Nanoscale mechanical properties of chitosan hydrogels as revealed by AFM.

In the context of tissue engineering, chitosan hydrogels are attractive biomaterials because they represent a family of natural polymers exhibiting several suitable features (cytocompatibility, bioresorbability, wound healing, bacteriostatic and fungistatic properties, structural similarity with glycosaminoglycans), and tunable mechanical properties. Optimizing the design of these biomaterials requires fine knowledge of its physical characteristics prior to assessment of the cell-biomaterial interactions. In this work, using atomic force microscopy (AFM), we report a characterization of mechanical and topographical properties at the submicron range of chitosan hydrogels, depending on physico-chemical parameters such as their polymer concentration (1.5%, 2.5% and 3.5%), their degree of acetylation (4% and 38.5%), and the conditions of the gelation process. Well-known polyacrylamide gels were used to validate the methodology approach for the determination and analysis of elastic modulus (i.e., Young's modulus) distribution at the gel surface. We present elastic modulus distribution and topographical and stiffness maps for different chitosan hydrogels. For each chitosan hydrogel formulation, AFM analyses reveal a specific asymmetric elastic modulus distribution that constitutes a useful hallmark for chitosan hydrogel characterization. Our results regarding the local mechanical properties and the topography of chitosan hydrogels initiate new possibilities for an interpretation of the behavior of cells in contact with such soft materials.

Repair strategies for traumatic spinal cord injury, with special emphasis on novel biomaterial-based approaches.

As a part of the central nervous system (CNS), the adult mammalian spinal cord displays only very poor ability for self-repair in response to traumatic lesions, which mostly lead to more or less severe, life-long disability. While even adult CNS neurons have a certain plastic potential, their intrinsic regenerative capacity highly varies among different neuronal populations and in the end, regeneration is almost completely inhibited due to extrinsic factors such as glial scar and cystic cavity formation, excessive and persistent inflammation, presence of various inhibitory molecules, and absence of trophic support and of a growth-supportive extracellular matrix structure. In recent years, a number of experimental animal models have been developed to overcome these obstacles. Since all those studies based on a single approach have yielded only relatively modest functional recovery, it is now consensus that different therapeutic approaches will have to be combined to synergistically overcome the multiple barriers to CNS regeneration, especially in humans. In this review, we particularly emphasize the hope raised by the development of novel, implantable biomaterials that should favor the reconstruction of the damaged nervous tissue, and ultimately allow for functional recovery of sensorimotor functions. Since human spinal cord injury pathology depends on the vertebral level and the severity of the traumatic impact, and since the timing of application of the different therapeutic approaches appears very important, we argue that every case will necessitate individual evaluation, and specific adaptation of therapeutic strategies.

Morphofunctional plasticity in the adult hypothalamus induces regulation of polysialic acid-neural cell adhesion molecule through changing activity and expression levels of polysialyltransferases.

Polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression in the adult nervous system is restricted to regions retaining a capacity for morphological plasticity. For the female rat hypothalamoneurohypophysial system (HNS), we have previously shown that lactation induces a dramatic decrease in PSA-NCAM, while leaving the level of total NCAM protein unchanged. Here, we wanted to elucidate the molecular mechanisms leading to a downregulation of PSA, thereby stabilizing newly established synapses and neurohemal contacts that accompany the increased activity of oxytocinergic neurons. First, we show that the overall specific activity of polysialyltransferases present in tissue extracts from supraoptic nuclei decreases by approximately 50% during lactation. So far, two polysialyltransferase enzymes, STX and PST, have been characterized for their capacity to transfer PSA onto NCAM in vitro. Using a competitive RT-PCR on RNA extracts from the HNS, we demonstrate furthermore a significant decrease in the expression levels of both STX and PST mRNAs in lactating versus virgin animals. Interestingly, this downregulation of NCAM polysialylation is not correlated with the post-transcriptional regulation of variable alternative spliced exon splicing, in contrast to neural development. The control of polysialylation via a regulation of both enzyme activity and expression underlines the important role of this post-translational modification of NCAM in morphofunctional plasticity in adult brain.

The mouse homeodomain protein OTX2 regulates NCAM promoter activity.

The homeodomain transcription factor OTX2 is involved in defining regional identities in developing rostral brain. It appears to participate in morphogenetic processes leading to the formation of boundaries and substrates for early axon growth, processes which are in the end largely based on site-specific expression of cell adhesion molecules. Here, we present evidence that a candidate target of OTX2 is the gene encoding the neural cell adhesion molecule, NCAM. When Otx2 is transfected into NIH3T3 cells, NCAM protein expression is upregulated. Moreover, while mock-transfected cells display only the 140 kDa-isoform of NCAM, Otx2 transfected cells express also the two other major isoforms (NCAM-120 and -180), in agreement with the presence of the corresponding transcripts in Northern blots. In addition, transient expression of Otx2 in COS7 cells is able to dramatically enhance the transcriptional activity of the NCAM promoter. Taken together, our results argue for a regulation of NCAM expression by OTX2.

A potential role for the OTX2 homeoprotein in creating early 'highways' for axon extension in the rostral brain.

Brain pattern formation starts with a subdivision of the neuroepithelium through site-specific expression of regulatory genes and, subsequently, the boundaries between presumptive neuromeres may provide a scaffold for early formation of axon tracts. In the mouse forebrain, the transcription factor OTX2 is strongly expressed at several such boundaries. Combining dye tracing and staining for OTX2 protein, we show that a number of early fibre tracts develop within stripes of OTX2 expression. To analyse a putative influence of OTX2 on the expression of molecules involved in neurite growth, we generated several clones of NIH3T3 cells stably expressing OTX2 protein at varying levels. As shown by immunoblotting, Otx2 transfection affects the expression of a variety of cell and substratum adhesion molecules, rendering the cells a favourable substratum in neurite outgrowth assays. Among the molecules upregulated with increasing levels of OTX2 are NCAM, tenascin-C and DSD-1-PG, which also in situ colocalize with zones of OTX2 expression at boundaries. These data suggest that Otx2 might be involved in defining local substrata for axon extension in the forebrain.

Modulation of NCAM polysialylation is associated with morphofunctional modifications in the hypothalamo-neurohypophysial system during lactation.

Post-transcriptional modification of the neural cell adhesion molecule (NCAM) by polysialic acid significantly decreases NCAM adhesiveness and more generally modifies cell-cell interactions. Polysialic acid-NCAM (PSA-NCAM) is mainly expressed in the developing nervous system. In the adult, its expression is restricted to regions that retain morphological plasticity, such as the hypothalamo-neurohypophysial system during lactation in rats. Since cell-cell interactions and synaptic contacts in the hypothalamo-neurohypophysial system are greatly increased during lactation, we examined whether PSA-NCAM expression is modified during this period. Immunohistochemistry and immunoblotting showed that, compared with virgin rats, PSA-NCAM dramatically decreased during lactation in both the supraoptic nuclei and the neurohypophysis, and returned to its initial level only after weaning. This decrease was progressive and became significant only at the end of the first week of lactation. By contrast, modifications in the level of NCAM protein or changes in the splicing pattern of NCAM mRNAs could not be detected. The decline in polysialic acid on the NCAM molecule could strengthen membrane appositions, thereby stabilizing the newly established synapses and neurohaemal contacts in the hypothalamo-neurohypophysial system that accompany the increased neuronal activity that occurs during lactation. We also studied the regulation of the phosphorylated microtubule-associated protein-1B (MAP1B-P), whose distribution pattern largely overlaps with that of PSA-NCAM in the adult brain. Expression of MAP1B-P was greatly increased during lactation in the hypothalamic axons projecting into the neurohypophysis. Thus, the expression patterns of both PSA-NCAM and MAP1B-P may reflect the permanent structural plasticity characterizing the hypothalamo-neurohypophysial system in the adult.

Affinity purification of monospecific antibodies from polyclonal sera as a means for the identification of differentially expressed genes and proteins.

The identification and characterization of genes and proteins that are either cell-type specific or exhibit an otherwise spatially or temporally restricted expression pattern is a crucial step toward understanding the complex interactions between the different cell types in an organism. We demonstrate here that monospecific polyclonal antibodies purified from rabbit antisera by a simple Western blotting and reelution technique can be successfully used for the detection of such proteins, as well as the corresponding genes by screening cDNA expression libraries. This strategy is discussed as a supplement to other approaches, such as the generation of tissue-specific monoclonal antibodies and the "subtractive hybridization technique."

Guidance and topographic stabilization of nasal chick retinal axons on target-derived components in vitro.

We studied mechanisms underlying the generation of topographic order within the developing chick retinotectal connection by combining the recently introduced stripe assay with a novel membrane protein fractionation technique. Our experiments show a preference of temporal and nasal retinal fibers for growing on cell membranes prepared from their proper target area. In addition, membrane preparations from posterior tectum were found to prolong substantially the survival of nasal neurites in vitro. We conclude that tropic as well as trophic interactions contribute to the generation of topographic maps during embryogenesis, in our case to the homing of nasal axons within the posterior tectum.

Differential reaction of crossing and non-crossing rat retinal axons on cell membrane preparations from the chiasm midline: an in vitro study.

In the rat, a small subpopulation of retinal ganglion cell axons forms a persistent projection to the ipsilateral half of the brain. These fibres originate almost exclusively from the ventrotemporal margin of the retina. In contrast to all other retinal axons they seem to be deflected from the midline of the optic chiasm and thereby led into the ipsilateral optic tract. In order to analyse the interactions between growing fibres and chiasm midline, we have developed the following in vitro model. Axons of the embryonic rat retina are grown on a carpet of tectal cell membranes used as a general growth-permissive substratum. At a certain distance from the explant (200-450 microns), the advancing fibres are confronted with two stripes of cell membranes prepared from the chiasm midline. Such chiasm membranes are shown to act as a barrier for the presumptive non-crossing axons, while they do not influence growth of fibres originating from any other regions of the retina, including the dorsotemporal part. The repulsion of non-crossing fibres by chiasm membranes is observed in vitro only when retinal explants from embryonic day (E) 17/18 and chiasm preparations from E14/15 are used. Fibres and tissue from different regions of the brain as well as from different developmental ages, and even from different species, can be combined in this assay system. In a first attempt to characterize the molecular basis of the repulsive effect of chiasm membranes on ventrotemporal fibres, similar assays were performed with membranes derived from other regions of the central nervous system midline, some of which are known to have repulsive properties against certain axon populations. Since these cell membranes did not act as a barrier for the ventrotemporal retinal axons, we suggest that the guidance cues at the chiasm are very specific. Our results are consistent with the hypothesis that certain cells at the chiasm midline (very likely radial glial cells) express 'repulsive or inhibitory' molecules, which act in a specific way on ipsilaterally projecting axons.

A method for the generation of monoclonal antibodies against rare cell-surface molecules.

Monoclonal antibodies provide a powerful tool for the identification and analysis of novel cell-surface molecules. We present here a method for antigen preparation and an immunization protocol that facilitates generation of mAb reactive with cell-surface molecules of low abundance and/or low antigenicity. The procedure involves isolation and extensive fractionation of cell-surface and detergent-soluble extracellular-matrix molecules prior to immunization. Cell-surface proteins on intact tissue are biotin-labeled using a reagent that does not penetrate cells. Avidin affinity chromatography is then used to purify these biotinylated molecules. Size-exclusion HPLC is used to separate these surface molecules on the basis of apparent molecular mass. Finally, immunization with antigen coupled to keyhole-limpet hemocyanin is combined with long-term booster immunizations to generate a hyperimmune response resulting in high-affinity IgG. A test application of this approach was aimed at the generation of mAb against cell-surface molecules of approximately 135 kDa in the developing chicken retinotectal system. Immunochemical analyses using antibodies produced by this approach which showed restricted patterns of tissue staining reveal that mAb were generated against all previously identified immunoglobulin superfamily molecules of this size in this system. Furthermore, we produced many additional antibodies that labeled retinotectal tissue in novel staining patterns. In the two cases analyzed in detail, these new patterns reflect the distributions of previously uncharacterized members of the immunoglobulin superfamily. The success of this initial study suggests that this method may represent a broadly applicable approach towards the preparation of extensive libraries of antibodies against cell-surface molecules expressed on cells from numerous sources.

Axonal Growth on Solubilized and Reconstituted Matrix from the Embryonic Chicken Retina Inner Limiting Membrane.

Basal laminae, thin sheets of extracellular matrix covering the basal side of all neuroepithelia, are strongly supportive for neurite outgrowth in vitro and may provide a permissive environment for growing neurites in vivo. To gain information about the biological activity and composition of in situ-derived basal laminae the inner limiting membranes from embryonic day (E) 7 to E11 chick and quail retinae were isolated. The basal laminae were solubilized with high-molar guanidine hydrochloride or urea, and the solubilized proteins reconstituted by dialysis. The matrix proteins were spotted or dried onto nitrocellulose or polylysine-coated dishes. When explants from retina or from dorsal root ganglia were incubated on the protein spots, neurite extension was very robust, at a level as high as on authentic basal lamina. Extracts from the pigment epithelial basement membrane did not support neurite extension. Western blot analysis showed that the explant from the retinal inner limiting membrane contained predominantly basal lamina-type proteins, such as laminin, collagen type IV and heparan sulphate proteoglycan, whereas the matrix extract from the pigment epithelium contained predominantly mesenchymal-type proteins, like collagen type I and tenascin. JG22, a beta1 integrin antibody that inhibited neurite extension on EHS tumour laminin substrate, had no effect on neurite outgrowth on retinal basal lamina matrix, indicating that embryonic basal laminae contain other or additional growth promoting substrate molecules.

Biochemical characterization of a putative axonal guidance molecule of the chick visual system.

Temporal retinal axons growing in vitro on carpets of tectal membranes are deflected by cell membranes of posterior tectum. The activity responsible for this deflection can be abolished by antibodies raised against tectal membranes and the corresponding Fab fragments. Analysis of tectal membranes by two-dimensional gel electrophoresis and immunoblotting reveals a 33 kd glycoprotein that has a higher concentration in posterior than in anterior tectum. Its expression is developmentally regulated, and it is sensitive to phosphatidylinositol-specific phospholipase C. These are properties expected for a molecule responsible for the phenomena observed in experiments on in vitro guidance of retinal axons.

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo.

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.

Use of the biotin-avidin system for labelling, isolation and characterization of neural cell-surface proteins.

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.

Factors influencing regeneration of retinal ganglion cell axons in adult mammals.

Adult retina ganglion cells regrow vigorously their lesioned axons in sciatic nerve segments transplanted at the site of optic nerve transection. In order to determine whether the transplanted peripheral nerve produces secretable substances involved in regeneration, a nerve exudate was collected from the adult sciatic nerve. Transection of the sciatic nerve in situ, implantation of silicone tubes around its proximal stump, and additional crush of the nerve further proximal permitted the reinnervation of the nerve stump inside the tube and the release of regeneration-induced substances. Biochemical analysis revealed that several proteins are secreted into the tubes. The fluid contents of the implanted tubes were removed 1 week after implantation and tested for neurotrophic activity in cultures of adult retinae. Massive regrowth of ganglion cell axons has been found in the presence of the nerve exudate. The numbers of axons were significantly higher than these obtained in the presence of nerve and fibroblast growth factors. The results suggest that the lesioned peripheral nerves deliberate during the period of reinnervation substances which also support axonal regrowth of injured central neurons.

Local embryonic matrices determine region-specific phenotypes in neural crest cells.

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions.