ABSTRACT
The blue crab (BC) Portunus segnis is considered an invasive species colonizing Tunisian coasts since 2014. This work aims to explore its associated bacteria potential to produce anionic exopolysaccharides (EPSs) in order to open up new ways of valorization. In this study, different BC samples were collected from the coastal area of Sfax, Tunisia. First, bacterial DNA was extracted from seven different fractions (flesh, gills, viscera, carapace scraping water, and three wastewaters from the production plant) and then sequenced using the metabarcoding approach targeting the V3-V4 region of the 16S rDNA to describe their microbiota composition. Metabarcoding data showed that the dominant bacterial genera were mainly Psychrobacter, Vagococcus, and Vibrio. In parallel, plate counting assays were performed on different culture media, and about 250 bacterial strains were isolated and identified by sequencing the 16S rDNA. EPS production by this new bacterial diversity was assessed to identify new compounds of biotechnological interest. The identification of the bacterial strains in the collection confirmed the dominance of Psychrobacter spp. strains. Among them, 43 were identified as EPS producers, as revealed by Stains-all dye in agarose gel electrophoresis. A Buttiauxella strain produced an EPS rich in both neutral sugars including rare sugars such as rhamnose and fucose and uronic acids. This original composition allows us to assume its potential for biotechnological applications and, more particularly, for developing innovative therapeutics. This study highlights bacterial strains associated with BC; they are a new untapped source for discovering innovative bioactive compounds for health and cosmetic applications, such as anionic EPS.
Subject(s)
Brachyura , Microbiota , Animals , Brachyura/genetics , Bacteria , Sugars , DNA, Ribosomal/genetics , Polysaccharides, BacterialABSTRACT
Diabolican is an exopolysaccharide (EPS) produced by Vibrio diabolicus HE800, a mesophilic bacterium firstly isolated from a deep-sea hydrothermal field. Its glycosaminoglycan (GAG)-like structure, consisting of a tetrasaccharide repeating unit composed of two aminosugars (N-acetyl-glucosamine and N-acetyl-galactosamine) and two glucuronic acid units, suggested to subject it to regioselective sulfation processes, in order to obtain some sulfated derivatives potentially acting as GAG mimics. To this aim, a multi-step semi-synthetic approach, relying upon tailored sequence of regioselective protection, sulfation and deprotection steps, was employed in this work. The chemical structure of the obtained sulfated diabolican derivatives was characterized by a multi-technique analytic approach, in order to define both degree of sulfation (DS) and sulfation pattern within the polysaccharide repeating unit, above all. Finally, binding affinity for some growth factors relevant for biomedical applications was measured for both starting diabolican and sulfated derivatives thereof. Collected data suggested that sulfation pattern could be a key structural element for the selective interaction with signaling proteins not only in the case of native GAGs, as already known, but also for GAG-like structures obtained by regioselective sulfation of naturally unsulfated polysaccharides.
Subject(s)
Polysaccharides , Sulfates , Sulfates/chemistry , Polysaccharides/chemistry , Glycosaminoglycans , Oligosaccharides , Intercellular Signaling Peptides and ProteinsABSTRACT
Glycosaminoglycans (GAGs) are essential constituents of the cell surface and extracellular matrix, where they are involved in several cellular processes through their interactions with various proteins. For successful tissue regeneration, developing an appropriate matrix supporting biological activities of cells in a similar manner than GAGs remains still challenging. In this context, this study aims to design a thermosensitive polysaccharide that could further be used as hydrogel for tissue engineering applications. For this purpose, infernan, a marine bacterial exopolysaccharide (EPS) endowed with GAG-mimetic properties was grafted with a thermosensitive polymer, poly(N-isopropylacrylamide) (pNIPAM). Eight grafted polysaccharides were obtained by varying EPS/pNIPAM molar ratio and the molecular weight of pNIPAM. Their physicochemical characteristics and their thermosensitive properties were determined using a multi-technique, experimental approach. In parallel, molecular dynamics and Monte Carlo simulations were applied at two different scales to elucidate, respectively, the molecular conformation of grafted infernan chain and their ability to form an infinite network undergoing a sol-gel transition near the percolation, a necessary condition in hydrogel formation. It comes out from this study that thermosensitive infernan was successfully developed and its potential use in tissue regeneration as a hydrogel scaffold will further be assessed.
Subject(s)
Glycosaminoglycans , Hydrogels , Temperature , Hydrogels/chemistry , PolysaccharidesABSTRACT
Bacteria are well-known to synthesize high molecular weight polysaccharides excreted in extracellular domain, which constitute their protective microenvironment. Several bacterial exopolysaccharides (EPS) are commercially available for skincare applications in cosmetic products due to their unique structural features, conferring valuable biological and/or textural properties. This review aims to give an overview of bacterial EPS, an important group of macromolecules used in cosmetics as actives and functional ingredients. For this purpose, the main chemical characteristics of EPS are firstly described, followed by the basics of the development of cosmetic ingredients. Then, a focus on EPS production, including upstream and downstream processes, is provided. The diversity of EPS used in the cosmetic industry, and more specifically of marine-derived EPS is highlighted. Marine bacteria isolated from extreme environments are known to produce EPS. However, their production processes are highly challenging due to high or low temperatures; yield must be improved to reach economically viable ingredients. The biological properties of marine-derived EPS are then reviewed, resulting in the highlight of the challenges in this field.
Subject(s)
Bacteria , Polysaccharides, Bacterial , Polysaccharides, Bacterial/pharmacology , Polysaccharides, Bacterial/chemistry , Molecular Weight , Cold Temperature , Extreme EnvironmentsABSTRACT
Mucopolysaccharidosis IIIA is a hereditary disease caused by mutations in the sulfamidase enzyme that participates in catabolism of heparan sulfate (HS), leading to HS fragment accumulation and multisystemic failure. No cure exists and death occurs around the second decade of life. Two low molecular weight highly sulfated compounds derived from marine diabolican and infernan exopolysaccharides (A5_3 and A5_4, respectively) with heparanase inhibiting properties were tested in a MPSIIIA cell line model, resulting in limited degradation of intracellular HS. Next, we observed the effects of intraperitoneal injections of the diabolican derivative A5_3 from 4 to 12 weeks of age on MPSIIIA mice. Brain metabolism and microstructure, levels of proteins and genes involved in MPSIIIA brain pathophysiology were also investigated. 1H-Magnetic Resonance Spectroscopy (MRS) indicated deficits in energetic metabolism, tissue integrity and neurotransmission at both 4 and 12 weeks in MPSIIIA mice, with partial protective effects of A5_3. Ex-vivo Diffusion Tensor Imaging (DTI) showed white matter microstructural damage in MPSIIIA, with noticeable protective effects of A5_3. Protein and gene expression assessments displayed both pro-inflammatory and pro-apoptotic profiles in MPSIIIA mice, with benefits of A5_3 counteracting neuroinflammation. Overall, derivative A5_3 was well tolerated and was shown to be efficient in preventing brain metabolism failure and inflammation, resulting in preserved brain microstructure in the context of MPSIIIA.
ABSTRACT
Sulfated glycosaminoglycans (GAGs) are fundamental constituents of both the cell surface and extracellular matrix. By playing a key role in cell-cell and cell-matrix interactions, GAGs are involved in many physiological and pathological processes. To design GAG mimetics with similar therapeutic potential as the natural ones, the specific structural features, among them sulfate content, sulfation pattern, and chain length, should be considered. In the present study, we describe a sulfation method based on microwave radiation to obtain highly sulfated derivatives as GAG mimetics. The starting low-molecular-weight (LMW) derivative was prepared from the infernan exopolysaccharide, a highly branched naturally slightly sulfated heteropolysaccharide synthesized by the deep-sea hydrothermal vent bacterium Alteromonas infernus. LMW highly sulfated infernan derivatives obtained by conventional heating sulfation have already been shown to display GAG-mimetic properties. Here, the potential of microwave-assisted sulfation versus that of the conventional method to obtain GAG mimetics was explored. Structural analysis by NMR revealed that highly sulfated derivatives from the two methods shared similar structural features, emphasizing that microwave-assisted sulfation with a 12-fold shorter reaction time is as efficient as the classical one.
Subject(s)
Glycosaminoglycans , Microwaves , Glycosaminoglycans/chemistry , Sulfates/chemistry , Magnetic Resonance Spectroscopy , Extracellular Matrix/metabolismABSTRACT
In the field of tissue engineering, in order to restore tissue functionality hydrogels that closely mimic biological and mechanical properties of the extracellular matrix are intensely developed. Mechanical properties including relaxation of the surrounding microenvironment regulate essential cellular processes. However, the mechanical properties of engineered hydrogels are particularly complex since they involve not only a nonlinear elastic behavior but also time-dependent responses. An accurate determination of these properties at microscale, i.e. as probed by cells, becomes an essential step to further design hydrogel-based biomaterials able to induce specific cellular responses. Atomic Force Microscopy (AFM) with contact sizes of the order of few micrometers constitutes an appropriate technique to determine the origin of relaxation mechanisms occurring in hydrogels. In the present study, AFM force relaxation experiments are conducted on chemically and physically crosslinked hydrogels respectively based on a synthetic polymer, polyacrylamide and a natural polymer, a bacterial exopolysaccharide infernan, produced by the deep-sea hydrothermal vent bacterium, Alteromonas infernus. Two distinct relaxation mechanisms are clearly evidenced depending on the nature of hydrogel network crosslinks. Chemically crosslinked hydrogel exhibits poroelastic relaxations, whereas physically crosslinked hydrogel shows time-dependent responses arising from viscoelastic effects. In addition, two relaxation processes are revealed in ionic physical hydrogel originating from chain rearrangement and breaking/reforming of the ionic crosslinks. The effect of the ionic strength on both the long-term elastic modulus and relaxation times of physical hydrogels was also shown. These findings highlight that physical hydrogels with well-defined time-dependent mechanical properties could be tuned for an optimized response of cells.
Subject(s)
Hydrogels , Tissue Engineering , Biocompatible Materials , Elastic Modulus , Extracellular Matrix , Hydrogels/chemistry , Tissue Engineering/methodsABSTRACT
With the increasing need for hydrogels with tunable properties for specific biomedical applications, a complete understanding of the structure-function relationship of polymers used for hydrogel development remains crucial for their optimal use. In the present study, by combining experimental and theoretical approaches, the structure-function relationship of a bacterial exopolysaccharide, infernan, displaying both glycosaminoglycan-mimetic and gelling properties, was investigated at molecular and microscopic levels. Atomic force microscopy (AFM) experiments and molecular dynamics simulations were applied to determine the persistence length of individual infernan chains before studying their association induced by calcium. Infernan-based microgels were then produced using microfluidics and their mechanical properties were characterized by AFM methods. The mechanical properties of EPS/calcium microgels were finely tuned by varying the crosslinking density of their network, either by calcium or EPS concentrations. The obtained set of viscoelastic microgels with different elastic modulus values opens several possibilities for their applications in tissue engineering.
Subject(s)
Microgels , Calcium , Hydrogels , Microscopy, Atomic Force , Tissue Engineering/methodsABSTRACT
Recent advances in glycobiotechnology show that bacterial exopolysaccharides (EPS) presenting glycosaminoglycan (GAG)-like properties can provide a valuable source of bio-active macromolecules for industrial applications. The HE800 EPS, named diabolican, is a marine-derived anionic high-molecular-weight polysaccharide produced by Vibrio diabolicus CNCM I-1629 which displays original structural features close to those of hyaluronic acid. We investigated the impact of carbon and nitrogen substrates on both Vibrio diabolicus growth and diabolican production. Both substrates were screened by a one-factor-at-a-time method, and experimental designs were used to study the effect of glucose, mannitol, and ammonium acetate various concentrations. Results showed that the medium composition affected not only the bacterium growth and EPS yield, but also the EPS molecular weight (MW). EPS yields of 563 and 330 mg L-1 were obtained in the presence of 69.3 g L-1 glucose and 24.6 g L-1 mannitol, respectively, both for 116.6 mM ammonium acetate. MW was the highest, with 69.3 g L-1 glucose and 101.9 mM ammonium acetate (2.3 × 106 g mol-1). In parallel, the bacterial maximum specific growth rate was higher when both carbon and nitrogen substrate concentrations were low. This work paves the way for the optimization of marine exopolysaccharide production of great interest in the fields of human health and cosmetics.
ABSTRACT
Bone Morphogenetic Protein (BMP-2) is an osteoinductive growth factor clinically used for bone regeneration. Tuneable sustained strategies for BMP-2 delivery are intensely developed to avoid severe complications related to supraphysiological doses applied. To address this issue, we investigated the ability of the bacterial exopolysaccharide (EPS) called Infernan produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus, exhibiting both glycosaminoglycan-mimetic and physical gelling properties, to efficiently bind and release the bioactive BMP-2. Two delivery systems were designed based on BMP-2 retention in either single or complex EPS-based microgels, both manufactured using a microfluidic approach. BMP-2 release kinetics were highly influenced by the ionic strength, affecting both microgel stability and growth factor/EPS binding, appearing essential for BMP-2 bioactivity. The osteogenic activity of human bone-marrow derived mesenchymal stem cells studied in vitro emphasized that Infernan microgels constitute a promising platform for BMP-2 delivery for further in vivo bone repair.
Subject(s)
Microgels , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 2/pharmacology , Bone Morphogenetic Proteins , Bone Regeneration , Glycosaminoglycans , Humans , OsteogenesisABSTRACT
Sulfated glycosaminoglycan (GAG) analogues derived from plant, algae or microbial sourced polysaccharides are highly interesting in order to gain bioactivities similar to sulfated GAGs but without risks and concerns derived from their typical animal sources. Since the exopolysaccharide (EPS) produced by the bacterium Vibrio diabolicus HE800 strain from deep-sea hydrothermal vents is known to have a GAG-like structure with a linear backbone composed of unsulfated aminosugar and uronic acid monomers, its structural modification through four different semi-synthetic sulfation strategies has been performed. A detailed structural characterization of the six obtained polysaccharides revealed that three different sulfation patterns (per-O-sulfation, a single N-sulfation and a selective primary hydroxyls sulfation) were achieved, with molecular weights ranging from 5 to 40 kDa. A Surface Plasmonic Resonance (SPR) investigation of the affinity between such polysaccharides and a set of growth factors revealed that binding strength is primarily depending on polysaccharide sulfation degree.
Subject(s)
Glycosaminoglycans/chemistry , Polysaccharides, Bacterial/chemistry , Vibrio , Amino Sugars/chemistry , Animals , Intercellular Signaling Peptides and Proteins/chemistry , Magnetic Resonance Spectroscopy/methods , Molecular Weight , Sulfates/chemistry , Surface Plasmon Resonance/methods , Uronic Acids/chemistryABSTRACT
The exopolysaccharide Infernan, from the bacterial strain GY785, has a complex repeating unit of nine monosaccharides established on a double-layer of sidechains. A cluster of uronic and sulfated monosaccharides confers to Infernan functional and biological activities. We characterized the 3-dimensional structures and dynamics along Molecular Dynamics trajectories and clustered the conformations in extended two-fold and five-fold helical structures. The electrostatic potential distribution over all the structures revealed negatively charged cavities explored for Ca2+ binding through quantum chemistry computation. The transposition of the model of Ca2+complexation indicates that the five-fold helices are the most favourable for interactions. The ribbon-like shape of two-fold helices brings neighbouring chains in proximity without steric clashes. The cavity chelating the Ca2+ of one chain is completed throughout the interaction of a sulfate group from the neighbouring chain. The resulting is a 'junction zone' based on unique chain-chain interactions governed by a heterotypic binding mode.
Subject(s)
Alteromonas/chemistry , Calcium/chemistry , Polysaccharides, Bacterial/chemistry , Models, Molecular , Molecular Conformation , Molecular Dynamics Simulation , Molecular Structure , Monosaccharides/chemistry , Quantum Theory , Sulfates/chemistryABSTRACT
Antimetastatic properties on both murine and human osteosarcoma cell lines (POS-1 and KHOS) have been evidenced using exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium. These derivatives had no significant effect on the cell cycle neither a pro-apoptotic effect on osteosarcoma cells. Based on this observation, these EPSs could be employed as new drug delivery systems for therapeutic uses. A theranostic approach, i.e., combination of a predictive biomarker with a therapeutic agent, has been developed notably by combining with true pair of theranostic radionuclides, such as scandium 47Sc/44Sc. However, it is crucial to ensure that, once complexation is done, the biological properties of the vector remain intact, allowing the molecular tropism of the ligand to recognize its molecular target. It is important to assess if the biological properties of EPS evidenced on osteosarcoma cell lines remain when scandium is complexed to the polymers and can be extended to other cancer cell types. Scandium-EPS complexes were thus tested in vitro on human cell lines: MNNG/HOS osteosarcoma, A375 melanoma, A549 lung adenocarcinoma, U251 glioma, MDA231 breast cancer, and Caco2 colon cancer cells. An xCELLigence Real Cell Time Analysis (RTCA) technology assay was used to monitor for 160 h, the proliferation kinetics of the different cell lines. The tested complexes exhibited an anti-proliferative effect, this effect was more effective compared to EPS alone. This increase of the antiproliferative properties was explained by a change in conformation of EPS complexes due to their polyelectrolyte nature that was induced by complexation. Alterations of both growth factor-receptor signaling, and transmembrane protein interactions could be the principal cause of the antiproliferative effect. These results are very promising and reveal that EPS can be coupled to scandium for improving its biological effects and also suggesting that no major structural modification occurs on the ligand.
Subject(s)
Alteromonas/metabolism , Cell Proliferation/drug effects , Neoplasms/drug therapy , Polysaccharides, Bacterial/pharmacology , Scandium/pharmacology , A549 Cells , Animals , Caco-2 Cells , Coordination Complexes , Heparin/pharmacology , Humans , Kinetics , Mice , Neoplasms/pathology , Polysaccharides, Bacterial/isolation & purificationABSTRACT
(1) Background: Exopolysaccharide (EPS) derivatives, produced by Alteromonas infernus bacterium, showed anti-metastatic properties. They may represent a new class of ligands to be combined with theranostic radionuclides, such as 47Sc/44Sc. The goal of this work was to investigate the feasibility of such coupling. (2) Methods: EPSs, as well as heparin used as a drug reference, were characterized in terms of molar mass and dispersity using Asymmetrical Flow Field-Flow Fractionation coupled to Multi-Angle Light Scattering (AF4-MALS). The intrinsic viscosity of EPSs at different ionic strengths were measured in order to establish the conformation. To determine the stability constants of Sc with EPS and heparin, a Free-ion selective radiotracer extraction (FISRE) method has been used. (3) Results: AF4-MALS showed that radical depolymerization produces monodisperse EPSs, suitable for therapeutic use. EPS conformation exhibited a lower hydrodynamic volume for the highest ionic strengths. The resulting random-coiled conformation could affect the complexation with metal for high concentration. The LogK of Sc-EPS complexes have been determined and showing that they are comparable to the Sc-Hep. (4) Conclusions: EPSs are very promising to be coupled with the theranostic pair of scandium for Nuclear Medicine.
Subject(s)
Alteromonas/chemistry , Coordination Complexes/chemistry , Polysaccharides/chemistry , Scandium/chemistry , Carbohydrate Conformation , Fractionation, Field Flow , Hydrodynamics , Light , Nuclear Medicine , Osmolar Concentration , Scattering, Radiation , Theranostic Nanomedicine , ViscosityABSTRACT
Bacteria from deep-sea hydrothermal vents constitute an attractive source of bioactive molecules. In particular, exopolysaccharides (EPS) produced by these bacteria become a renewable source of both biocompatible and biodegradable molecules. The low molecular weight (LMW) derivatives of the GY785 EPS produced by the deep-sea hydrothermal vent strain Alteromonas infernus have previously displayed some biological properties, similar to those of glycosaminoglycans (GAG), explored in cancer and tissue engineering. These GAG-mimetic derivatives are obtained through a free radical depolymerization process, which could, however, affect their structural integrity. In a previous study, we have shown that A. infernus produces depolymerizing enzymes active on its own EPS. In the present study, an enzymatic reaction was optimized to generate LMW derivatives of the GY785 EPS, which could advantageously replace the present bioactive derivatives obtained by a chemical process. Analysis by mass spectrometry of the oligosaccharide fractions released after enzymatic treatment revealed that mainly a lyase activity was responsible for the polysaccharide depolymerization. The repeating unit of the GY785 EPS produced by enzyme cleavage was then fully characterized.
Subject(s)
Alteromonas/chemistry , Oligosaccharides/chemistry , Polysaccharides, Bacterial/chemistry , Mass SpectrometryABSTRACT
Bacteria have developed a unique strategy to survive in extreme environmental conditions through the synthesis of an extracellular polymeric matrix conferring upon the cells a protective microenvironment. The main structural component of this complex network constitutes high-molecular weight hydrophilic macromolecules, namely exopolysaccharides (EPS). EPS composition with the presence of particular chemical features may closely be related to the specific conditions in which bacteria evolve. Deep-sea hydrothermal vent bacteria have already been shown to produce EPS rich in hexosamines and uronic acids, frequently bearing some sulfate groups. Such a particular composition ensures interesting functional properties, including biological activities mimicking those known for glycosaminoglycans (GAG). The aim of the present study was to go further into the exploration of the deep-sea hydrothermal vent IFREMER (French Research Institute for Exploitation of the Sea) collection of bacteria to discover new strains able to excrete EPS endowed with GAG-like structural features. After the screening of our whole collection containing 692 strains, 38 bacteria have been selected for EPS production at the laboratory scale. EPS-producing strains were identified according to 16S rDNA phylogeny. Chemical characterization of the obtained EPS highlighted their high chemical diversity with the presence of atypical compositional patterns. These EPS constitute potential bioactives for a number of biomedical applications, including regenerative medicines and cancer treatment.
Subject(s)
Bacteria/classification , Polysaccharides, Bacterial/metabolism , Seawater/microbiology , Sequence Analysis, DNA/methods , Bacteria/genetics , Bacteria/isolation & purification , Bacteria/metabolism , DNA, Bacterial/analysis , DNA, Ribosomal/analysis , France , Hexosamines/metabolism , Hydrothermal Vents , Phylogeny , RNA, Ribosomal, 16S/analysis , Uronic Acids/metabolismABSTRACT
Polysaccharides of marine origin are gaining interest as biomaterial components. Bacteria derived from deep-sea hydrothermal vents can produce sulfated exopolysaccharides (EPS), which can influence cell behavior. The use of such polysaccharides as components of organic, collagen fibril-based coatings on biomaterial surfaces remains unexplored. In this study, collagen fibril coatings enriched with HE800 and GY785 EPS derivatives were deposited on titanium alloy (Ti6Al4V) scaffolds produced by rapid prototyping and subjected to physicochemical and cell biological characterization. Coatings were formed by a self-assembly process whereby polysaccharides were added to acidic collagen molecule solution, followed by neutralization to induced self-assembly of collagen fibrils. Fibril formation resulted in collagen hydrogel formation. Hydrogels formed directly on Ti6Al4V surfaces, and fibrils adsorbed onto the surface. Scanning electron microscopy (SEM) analysis of collagen fibril coatings revealed association of polysaccharides with fibrils. Cell biological characterization revealed good cell adhesion and growth on bare Ti6Al4V surfaces, as well as coatings of collagen fibrils only and collagen fibrils enhanced with HE800 and GY785 EPS derivatives. Hence, the use of both EPS derivatives as coating components is feasible. Further work should focus on cell differentiation.
ABSTRACT
Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-ß1 (TGF-ß1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-ß1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-ß1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-ß1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.
Subject(s)
Alteromonas/chemistry , Drug Carriers/chemistry , Polysaccharides/chemistry , Transforming Growth Factor beta1/administration & dosage , Biological Availability , Cartilage, Articular/drug effects , Cartilage, Articular/physiology , Cell Line , Delayed-Action Preparations/administration & dosage , Delayed-Action Preparations/pharmacokinetics , Drug Carriers/isolation & purification , Drug Compounding/methods , Drug Implants , Drug Liberation , Humans , Hydrothermal Vents/microbiology , Microfluidics , Polysaccharides/isolation & purification , Regeneration/drug effects , Tissue Scaffolds/chemistry , Transforming Growth Factor beta1/pharmacokineticsABSTRACT
Sulfated polysaccharides, such as glycosaminoglycans (GAG) regulate various biological activities through their interactions with growth factors. Investigating these interactions becomes the key to understand the structure-function relationship of GAG. Highly sulfated derivatives prepared from the marine GY785 exopolysaccharide (EPS) produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus have previously shown to stimulate the chondrogenic differentiation of mesenchymal stem cells in the presence of Transforming Growth Factor-ß1 (TGF-ß1). Here, the interactions between the GAG-mimetic GY785 EPS derivatives and TGF-ß1 were investigated by Atomic Force Microscopy (AFM). The affinity between slightly sulfated or highly sulfated derivatives and TGF-ß1 was explored by AFM imaging and single-molecule force spectroscopy experiments. The number of measured interactions and the interaction strength were both higher for highly sulfated derivative compared to the slightly sulfated one. These results clearly emphasize the involvement of sulfate groups in the protein binding and open new ways to tune cellular processes by designing macromolecules with adjustable sulfate charge density.
ABSTRACT
Polysaccharides have attracted much attention due to their interesting physico-chemical and also biological properties that are explored in food, cosmetic and pharmaceutical industries. GY785 exopolysaccharide (EPS) presenting an unusual structure is secreted by the deep-sea hydrothermal bacterium, Alteromonas infernus. Low-molecular weight (LMW) derivatives obtained by chemical depolymerization of the native high molecular weight (HMW) EPS were previously shown to exhibit biological properties similar to glycosaminoglycans (GAG). In the present study, in order to generate well defined derivatives with a better control of the depolymerization, an enzymatic approach was applied for the first time. Various commercially available enzymes were firstly screened for their depolymerizing activities, however none of them was able to degrade the polysaccharide. Enzymatic assays performed with A. infernus protein extracts have shown that the bacterium produces by itself endogenous enzymes able to depolymerize its own EPS. The oligosaccharides released by the enzymes were analyzed and their structures allowed to assess that the protein extract contains several depolymerizing activities.