Publications

The extracellular matrix (ECM) is a dynamic and complex network of proteins and molecules that surrounds cells and tissues in the nervous system and orchestrates a myriad of biological functions. This review carefully examines the diverse interactions between cells and the ECM, as well as the transformative chemical and physical changes that the ECM undergoes during neural development, aging, and disease. These transformations play a pivotal role in shaping tissue morphogenesis and neural activity, thereby influencing the functionality of the central nervous system (CNS). In our comprehensive review, we describe the diverse behaviors of the CNS ECM in different physiological and pathological scenarios and explore the unique properties that make ECM-based strategies attractive for CNS repair and regeneration. Addressing the challenges of scalability, variability, and integration with host tissues, we review how advanced natural, synthetic, and combinatorial matrix approaches enhance biocompatibility, mechanical properties, and functional recovery. Overall, this review highlights the potential of decellularized ECM as a powerful tool for CNS modeling and regenerative purposes and sets the stage for future research in this exciting field. This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Implantable Materials and Surgical Technologies > Nanomaterials and Implants

JTD Keywords: Amyotrophic-lateral-sclerosis, Biologic scaffold, Central nervous system, Central-nervous-system, Chondroitin sulfate proteoglycans, Decellularization, Extracellular matrix, Motor-neurons, Neural disorders, Neural regeneratio, Perineuronal nets, Self-healing hydrogel, Spinal-cord-injury, Stem-cell, Vascular basement-membrane

The accurate spatial segregation into distinct phases within cell membranes coordinates vital biochemical processes and functionalities in living organisms. One of nature's strategies to localize reactivity is the formation of dynamic raft domains. Most raft models rely on liquid-ordered L-0 phases in a liquid-disordered L-d phase lacking correlation and remaining static, often necessitating external agents for phase separation. Here, we introduce a synthetic system of bicomponent glycodendrimersomes coassembled from Janus dendrimers and Janus glycodendrimers (JGDs), where lactose-lactose interactions exclusively drive lateral organization. This mechanism results in modulated phases across two length scales, yielding raft-like microdomains featuring nanoarrays at the nanoscale. By varying the density of lactose and molecular architecture of JGDs, the nanoarray type and size, shape, and spacing of the domains were controlled. Our findings offer insight into the potential primordial origins of rudimentary raft domains and highlight the crucial role of glycans within the glycocalyx.

JTD Keywords: Article, Artificial cells, Atomic force microscopy, Bicomponents, Bilayer, Bilayer membrane, Biochemical functionality, Biochemical process, Biological-membranes, Cell component, Cell membrane, Cellular parameters, Chemical interaction, Chemical structure, Chemistry, Cytology, Defined janus glycodendrimers, Dehydration, Dendrimer, Dendrimers, Dilution, Dimer, External agents, Fourier transform, Giant vesicles, Glycan, Glycans, Glycocalyx, Glycodendrimers, Janus dendrimer, Janus glycodendrimer, Lactose, Lateral organization, Lectin, Lipid rafts, Living organisms, Membrane damage, Membrane microdomain, Membrane microdomains, Membrane structure, Metabolism, Modulated phases, Molecule, Monomer, Nanoarrays, Oligosaccharide, Organization, Periodicity, Phase separation, Phase-separation, Phospholipids, Polysaccharide, Polysaccharides, Raft like domain, Relative humidity, Spatial segregation, Structure analysis, Sugars, Synthetic systems, Tetramer, Unclassified drug, Unilamellar vesicles, Water

Anisotropy is an important cue for neural organization during morphogenesis and healing, contributing to the mechanical and functional properties of neural tissues. The ability to replicate such anisotropy in vitro holds great promise for the development of effective regeneration strategies. In this work, interfacial polyelectrolyte complexation (IPC) is applied to fabricate microfibers from charged ECM components without any chemical modification. Using flow-focusing microfluidics, collagen (Col) and glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) or heparin (Hep), form Col/CS and Col/Hep interfacial complexes that coalesce as IPC microfibers. These fibers are flexible and absorb large amounts of water but remain stable under physiological conditions. At these conditions, the tensile strength of the assembled Col/GAG microfibers is similar to the strength of the neural tissue. The fibers are biocompatible and biofunctional; PC12 neural cells adhere and orient longitudinally to the fibers. Moreover, Col/CS microfibers promote the formation of neural processes. The results demonstrate that the microfluidic-assisted IPC complexation enables the assembly of ECM mimics by synergetic integration of anisotropic, chemical, and mechanical cues that boost the development of neural cells.

JTD Keywords: Cells, Chondroitin sulfate, Collagen, Fibers, Glycosaminoglycans, Heparin, Microfibers

Glycans are ubiquitously expressed sugars, coating the cell and protein surfaces. They are found on many proteins as either short and branched chains or long chains sticking out from special membrane proteins, known as proteoglycans. This sugar cushion, the glycocalyx, modulates specific interactions and protects the cell. Here it is shown that both the expression of proteoglycans and the glycans expressed on the surface of both the host and virus proteins have a critical role in modulating viral attachment to the cell. A mathematical model using SARS-Cov-2 as an archetypical virus to study the glycan role during infection is proposed. It is shown that this occurs via a tug-of-war of forces. On one side, the multivalent molecular recognition that viral proteins have toward specific host glycans and receptors. On the other side, the glycan steric repulsion that a virus must overcome to approach such specific receptors. By balancing both interactions, viral tropism can be predicted. In other words, the authors can map out the cells susceptible to virus infection in terms of receptors and proteoglycans compositions.© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.

JTD Keywords: binding, entry, glycocalyx, mechanisms, multiplexing, multivalency, nanoparticles, recognition, super-selectivity, viral infectivity, Functional receptor, Glycans, Glycocalyx, Multiplexing, Multivalency, Nanoparticles, Super-selectivity, Viral infectivity

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

JTD Keywords: expression, in-vitro, mouse model, muscle, mutations, phenotype, quantification, sarcolemma, therapy, 3' untranslated regions, Cells, cultured, Crispr-cas systems, Cytoskeletal proteins, Drug discovery, Dystroglycans, Dystrophin, Gene editing, Hek293 cells, Humans, Muscular dystrophy, duchenne, Myoblasts, Myogenic regulatory factor 5, Primary cell culture, Sarcoglycans, Utrophin, Utrophin up-regulation

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10 mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and β-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters.

JTD Keywords: Carbohydrate interactions, Evolutionary adaptation, Porifera, Proteoglycans, Sulfated polysaccharides

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.

JTD Keywords: Glycosaminoglycans, Malaria, Nanomedicine, Plasmodium, Targeted drug delivery

The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2β1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment. Statement of Significance: Natural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine.

JTD Keywords: Collagen reorganization, Glycosaminoglycans, Layer-by-layer technique, Mesenchymal stem cells, Osteogenic differentiation

Olfactory ensheathing cell (OEC) transplantation emerged some years ago as a promising therapeutic strategy to repair injured spinal cord. However, inhibitory molecules are present for long periods of time in lesioned spinal cord, inhibiting both OEC migration and axonal regrowth. Two families of these molecules, chondroitin sulphate proteoglycans (CSPG) and myelin-derived inhibitors (MAIs), are able to trigger inhibitory responses in lesioned axons. Mounting evidence suggests that OEC migration is inhibited by myelin. Here we demonstrate that OEC migration is largely inhibited by CSPGs and that inhibition can be overcome by the bacterial enzyme Chondroitinase ABC. In parallel, we have generated a stable OEC cell line overexpressing the Nogo receptor (NgR) ectodomain to reduce MAI-associated inhibition in vitro and in vivo. Results indicate that engineered cells migrate longer distances than unmodified OECs over myelin or oligodendrocyte-myelin glycoprotein (OMgp)-coated substrates. In addition, they also show improved migration in lesioned spinal cord. Our results provide new insights toward the improvement of the mechanisms of action and optimization of OEC-based cell therapy for spinal cord lesion.

JTD Keywords: Olfactory ensheathing cells, Traction force microscopy, Chondroitin sulphate proteoglycans, Cell migration, Nogo receptor ectodomain

One mechanism leading to neurodegeneration during Alzheimer's Disease (AD) is amyloid beta peptide (A beta)-induced neurotoxicity. Among the factors proposed to potentiate A beta toxicity is its covalent modification through carbohydrate-derived advanced glycation endproducts (AGEs). Other experimental evidence, though, indicates that certain polymeric carbohydrates like the glycosaminoglycan (GAG) chains found in proteoglycan molecules attenuate the neurotoxic effect of A beta in primary neuronal cultures. Pretreatment of the 42-residue A beta fragment (A beta(1-42)) with the ubiquitous brain carbohydrates, glucose, fructose, and the GAG chondroitin sulfate B (CSB) inhibits A beta beta(1-42)-induced apoptosis and reduces the peptide neurotoxicity on neuroblastoma cells, a cytoprotective effect that is partially reverted by AGE inhibitors such as pyridoxamine and L-carnosine. Thioflavin T fluorescence measurements indicate that at concentrations close to physiological, only CSB promotes the formation of A beta amyloid fibril structure. Atomic force microscopy imaging and Western blot analysis suggest that glucose favours the formation of globular oligomeric structures derived from aggregated species. Our data suggest that at short times carbohydrates reduce A beta(1-42) toxicity through different mechanisms both dependent and independent of AGE formation.

JTD Keywords: Alzheimer's disease, Advanced glycation endproducts, Amyloid fibrils, Amyloid beta peptide, Apoptosis, Carbohydrates, Glycosaminoglycans

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca2+-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of I-p = 0.31 +/- 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of tau = 7.9 +/- 4.9 s and a reaction bond length of x(beta) = 0.31 +/- 0.08 nm. Whereas the x(beta)-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.

JTD Keywords: Bovine nasal cartilage, Articular-cartilage, Sinorhizobium-meliloti, Proteoglycan, Microscopy, DNA, Macromolecules, Binding, Protein, Glycosaminoglycans