RÉSUMÉ
Sulfated cellulose (CS) represents an interesting biopolymer due to bioactivity comparable to heparin. However, use of CS for making surface coatings or hydrogels requires the presence of reactive groups for covalent reactions. Here, an approach is presented to oxidize cellulose sulfates for subsequent cross-linking reactions with amino groups to form imine bonds. Cellulose is sulfated by direct sulfation or acetosulfation, followed by a Malaprade oxidation. The CS obtained is characterized by elemental analysis and 13 C-NMR spectroscopy. The resulting oxidized cellulose sulfates (oxCS) have different degrees of sulfation ranging from 0.79 to 1.13 and oxidation degrees from 0.18 to 0.34, but also different mass average molecular mass (MW ). Toxicity studies are carried out with mouse 3T3 fibroblasts exposed to aqueous solutions of oxCS. The results show that all oxCS are non-toxic at lower concentrations (0.5 mg mL-1 ), but with both increasing degree of oxidation and concentrations, toxic effects are observed particularly for acetosulfated and lesser for direct sulfated oxCS, which is related to a decrease in the MW of the products. It is concluded that oxCS obtained by direct sulfation with MW above 70 kDa may represent a biocompatible material for the applications suggested above.
Sujet(s)
Matériaux biocompatibles , Cellulose/analogues et dérivés , Test de matériaux , Cellules 3T3-L1 , Animaux , Matériaux biocompatibles/composition chimique , Matériaux biocompatibles/pharmacologie , Cellulose/composition chimique , Cellulose/pharmacologie , Souris , OxydoréductionRÉSUMÉ
Control of the biomaterial properties through stimuli-responsive polymeric platforms has become an essential technique in recent biomedical applications. A multilayer system of thiolated chitosan (t-Chi) and thiolated chondroitin sulfate (t-CS), consisting of five double layers ([t-Chi/t-CS]5), was fabricated here by applying a layer-by-layer coating strategy. To represent a novel class of chemically tunable nanostructures, the ability to cross-link pendant thiol groups was tested by a rise from pH 4 during layer formation to pH 9.3 and a more powerful chemical stimulus by using chloramine-T (ChT). Following both treatments, the resulting multilayers showed stimuli-dependent behavior, as demonstrated by their content of free thiols, wettability, surface charge, elastic modulus, roughness, topography, thickness, and binding of fibronectin. Studies with human dermal fibroblasts further demonstrated the favorable potential of the ChT-responsive multilayers as a cell-adhesive surface compared to pH-induced cross-linking. Because the [t-Chi/t-CS]5 multilayer system is responsive to stimuli such as the pH and redox environment, multilayer systems with disulfide bond formation may help to tailor their interaction with cells, film degradation, and controlled release of bioactive substances like growth factors in a stimuli-responsive manner useful in future wound healing and tissue engineering applications.
Sujet(s)
Fibroblastes , Matériaux biocompatibles , Adhérence cellulaire , Chitosane , Humains , Ingénierie tissulaireRÉSUMÉ
Surface properties are believed to play important roles in initial inflammatory and subsequent wound healing/fibrotic responses after implantation of biomaterials. To investigate the surface property effect in mediating these host responses, we used an in vitro fibroblast/macrophage co-culture model established with a cell migration chamber, and a series of self-assembling monolayers (SAMs) bearing different terminal groups as model surfaces to study the effect of surface properties on macrophage fusion, fibroblast attachment, spreading morphology, proliferation, outgrowth, as well as pro-(interleukin-6) and anti-(interleukin-10) inflammatory cytokine production, expression of ED-A fibronectin (FN) and alpha-smooth muscle actin (α-SMA). The obtained results show that the hydrophobic CH3 surface caused high levels of inflammatory but low levels of wound healing/fibrotic responses, while the hydrophilic/anionic COOH surface resulted in both low levels of inflammatory and wound healing/fibrotic responses. Interestingly, the hydrophilic OH surface was found to possess a low potential of inducing inflammatory responses but high potential of inducing wound healing/fibrotic responses. These results reveal that the extent of inflammation and wound healing/fibrosis might not be always related in vitro. However, more important is the observation of the macrophage contributions in facilitating the wound healing and fibrotic responses by up-regulation of fibroblast outgrowth, cytokine production as well as ED-A FN and α-SMA expression. Overall, by linking the surface properties to cell activities with our established fibroblast/macrophage co-culture system, we could provide an useful model system for in vitro studies to design more biocompatible biomaterials for various biomedical and tissue engineering applications.
Sujet(s)
Matériaux biocompatibles , Fibroblastes/effets des médicaments et des substances chimiques , Macrophages/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Techniques de coculture , Fibroblastes/cytologie , Fibroblastes/immunologie , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Humains , Interleukine-10/métabolisme , Interleukine-6/métabolisme , Macrophages/cytologie , Macrophages/immunologieRÉSUMÉ
The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool. Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells. Here we used PuraMatrix (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types. PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free. Recently we have studied the influence of the PM-concentration on the formation of the scaffold. In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by ßIII-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells, however the concentration might be adapted to other cell types. The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.
Sujet(s)
Techniques cytologiques/méthodes , Hydrogels/composition chimique , Cellules souches neurales/cytologie , Cytométrie en flux/méthodes , HumainsRÉSUMÉ
Hydrogel-based three-dimensional (3D) scaffolds are widely used in the field of regenerative medicine, translational medicine, and tissue engineering. Recently, we reported the effect of scaffold formation on the differentiation and survival of human neural progenitor cells (hNPCs) using PuraMatrix™ (RADA-16) scaffolds. Here, we were interested in the impact of PuraMatrix modified by the addition of short peptide sequences, based on a bone marrow homing factor and laminin. The culture and differentiation of the hNPCs in the modified matrices resulted in an approximately fivefold increase in neuronal cells. The examination of apoptotic and necrotic cells, as well as the level of the anti-apoptotic protein Bcl-2, indicates benefits for cells hosted in the modified formulations. In addition, we found a trend to lower proportions of apoptotic or necrotic neuronal cells in the modified matrices. Interestingly, the neural progenitor cell pool was increased in all the tested matrices in comparison to the standard 2D culture system, while no difference was found between the modified matrices. We conclude that a combination of elevated neuronal differentiation and a protective effect of the modified matrices underlies the increased proportion of neuronal cells.
RÉSUMÉ
The transplantation of stem cells offers potential therapies for many neurodegenerative disorders that currently have limited or no treatment options. However, relatively little is known about how the host environment affects the development and integration of these cells. In this study we have engrafted immortalized human midbrain neural progenitor cells (NPCs) onto rat hippocampal brain slice cultures to examine the influence of a neural environment on differentiation. Patch clamp recordings revealed that the transplanted progenitor cells could express neuronal-type voltage-gated currents and rapidly receive synaptic input from the hippocampal brain slice. The distribution of progenitor cells across the hippocampal slices was strongly influenced by the neural architecture, with most cells located in the fissural regions and sending processes parallel to the laminar structure, while in contrast, cells located in the dentate gyrus showed no organized pattern. Almost no cells were found in the stratum radiatum or pyramidal cell layers. Together, these results demonstrate the potential for the architecture of the host environment to regulate the integration of transplanted cells, and highlight the utility of coculture systems for studying the mechanisms underlying the migration, integration, and differentiation of human NPCs in structured neural environments.
Sujet(s)
Différenciation cellulaire/physiologie , Gyrus denté/métabolisme , Cellules souches neurales/métabolisme , Neurones/métabolisme , Niche de cellules souches/physiologie , Transplantation de cellules souches , Animaux , Lignée de cellules transformées , Techniques de coculture , Gyrus denté/cytologie , Humains , Cellules souches neurales/cytologie , Cellules souches neurales/transplantation , Neurones/cytologie , Rats , Rat Wistar , Transplantation hétérologueRÉSUMÉ
BACKGROUND: 3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenviroment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. METHODS: In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. RESULTS: Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. CONCLUSIONS: Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.
Sujet(s)
Différenciation cellulaire/effets des médicaments et des substances chimiques , Cellules souches neurales/cytologie , Cellules souches neurales/effets des médicaments et des substances chimiques , Structures d'échafaudage tissulaires/composition chimique , Techniques de culture cellulaire , Survie cellulaire/effets des médicaments et des substances chimiques , Humains , Hydrogels/composition chimique , Hydrogels/pharmacologie , Laminine/composition chimique , Neurones/cytologie , Ingénierie tissulaireRÉSUMÉ
Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and ß-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized ß-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/ß-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized ß-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/ß-catenin pathway in ReNcell VM cells.