Regenerative Medicine Unit

Rome, Italy

Regenerative Medicine Unit

Rome, Italy
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Gamboa-Martinez T.C.,Polytechnic University of Valencia | Gomez Ribelles J.L.,Polytechnic University of Valencia | Gomez Ribelles J.L.,Regenerative Medicine Unit | Gomez Ribelles J.L.,CIBER ISCIII | And 3 more authors.
Journal of Bioactive and Compatible Polymers | Year: 2011

A hybrid scaffold was obtained by the deposition of a thin network of submicron fibrin fibrils on the microporous walls of a macroporous poly(L-lactide) (PLLA) three-dimensional structure. The fibrin coating is homogeneous across the entire substrate and allowed the pore structure remain open in the hybrid scaffold. The elastic modulus of the hybrid scaffold (0.65 MPa) was increased up to twofold compared to the pure PLLA scaffold (0.29 MPa). Mouse pre-osteoblastic cells, MC3T3, were seeded on both pure PLLA and hybrid scaffolds, and cultured for 3, 6, and 24 h. The coating enhanced the cell colonization and proliferation and provided a more homogeneous distribution of cells within the scaffolds. In addition, the coating improved the scaffold adhesion properties by supplying new binding sites to the cells that modify the transmembrane receptors involved in initial cell adhesion mechanism. The expression of the β3 integrin was observed in cells cultured on fibrin-coated scaffolds instead of the ?5 integrin, which was expressed in the uncoated scaffold. These hybrid PLLA/fibrin scaffolds have cell culture features suitable to promote early tissue regeneration. © The Author(s) 2011.

Salmeron-Sanchez M.,Polytechnic University of Valencia | Salmeron-Sanchez M.,CIBER ISCIII | Salmeron-Sanchez M.,Regenerative Medicine Unit | Rico P.,Polytechnic University of Valencia | And 5 more authors.
Biomaterials | Year: 2011

Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials. © 2010 Elsevier Ltd.

Gonzalez-Garcia C.,Polytechnic University of Valencia | Sousa S.R.,INEB Institute Engineering Biomedica | Sousa S.R.,Polytechnic Institute of Porto | Moratal D.,Polytechnic University of Valencia | And 5 more authors.
Colloids and Surfaces B: Biointerfaces | Year: 2010

Phase separation of PLLA/PS (50/50, w/w) solutions during a spin-casting process gives rise to well-defined nanotopographies of 14, 29 and 45. nm deep pits depending on the concentration of the solution. Their influence on the biological activity of fibronectin (FN) was investigated. FN adsorption was quantified by radiolabelling the protein. The amount of adsorbed FN was higher on the 14. nm deep pit nanotopography than on the other two surfaces. FN distribution between valleys and peaks was investigated by AFM combined with image analysis. FN tends to adsorb preferentially on the valleys of the nanotopography only for the 14. nm system and when adsorbed from solutions of concentration lower than 10μg/ml. Higher concentration of the FN solution leads to evenly distribution of the protein throughout the surface; moreover, there is no difference in the distribution of the protein between valleys and peaks for the other two systems (29 and 45. nm) irrespective of the concentration of the FN solution. The biological activity of the adsorbed protein layer was assessed by investigating MC3T3 osteoblast-like cells adhesion, FN reorganisation and late matrix formation on the different substrates. Even if initial cell adhesion is excellent for every substrate, the size of the focal adhesion plaques increases as the size of the pits in the nanotopography does. This is correlated to FN reorganisation, which only takes places on the 29 and 45. nm deep pits surfaces, where enhanced late matrix production was also found. © 2010 Elsevier B.V.

Ferreira A.,University of Minho | Silva J.,University of Minho | Sencadas V.,University of Minho | Ribelles J.L.G.,Polytechnic University of Valencia | And 3 more authors.
Macromolecular Materials and Engineering | Year: 2010

Electroactive macroporous poly[(vinylidene fluoride)-co-trifluoroethylene] membranes have been produced by solvent evaporation at room temperature, starting with a diluted solution of the copolymer in dimethylformamide. The pore architecture consists of interconnected spherical pores. This architecture is independent of the membrane thickness. The thickness of the membranes ranges from a few to several hundred mm, using spin coating and evaporation in static conditions, respectively. The pore structure is explained by a spinodal decomposition of the liquid/liquid phase separation and crystallization in the copolymer-rich phase.© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ballester-Beltran J.,Polytechnic University of Valencia | Rico P.,Polytechnic University of Valencia | Rico P.,CIBER ISCIII | Moratal D.,Polytechnic University of Valencia | And 7 more authors.
Soft Matter | Year: 2011

Protein adsorption and cellular behavior depend strongly on the wettability of substrates. Such studies are scarce for surfaces exhibiting extreme values of contact angles. Fibronectin (FN) adsorption and adhesion of MC3T3-E1 cells were investigated on superhydrophobic polystyrene (SH-PS) surfaces and compared with the corresponding smooth polystyrene (PS) substrate and the control glass. The FN surface density was lower on the SH-PS than on PS, and the adsorbed protein showed altered conformation of cell adhesion domains, as obtained by ELISA with monoclonal antibodies. Cell adhesion occurred on the SH-PS without the formation of mature focal adhesions, as assessed by immunofluorescence for vinculin, talin and paxillin. Correspondingly, the development of the actin cytoskeleton was delayed and without the presence of defined F-actin fibers. FAK phosphorylation was reduced on SH-PS, as compared with PS and the control glass. Also, cell contractility was diminished on the SH-PS as revealed by phosphorylation of myosin light chain (pMLC). Likewise, FN reorganization and secretion were impaired on the superhydrophobic surfaces. Cell proliferation was significantly lower in SH-PS as compared with PS up to 21 days of culture. © 2011 The Royal Society of Chemistry.

Llopis-Hernandez V.,Polytechnic University of Valencia | Rico P.,Polytechnic University of Valencia | Rico P.,CIBER ISCIII | Ballester-Beltran J.,Polytechnic University of Valencia | And 4 more authors.
PLoS ONE | Year: 2011

Background: The cell-material interaction is a complex bi-directional and dynamic process that mimics to a certain extent the natural interactions of cells with the extracellular matrix. Cells tend to adhere and rearrange adsorbed extracellular matrix (ECM) proteins on the material surface in a fibril-like pattern. Afterwards, the ECM undergoes proteolytic degradation, which is a mechanism for the removal of the excess ECM usually approximated with remodeling. ECM remodeling is a dynamic process that consists of two opposite events: assembly and degradation. Methodology/Principal Findings: This work investigates matrix protein dynamics on mixed self-assembled monolayers (SAMs) of -OH and -CH3 terminated alkanethiols. SAMs assembled on gold are highly ordered organic surfaces able to provide different chemical functionalities and well-controlled surface properties. Fibronectin (FN) was adsorbed on the different surfaces and quantified in terms of the adsorbed surface density, distribution and conformation. Initial cell adhesion and signaling on FN-coated SAMs were characterized via the formation of focal adhesions, integrin expression and phosphorylation of FAKs. Afterwards, the reorganization and secretion of FN was assessed. Finally, matrix degradation was followed via the expression of matrix metalloproteinases MMP2 and MMP9 and correlated with Runx2 levels. We show that matrix degradation at the cell material interface depends on surface chemistry in MMP-dependent way. Conclusions/Significance: This work provides a broad overview of matrix remodeling at the cell-material interface, establishing correlations between surface chemistry, FN adsorption, cell adhesion and signaling, matrix reorganization and degradation. The reported findings improve our understanding of the role of surface chemistry as a key parameter in the design of new biomaterials. It demonstrates the ability of surface chemistry to direct proteolytic routes at the cell-material interface, which gains a distinct bioengineering interest as a new tool to trigger matrix degradation in different biomedical applications. © 2011 Llopis-Hernández et al.

Lebourg M.,CIBER ISCIII | Anton J.S.,CIBER ISCIII | Anton J.S.,Polytechnic University of Valencia | Anton J.S.,Regenerative Medicine Unit | And 3 more authors.
Journal of Materials Science: Materials in Medicine | Year: 2010

Polymer-ceramic composites are favourite candidates when aiming to replace bone tissue. We present here scaffolds made of polycaprolactone-hydroxyapatite (PCL-HAp) composites, and investigate in vitro mineralisation of the scaffolds in SBF after or without a nucleation treatment. In vitro bioactivity is enhanced by HAp incorporation as well as by nucleation treatment, as demonstrated by simulated body fluid (SBF) mineralization. Surprisingly, we obtained a hybrid interconnected organic-inorganic structure, as a result of micropore invasion by biomimetic apatite, which results in a mechanical strengthening of the material after two weeks of immersion in SBF×2. The presented scaffolds, due to their multiple qualities, are expected to be valuable supports for bone tissue engineering. © 2009 Springer Science+Business Media, LLC.

Arnal-Pastor M.,Polytechnic University of Valencia | Valles-Lluch A.,Polytechnic University of Valencia | Valles-Lluch A.,Regenerative Medicine Unit | Keicher M.,Polytechnic University of Valencia | And 3 more authors.
Journal of Colloid and Interface Science | Year: 2011

A set of elastomeric scaffolds with a well defined porous structure was prepared with a template leaching procedure and coated with hyaluronic acid solutions. Depending on the coating process parameters the hyaluronic acid deposited on the pores had configurations ranging from thin disconnected aggregates to a thick continuous layer on the pore surface. The development of the coating layer was studied by scanning electron microscopy and the materials were subjected to dynamical and equilibrium swelling experiments in a water vapor ambient of fixed activity. The porosity change due to coating and to swelling of the coating layer were determined. The hyaluronic acid coating the pores has a different swelling capacity depending on the type of layer formed, as a consequence of the scaffold constraint and of the layer typology. These factors were investigated analytically by modifying the standard theory of gel swelling. An experimental quantity is introduced which reflects the constrainment build-up on gel swelling. © 2011 Elsevier Inc.

del Fattore A.,Regenerative Medicine Unit | Teti A.,University of L'Aquila
Inflammation and Allergy - Drug Targets | Year: 2012

Osteoimmunology is an interdisciplinary field addressing the interplay between the skeletal and the immune system. A substantial body of evidence demonstrated the existence of two-way regulatory mechanisms that affect both systems, placing them in much closer association to each other than one could ever predict. Inflammatory diseases have long been known to induce alterations in bone metabolism, and inflammatory cytokines play prominent roles in the control of bone resorption, representing communication pathways bridging the two systems. Osteoclasts are particularly linked to the immune cells because they belong to the monocyte/macrophage family, have tight relationships with B and T cells, and differentiate in response to RANKL which is also produced by lymphocytes and regulates lymphopoiesis. Osteoclasts are negatively regulated by cytokines and other factors known for their anti-inflammatory and immune regulatory activity. Finally, they express immune co-receptor typical of immune cells which are indispensable for their differentiation, thus leading to the hypothesis that osteoclasts are immune cells themselves. The underlying principle why an immune cell is required to resorb bone has not yet been elucidated. Data from early literature suggest that the bone matrix could trigger an innate immune response activating giant cells that could destroy large bone areas because of their unique property of resorbing bone extracellularly. Bone resorption could though be prevented by the physical barrier made by osteoblasts and lining cells, whose retraction would be required to give access to osteoclasts when specific pathways signal their precursors to differentiate and mature osteoclasts to reach the uncovered bone surface. © 2012 Bentham Science Publishers.

Annunziato F.,University of Florence | Annunziato F.,Regenerative Medicine Unit | Santarlasci V.,University of Florence | Maggi L.,University of Florence | And 5 more authors.
Seminars in Immunology | Year: 2013

T helper 17 (Th17) cells have been reported to be responsible for several chronic inflammatory diseases. However, a peculiar feature of human Th17 cells is that they are very rare in the inflammatory sites in comparison with Th1 cells. The first reason for this rarity is the existence of some self-regulatory mechanisms that limit their expansion. The limited expansion of human Th17 cells is related to the retinoic acid orphan (ROR)C-dependent up-regulation of the interleukin (IL)-4 induced gene 1 (IL4I1), which encodes for a l-phenylalanine oxidase, that has been shown to down-regulate CD3ζ expression in T cells. This results in abnormalities of the molecular pathway which is responsible for the impairment of IL-2 production and therefore for the lack of cell proliferation in response to T-cell receptor (TCR) signalling. IL4I1 up-regulation also associates with the increased expression of Tob1, a member of the Tob/BTG anti-proliferative protein family, which is involved in cell cycle arrest. A second reason for the rarity of human Th17 cells in the inflammatory sites is their rapid shifting into the Th1 phenotype, which is mainly related to the activity of IL-12 and TNF-α. We have named these Th17-derived Th1 cells as non-classic because they differ from classic Th1 cells for the expression of molecules specific for Th17 cells, such as RORC, CD161, CCR6, IL4I1, and IL-17 receptor E. This distinction may be important for defining the respective pathogenic role of Th17, non-classic Th1 and classic Th1 cells in many human inflammatory disorders. © 2013 Elsevier Ltd.

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