carboxy terminus of SDF-1ß. The biological significance of the existence of 2 splice forms of SDF-1 has remained unclear. SDF-1 consists of three structural regions: N-terminus, a central beta sheet core, and C-terminus. Structure-function analysis of chemokine identified the NH2-terminal amino acids as critical to CXCR4 binding and activation, and suggested that C-terminus of protein, is one of interaction sites with glycosaminoglycans (GAGs) and may exert a modulation to biological function of SDF-1. This research verified that the C-terminus could be responsible of the pro-angiogenic effects of SDF-1. The designed peptides correspond to the C-terminus sequences of the two natural isoforms of splicing, mutated analog peptides of SDF-1ßT C-terminal region, and fusion forms containing both N- and C-terminus. The collected data indicated that C-terminal region is functional implicated in pro-angiogenic SDF-1 activity, but only the wild-type sequence were able to induce both in vitro and in vivo an angiogenic response. In 1971, Folkman proposed that tumour growth and metastasis are angiogenesis-dependent, and hence, blocking angiogenesis could be a strategy to arrest tumour growth. This possibility stimulated an intensive search for anti-angiogenic molecules. Cell adhesion to macromolecules of the extracellular matrix is needed to allow EC proliferation and migration inside the tumor. EC adhesion can be mediated by i) RGD-dipendent integrin receptors or ii) heparin sulphate proteoglycans (HSPGs), which recognized heparin-binding sites on extracellular matrix proteins. Since the loss of anchorage can induce pro-apoptotic signals leading to an inhibition of angiogenic process, RGD-peptides and vitronectin heparin-binding domain (HVP) were assayed as soluble agents to evaluate the effects on angiogenesis. Experimental evidences indicate that some designed peptides, i.e. (GRGDSP)4K and HVP, may represent promising anti-angiogenic agents whose effects seems to be related to an inhibition of both EC morphogenesis and FGF-2-induced angiogenic effects

An XPS study on the covalent immobilization of adhesion peptides on a glass surface

Covalent attachment of adhesive peptides to biomaterials surfaces can result in the formation of a bioactive and biomimetic surface. We have demonstrated that titanium surfaces grafted with adhesion peptides, reproducing sequences of fibronectin and vitronectin, can increase osteoblast adhesion compared to non-treated surfaces. We now extend our investigation to peptide immobilization on glass for studying human osteoblast adhesion and spreading. Silanization was used to anchor adhesion peptides to the glass surface through a selective or a non-selective immobilization. Investigated samples were analysed by XPS spectroscopy. Comparison between the results obtained using two different peptides and applying selective and non-selective immobilization will be discussed. -------------------------------------------------------------------------------

Biomimetic Peptide-Enriched Electrospun Polymers: A Photoelectron and Infrared Spectroscopy Study

Biomimetic polymer nanofibers of poly(ε-caprolactone) and poly(L-lactide–caprolactone) copolymer were prepared by electrospinning. Modifications of the polymer nanofibers aimed at improving their biomimetic properties were performed by two different routes: (1) immobilization of an adhesion peptide, which mimicked the adhesion sequence of the extracellular matrix protein fibronectin, on the polymer surface and (2) incorporation of self-complementary oligopeptides, which showed alternated hydrophilic and hydrophobic side chain groups and was capable of generating extended ordered structures by self-assembling, into the polymer nanofibers. The structure of the polymer/peptide nanofibers was investigated by X-ray photoelectron and Fourier transform infrared spectroscopies