Manuel Salmerón Sánchez

Chitosan microparticles as injectable scaffolds for tissue engineering

The use of chitosan microparticles as injectable carriers for cell transplantation represents a promising alternative to avoid the drawbacks of the implantation of other forms of three‐dimensional (3D) scaffolds seeded with cells. In this study, a 3D construct is obtained in vitro by combining chitosan microparticles crosslinked with genipin and goat bone marrow stromal cells (GBMCs). Cell viability and the morphology of GBMCs were evaluated after culture for 7 and 14 days. Our results show the feasibility of chitosan microparticles as potential injectable scaffolds for tissue engineering and regenerative medicine. Copyright

Analysis of the Biological Response of Endothelial and Fibroblast Cells Cultured on Synthetic Scaffolds with Various Hydrophilic/Hydrophobic Ratios: Influence of Fibronectin Adsorption and Conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.

Fibronectin activity on substrates with controlled OH density

Adhesion of human fibroblast to a family of fibronectin (FN) coated model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios to obtain a controlled surface density of --OH groups was investigated. Cell adhesion and spreading surprisingly decreased as the fraction of --OH groups on the surface increased. AFM studies of FN conformation revealed formation of a protein network on the more hydrophobic surfaces. The density of this network diminished as the fraction of --OH groups in the sample increased, up to a maximal --OH concentration at which, instead of the network, only FN aggregates were observed. The kinetics of network development was followed at different adsorption times. Immunofluorescence for vinculin revealed the formation of well-developed focal adhesion complexes on the more hydrophobic surface (similar to the control glass), which became less defined as the fraction of --OH groups increased. Thus, the efficiency of cell adhesion is enhanced by the formation of FN networks on the substrate, directly revealing the importance of the adsorbed protein conformation for cell adhesion. However, cell-dependent reorganization of substrate-associated FN, which usually takes place on more hydrophilic substrates (as do at the control glass slides), was not observed in this system, suggesting the increased strength of protein-to-substrate interaction. Instead, the late FN matrix formation--after 3 days of culture--was again better pronounced on the more hydrophobic substrates and decreased as the fraction of --OH groups increase, which is in a good agreement with the results for overall cell morphology and focal adhesion formation.

Differentiation of Postnatal Neural Stem Cells into Glia and Functional Neurons on Laminin-Coated Polymeric Substrates

A series of polymeric biomaterials, including poly(methyl acrylate), chitosan, poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate, hydroxyethyl acrylate, and methyl acrylate were tested in vitro as culture substrates and compared for their effect on the differentiation of neural stem cells (NSCs) obtained from the subventricular zone of postnatal rats. Immunocytochemical assay for specific markers and scanning electron microscopy techniques were employed to determine the adhesion of the cultured NSCs to the different biomaterials and the respective neuronal differentiation. The functional properties and the membrane excitability of differentiated NSCs were investigated using a patch-clamp. The results show that the substrates surface chemistry influences cell attachment and neuronal differentiation, probably through its influence on adsorbed laminin, and that copolymers based on PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and differentiation of adult NSCs into functional neurons and glia.

Human Chondrocyte Morphology, Its Dedifferentiation, and Fibronectin Conformation on Different PLLA Microtopographies

Surfaces of poly(L-lactic acid) (PLLA) of well-defined microtopography were prepared by making use of the semicrystalline character of PLLA. Different thermal treatments before isothermal crystallization (which include nucleation steps) permit to obtain a controlled number of simultaneously growing spherulites, which, in the end, modulate the topography at the microscale. Four qualitatively different surfaces were prepared. The dynamics of primary human chondrocyte adhesion and cytoskeleton organization was investigated on the different surfaces. Chondrocyte morphology is shown to be influenced by the microtopography of the system as obtained by scanning electron microscopy and atomic force microscopy (AFM). The cytoplasmatic distribution of a focal adhesion protein, tensin, is followed as a function of time. Since the effect of surface topography on cell morphology is a consequence of the process of interaction between the extracellular matrix (ECM) proteins, adsorbed on the surface of the material, and related cell adhesion molecules, the conformation of one ECM protein, fibronectin, adsorbed on the different substrates was investigated by means of AFM.

Thermodynamics and statistical mechanics of multilayer adsorption

The multilayer adsorption models of Brunauer-Emmett-Teller and Guggenheim-Anderson-de Boer are reconsidered. The relationship between the fitting parameters and the physical parameters of the equation is discussed. The preexponential factors of the parameters are shown to be in general far different from unity, contrary to a widespread use. A thermodynamical derivation illuminates the hypothesis on which the multilayer sorption equation is dependent and frees it from too restrictive hypothesis usually taken as necessary for its validity. Equations are derived for the number fraction of sorption sites occupied by different numbers of molecules. The Guggenheim-Anderson-de Boer equation is shown to imply incomplete occupation (jamming) of the first sorption layer at saturation.

Blending polysaccharides with biodegradable polymers. II. Structure and biological response of chitosan/polycaprolactone blends

Blends of polycaprolactone (PCL) and chitosan (CHT) were prepared by casting from the mixture of solutions of both components in suitable solvents. PCL, and CHT, form phase separated blends with improved mechanical properties and increased water sorption ability with respect to pure PCL. The morphology of the system was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and confocal microscopy. Dispersed domains of CHT in the semicrystalline PCL matrix were found in samples with less than 20% CHT but cocontinuous phase morphologies are found in blends with 20% or more CHT. This feature was corroborated by the temperature dependence of the elastic modulus measured by dynamic mechanical properties as a function of temperature. It was observed that for those blends above 20 wt% CHT, the mechanical stability of the system was kept even after melting of the PCL phase. Primary human chondrocytes were cultured on the different substrates. Cell morphology was studied by SEM and the viability and proliferation was investigated by the colorimetric MTT assay. Different protein conformations were found by AFM on CHT and PCL samples which were related to the biological performance of the substrates. Hydrophilicty of the material is not directly related to the biological response and the sample with 20 wt% CHT shows better results than the other blends with respect to chondrocyte viability and proliferation. However, the results obtained in the blends are worse than in pure PCL. It seems to be correlated with the surface energy of the different blends rather than hydrophilicity.

Acrylic scaffolds with interconnected spherical pores and controlled hydrophilicity for tissue engineering

Polymer scaffolds are obtained in which the geometric characteristics (pore size, connectivity, porosity) and the physico-chemical properties of the resulting material can be controlled in an independent way. The interconnected porous structure was obtained using a template of sintered PMMA microspheres of controlled size. Copolymerization of hydrophobic ethyl acrylate and hydrophilic hydroxyethyl methacrylate comonomers took place in the free space of the template, different comonomer ratio gave rise to different hydrophilicity degrees of the material keeping the same pore architecture. The morphology of the resulting scaffolds was investigated by scanning electron microscopy (SEM), the porosity of the material calculated, and the mechanical properties compared with those of the bulk (non porous) material of the same composition.

Molecular Dynamics of Ethylene Glycol Dimethacrylate Glass Former: Influence of Different Crystallization Pathways

The crystallization induced by different thermal treatments of a low molecular weight glass former, ethylene glycol dimethacrylate (EGDMA), was investigated by dielectric relaxation spectroscopy (DRS) and differential scanning calorimetry (DSC). The fully amorphous material, dielectrically characterized for the first time, exhibits three relaxation processes: the alpha-relaxation related to dynamic glass transition whose relaxation rate obeys a Vogel-Fulcher-Tamman-Hesse (VFTH) law and two secondary processes (beta and gamma) with Arrhenius temperature dependence. Therefore, the evaluation of distinct crystallization pathways driven by different thermal histories was accomplished by monitoring the mobility changes in the multiple dielectric relaxation processes. Besides isothermal cold-crystallization, nonisothermal crystallizations coming from both the melt and the glassy states were induced. While an amorphous fraction, characterized by a glass transition, remains subsequent to crystallization from the melt, no alpha-relaxation is detected after the material undergoes nonisothermal cold-crystallization. In the latter, the secondary relaxations persist with a new process that evolves at low frequencies, designated as alpha that was also detected at advanced crystallization states under isothermal cold-crystallization. Under the depletion of the alpha-relaxation, the beta-process when detected becomes better resolved keeping the same location prior to crystallization leading to a decoupled temperature dependence relative to the alpha-process.

Differences in photosynthetic performance and its correlation with growth among tomato cultivars in response to different salts.

Previous works into photosynthesis regulation under salt stress have focused on the effect of NaCl, although other salts may significantly contribute to the toxicity of saline soils. In this paper, the effects of different salt sources (NaCl, Na(2)SO(4), MgCl(2) and MgSO(4)) on photosynthesis and vegetative growth in three tomato (Solanum lycopersicum L.) cultivars (Marmande RAF, Leader and Daniela) are presented. Differences were found in the net photosynthetic rate and vegetative growth among the studied cultivars and salinity treatments. Cultivar photosynthetic performance related not only to capability for toxic ion exclusion, but also to the maintenance of appropriate essential macronutrient concentrations in leaves. In addition, the role of metabolic and diffusion limitations in regulating photosynthesis varied depending on the studied genotypes. These data, along with variation in biomass and ion distribution in leaves and roots, show that distinct tomato cultivars can address salt tolerance differently, which should be considered when designing strategies to overcome plant sensitivity to salt stress.