Osteoarthritis is the most prevalent musculoskeletal disease in humans, severely reducing the standard of living of millions of people. Osteoarthritis is characterized by degeneration and loss of articular cartilage which leads to pain, and loss of joint motility and function. Individuals suffering from severe osteoarthritis are commonly treated with full knee replacements. The procedure does eliminate the problem of degrading cartilage tissue; however, it does not fully restore function and its lifetime can be limited. To overcome the disadvantages of current treatments, tissue engineering has become a focus of research to regenerate cartilage. Tissue engineering attempts to repair or replace damaged tissue with cells, biomaterials, and/or molecular signals. Biodegradable scaffolds serve as a temporary replacement for the tissue until it has regenerated. Two types of scaffolds that have been used in tissue engineering are electrospun scaffolds and hydrogels. We have proposed and fabricated a scaffold for cartilage tissue engineering that incorporates an electrospun cylinder and a thermosetting hydrogel in order to provide improved properties compared to either individual material. Electrospun cylinders were created by sintering electrospun mats that include salt pores. The addition of salt pores decreased the mechanical properties of the electrospun materials while also improving the capability of cells to infiltrate into the scaffold. The sintering process involved the connecting of one electrospun mat to an adjacent one. Specifically, poly(d,l-lactide) was capable of sintering to an adjacent electrospun mat when exposed to either heat (near the glass transition temperature) or tetrahydrofuran vapor. The sintering process did not deteriorate the structure or function of the electrospun material. Sintering allowed the creation of unique structures of electrospun material that previously could not be produced. A thermosetting hydrogel was added to the scaffold to replicate the function of proteoglycans present in articular cartilage. A composite scaffold of electrospun polymer and hydrogel showed improved mechanical properties and better integration of the scaffold in vivo compared to an electrospun scaffold with no hydrogel. In conclusion, the composite electrospun and hydrogel scaffold could become an excellent tissue engineering scaffold to treat patients suffering from osteoarthritis. / Ph. D.

Poly(2-hydroxyethyl) methacrylate, (polyHEMA), hydrogels are synthesized for tribological study to investigate their potential for use as synthetic articular cartilage. A four factor, two level designed experiment was performed to evaluate friction and wear of polyHEMA. Tests were carried out using a friction and wear test device developed for biotribology research. The geometry consisted of a ball on flat; 6mm stainless steel ball and flat polyHEMA discs. Test factors were load, lubrication, hydration and material crosslink density. Linear oscillating sliding contact tests were performed on each polyHEMA disc for approximately 30 minutes per test. Friction coefficients found ranged from 0.05 to 1.7. Linear wear measured ranged from 0.02 mm to 1.32 mm. / Master of Science

In our aging population, arthritis is becoming an increasingly common problem. Pain, loss of joint function and other negative affects make arthritis a major health problem. The most common form of arthritis, osteoarthritis, is caused by the "wear and tear" of articular cartilage on the surface of bones in synovial joints. It is a chronic problem that is slowed with different types of therapies, including pharmaceutical, nutritional and surgical, but to date the wearing down of the cartilage cannot be stopped or reversed. Normal, mature, articular cartilage does not spontaneously repair itself after an injury. In light of this, several surgical techniques are being developed to repair degenerate and/or osteoarthritic cartilage. One such approach uses Autologous Chondrocyte Implantation (ACI). Dr. Mats Brittberg, and associates at Goteborg University in Sweden began using this cartilage repair procedure in 1987. Other techniques attempt to stimulate the subchondral bone to generate cartilage, such as Abrasion Arthroplasty. Still others use tissue grafts to attempt to repair lesions in cartilage. The surface biomechanics of these repaired tissues have not yet been studied. How well does the repaired cartilage resist wear? How long will it last? How does the repaired cartilage compare to "normal" cartilage in terms of wear-resistance? It is the goal of this research to gain initial knowledge to help answer these questions. Dr. Brittberg has provided 17 sample of cartilage, from 9 Swedish patients, including repaired and normal pairs using the aforementioned repair techniques and others, as well as a degenerate and normal cartilage pair. The intention of this paper is to report the findings of experiments performed using these samples, and compare the wear-resistance of repaired and degenerate cartilage to that of normal cartilage. Wear and friction tests were carried out on 2 mm diameter specimens using a biotribology device and a new, modified technique developed specifically for these small samples. The cartilage samples were mounted, using specially designed adapters, in our biotribology device for oscillating contact against polished stainless steel disks at a constant applied normal load, oscillating frequency, and test time. A buffered saline solution was used as the lubricant. Cartilage wear was determined from hydroxyproline analysis of the test fluid and washings from the wear test. Thin layers of transferred cartilage-like films to the stainless steel disks were also analyzed. Also, friction data was recorded throughout the tests. The results of these experiments show that: 1) For the two pairs of ACI repaired cartilage, the repaired cartilage gave substantially less wear than that of normal cartilage. 2) For all other repair techniques tested, the repaired cartilage produced more wear than normal cartilage. 3) The single osteoarthritic cartilage tested produced similar wear to that of normal cartilage. This is surprising since the current thought is osteoarthritic cartilage is more susceptible to wear. 4) The hydroxyproline concentration, by weight, of cartilage increases after the wear test. 5) Friction levels were in the boundary lubrication regime, and had no correlation with the amount of wear. To our knowledge, this research represents the first controlled "in vitro" study of an important unknown in cartilage repair, i.e., the wear-resistance of the repaired cartilage. It shows that ACI produces a cartilage with very good wear-resistance, better than that of other repair techniques, and possibly better than normal, healthy cartilage. ACI and its applications to the treatment of degenerate and osteoarthritic joints are promising, and studies will continue to investigate this and other types of cartilage repair. / Master of Science

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

<p>The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.</p><p> Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy. </p><p> In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct. </p><p> Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.</p> / Dissertation

Les lésions de cartilage ne cicatrisent pas spontanément et la réparation de ce tissu est un challenge. Les techniques chirurgicales restant insatisfaisantes, la thérapie cellulaire et l'ingénierie tissulaire sont maintenant envisagées. La transplantation de chondrocytes autologues (TCA) existe déjà mais cette procédure nécessite l'amplification des chondrocytes qui s'accompagne d'une perte du phénotype différencié (dont l'indicateur est le collagène de type II), au profit d'un phénotype fibroblastique (dont l'indicateur est le collagène de type I, retrouvé dans les tissus fibreux). La TCA conduit donc à une greffe de chondrocytes dédifférenciés produisant un fibrocartilage, dont les propriétés mécaniques sont différentes du cartilage hyalin natif. L'objectif de mes travaux était de développer un nouveau kit d'ingénierie tissulaire du cartilage par association de chondrocytes humains, de biomatériaux et d'une sélection de facteurs solubles. Nous avons utilisé le cocktail FGF-2/insuline (FI) pour l'amplification cellulaire et le cocktail BMP-2/insuline/T3 (BIT) pour redifférencier les chondrocytes dans des éponges de collagène. Nos résultats ont montré que cette combinaison permet la synthèse d'une matrice cartilagineuse dans les supports collagène. Cependant, cette synthèse s'est trouvée favorisée en périphérie des éponges cultivées en conditions statiques. Nous avons ensuite utilisé un bioréacteur pour perfuser les éponges et nos résultats ont révélé alors un dépôt plus homogène de cartilage dans ces supports. De manière très intéressante, nous avons aussi observé l'arrêt de l'expression du collagène de type I. Ainsi, notre approche multifactorielle combinant des chondrocytes humains, des biomatériaux collagène, une combinaison FI-BIT et une culture en perfusion permet la reconstruction d'un cartilage non fibrotique / Cartilage lesions are irreversible and cartilage repair is challenging. Actual surgical techniques remain unsatisfactory and therefore, cell therapy and tissue engineering approaches are now considered. The Autologous Chondrocytes Transplantation (ACT) already exists but this procedure requires chondrocytes amplification. During this amplification, a dedifferentiation process occurs: chondrocytes lose their differentiated phenotype (characterized by type II collagen) towards a fibroblastic phenotype (characterized by type I collagen, a component of fibrous tissues). ACT leads to the graft of dedifferentiated chondrocytes, hence provoking the production of a fibrocartilage that presents different mechanical properties than native hyaline cartilage. The aim of my work was to develop a new kit of tissue engineering for cartilage repair using human chondrocytes, biomaterials and a selection of soluble factors. We used a cocktail of FGF-2 and insulin (FI) for cell amplification and a cocktail of BMP-2, insulin and T3 (BIT) for chondrocyte redifferentiation in collagen sponges. Our results showed that the combination allows the synthesis of a cartilaginous matrix in collagen scaffolds. However, matrix production is favored in periphery of the sponges cultivated in static conditions. A perfusion bioreactor was then used to perfuse the sponges and our results revealed a more homogeneous deposition of cartilage in the scaffolds. Very interestingly, we also noticed a stop of type I collagen expression. Thus, our multifactorial approach combining human chondrocytes, collagen scaffold, the combination FI-BIT and culture under perfusion allows the reconstruction of a non-fibrotic cartilage

Des mutations faux-sens au niveau du récepteur à activité tyrosine kinase FGFR3 (Fibroblast Growth Factor Receptor 3) entrainent sa suractivation qui apporte des dysfonctions biologiques dans plusieurs maladies. L’achondroplasie, la forme la plus commune de chondrodysplasie liée à Fgfr3, est une maladie génétique rare, touchant 1 nouveau-né sur 20 000, caractérisée par des signes cliniques spécifiques : nanisme rhizomélique, membres courts, macrocéphalie, hypoplasie de l’étage moyen de la face, compression cervico-médullaire. L’activité anormale du récepteur induit des défauts de l’ossification endochondrale responsables du phénotype pathologique. Pendant longtemps le seul traitement pour cette maladie a été l’allongement chirurgical des membres, cependant au cours des dernières années de nombreux chercheurs ont développé des potentielles stratégies thérapeutiques basées sur des études moléculaires. L’objectif de ma thèse était d’évaluer une nouvelle approche thérapeutique pour l’achondroplasie. Une stratégie thérapeutique prometteuse prévoit l’utilisation de petits inhibiteurs chimiques, connus sous le nom d’inhibiteurs de tyrosine kinases, qui sont capables d’arrêter l’activité de FGFR3. J’ai estimé les effets d’un de ces composés, NVP-BGJ398, dans un modèle murin mimant le nanisme achondroplase (Fgfr3Y367C/+). Des expérimentations effectuées ont montré une amélioration des caractéristiques pathologiques dans les souris traitées avec NVP-BGJ398. Nous avons également examiné l’impact de la mutation activatrice de FGFR3 sur le développement mandibulaire. L’étude a reconnu un défaut dans la croissance mandibulaire chez l’homme et la souris atteints. En outre nous avons pu investiguer la croissance osseuse de la mandibule et corriger le défaut pathologique avec NVP-BGJ398. Enfin j’ai participé à des analyses moléculaires pour décrire comment trois mutations de FGFR3 localisées à la même position (Lys650) peuvent induire trois différents nanismes avec sévérité croissante. Les résultats ont fourni une meilleure compréhension des mécanismes moléculaires pathologiques et pourront mener à des nouvelles cibles pour des approches thérapeutiques. / Missense mutations in the tyrosine kinase receptor FGFR3 (Fibroblast Growth Factor Receptor 3) lead to its overactivation causing biological dysfunctions in several diseases. Achondroplasia, the most common Fgfr3-related chondrodysplasia, is a rare genetic disorder, affecting 1 in 20000 live births, characterized by particular clinical features: rhizomelic dwarfism, short limbs, macrocephaly, midface hypoplasia, cervicomedullary compression. The abnormal activity of the receptor induces endochondral ossification defects that are responsible for the pathological phenotype. For a long time the only treatment for this disease was the limb lengthening surgery, however in recent years several researchers have developed potential therapeutic strategies based on molecular studies. The objective of my thesis was to evaluate a novel therapeutic approach for achondroplasia. A promising therapeutic strategy involved the use of small chemical inhibitors, known as tyrosine kinase inhibitors, that are able to arrest the FGFR3 activity. I have assessed the effects of one of these compounds, NVP-BGJ398, in a mouse model mimicking the acondroplastic dwarfism (Fgfr3Y367C/+). The experiments performed showed an improvement of all pathological hallmarks in NVP-BGJ398 treated mice. We have also inspected the impact of the activating FGFR3 mutation on the mandibular development. The study established a defect in mandibular growth in both affected patients and mice. Furthermore we could investigate the mandibular bone growth and correct the pathological defect with NVP-BGJ398. Finally I have participated in molecular analyses to describe how three FGFR3 mutations at the same position could lead to three different dwarfisms with increasing severity. The results provided a better understanding of FGFR3 pathological molecular mechanisms and could lead to new targets for therapeutic approaches.

Lorsque le cartilage est altéré, la réponse des chondrocytes est insuffisante pour stopper ce processus et réparer le tissu. Les thérapies non chirurgicales actuelles sont basées sur les anti-inflammatoires pour soulager la douleur. Cependant, certaines molécules thérapeutiques ne sont pas utilisées en raison d'une faible biodisponibilité ou d'effets secondaires importants. Le but est de préparer des particules polymère pour améliorer la biodisponibilité dans l'articulation de principes actifs (PA). Ceci est obtenu par le recouvrement des particules par des dérivés de l'acide hyaluronique (HA), naturellement présent dans le cartilage et qui possède une affinité pour un récepteur (CD44) à la surface des chondrocytes. Ce ciblage actif permet de concentrer les PA à proximité ou dans (internalisation) ces cellules et donc, d'obtenir un effet à des concentrations plus faibles en PA. Le HA est estérifié par des groupements hydrophobes pour obtenir des propriétés tensio-actives. Puis ces dérivés servent de stabilisant pour la formulation d'émulsions conduisant à l'obtention de particules polymère. Elles sont alors caractérisées: taille, distribution et quantité de HA modifié en surface. L'encapsulation de traceurs fluorescents démontre une internalisation in vitro et l'administration par injection intra-articulaire (ia) chez le rat n'engendre pas d'inflammation. L'encapsulation de PA (HA, chondroïtine sulfate) est possible, avec des tailles de particules plus importantes. Ceci ouvre de nouvelles approches dans le traitement des atteintes articulaires: l'injection ia de particules polymère recouvertes de HA permet un adressage des molécules qu'elles contiennent dans les cellules / A defect in articular cartilage yield to a biological response of the chondrocytes but it is not sufficient to stop the damage or heal the tissue. The non-chirurgical therapies available are based on anti-inflammatory, to relieve the pain. But, many molecules which might have therapeutic effects are not used because of poor bioavailability or severe side effects. Here, we described the elaboration of polymeric nanoparticles that can enhance articular bioavailability of entrapped drugs. One can reach this goal by using HA derivatives at the surface of such particles. HA is a polysaccharide naturally present in the joint, with a strong affinity for one receptor expressed by the chondrocytes: CD44. Such an active targeting will allow the accumulation of drugs near or in the cells (internalization) and therefore, lead to biological effects with lower concentration. We chemically modified the HA by esterification with hydrophobic chains to confer tensio-actives properties. Then, those derivatives were used to stabilize the formulation of polymeric particles and the characterization (size, distribution and amount of HA derivatives present) was determined. The encapsulation of fluorescent probes proved in vitro an internalization by the chondrocytes. Moreover, intra-articular (ia) injection of particles in healthy rats did not generate any inflammation. Entrapment of molecules (HA or Chondroitin Sulfate) was possible but the size was higher and the encapsulated amount could not be precisely measured. This work is promising for new therapies in articular diseases, the ia injection of polymeric particles covered by HA derivatives allow an active targeting of the entrapped drugs

La trachée est une structure très complexe des voies respiratoires, qui est composée d'anneaux cartilagineux, fait de cartilage hyalin, et de bandes musculaires, formées de cellules musculaires lisses, dont l'architecture confère à la fois rigidité et souplesse au canal trachéen. Contrairement à d'autres cartilages, tels que ceux trouvés dans la plaque de croissance en développement et dans les articulations adultes, ou aux cellules musculaires lisses des vaisseaux, très peu d'informations sont disponibles sur le développement du cartilage et du tissu musculaire trachéal et sur leur capacité à se minéraliser, bien que la calcification de la trachée soit un événement commun dans la population âgée et plus rare dans certaines pathologies. Dans ce contexte, ce travail de thèse a cherché dans le modèle souris à mieux caractériser le cartilage et le tissu musculaire lisse de la trachée et également comprendre les mécanismes moléculaires jusqu'alors inexplorés, régulant la minéralisation de la trachée. Grâce à une nouvelle technique de culture de cellules provenant de la trachée, nous avons démontré que les chondrocytes et les cellules musculaires lisses trachéaux sont tous deux capables de minéraliser lorsqu'ils sont traités avec un haut niveau de Pi, mais via des mécanismes moléculaires différents. En parallèle, une étude in vivo nous a permis de démontrer que la minéralisation de la trachée se produit uniquement dans les anneaux cartilagineux dès 30 jours après la naissance. Des analyses histologiques et moléculaires ont permis d'affiner ces résultats et de proposer un modèle de minéralisation de la trachée via une progression rostro-caudale dépendante de BMP2 / The trachea is a very complex structure of the respiratory tract, composed of C-shaped cartilaginous rings, made of hyaline cartilage, and muscular bands, made of smooth muscle cells, conferring rigidity and compliance to the windpipe, respectively. In contrast to other intensely studied cartilages such as the ones found in the developing growth plate and in the adult joints or smooth muscle cells from the vasculature, very little information is available on the development of the tracheal cartilage and smooth muscle tissues and on their innate propensity to mineralize, although calcification of the trachea is a common finding in the elderly population and also a rare manifestation of pathologic conditions. In this context, this PhD work sought to better characterized the poorly studied tracheal cartilage and smooth muscle tissue and understand the molecular mechanisms regulating tracheal mineralization that has been unexplored so far. We tackle these questions in the mouse model. Setting up a novel in-vitro culture of tracheal cells, we demonstrated that tracheal chondrocytes and smooth muscles cells are prone to mineralize when treated with high level of Pi, through different molecular mechanisms. In parallel, we found that in vivo mineralization of the trachea only happens in the cartilaginous rings, as early as 30 days after birth. Histological and molecular evidence suggest that tracheal mineralization occurs through a BMP-dependent rostro-caudal progression

Articular cartilage damage and degeneration is a siginficant clinical problem which no technique has been able to adequately and reliably repair or regenerate. Recent research has investigated the use of cell-based therapies to treat focal cartilage lesions. In clinical practice proliferated autologous chondrocytes are used and clinical trials are investigating the use of mesenchymal stem cells. The aim of this thesis was to assess aspects of current cell-based therapy and to investigate the potential of perivascular stem cells for articular cartilage repair. The phenotype of expanded matrix-applied autologous chondrocytes utilised in current cell therapies was confirmed using immunocytochemistry and polymerase chain reaction (PCR) expression of hyaluronan and proteoglycan link protein 1 (HAPLN1), transcription factor sox-9 (SOX9) and aggrecan (ACAN). Quantitative real-time PCR demonstrated that they were down-regulated for expression of COL2A1, SOX9 and ACAN but up-regulated for COL1A1 compared to unproliferated chondrocytes. Confocal laser-scanning microscopy (CLSM) demonstrated a significant decrease in cell viability and density when the membranes were subjected to levels of trauma similar to those that could be experienced during surgery. Hyperosmolar solutions did not confer a chondroprotective effect. Pericytes and adventitial cells, collectively termed perivascular stem cells (PSCs), from the infra-patellar fat pad were identified using immunohistochemistry and isolated using enzymatic digestion and fluorescence-activated cell sorting (FACS). Cell identity was ascertained using PCR, FACS and mesenchymal differentiation (osteogenesis, adipogenesis and chondrogenesis). Quantitative real-time PCR analysis of micromass cultures indicated that PSCs displayed increased chondrogenic potential compared to mesenchymal stem cells. An ovine model of perivascular stem cells was developed and a pilot study using three sheep was undertaken to confirm the viability of the model. Autologous ovine PSCs were isolated and re-implanted into articular cartilage defects. Green fluorescent protein transfected cells were identified in the cartilage defect four weeks following re-implantation using CLSM. This thesis has examined aspects of matrix-applied autologous chondrocyte implantation for cell based cartilage repair and has identified a new source of prospectively identified and purified stem cells that have demonstrated increased chondrogenic potential compared to mesenchymal stem cells, which are commonly used in clinical research. The methods to identify and purify ovine perivascular stem cells were developed to investigate the use of autologous PSCs and to track the cells following implantation.