Ce travail est consacré à l'élaboration de nano-complexes polyélectrolytes (CPEs) ayant une stabilité améliorée en milieux physiologiques et à l'exemplification de leur fort potentiel d'application comme système de délivrance de (macro) molécules bioactives. Le chitosane comme polycation a été compléxé avec quatre polyanions naturels ayant différents densités de charges et groupements fonctionnels(-COO- et SO3-) à savoir l'acide hyaluronique (HYA), le chondroïtine sulfate (ChonS), le sulfate de dextrane (DS) et l'héparine (HEP). Les facteurs qui influent sur la formation et les propriétés physico-chimiques des nano-complexes chitosane-HYA ont été étudiés. Ces nanovecteurs perdent leur caractère colloïdal en milieux physiologiques. Pour améliorer leur stabilité dans ces conditions, une stratégie innovante qui implique l'ajout de zinc a été conçue. Cette stratégie de stabilisation a été démontrée comme étant polyvalente et a été étendue aux complexes polyélectrolytes (CPEs) chitosane-ChonS. Même si de cette manière une stabilité à long terme a été observée, cette stratégie reste uniquement applicable aux CPEs cationiques. Pour cette raison, une approche alternative permettant l'amélioration de la stabilité des colloïdes à charges positives ou négatives a été mise en oeuvre en concevant des nano-complexes de type coeur-couronne ternaires composés de polyacides forts c'est-à-dire de DS ou d'HEP associés au chitosane en coeur et un complexe chitosane-HYA en couronne. Tous les nano-complexes stables obtenus peuvent encapsuler le ténofovir, une molécule antirétrovirale et être fonctionnalisés par des IgAs de ciblage. En in vitro, ces nanovecteurs montrent une inhibition de l'infection des PBMC par le virus VIH-1 supérieure à l'antirétrovirale seule / This work is devoted to the elaboration of nano-polyelectrolyte complexes (PECs) systems with improved stability in physiological media and to the establishment of their high potential of applications as bioactive (macro) molecule delivery systems. Chitosan as polycation were complexed with four natural polyanions of different charged groups and densities (-COO- and SO3 - as negative charges), namely hyaluronan (HYA), chondroitin sulfate (ChonS), dextran sulfate (DS) and heparin (HEP). The factors impacting the formation and physical-chemical properties of chitosan-HYA nanocomplexes were investigated. However, these nanovectors lost their colloidal character in physiological media. To improve their colloidal stability in physiological conditions, an innovative stabilization strategy was designed, involving zinc ion. This stabilization strategy proved versatile and was extended to chitosan-ChonS PECs. Though a long-term stability was achieved, this strategy was only applicable to cationic PECs. Therefore, an alternate approach enabled the improvement of the colloidal stability in physiological media of both positive and negative colloids by designing core-shell ternary polyelectrolyte nanocomplexes composed of strong polyacid (DS or HEP)-chitosan PECs as core and a chitosan-HYA complex as shell. Furthermore, all of the stabilized nanocomplexes allowed the encapsulation of active molecules anti-retroviral drug tenofovir and surface functionalization with targeting IgAs. In vitro, these nanovectors exhibited an inhibition of infection of PBMCs by HIV-1 virus which could be superior to the free drug

This work focuses on examination of hyaluronan-sulfactant aggregates in term of determination of aggregate number. The value of critical micellar concentration (CMC) cetyltrimethylammonium bromide (CTAB) in three different solvents (water, phosphate buffer and physiological solution). Next the effect of the native hyaluronan supplement on the value of CMC was examined. It wasfound out that the solvent has the biggest effect on the value of CMC whilst the hyaluronan supplement affects CMC only a little. The aggregate number (Nagg) CTAB and the effect of the native hyaluronan supplement were determined out by means of fluorescence quenching. Pyrene was used as a fluorescence probe. Iodine and cetylpyridinium chloride (CPC) were used as quenchers. Sulfactant was dissolved in three different solvents (water, phosphate buffer and physiological solution). Not only the hyaluronan supplement but also the used solvent has the strong effect on the value of the aggregate number. When using 10mM CTAB dissolved in physiological solution the value of Nagg was 119 ± 4 while the value was half in buffer. Then we found out that in most cases the hyaluronan supplement reduces the value of the aggregate number.

In this diploma thesis, the interactions between polyelectrolyte and surfactant at low and also at high concentration were studied. There was used pyrene as fluorescent probe during the fluorescence spectroscopy measurement, a cationic surfactant cetyltrimethylammonium bromide and as a main polyelectolyte has been chosen sodium polystyrene sulfonate at 1 MDa molecular size. In the medium containing 0.15 M NaCl we could observed a creation of the complexes – precipitates in the surrounding of CMC concentration and behind of this concentration. In the mixtures containing sodium polystyrene sulfonate and hyaluronan together, there was stronger tend to keep aggregation properties of sodium polystyrene sulfonate during difference concentration ratios. Beyond CMC concentration, hyaluronan starts to influence the aggregation properties of the system as well.

The presented dissertation focuses on non-covalent interactions of hyaluronan of different molecular weights (9–1540 kDa) with basic (oligo)-amino acids (especially arginine) and the antimicrobial peptide cecropin B. High-resolution ultrasonic spectroscopy (HR-US), isothermal titration calorimetry (ITC) and potentiometric titration techniques were chosen to investigate the interactions. The thesis focuses on the characterization of interactions, especially with respect to the used molecular weight of interacting polymers and the ionic strength of the environment. Whether interactions occur or not was determined primarily by the length of the arginine oligomer chain. For monomeric amino acids, the interactions were investigated mainly by potentiometric titrations. Interactions were observable from arginine oligomers with eight monomer units. The molecular weight of hyaluronan mainly affected the intensity of the interactions. The transition between the individual conformations of hyaluronan (rod and random coil) was especially significant. Investigation of interactions was performed in water, in solutions with different concentrations of sodium chloride and in PBS. The sufficiently high ionic strength of the solution was able to suppress the interactions in water between the oligomers of arginine and hyaluronan. The basic antimicrobial peptide cecropin B has been shown to interact with hyaluronan in water but not in PBS. Based on these results, it was possible to conclude that the hyaluronan-cecropin B system would be particularly suitable for topical applications.

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

The main objective of this thesis work was to gain understanding of the principles of biolubrication, focusing on synergistic effects between biolubricants. To this end surface force and friction measurements were carried out by means of Atomic Force Microscopy, using hydrophilic and hydrophobic model surfaces in salt solutions of high ionic strength (≈ 150 mM) in presence of different biolubricants. There was also a need to gain information on the adsorbed layers formed by the biolubricants. This was achieved by using a range of methods such as Atomic Force Microscopy PeakForce imaging, Quartz Crystal Microbalance with Dissipation, Dynamic Light Scattering and X-Ray Reflectometry. By combining data from these techniques, detailed information about the adsorbed layers could be obtained.The biolubricants that were chosen for investigation were a phospholipid, hyaluronan, lubricin, and cartilage oligomeric matrix protein (COMP) that all exist in the synovial joint area. First the lubrication ability of these components alone was investigated, and then focus was turned to two pairs that are known or assumed to associate in the synovial area. Of the biolubricants that were investigated, it was only the phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) that was found to be an efficient lubricant on its own. Deposited DPPC bilayers on silica surfaces were found to be able to provide very low friction coefficients (≈ 0.01) up to high pressures, ≈ 50 MPa. A higher load bearing capacity was found for DPPC in the liquid crystalline state compared to in the gel state.The first synergy pair that was explored was DPPC and hyaluronan, that is known to associate on the cartilage surface, and we also noticed association between hyaluronan and DPPC vesicles as well as with adsorbed DPPC bilayers. By combining these two components a lubrication performance similar to that of DPPC alone could be achieved, even though the friction coefficient in presence of hyaluronan was found to be slightly higher. The synergy here is thus not in form of an increased performance, but rather that the presence of hyaluronan allows a large amount of the phospholipid lubricant to accumulate where it is needed, i.e. on the sliding surfaces.The other synergy pair was lubricin and COMP that recently has been shown to be co-localized on the cartilage surface, and thus suggested to associate with each other. Lubricin, as a single component, provided poor lubrication of PMMA surfaces, which we utilized as model hydrophobic surfaces. However, if COMP first was allowed to coat the surface, and then lubricin was added a low friction coefficient (≈ 0.03) was found. In this case the synergy arises from COMP facilitating strong anchoring of lubricin to the surface in conformations that provide good lubrication performance. / Huvudsyftet med det här avhandlingsarbetet var att öka förståelsen för den låga friktion som finns i vissa biologiska system, med fokus på synergistiska effekter mellan de smörjande molekylerna. För detta ändamål studerades ytkrafter och friktion med hjälp av atomkraftsmikroskopi. Mätningarna utfördes med hydrofila och hydrofoba modellytor i lösningar med hög salthalt (≈ 150 mM) i närvaro av smörjande biomolekyler. Det var också nödvändigt att få information om de adsorberade skikten av biomolekyler. Det åstadkoms med hjälp av en rad tekniker så som AFM PeakForce avbildning, kvartskristallmikrovåg, dynamisk ljusspridning och röntgen reflektometri. Genom att kombinera data från dessa tekniker erhölls detaljerad information om de smörjande skikten.De smörjande biomolekyler som valdes ut för studierna var en fosfolipid, hyaluronan, lubricin, and cartilage oligomeric matrix protein (COMP) vilka alla finns i synovialledsområdet. Först undersöktes den smörjande förmågan hos dessa komponenter var för sig, och sedan fokuserade vi på två par av biomolekyler som man vet eller antar bildar associationsstrukturer i synovialleder. Av de enskilda biomolekyler som undersöktes var det endast fosfolipiden 1,2-dipalmitoyl-sn-glycero-3-fosfokoline (DPPC) som visade sig vara en effektivt smörjande molekyl. Deponerade biskikt av DPPC på silikaytor gav upphov till mycket låga friktionskoefficienter (≈ 0.01) upp till höga pålagda tryck, ≈ 50 MPa. DPPC bilager i flytande kristallin fas visade sig ha högre lastbärande förmåga än DPPC bilager i geltillstånd.Det första synergistiska par som undersöktes var DPPC och hyaluronan vilka man vet associerar på broskytan, och vi visade att hyaluronan associerar med såväl DPPC vesiklar som med DPPC bilager. Genom att kombinera dessa två komponenter uppmättes en smörjande förmåga som var jämförbar med den som DPPC ensam uppvisar. Även om friktionskoefficienten var något högre i närvaro av hyaluronan. Synergieffekten här består inte av en bättre smörjande förmåga, utan istället gör närvaron av hyaluronan att de smörjande fosfolipiderna kan ansamlas i stora mängder där de behövs, dvs. på de glidande ytorna.Det andra synergiparet var lubricin och COMP vilka nyligen har visats vara lokaliserade på samma platser på broskytan, vilket tyder på att de associerar med varandra. På egen hand var lubricins smörjande förmåga av PMMA, våra hydrofoba modellytor, dålig. Emellertid, om COMP först adsorberades på PMMA och sedan lubricin tillsattes uppmättes en låg friktionskoefficient (≈ 0.03). I det här fallet består synergin av att COMP möjliggör en stark inbindning till ytan av lubricin i konformationer som ger god smörjande förmåga. / <p>QC 20141202</p> / Stiftelsen för strategisk forskning - SSF

Collagen and glycosaminoglycans, such as hyaluronan and chondroitin sulfate, are the major components of bone extracellular matrix, and extracellular matrix composites are being evaluated for a wide range of clinical applications. The molecular and cellular effects of native and sulfatemodified glycosaminoglycans on osteocytes were investigated as critical regulators of bone remodeling. The effects of glycosaminoglycans on viability, necrosis, apoptosis, and regulation of gene expression were tested in two osteocyte-like cell lines, the murine MLO-Y4 and the rat UMR 106-01 cells. Glycosaminoglycans were non-toxic and incorporated by osteocytic cells. In MLO-Y4 cells, sulfation of glycosaminoglycans led to a significant inhibition of osteocyte apoptosis, 42% inhibition for highly sulfated chondroitin sulfate and 58% for highly sulfated hyaluronan, respectively. Cell proliferation was not affected. While treatment with highly sulfated chondroitin sulfate increased cell viability by 20% compared to the native chondroitin sulfate. In UMR 106- 01 cells, treatment with highly sulfated hyaluronan reduced the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio by 58% compared to the non-sulfated form, whereas highly sulfated chondroitin sulfate led to 60% reduction in the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio in comparison to the native chondroitin sulfate. The expression of SOST, the gene encoding sclerostin, was reduced by 50% and 45% by highly sulfated hyaluronan and chondroitin sulfate, respectively, compared to their native forms. The expression of BMP- 2, a marker of osteoblast differentiation, was doubled after treatment with the highly sulfated hyaluronan in comparison to its native form. In conclusion, highly sulfated glycosaminoglycans inhibit osteocyte apoptosis in vitro and promote an osteoblast-supporting gene expression profile.

L’entreprise SEPROSYS est une société spécialisée dans le développement de solutions d'extraction et de séparation de molécules. Au cours de l’année 2011, l’entreprise a développé un procédé innovant de séparation et de purification fractionnée de biomolécules issues de macroalgues marines. Ce travail de thèse a pour objectif de trouver des applications aux fractions extraites selon le procédé d’extraction et de purification de la société SEPROSYS, et en particulier la fraction des polysaccharides sulfatés, dans deux domaines distincts : en pharmaceutique pour leur activité anti-coagulante et en cosmétique pour leur activité anti-âge. La première partie de nos travaux se penche sur le potentiel des polysaccharides sulfatés de macroalgues comme anti-coagulants. Pour cela, des extraits aqueux de différentes macroalgues brunes, rouges et vertes ont été préparés et leur activité anti-coagulante a été mesurée. Les algues présentant le meilleur potentiel ont ensuite été traitées au moyen du procédé SEPROSYS® afin de purifier leurs polysaccharides sulfatés et d’étudier leur activité anti-coagulante. De plus, l’objectif de ce travail consiste également à mieux comprendre la relation structure-fonction de l'activité anticoagulante d'ulvanes. A cette fin, des ulvanes issus d’Ulva sp. de pureté élevée ont été produits par le procédé SEPROSYS®, soumis à des modifications chimique (hypersulfatation) et physique (dépolymérisation) et leur activité anticoagulante a été étudiée. La deuxième partie de nos travaux concerne l’évaluation du potentiel de ces macroalgues comme agents actifs utilisables en dermo-cosmétique, en particulier pour leur activité anti-âge. Dans ce but, différentes fractions de macroalgues brunes, rouges et vertes ont été testées sur des lignées cellulaires de fibroblastes dermiques humains et leur effet sur la production de collagène par ces fibroblastes a été étudié. Enfin, le potentiel d’ulvanes extraits d’Uva sp. par le procédé de l’entreprise SEPROSYS pour stimuler la biosynthèse de collagène et d’acide hyaluronique par ces mêmes fibroblastes a été déterminé. / SEPROSYS is a company specialized in the development of solutions for the extraction and separation of molecules. In 2011, the company has developed an innovative process of fractionated separation and purification of biomolecules from macroalgae. The purpose of this thesis work is to find applications for the purified fractions from the SEPROSYS® process and in particular the sulfated polysaccharides, for two distinct biological activities : as pharmaceutical for their anti-coagulant activity and as dermo-cosmetics for their anti-aging activity. The first part of our work focus on the potential of sulfated polysaccharides from macroalgae as anti-coagulant agents. To this end, aqueous extracts from brown, red and green macroalgae were prepared and their anti-coagulant activity was measured. The best macroalgae were then processed with the SEPROSYS procedure in order to purify the sulfated polysaccharides and study their activity. Furthermore, one of the objectives of this work was to acquire a better understanding of the structure/anti-coagulant function relationship of ulvans. Ulvans of high purity were extracted from Ulva sp. with the SEPROSYS® process, submitted to chemical (hypersulfatation) and physical (depolymerization) modifications and their anti-coagulant activity was measured. The second part of our work relates to the potential of the macroalgae as active agents to be used in dermo-cosmetics, and, in particular, for their anti-aging activity. Different brown, red and green macroalgae fractions were thus tested on normal human dermal fibroblasts cell lines and their effects on the collagen production were measured. Furthermore, the capacity of different molecular weight ulvans extracted from Ulva sp. to stimulate the collagen and hyaluronan biosynthesis was studied. To this end, we developed an efficient depolymerization procedure using ion exchange resins.

No description available.

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.