Papers by Cristina Barrias
Biomaterials, 2009
The objective of this study was to compare the biological effects of two key cell-adhesive protei... more The objective of this study was to compare the biological effects of two key cell-adhesive proteins, fibronectin (FN) and vitronectin (VN), upon adsorption onto molecularly-designed model surfaces. Single-component and mixed self-assembled monolayers (SAMs) of alkanethiols on gold with OH and CH 3 terminal groups were prepared at 100%, 65%, 36% and 0% of OH at the surface, to generate a range of surfaces with a simple chemistry and a wettability gradient. FN and VN were adsorbed under noncompetitive (single-protein solutions) and competitive (multi-protein solutions) conditions, and compared at different levels: adsorbed amount (radiolabelling), elution, functional presentation of cellbinding domains (ELISA), and role in mediating cell adhesion (antibody-based assay). The observed trends were related to mesenchymal stem cell response in terms of adhesion and overall cell morphology. Under non-competitive conditions, adsorption of both proteins increased with surface hydrophobicity. The presence of competitive proteins significantly decreased the adsorbed amounts, although both proteins were still detected in all SAMs. Adsorption of FN followed a trend similar to that of non-competitive conditions, while adsorption of VN was higher on 100%OH-SAMs. Concerning elution, retention of adsorbed VN was always higher than that of FN. For both proteins, functional presentation of cell-binding domains was more effective on the more hydrophilic 100%OH-SAMs. This fact, coupled to the ability of this type of SAMs to selectively recruit and retain VN in the presence of competitive serum proteins, seems to correlate with the better cell response observed on these surfaces, as compared with hydrophobic 0%OH(100%CH 3 )-SAMs.
Acta Biomaterialia, 2005
Poly(a-hydroxy acids), and in particular polylactic acid (PLA), are nowadays amongst the most use... more Poly(a-hydroxy acids), and in particular polylactic acid (PLA), are nowadays amongst the most used bioabsorbable materials. However, this polymer may not meet some application requirements due to inadequate mechanical properties and or its degradation characteristics. A possible strategy to tackle this problem is the incorporation of an inorganic phase into the polymeric matrix. In this work a new fully biodegradable composite material made with PLA and calcium phosphate soluble glass particles has been developed. The behaviour of the PLA/glass composite has been analysed during its degradation in simulated physiological conditions by means of weight loss, molecular weight and thermal properties analysis and electron microscopy observation. The results showed that the incorporation of phosphate glass particles into the polymer significantly accelerated the degradation of the PLA and induced the formation of calcium phosphate precipitates at the composite surface.
Scientific Reports, 2015
Mesenchymal Stem/Stromal Cells (MSC) are a promising cell type for cell-based therapies -from tis... more Mesenchymal Stem/Stromal Cells (MSC) are a promising cell type for cell-based therapies -from tissue regeneration to treatment of autoimmune diseases -due to their capacity to migrate to damaged tissues, to differentiate in different lineages and to their immunomodulatory and paracrine properties. Here, a simple and reliable imaging technique was developed to study MSC dynamical behavior in natural and bioengineered 3D matrices. Human MSC were transfected to express a fluorescent photoswitchable protein, Dendra2, which was used to highlight and follow the same group of cells for more than seven days, even if removed from the microscope to the incubator. This strategy provided reliable tracking in 3D microenvironments with different properties, including the hydrogels Matrigel and alginate as well as chitosan porous scaffolds. Comparison of cells mobility within matrices with tuned physicochemical properties revealed that MSC embedded in Matrigel migrated 64% more with 5.2 mg protein/mL than with 9.6 mg/mL and that MSC embedded in RGDalginate migrated 51% faster with 1% polymer concentration than in 2% RGD-alginate. This platform thus provides a straightforward approach to characterize MSC dynamics in 3D and has applications in the field of stem cell biology and for the development of biomaterials for tissue regeneration.
Biomaterials, 2015
Absorption evaluation plays an increasingly important role at the early stage of drug discovery d... more Absorption evaluation plays an increasingly important role at the early stage of drug discovery due to its potential to scan the ADME (absorption, distribution, metabolism and excretion) properties of new drug candidates. Therefore, a new three-dimensional (3D) in vitro model replicating the intestinal functioning is herein proposed aiming to dissect the stromal-epithelial interactions and evaluate the permeation of a model drug, insulin. Inspired on the intestinal mucosal architecture, the present model comprises intestinal myofibroblasts (CCD18-Co cells) embedded in Matrigel, onto which epithelial enterocytes (Caco-2 cells) and mucus-producing cells (HT29-MTX cells) were seeded. CCD18-Co myofibroblasts showed to have a central role in the remodeling of the surrounding matrix confirmed by the production of fibronectin. Subsequently, this matrix revealed to be essential to the maintenance of the model architecture by supporting the overlying epithelial cells. In terms of functionality, this model allowed the efficient prediction of insulin permeability in which the presence of mucus, the less tight character between Caco-2 and HT29-MTX epithelial cells and the 3D assembly were critical factors. Concluding, this model constitutes a robust tool in the drug development field with potential to bridge the traditional 2D cell culture models and in vivo animal models. Biomaterials j o u r n a l h o m e p a g e : w w w .e l se v i e r. co m/ lo ca t e / b i o m a t e ri a l s http://dx.
The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functiona... more The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functionalities (X=CH3-, OH- and COOH-) was,evaluated,as a function,of solution,pH. Radiolabelling studies showed,that the enzyme,has higher,affinity for hydrophobic surfaces than for hydrophilic surfaces, and that the highest adsorption was obtained at the more acidic pH values (4.5 and 5.5), despite the type of surface. IRAS and XPS measurements
Key Engineering Materials, 2009
The aim of this study was to analyze the effect of starting powder granulometry and sintering con... more The aim of this study was to analyze the effect of starting powder granulometry and sintering conditions on the morphological structure and mechanical properties of injectable hydroxyapaptite (HAp) microspheres. The mechanical properties of the microspheres were evaluated, to investigate if their integrity could be maintained during the injection process. To obtain microspheres, HAp powders were dispersed in a sodium alginate solution and spherical particles were prepared by droplet extrusion under a co-axial air stream, coupled with ionotropic gelation in the presence of Ca 2+ . This was followed by a sintering process at various temperatures and times. The morphology of microspheres was observed under SEM, diameter measurements were performed in an optical microscope and the compression strength was evaluated using a texture analyzer. Finally, microspheres prepared using lower granulometry HAp powders and sintered at 1200 ºC for 1 hour presented the best properties and were selected as the most suitable for the envisaged application. Key Engineering Materials Vols. 396-398 (2009) pp 691-694 online at http://www.scientific.net
Soft Matter, 2013
Protease-sensitive hydrogels that recapitulate the mechanisms of cell-driven enzymatic remodellin... more Protease-sensitive hydrogels that recapitulate the mechanisms of cell-driven enzymatic remodelling of the natural extracellular matrix (ECM) have been gaining popularity as artificial 3D cell-microenvironments.
Macromolecular Bioscience, 2014
Cellular activities in 3D are differentially affected by several matrix-intrinsic and extrinsic f... more Cellular activities in 3D are differentially affected by several matrix-intrinsic and extrinsic factors. This study highlights the relevance of optimizing initial cell densities when establishing 3D cultures for specific applications. Independently of the entrapping density, MSCs cultured within RGD-alginate hydrogels showed steady-state levels of metabolic activity and were in a nearly non-proliferative state, but recovered ''normal'' activity levels when retrieved from 3D matrices and recultured as monolayers. Importantly, high-densities promoted the establishment of cell-cell contacts with formation of multicellular clusters stabilized by endogenous ECM, and also stimulated MSCs osteogenic differentiation. These MSC-ECM microtissues may be used as building blocks for tissue engineering.
Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2008
The aim of this study was to test the injectability of a bone filler system based on the combinat... more The aim of this study was to test the injectability of a bone filler system based on the combination of ceramic microspheres with a gel-like vehicle, for noninvasive surgery. Porous hydroxyapatite microspheres with a uniform size and an average diameter of 535 +/- 38 mum were prepared, and their compression strength and friability were tested. The sodium-alginate solution with a concentration of 7.25% (w/v) was used as the vehicle. To promote its in situ gelation, calcium carbonate and D-gluconic-delta-lactone were added to the solution. Microspheres were mixed with the vehicle at different percentages (20-40 wt %). Gelation times in the range of 8-20 min, were obtained, depending on the formulation. Mixtures of HAp microspheres with alginate solution at 7.25% originating a gel in 11 min present an adequate handling time to perform an injection. Their injectability was evaluated using an injection device commonly employed in vertebroplasty surgical procedures, coupled to a texturometer in compression mode. Using an extrusion rate of 0.1 mm/s, the force required to extrude any of the mixtures tested was lower than 100 N. For an extrusion rate of 1 mm/s mixtures with 40 wt % of microspheres were very difficult to inject. Mixtures with 35 wt % of microspheres presented the best compromise between injectability and compression strength of the gelled system. MicroCT analysis revealed a homogeneous distribution of the microspheres inside the vehicle, as well as full interconnection of the intra-microspheres spaces. The compression strength for the gelled systems ranged from 80 kPa (gel itself) to 600 kPa (composite with 40 wt % of microspheres).
Journal of Biomedical Materials Research, 2005
In this study, the behavior of bone marrow stromal cells cultured on calcium titanium phosphate (... more In this study, the behavior of bone marrow stromal cells cultured on calcium titanium phosphate (CTP) microspheres was analyzed. Cell adhesion and proliferation were estimated by the neutral red assay and by total DNA quantification. Morphology and deposition of extracellular matrix were assessed by confocal laser scanning microscopy and/or scanning electron microscopy. The expression of the osteoblastic phenotype was evaluated by monitoring alkaline phosphatase activity and osteocalcin secretion. Results revealed that cells were able to attach and spread on the surface of CTP microspheres, and gradually grow into nearly confluent monolayers. Moreover, cells were able to bridge adjacent microspheres forming microsphere-cell clusters. Cells produced an abundant amount of fibrillar extracellular matrix that covered the substrate surface. Alkaline phosphatase activity peaked around days 7-14 and then decreased until day 21. Cells secreted osteocalcin, with higher levels being detected at day 14 than at day 21. Taken together, these results suggest that CTP microspheres are appropriate scaffolds for the growth and differentiation of cells along the osteoblastic lineage.
Journal of Biomedical Materials Research Part A, 2008
This study concerns the preparation, physical, and in vitro characterization of two different typ... more This study concerns the preparation, physical, and in vitro characterization of two different types of hydroxyapatite (HA) microspheres, which are intended to be used as drug-delivery systems and bone-regeneration matrices. Hydroxyapatite nanoparticles (HA-1 and HA-2) were prepared using the chemical precipitation synthesis with H 3 PO 4 , Ca(OH) 2 , and a surfactant, SDS (sodium dodecyl sulfate), as starting reagents. The HA powders were dispersed in a sodium alginate solution, and spherical particles were obtained by droplet extrusion coupled with ionotropic gelation in the presence of Ca 2þ . These were subsequently sintered to produce HA-1 and HA-2 microspheres with a uniform size and interconnected microporosity. Both powders and microspheres were characterized using FTIR and X-ray diffraction. Moreover, SEM and mercury intrusion porosimetry were used to analyze the microspheres, and TEM was used to analyze the powders. Results showed that pure HA and mixtures of HA/b-TCP in the nanometer range and needlelike shape were obtained for HA-1 and HA-2 powders, respectively. Neutral Red, scanning electron microscopy and confocal microscopy were used to evaluate the behavior of osteoblastic-like MG-63 cells cultured on HA microspheres surfaces for 7 days. Results showed that good adhesion and proliferation of osteoblasts on the HA microspheres surface. Cells built bridges between adjacent microspheres, forming microspheres-cells clusters in both types of materials.
Nanomedicine (London, England), 2011
To study the mechanism of transfection mediated by imidazole-grafted chitosan (CHimi) nanoparticl... more To study the mechanism of transfection mediated by imidazole-grafted chitosan (CHimi) nanoparticles, to propose new strategies to control and improve the expression of a delivered gene in the context of regenerative medicine.
Biomaterials, 2009
The objective of this study was to compare the biological effects of two key cell-adhesive protei... more The objective of this study was to compare the biological effects of two key cell-adhesive proteins, fibronectin (FN) and vitronectin (VN), upon adsorption onto molecularly-designed model surfaces. Single-component and mixed self-assembled monolayers (SAMs) of alkanethiols on gold with OH and CH 3 terminal groups were prepared at 100%, 65%, 36% and 0% of OH at the surface, to generate a range of surfaces with a simple chemistry and a wettability gradient. FN and VN were adsorbed under noncompetitive (single-protein solutions) and competitive (multi-protein solutions) conditions, and compared at different levels: adsorbed amount (radiolabelling), elution, functional presentation of cellbinding domains (ELISA), and role in mediating cell adhesion (antibody-based assay). The observed trends were related to mesenchymal stem cell response in terms of adhesion and overall cell morphology. Under non-competitive conditions, adsorption of both proteins increased with surface hydrophobicity. The presence of competitive proteins significantly decreased the adsorbed amounts, although both proteins were still detected in all SAMs. Adsorption of FN followed a trend similar to that of non-competitive conditions, while adsorption of VN was higher on 100%OH-SAMs. Concerning elution, retention of adsorbed VN was always higher than that of FN. For both proteins, functional presentation of cell-binding domains was more effective on the more hydrophilic 100%OH-SAMs. This fact, coupled to the ability of this type of SAMs to selectively recruit and retain VN in the presence of competitive serum proteins, seems to correlate with the better cell response observed on these surfaces, as compared with hydrophobic 0%OH(100%CH 3 )-SAMs.
Biomaterials, 2005
The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functiona... more The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functionalities (X=CH 3 -, OH-and COOH-) was evaluated as a function of solution pH. Radiolabelling studies showed that the enzyme has higher affinity for hydrophobic surfaces than for hydrophilic surfaces, and that the highest adsorption was obtained at the more acidic pH values (4.5 and 5.5), despite the type of surface. IRAS and XPS measurements confirmed this tendency. Dye-binding studies and fluorescence quenching were used to investigate if a pH variation induces any conformational changes on the enzyme. Both methods suggest that lowering the pH from physiological to acidic values triggers an increased exposure of non-polar sites in the enzyme, which may modulate its adsorption behaviour to the more hydrophobic surfaces. At pH 4.5, the enzyme carries a substantial positive net charge and therefore relatively low native-state stability. As a consequence, surface binding may be favoured, irrespective of the type of surface, by providing increased conformational entropy to the enzyme. The specific activity (SA) of the adsorbed enzyme was strongly dependent on the conditions used. A decrease in SA (ca. 30% of control) was observed after adsorption on CH 3 -SAMs for all the pH tested. Adsorption on gold and on the more hydrophilic SAMs (OH-and COOH-) resulted in different degrees of inactivation at the more acidic pH (4.5), and in enzyme activation (up to ca. 230% of control) at higher pH (7-8), near the isoelectric point of the enzyme. r
Biomaterials, 2011
Cell-based therapies offer an attractive approach for revascularization and regeneration of tissu... more Cell-based therapies offer an attractive approach for revascularization and regeneration of tissues. However, and despite the pressing clinical needs for effective revascularization strategies, the successful immobilization of viable vascular cells within 3D matrices has been difficult to achieve. In this paper the in vitro potential of a natural, injectable RGD-alginate hydrogel as an in situ forming matrix to deliver endothelial cells was evaluated. Several techniques were employed to investigate how these microenvironments could influence the behavior of vascular cells, namely their ability to promote the outward migration of viable, proliferative cells, retaining the ability to form a 3D arrangement. Cells within RGD-grafted alginate hydrogel were able to proliferate and maintained 80% of viability for at least 48 h post-immobilization. Additionally, entrapped cells created a 3D organization into cellular networks and, when put in contact with matrigel, cells migrated out of the RGD-matrix. Overall, the obtained results support the idea that the RGD peptides conjugated to alginate provide a 3D environment for endothelial cells adhesion, survival, migration and organization.
Biomacromolecules, 2014
Hydrogels with the potential to provide minimally invasive cell delivery represent a powerful too... more Hydrogels with the potential to provide minimally invasive cell delivery represent a powerful tool for tissue-regeneration therapies. In this context, entrapped cells should be able to escape the matrix becoming more available to actively participate in the healing process. Here, we analyzed the performance of proteolytically degradable alginate hydrogels as vehicles for human mesenchymal stem cells (hMSC) transplantation. Alginate was modified with the matrix metalloproteinase (MMP)-sensitive peptide Pro-Val-Gly-Leu-Iso-Gly (PVGLIG), which did not promote dendritic cell maturation in vitro, neither free nor conjugated to alginate chains, indicating low immunogenicity. hMSC were entrapped within MMP-sensitive and MMP-insensitive alginate hydrogels, both containing cell-adhesion RGD peptides. Softer (2 wt % alginate) and stiffer (4 wt % alginate) matrices were tested. When embedded in a Matrigel layer, hMSC-laden MMP-sensitive alginate hydrogels promoted more extensive outward cell migration and invasion into the tissue mimic. In vivo, after 4 weeks of subcutaneous implantation in a xenograft mouse model, hMSC-laden MMP-sensitive alginate hydrogels showed higher degradation and host tissue invasion than their MMP-insensitive equivalents. In both cases, softer matrices degraded faster than stiffer ones. The transplanted hMSC were able to produce their own collagenous extracellular matrix, and were located not only inside the hydrogels, but also outside, integrated in the host tissue. In summary, injectable MMP-sensitive alginate hydrogels can act as localized depots of cells and confer protection to transplanted cells while facilitating tissue regeneration.
AAPS PharmSciTech, 2010
A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone... more A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone defects. However, the properties of the solution should be suitable to enable the transport of that extra phase. Additionally, the use of biocompatible materials is a requirement for tissue regeneration. Thus, we intended to optimize a biocompatible polymeric solution able to carry hydroxyapatite microspheres into bone defects using an orthopedic injectable device. To achieve that goal, polymers usually regarded as biocompatible were selected, namely sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and Na-alginate (ALG). The rheological properties of the polymeric solutions at different concentrations were assessed by viscosimetry before and after moist heat sterilization. In order to correlate rheological properties with injectability, solutions were tested using an orthopedic device applied for minimal invasive surgeries. Among the three polymers, ALG solutions presented the most suitable properties for our goal and a non-sterile ALG 6% solution was successfully used to perform preliminary injection tests of hydroxyapatite microspheres. Sterile ALG 7.25% solution was found to closely match non-sterile ALG 6% properties and it was selected as the optimal vehicle. Finally, sterile ALG 7.25% physical stability was studied at different temperatures over a 3-month period. It was observed that its rheological properties presented minor changes when stored at 25 degrees C or at 4 degrees C.
Advances in Applied Ceramics, 2011
In the present work, a novel route for the preparation of porous ceramic microspheres is describe... more In the present work, a novel route for the preparation of porous ceramic microspheres is described. Two ceramic powders, calcium-titanium-phosphate (CTP) and hydroxyapatite (HAp), were mixed with a sodium alginate solution that enabled the preparation of spherical particles, using the droplet extrusion method combined with ionotropic gelation in the presence of Ca 2+ . The spherical particles were subsequently sintered, to burn-off the polymer and obtain calcium-phosphate microspheres with a uniform size and an interconnected porous network. CTP microspheres with diameters ranging from 513 ± 24 μm to 792 ± 35 μm and with pores of approximately 40 μm were obtained. HAp microspheres presented diameters of 429 ± 46 μm and 632 ± 40 μm and pores of ca. 2 μm. Depending on the formulations tested, the structure of both calcium phosphates may become altered during the sintering process, suggesting that the ratio between the ceramic phase and the polymer solution is a critical parameter. Porous microspheres prepared using the described methodology are promising candidates as bone defect fillers and scaffolds for bone tissue regeneration.
Acta Biomaterialia, 2013
Human mesenchymal stem cells (MSCs) are currently recognized as a powerful cell source for regene... more Human mesenchymal stem cells (MSCs) are currently recognized as a powerful cell source for regenerative medicine, notably for their capacity to differentiate into multiple cell types. The combination of MSCs with biomaterials functionalized with instructive cues can be used as a strategy to direct specific lineage commitment, and can thus improve the therapeutic efficacy of these cells. In terms of biomaterial design, one common approach is the functionalization of materials with ligands capable of directly binding to cell receptors and trigger specific differentiation signaling pathways. Other strategies focus on the use of moieties that have an indirect effect, acting, for example, as sequesters of bioactive ligands present in the extracellular milieu that, in turn, will interact with cells. Compared with complex biomolecules, the use of simple compounds, such as chemical moieties and peptides, and other small molecules can be advantageous by leading to less expensive and easily tunable biomaterial formulations. This review describes different strategies that have been used to promote substrate-mediated guidance of osteogenic differentiation of immature osteoblasts, osteoprogenitors and MSCs, through chemically conjugated small moieties, both in two-and three-dimensional set-ups. In each case, the selected moiety, the coupling strategy and the main findings of the study were highlighted. The latest advances and future perspectives in the field are also discussed.
Acta Biomaterialia, 2011
The development of sophisticated three-dimensional (3-D) cell culture microenvironments that recr... more The development of sophisticated three-dimensional (3-D) cell culture microenvironments that recreate some of the complexity of the natural extracellular matrix (ECM) remains a challenging task. Here, the modification of alginate through partial crosslinking with a matrix metalloproteinase (MMP) cleavable peptide (proline-valine-glycine-leucine-isoleucine-glycine, PVGLIG) is described, and its use in the preparation of injectable, in situ crosslinkable hydrogel-like matrices is proposed. PVGLIG-grafted alginates were synthesized by carbodiimide chemistry and characterized. Their biological performance was evaluated by comparing the response of 3-D cultured mesenchymal stem cells (MSCs) to alginate hydrogels containing only cell-adhesion peptides (RGD-alginate) or both peptides (PVGLIG/RGD-alginate). After 1 week, cells remained essentially round within RGD-alginate, while they exhibited an elongated morphology within PVGLIG/RGD-alginate hydrogels, forming cellular networks. This suggests that cells were able to structurally reorganize the matrix, through enzymatic hydrolysis of PVGLIG residues, overcoming biophysical hydrogel resistance. As shown by gelatine-zymography, MSC presented higher activity of MMP-2 when cultured within alginate functionalized with MMP-sensitive peptide, suggesting that the cell's proteolytic phenotype was modulated by the matrix composition. Additionally, PVGLIG/ RGD-alginate hydrogels were clearly degraded in cell culture. Taken together, the results demonstrate that the co-incorporation of MMP-labile peptides in cell-adhesive RGD-alginate hydrogels improved their performance as ECM analogues, providing a more dynamic and physiological 3-D cellular microenvironment.
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Papers by Cristina Barrias