Hyaluronan (HA) is a negatively charged extracellular matrix (ECM) component with water-attracting properties. It is the dominating ECM component in the renal medullary interstitium, where the amount changes in relation to hydration status: it increases during hydration and decreases during dehydration. It has, therefore, been suggested that HA participates in the regulation of renal fluid handling by changing the permeability properties of the interstitial space. This thesis investigates potential mechanisms for such a role in renal fluid regulation. The results demonstrate that the high renal HA content of late nephrogenesis decreases during the completion of kidney development in the rat, which takes place in the neonatal period. The heterogenous distribution of HA is mainly established during the first three weeks after birth. On day 21, the HA content is similar to that in the adult rat. The process is dependent on normal Ang II function. It primarily involves a reduction of HA synthase 2 expression and an increase of medullary hyaluronidase 1.  The cortical accumulation of HA that results from neonatal ACE inhibition can partly explain the pathological condition of the adult kidney, which causes reduced urinary concentration ability and tubulointerstitial inflammation. It is possible to reduce renomedullary HA with the HA synthesis inhibitor 4-MU, and the kidney’s ability to respond to a hydration challenge will then be suppressed, without affecting GFR.  The investigation of renomedullary interstitial cells (RMIC) in culture, shows that media osmolality and hormones of central importance for body fluid homeostasis, such as angiotensin II, ADH and endothelin, affect HA turnover through their effect on the RMICs, in a manner comparable to that found in vivo during changes in hydration status.  In established streptozotocin-induced diabetes, HA is regionally accumulated in the kidney, proteinuria and polyuria, reduced urine osmolality, and reduced response to ADH V2 activation will occur. As opposed to the proteinuria, the HA accumulation is not sensitive to mTOR inhibition, suggesting an alternate pathway compared to other ECM components  Taken together, the data suggest that during normal physiological conditions, renomedullary interstitial HA participates in renal fluid handling by affecting the interstitial prerequisites for fluid flux across the interstitial space. This is possible due to the water-attracting and physicochemical properties of this glycosaminoglycan. During pathological conditions, such as diabetes, the elevated interstitial HA can contribute to the defective kidney function, due to the proinflammatory and water-attracting properties of HA.

En accordance avec son haut prévalence dans le monde, parmi tous les cas de démence, la maladie d'Alzheimer (MA) est considérée comme la principal pathologie affectant les personnes plus âgées que 65 ans. Depuis son première description en 1907, de la recherche important et des observations innovants ont été faites concernant des aspects histopathologiques et moléculaires la neurodégénération associée à la maladie. Cependant, les mécanismes moléculaires de la pathogenèse et de la progression de la MA restent toujours partiellement compris. Outre, des stratégies thérapeutiques efficaces soit pour la prévention, soit pour l'arrêt de la progression de la maladie ne sont pas encore développées. Il semble donc crucial le développement de la recherche dans des domaines émergeants, nés à partir des concepts innovants et basés sur des approches mécanistiques novateurs à fin de découvrir des aspects dans la physiopathologie de la neurodégénérescence qui puissent conduire à des stratégies thérapeutiques pour soigner ces maladies.Les études présentées ici sont centrées dans le rôle des héparanes sulfates (HS), un membre particulier de la famille des glycosaminoglycannes, dans la physiopathologie des troubles neurodégénératifs, tels que la MA et démences associées, nommées taupathies. Ce travail de recherche, basé sur plusieurs observations isolées suggérant une association entre la pathologie de tau caractéristique des taupathies et les HS, explore par des moyens de études moléculaires, cellulaires et animaux les implications pathologiques de telle interaction. Comme résultat, je montre ici des évidences suggérant une participation clé des HS dans les évènements pathologiques de tau, tels que la phosphorylation anormale, la formation des inclusions intracellulaires, et la propagation des amas de tau.Globalement, le travail présenté ici dévoile une implication importante des HS hautement sulfatés dans la pathologie de tau associée à la MA, et au même temps ouvre une gamme de voies de recherche novatrices pour approfondir dans la caractérisation de l'interaction tau/HS et ses consequences physiopathologiques. De plus, ceci suggère des cibles pharmacologiques alternatives qui puisèrent donner d'espoir pour trouver un traitement effectif pour la MA. / According to its higher prevalence worldwide among all dementia cases, Alzheimer's disease (AD) is placed as the first pathology affecting people aged of more than 65 years old. Since it first description in 1907, profound research and groundbreaking observations have been made concerning the histopathological and molecular aspects of its associated neurodegeneration. However, the molecular mechanisms of AD pathogenesis and progression remain still poorly understood. In addition, an efficient therapeutic approach to either prevent or stop the disease progression has not yet been developed. It becomes hence crucial to develop research in emerging areas raising from groundbreaking concepts and supported by new mechanistic approaches in order to unveil novel aspects of the physiopathology of neurodegeneration and therefore design new therapeutic approaches to treat these pathologies.The present study is focused on the role of heparan sulfate (HS), a particular member of the glycosaminoglycan family, in the physiopathology of neurodegenerative disorders, such as AD and related dementias, termed tauopathies. Based on numerous separate observations suggesting an association between tau pathology characteristic of tauopathies and HS, this research explores the pathological implications of such interaction by the means of molecular, cellular, and animal studies. As a result, I hereby present evidence suggesting a crucial involvement of HS in tau pathological events, such as abnormal phosphorylation, inclusion formation, and assembly propagation.Globally, the present work unveils a strong implication of highly sulfated HS in tau pathology associated to AD and related tauopathies, and opens a wide array of novel research pathways to deepen into the characterization of tau /HS interplay and its pathophysiologic consequences. In addition, it suggests alternative pharmacological targets that could bring some hope in finding an effective treatment for AD.

Les chaînes de glycosaminoglycanes (GAGs) des protéoglycanes (PGs) jouent un rôle majeur dans la régulation de multiples événements cellulaires et le maintien de l'architecture des tissus. Des perturbations de la synthèse des GAGs sont impliquées dans des pathologies d'origine dégénérative, tumorale et génétique, tel que le syndrome progéroïde d'Ehlers-Danlos (ED). Ce déficit résulte de mutations de la [bêta]1,4-galactosyltransférase 7 ([bêta]4GalT7) humaine associées à des atteintes sévères du système musculo-squelettique. En effet, cette enzyme catalyse une étape essentielle de l?initiation de la synthèse des GAGs à partir de la protéine "core" des PGs et de xylosides exogènes. Notre travail a porté sur l'étude structure-fonction de la [bêta]4GalT7 recombinante humaine. Nous avons associé des approches in vitro et ex vivo afin d?explorer le rôle des acides aminés des motifs 163DVD165, 221FWGWGREDDD230 et 257HLH259, strictement conservés au sein des [bêta]4GalTs. L'étude des conséquences de mutations systématiques sur les propriétés cinétiques et fonctionnelles de la [bêta]4GalT7 recombinante a permis d'identifier des acides aminés essentiels du site actif. Nous avons montré que les résidus D165 et H257 forment des liaisons de coordination avec le cation Mn2+ et proposé le rôle du résidu D228 dans la catalyse. Nous avons mis en évidence un rôle central du résidu W224 dans les interactions avec les substrats donneur et accepteur. Nous avons également établi les bases moléculaires des mutations de la [bêta]4GalT7 associées au syndrome ED. Enfin, l'étude de mécanismes de régulation épigénétique des voies de biosynthèse des GAGs dans les cellules H-EMC-SS de chondrosarcome humain a mis en évidence une hyperméthylation spécifique des gènes de la famille des 3-O-sulfotransférases, associée à un phénotype invasif. L'ensemble de ce travail ouvre des perspectives vers de nouvelles stratégies thérapeutiques dans le traitement des arthropathies / Proteoglycans (PGs) and their glycosaminoglycan chains (GAGs), play a major role in the architecture of extracellular matrices and are implicated in numerous cell events. The impairment of GAG synthesis and sulfation is involved in degenerative, tumor and genetic diseases, such as the progeroid form of Ehlers-Danlos (ED) syndrome. This inherited disorder is due to mutations of human [bêta]4GalT7 ([bêta]4GalT7) causing a defect in GAG synthesis, associated with severe musculo-skeletal alterations. Indeed, this enzyme catalyzes a key step in GAG synthesis linked to the core protein of PGs and from exogenous xylosides. Our work has been focused on the structural and functional characterization of human recombinant [bêta]4GalT7 enzyme. We combined in vitro and ex vivo approaches to explore the role of amino acids located in 163DVD165, 221FWGWGREDDD230 and 257HLH259 motifs, which are highly conserved within [bêta]4GalTs. The study of the consequences of site-directed mutations on kinetic and functional properties of the [bêta]4GalT7 enzyme allowed us to identify key active site amino acids. Our results indicate that D165 and H257 residues form coordination bonds with Mn2+ divalent cations. Furthermore, we suggested a catalytic role for D228 residue and highlighted a central role of W224 residue via interactions with the donor and acceptor substrates. We also determined the molecular basis of [bêta]4GalT7 mutations associated with ED syndrome. Finally, the study of epigenetic regulation mechanisms by DNA methylation of GAG biosynthesis in human chondrosarcoma cells (H-EMC-SS) revealed the specific hypermethylation of the 3-O-sulfotransferase gene family, associated with the invasive phenotype of these cells. Together, this work paves the way towards innovative strategies in the treatment of arthropathies

Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that assist the catalytic domains to recognize the target substrate and keep it in proximity. Understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. We focus on CBMs that target both oligomeric and non-crystalline cellulose while exhibiting various similarities and differences in binding specificity and structural properties; such CBMs are classified as Type B CBMs. We show that all six cellulose-specific Type B CBMs studied in this dissertation can recognize the cello-oligomeric ligands in bi-directional fashion, meaning there was no preference towards reducing or non-reducing end of ligand for the cleft/groove like binding sites. Out of the two sandwich and twisted forms of binding site architectures, twisted platform turned out to facilitate tighter binding also exhibiting longer binding sites. The exterior loops of such binding sites were specifically identified by modeling the CBMs with non-crystalline cellulose showing that high- and low-affinity binding site may arise based on orientation of CBM while interacting with non-crystalline substrate. These findings provide various insights that can be used for further understanding of tandem CBMs and for various CBM based biotechnological applications. The later part of this dissertation reports the identification of a physiological ligand for a mammalian glycoprotein YKL-40 that has been only known as a biomarker in various inflammatory diseases and cancers. It has been shown to bind to oligomers of chitin, but there is no known function of YKL-40, as chitin production in the human body has never been reported. Possible alternative ligands include proteoglycans, polysaccharides, and fibers such as collagen, all of which make up the mesh comprising the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. We considered the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. Our simulation results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, as the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non- specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to the associated increases in hyaluronan.

<p>Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-<i>O</i>-sulfotransferase (2OST) and HS glucosaminyl 6-<i>O</i>-sulfotransferases (6OSTs) were investigated. </p><p>Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-<i>O</i>-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-<i>O</i>-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[³⁵S]sulfate (³⁵S-labeled PAPS). All three enzymes catalyzed 6-<i>O</i>-sulfation of both <i>N</i>-acetylated (GlcNAc) as well as <i>N</i>-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.</p><p>To understand how pre-existing 2-<i>O</i>-sulfates affects 6-<i>O</i>-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-<i>O</i>-sulfates. The specificities for substrates with two or three 2-<i>O</i>-sulfates were higher compared to octasaccharides with no or one 2-<i>O</i>-sulfate indicating that 2-<i>O</i>-sulfate groups substantially promote the subsequent 6-<i>O</i>-sulfation. </p><p>Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-<i>O</i>-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. </p><p>The 2OST catalyzes 2-<i>O</i>-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-<i>O</i>-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and ³⁵S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-<i>O</i>-sulfation of GlcA occurs before IdoA is formed.</p>

Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-O-sulfotransferase (2OST) and HS glucosaminyl 6-O-sulfotransferases (6OSTs) were investigated. Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-O-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-O-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (35S-labeled PAPS). All three enzymes catalyzed 6-O-sulfation of both N-acetylated (GlcNAc) as well as N-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted. To understand how pre-existing 2-O-sulfates affects 6-O-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-O-sulfates. The specificities for substrates with two or three 2-O-sulfates were higher compared to octasaccharides with no or one 2-O-sulfate indicating that 2-O-sulfate groups substantially promote the subsequent 6-O-sulfation. Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-O-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. The 2OST catalyzes 2-O-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-O-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and 35S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-O-sulfation of GlcA occurs before IdoA is formed.

Les grandes propriétés interactives des HS dépendent de leur profil de sulfatation, finement régulé au cours de la biosynthèse du polysaccharide ainsi qu'à la surface cellulaire par l'action d'endosulfatases nommées Sulfs. Ces enzymes ôtent spécifiquement les groupements 6-O-sulfates d'un certain nombre de disaccharides trisulfatés, éléments clés de nombreuses interactions protéines/HS. Ainsi, ces enzymes sont impliquées dans de nombreux processus, autant physiologiques que pathologiques. En utilisant différentes approches (biochimique et biophysique), nous avons révélé récemment que les isoformes humain HSulf-1 et -2 agissent de façon processif et orientées pour désulfater les chaines d’HS ce qui régule différemment les facteurs de croissances FGF-1 et -2 (Seffouh et al., FASEB J. 2013). Nous avons également identifié deux motifs impliqués dans l’interaction HSulf-2/HS et qui se sont avérés importants pour l’activité 6-O-endosulfatase [Manuscrit en préparation]. Par ailleurs, HSulf-2, et contrairement à HSulf-1, s’est révélé porteuse d’une O-glycosylation capable de moduler considérablement son activité à la surface cellulaire [Manuscrit en préparation]. Enfin, et en collaboration avec l’'institut Karolinska (Stockholm, Sweden), une éventuelle implication de Sulf-1 dans le mésothéliome malin a été suggéré (Heidari-Hamedani et al., Cellular Signalling J. 2015). / Heparan sulfate (HS) is a polysaccharide able to bind and modulate a wide variety of proteins. HS large interactive properties are essentially governed by precise sulfation patterns of the polysaccharide. Sulf-1 and Sulf-2 are two endosulfatases able to cleave specific 6-O sulfate groups within HS chains. Their action can modulate critical intracellular signaling processes, many of which with key relevance for cancer development. However, little is known on their structure, substrate specificities, and on the way their catalytic activity directs the subtle modifications of HS 6-O-sulfation profile.In this work, we have identified an original enzymatic mechanism by which Sulfs catalyze the processive and orientated desulfation of HS and finely regulate the polysaccharide biological properties. We also identified two basic motifs within the N-terminal domain of HSulf-2. Recombinant enzymes mutated for these sites were then produced and characterized. We found that these epitopes are necessary for its endosulfatase activity. Altogether, these results provide further insights into the enzyme/substrate recognition process and contribute to the understanding of the fundamental role played by these enzymes in the regulation of HS activity. The biochemical study of the purified HSulf-2 allowed to highlight a O-glycosylation able to significantly modulate its activity at the cell surface. Otherwise, and in collaboration with the Karolinska Institute (Stockholm, Sweden), the study of malignant mesothelioma (MM) provided new evidence that enhanced Syndecan-1 expression also finely modulates HS structure by interfering with HS-modifying enzymes HSulf-1. These results suggest important roles for Syndecan-1 and HSulf-1 in MM.

Background: Tissue engineering and its contribution to regenerative medicine has advanced through the years. It has proven its efficacy especially in the treatment of advanced full thickness burn wounds. Tissue engineering is the synergy between biology and engineering. This fairly young science has one common goal and that is to regenerate new tissue. Various commercially available products have appeared on the market and this due to the ground-breaking work of many. One such well known product is Integra® which is the brain child of Yannas and Burke. This is a collagen-glycosaminoglycan copolymer which serves as a bioactive regeneration template or extracellular matrix analogue. Advanced wound healing is promoted along with the prevention of scar tissue formation and consequent contractures. Aims:</p This study provides an extensive review on the development of this dermal regeneration matrix and also aims to develop an equivalent product. Attention will be paid to: the biological building blocks and the motivation for their use; the essential production steps; and the final processing required in order to deliver a sterile product. Materials and Methods: A collagen and chondroitin 6-sulphate coprecipitate was prepared and subjected to either controlled or uncontrolled freezing. The frozen slurry was dried under vacuum for 17 hours after which each sample was coated with a thin silicone film. Glutaraldehyde crosslinking followed after which the product was thoroughly rinsed. The packaged products were then subjected to terminal sterilisation via gamma irradiation under various conditions. Various tests were conducted to evaluate the newly formed regeneration matrices and included scanning electron microscopy, enzymatic degradation by collagenase, and a cytotoxicity assay. Scanning electron microscopic analysis was done in order to reveal the adequacy of the scaffold architecture. Collagenase degradation of the scaffolds was used to project the rate of degradation of each template. Integra® served as the gold standard for each test. Quantifiable data was statistically analysed and any comparison made included the calculation of means, standard deviations and p-values (confidence interval of 95%). Results: Results indicated that highly porous bioactive tissue engineering matrices were obtained by either controlled freezing or uncontrolled freezing. The average pore diameter of the most homogenous scaffolds ranged between 52.47 and 136.44 µm with a mean of 87.34 µm. These templates were formed by using a 0.5% collagen concentration and a controlled freeze rate of 0.92 °C/min. Uncontrolled freezing (1.3 °C/min) of a 0.5% collagen concentration resulted in the formation of an irregular scaffold with an average pore diameter of 174.08 µm. It was found that the architecture of the most equivalent scaffold compared well with that of Integra® with p = 0.424. Scaffolds prepared using higher collagen concentrations (1.0%) and controlled freezing resulted in dense sponges with average pore diameters of 56.51 µm. Statistical analysis upon comparison indicated a significant difference p = 0.000 in the micro architecture. The rate of degradation of the most equivalent scaffold was 1.9 times that of Integra®. This implicates that the crosslinking was insufficient and due to one of the following: poor collagen quality; method of crosslinking; and degradation due to terminal sterilization. The rate of scaffold degradation can be extended, either by additional crosslinking or the prevention of degradation induced by irradiation. Temperature vacuum dehydration crosslinking through esterification or amide formation can be used as an initial crosslinking method in further studies. This form of crosslinking will complete the conventional glutaraldehyde crosslinking that reacts with the free amine groups of lysine or hydroxylysine of the protein backbone of collagen. It should be stressed that the determination of an in vivo degradation rate, in the form of an animal study, will aid to confirm the efficacy of the biologically active regeneration matrix. / Dissertation (MSc)--University of Pretoria, 2008. / Anatomy / unrestricted

Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host...

Dynamic alterations of composition and mechanics of the extracellular matrix (ECM) are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) were used to mimic matrix stiffening as well as modification by sulfated and non-sulfated GAGs at different stages of wound healing. Human MPhs were found to sensitively respond to these microenvironmental cues in terms of polarization towards pro-inflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibited a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, TNF). Presence of sulfated and non-sulfated GAGs inhibited this polarization effect. Furthermore, control experiments on 2D matrices stressed the relevance of using stiffness-controlled 3D matrices, as MPhs showed a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.