Staff member

Marta Pegueroles

Postdoctoral Researcher
RA_Biomaterials, Implants i Engenyeria de Teixits

Staff member publications

Schieber, R., Lasserre, F., Hans, M., Fernández-Yagüe, M., Díaz-Ricart, M., Escolar, G., Ginebra, M. P., Mücklich, F., Pegueroles, M., (2017). Direct laser interference patterning of CoCr alloy surfaces to control endothelial cell and platelet response for cardiovascular applications Advanced Healthcare Materials Early View (Online Version of Record published before inclusion in an issue)

The main drawbacks of cardiovascular bare-metal stents (BMS) are in-stent restenosis and stent thrombosis as a result of an incomplete endothelialization after stent implantation. Nano- and microscale modification of implant surfaces is a strategy to recover the functionality of the artery by stimulating and guiding molecular and biological processes at the implant/tissue interface. In this study, cobalt-chromium (CoCr) alloy surfaces are modified via direct laser interference patterning (DLIP) in order to create linear patterning onto CoCr surfaces with different periodicities (≈3, 10, 20, and 32 μm) and depths (≈20 and 800 nm). Changes in surface topography, chemistry, and wettability are thoroughly characterized before and after modification. Human umbilical vein endothelial cells' adhesion and spreading are similar for all patterned and plain CoCr surfaces. Moreover, high-depth series induce cell elongation, alignment, and migration along the patterned lines. Platelet adhesion and aggregation decrease in all patterned surfaces compared to CoCr control, which is associated with changes in wettability and oxide layer characteristics. Cellular studies provide evidence of the potential of DLIP topographies to foster endothelialization without enhancement of platelet adhesion, which will be of high importance when designing new BMS in the future.

Keywords: CoCr, Direct laser interference patterning, Endothelial cells, Linear surface pattern, Platelets

Castellanos, M. I., Mas-Moruno, C., Grau, A., Serra-Picamal, X., Trepat, X., Albericio, F., Joner, M., Gil, F. J., Ginebra, M. P., Manero, J. M., Pegueroles, M., (2017). Functionalization of CoCr surfaces with cell adhesive peptides to promote HUVECs adhesion and proliferation Applied Surface Science 393, 82-92

Biomimetic surface modification with peptides that have specific cell-binding moieties is a promising approach to improve endothelialization of metal-based stents. In this study, we functionalized CoCr surfaces with RGDS, REDV, YIGSR peptides and their combinations to promote endothelial cells (ECs) adhesion and proliferation. An extensive characterization of the functionalized surfaces was performed by XPS analysis, surface charge and quartz crystal microbalance with dissipation monitoring (QCM-D), which demonstrated the successful immobilization of the peptides to the surface. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represents the most powerful strategy to enhance the early stages of ECs adhesion and proliferation, indicating a positive synergistic effect between the two peptide motifs. Although these peptide sequences slightly increased smooth muscle cells (SMCs) adhesion, these values were ten times lower than those observed for ECs. The combination of RGDS with the REDV sequence did not show synergistic effects in promoting the adhesion or proliferation of ECs. The strategy presented in this study holds great potential to overcome clinical limitations of current metal stents by enhancing their capacity to support surface endothelialization.

Keywords: Cell adhesive peptides, CoCr alloy, Endothelialization, HUVEC proliferation, SMCs adhesion, Surface functionalization

Castellanos, M. I., Guillem-Marti, J., Mas-Moruno, C., Díaz-Ricart, M., Escolar, G., Ginebra, M. P., Gil, F. J., Pegueroles, M., Manero, J. M., (2017). Cell adhesive peptides functionalized on CoCr alloy stimulate endothelialization and prevent thrombogenesis and restenosis Journal of Biomedical Materials Research - Part A 105, (4), 973-983

Immobilization of bioactive peptide sequences on CoCr surfaces is an effective route to improve endothelialization, which is of great interest for cardiovascular stents. In this work, we explored the effect of physical and covalent immoblization of RGDS, YIGSR and their equimolar combination peptides on endothelial cells (EC) and smooth muscle cell (SMC) adhesion and on thrombogenicity. We extensively investigated using RT-qPCR, the expression by ECs cultured on functionalised CoCr surfaces of different genes. Genes relevant for adhesion (ICAM-1 and VCAM-1), vascularization (VEGFA, VEGFR-1 and VEGFR-2) and anti-thrombogenicity (tPA and eNOS) were over-expressed in the ECs grown to covalently functionalized CoCr surfaces compared to physisorbed and control surfaces. Pro-thrombogenic genes expression (PAI-1 and vWF) decreased over time. Cell co-cultures of ECs/SMCs found that functionalization increased the amount of adhered ECs onto modified surfaces compared to plain CoCr, independently of the used peptide and the strategy of immobilization. SMCs adhered less compared to ECs in all surfaces. All studied peptides showed a lower platelet cell adhesion compared to TCPS. Covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represented prevailing strategy to enhance the early stages of ECs adhesion and proliferation, while preventing SMCs and platelet adhesion.

Keywords: Cell coculture, CoCr alloy, Functionalization, Gene expression, Platelet adhesion

Pegueroles, M., Tonda-Turo, C., Planell, J. A., Gil, F. J., Aparicio, C., (2012). Adsorption of fibronectin, fibrinogen, and albumin on TiO2: Time-resolved kinetics, structural changes, and competition study Biointerphases 7, (48), 13

An understanding of protein adsorption process is crucial for designing biomaterial surfaces. In this work, with the use of a quartz-crystal microbalance with dissipation monitoring, we researched the following: (a) the kinetics of adsorption on TiO2 surfaces of three extensively described proteins that are relevant for metallic implant integration [i.e., albumin (BSA), fibrinogen (Fbg), and fibronectin (Fn)]; and (b) the competition of those proteins for adsorbing on TiO2 in a two-step experiment consisted of sequentially exposing the surfaces to different monoprotein solutions. Each protein showed a different process of adsorption and properties of the adlayer-calculated using the Voigt model. The competition experiments showed that BSA displaced larger proteins such as Fn and Fbg when BSA was introduced as the second protein in the system, whereas the larger proteins laid on top of BSA forming an adsorbed protein bi-layer when those were introduced secondly in the system.

Keywords: QCM, Human plasma fibronectin, Induced conformational-changes, Von-willebrand-factor, BSA, Protein adsortion, Polymer surfaces, Solid-surfaces, Viscoelastic properties, Globular-proteins

Pegueroles, M., Aguirre, A., Engel, E., Pavon, G., Gil, F. J., Planell, J. A., Migonney, V., Aparicio, C., (2011). Effect of blasting treatment and Fn coating on MG63 adhesion and differentiation on titanium: a gene expression study using real-time RT-PCR Journal of Materials Science: Materials in Medicine 22, (3), 617-627

Biomaterial surface properties, via alterations in the adsorbed protein layer, and the presence of specific functional groups can influence integrin binding specificity, thereby modulating cell adhesion and differentiation processes. The adsorption of fibronectin, a protein directly involved in osteoblast adhesion to the extracellular matrix, has been related to different physical and chemical properties of biomaterial surfaces. This study used blasting particles of different sizes and chemical compositions to evaluate the response of MG63 osteoblast-like cells on smooth and blasted titanium surfaces, with and without fibronectin coatings, by means of real-time reverse transcription-polymerase chain reaction (qRT-PCR) assays. This response included (a) expression of the alpha(5), alpha(v) and alpha(3) integrin subunits, which can bind to fibronectin through the RGD binding site, and (b) expression of alkaline phosphatase (ALP) and osteocalcin (OC) as cell-differentiation markers. ALP activity and synthesis of OC were also tested. Cells on SiC-blasted Ti surfaces expressed higher amounts of the alpha(5) mRNA gene than cells on Al2O3-blasted Ti surfaces. This may be related to the fact that SiC-blasted surfaces adsorbed higher amounts of fibronectin due to their higher surface free energy and therefore provided a higher number of specific cell-binding sites. Fn-coated Ti surfaces decreased alpha(5) mRNA gene expression, by favoring the formation of other integrins involved in adhesion over alpha(5)beta(1). The changes in alpha(5) mRNA expression induced by the presence of fibronectin coatings may moreover influence the osteoblast differentiation pathway, as fibronectin coatings on Ti surfaces also decreased both ALP mRNA expression and ALP activity after 14 and 21 days of cell culture.

Keywords: -----

Pegueroles, M., Aparicio, C., Bosio, M., Engel, E., Gil, F. J., Planell, J. A., Altankov, G., (2010). Spatial organization of osteoblast fibronectin matrix on titanium surfaces: Effects of roughness, chemical heterogeneity and surface energy Acta Biomaterialia 6, (1), 291-301

We investigated the early events of bone matrix formation, and specifically the role of fibronectin (FN) in the initial osteoblast interaction and the subsequent organization of a provisional FN matrix on different rough titanium (Ti) surfaces. Fluorescein isothiocyanate-label led FN was preadsorbed on these surfaces and studied for its three-dimensional (3-D) organization by confocal microscopy, while its amount was quantified after NaOH extraction. An irregular pattern of adsorption with a higher amount of protein on topographic peaks than on valleys was observed and attributed to the physicochemical heterogeneity of the rough Ti surfaces. MG63 osteoblast-like cells were further cultured on FN-preadsorbed Ti surfaces and an improved initial cellular interaction was observed with increasing roughness. 3-D reconstruction of the immunofluorescence images after 4 days of incubation revealed that osteoblasts deposit FN fibrils in a specific facet-like pattern that is organized within the secreted total matrix overlying the top of the samples. The thickness of this FN layer increased when the roughness of the underlying topography was increased, but not by more than half of the total maximum peak-to-valley distance, as demonstrated with images showing simultaneous reconstruction of fluorescence and topography after 7 days of cell culture.

Keywords: Fibronectin, Extracellular matrix organization, Titanium, Surface topography, Surface energy

Altankov, George, Groth, Thomas, Engel, Elisabeth, Gustavsson, Jonas, Pegueroles, Marta, Aparicio, Conrado, Gil, Francesc J., Ginebra, Maria-Pau, Planell, Josep A., (2010). Development of provisional extracellular matrix on biomaterials interface: Lessons from in vitro cell culture NATO Science for Peace and Security Series A: Chemistry and Biology Advances in Regenerative Medicine: Role of Nanotechnology, and Engineering Principles (ed. Shastri, P., Altankov, G., Lendlein, A.), Springer Netherlands (Dortrecht, The Netherlands) , 19-43

The initial cellular events that take place at the biomaterials interface mimic to a certain extent the natural interaction of cells with the extracellular matrix (ECM). The cells adhering to the adsorbed soluble matrix proteins, such as fibronectin (FN) and fibrinogen (FNG) tend to re-arrange them in fibril-like pattern. Using model surfaces we have demonstrated that this cellular activity is abundantly dependent on the surface properties of materials, such as wettability, surface chemistry, charge and topography. This raises the possibility that tissue compatibility of materials is connected with the allowance of cells to remodel substratum associated proteins presumably to form provisional ECM. We have further shown that antibodies which bind β1 and αv integrins (subunits of the FN and FNG receptors respectively) may induce their linear rearrangement on the dorsal surface of living cells – a phenomenon presumably related to the same early molecular events of fibrillar matrix assembly. Because the quantitative measurements revealed that this receptor dynamics is strongly altered on the low compatible (hydrophobic) substrata we hypothesized that in order to be biocompatible, materials need to adsorb matrix proteins loosely, i.e. in such a way that the cells can easily remove and organize them in matrix-like fibrils via coordinated functioning of integrins. More recent studies on the fate of FN on some real biomaterial surfaces, including different rough titanium (Ti) and hydroxyapatite (HA) cements and the surface of biosensors confirmed this point of view. They also show that quantitative measurements of adsorbed matrix proteins and their dynamic rearrangement at cell-material interface might provide insight to the biocompatibility of given material and even predict its tissue integration.

Keywords: Materials Science

Aparicio, C., Salvagni, E., Werner, M., Engel, E., Pegueroles, M., Rodriguez-Cabello, C., Munoz, F., Planell, J. A., Gil, J., (2009). Biomimetic treatments on dental implants for immediate loading applications Journal of Medical Devices 3, (2), 027555

Summary form only given. Commercially pure titanium (cp Ti) dental implants have been widely and successfully used with high rates of clinical success in normal situations. However, there is still a lack of reliable synthetic materials to be used either a) when immediate loading of the implant is desired or b) when bone presents compromised conditions due to trauma, infection, systemic disease and/or lack of significant bone volume. Our group has aimed the development of biomimetic strategies of surface modification to obtain metallic implants with osteostimulative capabilities. These surface modifications will provide implants with a rapid rate of newly-formed bone growth and with ossecoalescence, i.e., direct chemical contact with the surrounding tissues. Consequently, the biomimetically-modified implants will be reliably used on those more demanding clinical situations, cp Ti surfaces treated to obtain a combination of an optimal random surface topography (in the micro and nanolevels) with a chemical modification of the naturally-formed titania layer have been proved bioactive. These rough and bioactive surfaces nucleate and grow a homogeneous hydroxyapatite layer both in vitro and in vivo. They stimulate the osteoblasts differentiation and trigger a rapid bone formation that mechanically fixes implants under immediate-loading conditions. A simple process using silane chemistry has been proved specific, rapid, and reliable to covalently immobilize biomolecules on cp Ti surfaces. This methodology can be used to develop biofunc- tionalized implant surfaces with different or combined bioactivities. The biofunctional molecules can be biopolymers, proteins, growth factors, and synthetic peptides specifically designed to be attached to the surface. The bioactive properties of the molecules designed and used can be mineral growing and nucleation, osteoblast differentiation (bone regeneration), fibroblasts differentiation (biological sealing), antibiotic,... Specifically, we have obtained mechanically and thermochemically stable coatings made of recombinant elastin-like biopolymers. The biopolymers bear either a) the RODS peptide, which is a highly-specific cell-adhesion motif present in proteins of the extracellular matrix for different tissues including bone, or b) an acidic peptide sequence derived from statherin, a protein present in saliva with high affinity for calcium-phosphates and with a leading role in the remineralization processes of the hard tissues forming our teeth. Two different biomimetic strategies have been successfully developed combining topographical modification, inorganic treatments and/or biofunctionalization for improving bioactive integrative properties of cp Ti implants.

Keywords: Biomedical materials, Bone, Cellular biophysics, Dentistry, Molecular biophysics, Prosthetics, Proteins, Surface treatment, Titanium

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