Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.

Les hydrogels injectables et auto-réparants suscitent un intérêt particulier dans les domaines de l'ingénierie tissulaire et de la médecine régénératrice. En raison de la nature dynamique de leurs réticulations, ces gels peuvent être pré-formés dans des seringues, extrudés sous cisaillement et s’auto-réparer spontanément après arrêt de la déformation mécanique. Au vu du potentiel que peuvent offrir les polymères fonctionnalisés par des acides boroniques pour la fabrication de gels covalents réversibles, nous avons développé des hydrogels injectables d’acide hyaluronique (HA) réticulés par des liaisons esters boronates pour des applications esthétiques et biomédicales. Pour élaborer de tels gels avec des propriétés optimisées, nous avons exploré l’effet de la nature du dérivé d’acide boronique ainsi que du motif saccharidique greffés sur le HA sur les propriétés rhéologiques dynamiques des gels. Parmi les différents dérivés d'acide boronique, le benzoboroxole (BOR) a été choisi en plus de l'acide phénylboronique (PBA) pour complexer différents motifs saccharidiques greffés sur le HA. Comparé au PBA, le dérivé BOR est, en effet, capable de se complexer de manière plus efficace à pH neutre et avec une plus grande variété de composés saccharidiques, en particulier les glycopyranosides. Cette étude a démontré que les propriétés rhéologiques dynamiques des assemblages de HA formés par complexation des unités de BOR ou PBA avec les différents sucres sont étroitement liées à la dynamique des échanges moléculaires et à la thermodynamique des pontages. En outre, nous avons également établi pour la première fois la possibilité d’obtenir des hydrogels de HA auto-réticulants à pH physiologique via des interactions multivalentes entre les unités de BOR greffées sur le HA et des groupements diols se répétant sur la chaîne polysaccharidique. Outre le BOR, la capacité de son homologue cyclique à six chaînons, la benzoxaborine, et d’un nouveau dérivé original similaire à ce composé a été explorée en tant que nouveaux sites de complexation de saccharides pour la formation de réseaux de HA réversibles. Compte-tenu des propriétés injectables, d'auto-réparation et de réponse à différents stimuli démontrées par ces nouveaux hydrogels de HA, ces biomatériaux apparaissent comme des candidats prometteurs pour de nombreuses applications innovantes dans le domaine biomédical, notamment pour l'ingénierie tissulaire et la thérapie cellulaire. / Injectable and self-healing hydrogels have recently drawn great attention in the fields of tissue engineering and regenerative medicine. Such gels can be pre-formed into syringes, be extruded under shear stress and show rapid recovery when the applied stress is removed due to the dynamic nature of their crosslinks. Given the exciting potential benefit of using boronic acid-containing polymers to construct dynamic covalent hydrogels, we explored this attractive strategy to design injectable boronate-crosslinked hydrogels based on hyaluronic acid (HA) for aesthetic and other biomedical applications. To design hydrogels with optimized properties, we investigated the effect of the nature of the boronic acid moiety as well as the sugar molecule grafted onto the HA backbone on the gel properties. Among arylboronic acid derivatives, benzoboroxole (BOR) was selected in addition to phenylboronic acid (PBA) as the binding site for sugar moieties grafted on HA. This choice was based on the efficient binding capability of BOR at neutral pH compared to PBA, and on its ability to complex glycopyranosides. With this study, we demonstrated that the dynamic rheological properties of the HA networks based on BOR- or PBA-saccharide complexation are closely linked to the molecular exchange dynamics and thermodynamics of the small molecule crosslinkers. Besides, we also established for the first time the feasibility of self-crosslinking HA hydrogels with extremely slow dynamics at physiological pH through multivalent interactions between BOR derivatives grafted on HA and diols from the polysaccharide chains. Finally, in addition to BOR, we demonstrated the unprecedented capacity of its six-membered ring homologue, benzoxaborin, and a new original benzoxaborin-like derivative as new carbohydrate binding sites for the formation of reversible HA networks. Taking into account the injectable, self-healing and stimuli-responsive properties showed by these new HA hydrogels, these biomaterials appear as promising injectable scaffolds for many innovative applications in the biomedical field, including in tissue engineering and cell therapy.

Ce travail est consacré à l'élaboration de complexes polyélectrolytes combinant deux polyélectrolytes de charges opposées ainsi que l'étude de leur potentiel en tant que biomatériaux injectables pour du comblement de ride. L'acide hyaluronique (portant des - charges négatives sur ses groupements carboxyliques -COO ) a été complexé avec l'unique polycation d'origine naturelle appelé chitosane (portant des charges positives de + par ses groupements amines -NH3 ). Les paramètres influençant la formation et les propriétés physico-chimiques des complexes acide hyaluronique – chitosane ont été étudiés. Nous avons utilisé une nouvelle technique de complexation développée au laboratoire mettant en œuvre la diminution de la force ionique de mélanges acide hyaluronique – chitosane – chlorure de sodium par dialyse dans le domaine de complexation de l'acide hyaluronique et du chitosane (pH approximativement compris entre 2.5 et 6.5). Ce procédé permet l'élimination progressive des sels et une association lente. Nous avons par ce biais été capable d'induire et de contrôler l'auto-assemblage de ces deux polyélectrolytes. Plusieurs formes ont ainsi été obtenues comme des agrégats, des complexes solubles, des suspensions colloïdales ou des coacervats. Au cours de ce travail, nous avons obtenu des hydrogels mixtes d'acide hyaluronique et de chitosane ayant d'exceptionnelles propriétés d'étirabilité à pH acide. D'autre part, une approche alternative a été envisagée, visant à utiliser les propriétés intrinsèques du chitosane, en particulier son aptitude à gélifier au contact de milieux alcalins. Ainsi, par un procédé similaire, nous avons pu former des hydrogels acide hyaluronique – chitosane réticulés physiquement, stable à pH et osmolarité physiologiques, et pouvant endurer des déformations importantes. De plus, ces systèmes peuvent être stérilisés par autoclave et peuvent être formulés afin d'être injectables. Réunissant toutes les conditions pour être de bons candidats au développement de biomatériaux injectables, ces hydrogels ont été testés in vivo sur un modèle lapin afin d‘évaluer leur biocompatibilité et leur applicabilité en tant que produits injectables en intradermique / This work is devoted to the elaboration of polyelectrolyte complexes systems combining two oppositely-charged polyelectrolytes and to the study of their potential application as - injectable dermal fillers. Hyaluronic acid as polyanion (carboxylic groups -COO as negative charges) was complexed with the only naturally-occuring polycation named + chitosan (amine groups -NH3 as positive charges). The factors impacting the formation of hyaluronic acid - chitosan complexes and their physico-chemical properties were investigated. We used a new technique of complexation developed in the laboratory through the desalting of highly salted mixtures, and systematically investigated the impact of pH in the range 2.5 - 6.5, corresponding to the complexation domain of hyaluronic acid and chitosan. This process allowed the progressive elimination of the salts and the slow restoration of the attractive electrostatic interactions resp onsible for the self-assembly of the two polyelectrolytes. Various physical forms were obtained: macroscopic aggregates, soluble complexes, colloidal suspensions or hydrogels. During this work, we observed for the first time the formation of hyaluronic acid-chitosan hydrogels exhibiting a very unusual hyper-stretchability, only at acidic pH. Therefore, an alternate approach consisted in taking advantage of the chitosan ability to gel in alkaline medium. By using a similar process, we were then able to form physically-crosslinked hyaluronic acid-chitosan hydrogels stable at physiological pH and osmolarity and still able to undergo high deformations. Moreover, these systems could be submitted to steam sterilization and could be formulated so as to be injectable. Hence, these hydrogels gathered all the conditions to be good candidates as injectable biomaterials, these hydrogels were then tested in vivo on a rabbit model to evaluate their biocompatibility and suitability for intradermal applications

The building blocks of human tissues are cells. The cells interact and respond to the characteristics of their local microenvironment. The cellular microenvironment is formed by three main components, the extracellular matrix, neighbouring cells and signalling molecules. Particularly, the extracellular matrix and neighbouring cells impose boundary conditions that limits the cell volume and cell spreading. However, these characteristics are often not present in traditional in vitro models, where cells experience a stiff and vast environment.   An approach to improve in vitro models is to use hydrogels, soft and highly hydrated polymers. Through chemical modifications, polymers naturally found in the extracellular matrix can be functionalized to form crosslinked hydrogels. Moreover, these functionalities can also be used to prepare micropatterns, micrometre sized cell adhesive areas on the hydrogels. These micropatterns guide the cell shape and permit the study of the cell response to these changes in shape, which has been observed in e.g. endothelial cells from various origins.   Taken all together, the aim of this work was to develop a hydrogel-based cell culture scaffold that permits the control of the spatial adhesion of brain endothelial cells in order to study the morphological effects on these cells and contribute to the understanding of the function of brain endothelial cells in health and disease.   This thesis demonstrates the functionalization of hyaluronic acid, a naturally occurring extracellular matrix polymer, to prepare photocrosslinkable hydrogels. Furthermore, through photolithography, micropatterns of cell adhesive peptides were prepared on these hydrogels. Brain microvascular endothelial cells, a highly specialized type of endothelial cells, adhered to the micropatterns, and the effect on their alignment depending on the micropatterned sized was studied. Furthermore, changes in their alignment were also observed when exposed to different glucose concentration.

Hyaluronic acid is an organic polysaccharide with a wide range of uses in medical and cosmetic industries due to its physiological properties. Crosslinked hyaluronic acid is a commonly used filler agent because of its water retention capabilities. N-deacetylation can be performed to enable new derivatives of hyaluronic acid. Both chemical and enzymatical approaches were investigated in this literature study to find methods retaining a high molecular weight product. Chemical N-deacetylation of hyaluronic acid has significant challenges with being treated by acid or base while both preventing degradation and maintaining its molecular weight. The method that seems the most promising is treating hyaluronic acid with hydroxylamine. Another method is enzymatic N-deacetylation. It was found that an enzyme N-deacetylated hyaluronic acid in female breast skin from 69-year-olds and above. The isolated enzyme had molecular weights ranging from 63 kDa to 79 kDa. Another enzyme that was produced recombinantly proved to be efficient since it retained high molecular weight and had a degree of deacetylation of 10.1 %. Today there exists only a few methods for crosslinking deacetylated hyaluronic acid. However, for chitosan, there are multiple methods available for crosslinking. Since it uses similar reactions that could be applicable to that of deacetylated hyaluronic acid. Reacetylation of the free amino groups has proven to be possible after crosslinking with a simple and cheap method resulting in an almost complete reacetylation. NMR proved to be an adequate method for analyzing the degree of deacetylation and higher-order structures. HPLC-UV spectroscopy may be used to increase the credibility of the analysis.

Intratumor heterogeneity poses a significant challenge for the diagnosis and treatment of patients with breast cancer because distinct sub-populations of tumor cells contribute significantly more to therapy resistance and tumor recurrence than others. Consequently, understanding the mechanisms that contribute to this heterogeneity and identifying sub-populations responsible for aggressive behavior is a significant and timely problem. Considerable evidence indicates that a subpopulation of tumor cells with stem/progenitor-like characteristics, termed cancer stem cells (CSCs), is responsible for therapy resistance and recurrence, sparking interest in characterizing novel biomarkers and therapeutic targets for this aggressive population of cells. Unfortunately, CSCs share many protein markers with normal mammary stem/progenitor populations, minimizing potential targets for diagnostic and therapeutic purposes. Therefore, in my thesis research, I investigated novel ways to identify CSC populations based on their glycome. I observed that breast CSCs have a unique glycosylation pattern that can be used to distinguish them from other tumor populations. Specifically, I discovered a novel α2,3 sialoglycan on Core2 O-linked glycans expressed on CSCs that can identified using the lectin SLBR-N. I found that SLBR-N can be used to distinguish CSCs from bulk tumor cells in multiple in vitro and in vivo models. I also discovered that the CSC marker, CD44s, expresses O-linked α2,3 sialoglycan and that this glycan alters CD44s function by promoting the activation of the PDGFRβ/STAT3 pathway. In contrast, the fucosyltransferase FUT3 and its glycan sialyl Lewis X (sLeX) are expressed on non-CSCs and they function to impede stemness by inhibiting CD44s-mediated PDGFRβ/STAT3 signaling. In summary, this thesis provides insights into glycan heterogeneity in breast cancer and novel ways to identify CSCs using the glycome.

Disertační práce se zabývá experimentálním studiem viskosuplementů na bázi kyseliny hyaluronové, které se aplikují do synoviálních kloubů postižených osteoartrózou. Hlavní pozornost byla věnována objasnění vlivu koncentrace a molekulové hmotnosti kyseliny hyaluronové na tření v kontaktu kloubí chrupavky resp. změnám tření v kontaktu po smíchání osteoartritické synoviální kapaliny s exogenní kyselinou hyaluronovou. Důležitou součástí experimentů bylo rovněž studium reologických vlastností synoviální kapaliny a kyseliny hyaluronové. Výsledky ukázaly, že molekulová hmotnost kyseliny hyaluronové významně ovlivňuje viskozitu a viskoelastické vlastnosti roztoku. Výrazná závislost mezi reologickými vlastnostmi kyseliny hyaluronové a třením v kontaktu však nebyla pozorována. Přimíchání kyseliny hyaluronové do synoviální kapaliny způsobí výrazný pokles součinitele tření v kontaktu. Rozdíly mezi viskosuplementy obsahující kyselinu hyaluronovou s různou molekulovou hmotností ale nijak výrazné nejsou. Nicméně, výsledky poukazují na možné ovlivnění režimu mazání v důsledku vysoké molekulové hmotnosti kyseliny hyaluronové. Tyto původní výsledky rozšiřují pochopení mechanizmů, ke kterým dochází v kloubu bezprostředně do vstříknutí kyseliny hyaluronové a mohou být použity při dalším vývoji viskosuplementů či v klinické praxi.

No description available.

No description available.

No description available.