Chitosan-collagen-hydroxyapatite membranes for tissue engineering.

Tissue engineering is growing in developing new technologies focused on providing effective solutions to degenerative pathologies that affect different types of connective tissues. The search for biocompatible, bioactive, biodegradable, and multifunctional materials has grown significantly in recent years. Chitosan, calcium phosphates collagen, and their combination as composite materials fulfill the required properties and could result in biostimulation for tissue regeneration. In the present work, the chitosan/collagen/hydroxyapatite membranes were prepared with different concentrations of collagen and hydroxyapatite. Cell adhesion was evaluated by MTS assay for two in vitro models. Additionally, cytotoxicity of the different membranes employing hemolysis of erythrocytes isolated from human blood was carried out. The structure of the membranes was analyzed by X-rays diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and thermal stability properties by thermogravimetric methods (TGA). The highest cell adhesion after 48 h was obtained for chitosan membranes with the highest hydroxyapatite and collagen content. All composite membranes showed good cell adhesion and low cytotoxicity, suggesting that these materials have a significant potential to be used as biomaterials for tissue engineering. Graphical abstract.

Estudio de biocompatibilidad de membranas ultradelgadas de quitosano como sustituto dérmico, empleando células madre mesenquimales y queratinocitos / Biocompatibility study of ultrathin membranes of chitosan as a dermal substitute, using mesenchymal stem cells and keratinocytes

Introducción y Objetivo. Existe una importante demanda mundial para desarrollar sustitutos biológicos que restauren, mantengan o mejoren la función tisular, basándose en el uso combinado de biomateriales que funcionen como andamios y empleando células que induzcan regeneración para reparar los tejidos lesionados o enfermos. El objetivo de este estudio es cultivar y caracterizar células epidérmicas y mesenquimales de gelatina de Wharton sobre matrices de quitosano ultradelgadas con la finalidad de evaluar la biocompatibilidad y su potencial uso como sustitutos dérmicos. Material y Método. Empleamos 2 modelos celulares de origen humano previo consentimiento informado de los donantes sanos, células madre mesenquimales obtenidas a partir de gelatina de Wharton y células epidérmicas aisladas mediante digestión con dipasa de muestras de piel. Sembramos ambos tipos celulares en proporción de 5.000 cel/cm2 y evaluamos la adhesión celular por método cuantitativo CellTiter 96® (MTS) y cualitativamente empleando marcaje con DAPI de los núcleos celulares. Resultados. Logramos establecer cultivos homogéneos de ambos tipos celulares, queratinocitos y células madre mesenquimales a partir de gelatina de Wharton. Los dos tipos celulares mostraron una apropiada adhesión celular y viabilidad, tanto por el ensayo de MTS como por la evaluación cualitativa con DAPI, sobre las membranas ultradelgadas de quitosano. Conclusiones. Demostramos que las membranas de quitosano pueden emplearse como soportes apropiados para estos tipos celulares, los cuales podrían utilizarse como apósitos dérmicos vivos en el tratamiento de úlceras crónicas y quemaduras, ya que el sistema combina las propiedades regenerativas y bacteriostáticas del quitosano con el alto potencial regenerativo cutáneo que presentan tanto las células madre mesenquimales como las células epidérmicas

Introduction and Objective. There is an important worldwide demand to develop biological substitutes that restore, maintain or improve tissue function based on the combined use of biomaterials that function as scaffolds, using cells that induce regeneration, to repair injured or diseased tissues. Our objective is to culture and characterize the Wharton gelatin epidermal and mesenchymal cells on ultrathin chitosan matrices in order to evaluate the biocompatibility and its potential use as dermal substitutes. Methods. Two cellular models of human origin were used with the prior informed consent of healthy donors, mesenchymal stem cells obtained from Wharton's gelatin and epidermal cells, isolated by digestion with dispase of skin samples. Both cell types planted at a rate of 5,000 cel/cm2 and cell adhesion was evaluated by the quantitative CellTiter 96® (MTS) method and qualitatively using the DAPI labeling of the cell nuclei. Results. Through this study, homogenous cultures of both cell types, keratinocytes and mesenchymal stem cells were established. The 2 cell types showed an appropriate cellular adhesion and viability on the ultrathin membranes of chitosan, both by the MTS assay and by the qualitative evaluation with DAPI. Conclusions. It was demonstrated that chitosan membranes can be used as appropriate supports for these cell types, which could be used as living dermal dressings in the treatment of chronic ulcers and burns, since the system combines the regenerative and bacteriostats properties of chitosan with high cutaneous regenerative potential presented by both mesenchymal stem and epidermal cells

Growth arrest and morphological changes triggered by emodin on Trypanosoma cruzi epimastigotes cultivated in axenic medium.

Emodin is an anthraquinone obtained from Rheum palmatum rootstocks. Here we tested the cytotoxic effects of emodin on Trypanosoma cruzi epimastigotes, as well as the morphological changes that were induced by this compound in the parasite. Emodin was permeable and blocked in vitro cell division of T. cruzi epimastigotes in axenic medium, causing growth arrest in a dose-dependent but reversible manner. Emodin-exposed epimastigotes underwent duplication of organelles, such as the nucleus, kinetoplast and flagellum, but were incapable of completing cytokinesis. Neither elongation of the parasite body nor appearance of the regular longitudinal cleavage furrow was displayed, suggesting that emodin is most likely affecting components of the parasite cytoskeleton. Moreover, drug-treated parasites acquired alterations such as protuberances, folds and indentations on their membrane surface. Since emodin has been shown to be a potent protein kinase CK2 inhibitor, and we have previously described an association between tubulin and CK2 in T. cruzi epimastigotes (De Lima et al. Parasitology132, 511-523, 2006), we also measured the indirect effect of the drug on tubulin. Incubation of epimastigotes with axenic medium containing emodin hindered the endogenous phosphorylation of tubulin in whole-cell parasite extracts. All our results suggested that the parasite CK2 may be important for the maintenance of the morphology and for the regulation of mitosis-cytokinesis transition in T. cruzi epimastigotes.

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity.

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.