Peptides and peptide derivatives have shown great scope as biomaterials and for biomedicaltherapy application. It has been demonstrated that classes of these peptides can form fibrillar hydrogels making them a good candidate for ECM mimics. In particular, the ionic complementary peptides, composed of alternating hydrophobic and hydrophilic amino acidshave been reported as successful cell scaffolds. The simple structure of such ionic complementary peptides is generally seen to spontaneously self-assemble into β-sheet richfibrils in the presence of water. The highly aqueous environment, along with the inter meshing of fibres, results in an architecture akin to the natural ECM of the body, making peptide hydrogels highly suitable as cell culture scaffolds. The structure of such hydrogels, usually comprising 8-32 amino acids, has been widely reported as easily modifiable, thus, allowing for control of the final material properties. This study explores the potential use of a range of ionic-complementary peptides for the culture of primary bovine chondrocytes. Modifications and additions to peptide sequence, such as charge and amino acid substitution, were investigated. In all studies only 1 design parameter (sequence, charge etc.) was varied, to allow for better understanding of the effect of materials properties upon cell response. The encapsulation of primary bovine chondrocytes was undertaken, with the aim of providing a suitable cell scaffold capable of maintaining chondrocyte viability and function in vitro. Despite in vivo work being beyond the scope of this thesis, the properties of the hydrogel scaffold were designed with final aim of being suitable for use with matrix associated autologous chondrocyte implantation (MACI) in clinical therapy.

Amikacin (AK) and enrofloxacin (EF) concentrations consistent with intra-articular and regional limb perfusion were evaluated for their effects on equine chondrocytes. We evaluated the production of prostaglandin E2 (PGE2) by equine chondrocytes in response to AK and EF administration,and if the combination of avocado soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) could reduce the production of PGE2. Monolayer cell cultures of equine chondrocytes were treated with clinically relevant concentrations of AK and EF plus combinations of ASU, GLU, and CS.AK and EF generated a dose dependent cytotoxicity. The induction of PGE2 following EF administration was significantly greater than PGE2 levels induced by positive controls. Induction of PGE2 by EF was significantly reduced in chondrocytes pretreated with ASU, GLU, and CS. We have demonstrated for the first time that EF can induce production of PGE2 in equine chondrocytes and that this effect can be attenuated with the combination of ASU, GLU, and CS.

Avec l'augmentation de l'espérance de vie, les pathologies ostéo-articulaires comme l'arthrose ou la polyarthrite rhumatoïde, caractérisées par la dégradation du cartilage articulaire, deviennent de réels problèmes de santé publique. Les traitements actuels sont essentiellement symptomatiques et aboutissent en ultime recours à la pose de prothèses. En absence de réparation spontanée du tissu et de traitement efficace, des approches d'ingénierie tissulaire du cartilage sont envisagées. Les techniques actuelles reposent sur la transplantation de chondrocytes autologues mais dans la majorité des cas, cette approche n'apporte pas de résultats supérieurs aux techniques chirurgicales utilisées actuellement. Grâce à leurs propriétés de différenciation, les cellules souches mésenchymateuses (CSM) représentent une nouvelle source de cellules ayant des potentiels thérapeutiques intéressants. Cependant, la complexité du processus de différenciation des CSMs vers des chondrocytes articulaires matures rend difficile l'obtention de cartilage fonctionnel après implantation. Il est donc important de mieux comprendre le processus de différenciation de ces cellules afin de mieux contrôler leur devenir in vivo. C'est pourquoi, le laboratoire s'intéresse au rôle des micro-ARNs (miARNs) dans la régulation du processus de différenciation des CSMs. L'objectif de mon projet de thèse a consisté à identifier des miARNs modulés dans la différenciation chondrocytaire des CSM humaines primaires et à étudier leur rôle et leur régulation au cours de la chondrogenèse. Nous avons identifié deux miARNs : miR-29a dont l'expression diminue progressivement au cours de la différenciation et miR-574-3p dont l'expression augmente rapidement puis est maintenue jusqu'à la fin de la différenciation. Ces deux miARNs sont régulés par le facteur de transcription SOX9 mais de manière opposée : SOX9 inhibe miR-29a et induit miR-574-3p. Nous montrons que SOX9 interagit avec YY1 pour réguler miR-29a mais pas miR-574-3p, ce qui pourrait expliquer les effets opposés de SOX9 sur l'expression des deux miARNs. Nous montrons également que ces miARNs sont des inhibiteurs de la différenciation chondrocytaire et avons identifié FOXO3A et RXRα comme cibles respectives de miR-29a et miR-574-3p. L'inhibition de FOXO3A ou RXRα avant l'induction de la différenciation, en utilisant des siARNs spécifiques ou en sur-exprimant les miARNs correspondants, bloque la différenciation des CSM. Ces résultats confirment sur des CSMs adultes, que ces protéines jouent un rôle important dans la chondrogenèse et que miR-29a et miR-574-3p participent aux processus de régulation de la différenciation chondrocytaire. En conclusion, nous avons identifié deux nouveaux miARNs contrôlés par SOX9 et régulant négativement la chondrogenèse grâce à la modulation de deux gènes cibles, dont l'expression est nécessaire avant d'induire la différenciation chondrocytaire. / Roles of miR-29a and miR-574-3p during the chondrogenic differentiation of mesenchymal stem cells. With the constant increase of the lifespan, osteoarticular pathologies such as osteoarthritis or rheumatoid arthritis, characterized by articular cartilage degradation, are important public health problems. In absence of spontaneous regeneration, cartilage engineering approaches are being considered. Current techniques rely on autologous chondrocyte transplantation but in the majority of cases, this approach gives similar results as current surgeries. Due to their capacity of differentiation toward chondrocytes, mesenchymal stem cells (MSC) represent a new source of cells with therapeutic potential. However, production of a functional cartilage in vivo after implantation of expanded MSC is hampered by the difficulty to reproduce the complexity of the differentiation process to get mature chondrocytes from MSC. The objective of my Ph.D thesis aimed to identify micro-RNAs (miRNAs) modulated during chondrogenic differentiation of primary human MSCs and to study their role as well as their regulation in this process. We identified two miRNAs: miR-29a whose expression decreases progressively during the differentiation and miR-574-3p whose expression rapidly increases and stays constant until the end of the differentiation. Both miRNAs are regulated by the transcription factor Sox9 but in an opposite manner: Sox9 inhibits miR-29a and induces miR-574-3p. We show that YY1 directly interact with Sox9 to regulate miR-29a but not miR-574-3p; this interaction likely explaining the opposite effects of Sox9 on miR-29a and miR-574-3p expression. Moreover we showed that miR-29a and miR-574-3p are both inhibitors of chondrogenesis and we identified FOXO3A and RXRα as their respective targets. In conclusion, we identified two new miRNAs which are regulated by Sox9 and inhibitors of chondrogenesis. They act through the modulation of two target genes, whose role during chondrogenic differentiation of adult MSC was previously not characterized.

Th.--Pharm.--Paris 5, 1982. N°: 79.

Bone dysplasia’s affect every 1 in 5,000 babies; most of these dysplasia’s are incurable, and some are even lethal (Stembalska et al., 2021). Hundreds of skeletal dysplasia’s are heritable, yet the genes involved are not well defined (Krakow, 2015). Most of the skeleton forms through a process called endochondral ossification (EO). There are three parts of EO: chondrogenesis, maturation, and ossification. During chondrogenesis, mesenchymal progenitor cells condense and then differentiate into chondrocytes. After differentiation, chondrocytes will elongate, then proliferate and mature to set up for primary ossification. We know that this process happens through many activated genes, but the sequential steps through which this is achieved has yet to be elucidated. In order to understand the cause of skeletal dysplasia’s and find new treatments, the molecular mechanisms controlling EO requires further investigation. This study focuses on one gene, CXXC1 Finger Protein, Cfp1, and its role in chondrocyte maturation during skeletal mouse development. Cfp1 was specifically deleted in chondrocytes, and the resultant effects on cartilage and bone were analyzed. A mild phenotype was observed in the knockout mouse model. It was found that loss of Cfp1 in chondrocytes leads to delayed ossification in the vertebrae, tibias, metatarsals, and metacarpals. Therefore, Cfp1 is necessary for normal chondrocyte maturation.

Long bone lengthening occurs at the growth plate (GP) by well-regulated chondrocyte proliferation, hypertrophy and terminal matrix deposition. GP chondrocyte (GPC) hypertrophy has been implicated to be the main determinant of bone growth rate; however the mechanism is poorly understood. The work of this thesis examined some of the cellular process that drives the chondrocyte swelling or hypertrophy particularly in a mammalian post natal GPs using living in situ GPC and fixed GP tissues. Confocal scanning microscopy (CLSM) was used to determine living in situ GPC volume and dimension changes in proliferative zone (PZ) through to hypertrophic zone (HZ) chondrocytes of different GPs of various bones. While PZ cells showed similar volumes and dimensions, HZ cells varied in different GPs, even within the same long bone but at opposite ends. However, the hypertrophic cell volume measured at a single post natal age (day 7) was independent of the corresponding bone length. This could reflect a complex interplay between local and systemic factors in different GPs, which occurs throughout the active phase of bone growth. Maintaining GPC morphology was critical in studying GPC hypertrophy using fixed tissues. This work highlighted a problem caused by conventional fixative solutions, which caused up to 44% hypertrophic GPC shrinkage following GP fixation. This artifact appeared to be associated with the hyperosmotic nature of the fixatives used and could be abolished by adjusting the fixative osmolarity close to physiological level (280 mOsm), or could be significantly reduced by bisecting bone tissues prior to tissue fixation. This thesis proposed roles for plasma membrane transporter(s) in mediating GPC hypertrophy. This hypothesis was tested by examining roles of sodium-hydrogen exchanger (NHE) and anion exchanger (AE) in GPC hypertrophy using an ex vivo bone growth inhibition model. Inhibition of bone growth by inhibitors of NHE (EIPA) and AE (DIDS) respectively was shown to be dose-dependent. The histology of bones demonstrated that the late HZ width was significantly reduced in GPs treated with EIPA or DIDS. Although in situ GPC volumes in the PZ and HZ were not notably different in DIDS-treated GP, the cell volumes in both zones were significantly reduced by EIPA treatment. Fluorescence immunohistochemistry revealed distinctive cellular localisations of NHE1 and AE2 in the PZ and early HZ. These results suggest a possible role of AE in mediating GPC volume increase in PZ chondrocytes and those in the early stages of cell hypertrophy, whereas NHE could possibly maintain intracellular pH of GPC throughout all GP zones. This thesis has characterized various changes in volume and dimensions of living in situ GPC from PZ through to HZ of different GPs of postnatal rats. This work emphasized the importance of fixative osmolarity in order to accurately preserve the normal volume/morphology of cells within tissues. Most importantly, this thesis confirmed a potential role of the plasma membrane transporters, AE and NHE in GPC hypertrophy of growing bones.

Les chondrocalcinoses articulaires (CCA) sont des arthropathies microcristallines caractérisées par la présence de cristaux de pyrophosphate de calcium dihydratés (PPCD) au sein du cartilage. Il existe trois formes de CCA : les formes familiales, transmises de façon autosomique dominante, et les formes sporadiques, dont la probabilité d'apparition augmente avec l'âge, et les formes secondaires à d'autres pathologies, comme l'hyperparathyroïdie. Le gène Ank code un transporteur exportant le pyrophosphate inorganique (PPi) hors des cellules. Un lien est clairement établi entre des mutations dans la séquence codante du gène Ank et les formes familiales de CCA Nous avons étudié la contribution du gène Ank dans la production de PPi par les chondrocytes articulaires stimulés avec du transforming growth factor-ß1 (TGF-ß1), leur sensibilité a ce facteur de croissance augmentant avec le l'âge des patients. Nous avons également, vu la présence fréquente des cristaux de PPCD chez les patients arthrosiques, et l'influence du niveau d'expression d'Ank dans la chondrogenèse, voulu élucider la possible implication d'Ank dans la perte du phénotype du chondrocyte articulaire, retrouvée dans l'arthrose. Enfin, nous avons étudié le rôle du calcium sur les effets du TGF-ß1 dans la production d'ePPi, l'hypercalcémie étant souvent retrouvée dans les tableaux cliniques d'hyperparathyroïdisme. Ces travaux ont permis de mettre en évidence le rôle majeur d'ANK en cas sensibilité accrue du chondrocyte au TGF-ß1. De plus, le PPi transporté par ANK influence le phénotype du chondrocyte articulaire. Enfin, le calcium joue un rôle crucial sur les effets inducteurs du TGF-ß1 dans la production de PPi. / Articular chondrocalcinosis (CCA) are microcrystalline arthtopathies characterized by the presence of calcium pyrophosphate dihydrate crystals (CPPD) in the cartilage. There are 3 forms of CCA, including the familal forms, dependent upon an autosomal dominant transmission mechanism, the sporadic forms, whose probability of occurrence increases with ageing, and the metabolic forms, secondary to other pathologies like hyperparathyroidism. The Ank gene encodes a transporter which exports inorganic pyrophosphate (PPi) outside the cells. A link is well established between mutations in the coding sequence of the Ank gene and the familial forms of CCA. We studied, on one hand, the contribution of the Ank gene in the production of PPi by articular chondrocytes stimulated with transforming growth factor-ß1 (TGF-ß1), since the sensitivity of chondrocytes to this growth factor increases with ageing. On the other hand, since CPPD are often associated with osteoarthritis (OA), and since Ank expression levels influences the chondrogenesis, we elucidated the possible implication of Ank in the loss of the articular chondrocytes phenotype observed during OA. Finally, we analyzed the role of calcium on the TGF-ß1 effects on ePPi production, hypercalcaemia being often found during hyperparathyroidism. This work allowed demonstrating, on one hand, the predominant role of ANK in the situations of chondrocytic hypersensitivity to TGF-ß1. On the other hand, the PPi exported by ANK influences the expression of articular chondrocytic markers. Finally, calcium plays a crucial role in the inducer effects of TGF-ß1 in the production of PPi.

Experimental studies indicate that culturing chondrocytes on biodegradable polymeric scaffolds may yield“engineered" cartilage for the replacement of tissue lost to injury or diseases such as osteoarthritis. A method of estimating the outcome of cell-polymer cultures would aid in the design and evaluation of engineered tissue for therapeutic use. The goals of this project were to develop, validate, and apply first-generation mathematical models that describe the kinetics of extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs cultured in vitro. The ECM deposition model is based on a product-inhibition mechanism and predicts an asymptotic, exponential increase in the concentration of ECM molecules found in cartilage, including collagen and glycosaminoglycans (GAG). The scaffold degradation model uses first-order kinetics to describe the hydrolysis of biodegradable polyesters in systems not limited by diffusion. Each model was fit to published data describing the accumulation of GAG and collagen, as well as the degradation of poly glycolic acid (PGA) and poly lactic acid (PLA), respectively. As experimental validation, cell-polymer constructs (n = 24) and unseeded scaffolds (n = 24) were cultured in vitro, and biochemical assays for GAG and collagen content, as well as scaffold mass measurements, were performed at 1, 2, 4, 6, 8, or 10 weeks of culture (n = 8 per time point). The mathematical models demonstrate a moderate to strong goodness of fit with the previously published data and our experimental results (R2=0.75-0.99). These models were also combined to predict the temporal evolution of total construct mass with reasonable accuracy (30% RMS deviation). In ongoing work, estimates of biochemical composition derived from these models are being proposed to predict the mechanical properties and functionality of the constructs. This modeling scheme may be useful in elucidating more specific mechanisms governing ECM accumulation. Given their potential predictive power, these models may also reduce the cost of performing long-term culture experiments.

Dietary phosphate is essential for normal fracture healing and bone growth. Previous studies have established that mice given a phosphate deficient diet after a fracture demonstrate delayed cartilage maturation and callus mineralization, as well as changes in gene expression consistent with oxidative phosphorylation dysfunction. This study was undertaken in order to examine the role of inorganic and organic phosphate availability on chondrocyte differentiation and mineralization, and to define the relationship between these processes and changes in chondrocyte metabolic function. ATDC5 murine chondroprogenitor cell line, which has been shown to undergo in vitro differentiation and extracellular matrix mineralization, was cultured under both differentiating and non-differentiating media conditions under conditions in 1mM -0.25mM sodium phosphate monobasic (inorganic phosphate) in the presence or absence of 4mM β-glycerol phosphate (organic phosphate). In the first series of studies, overall cell growth (total DNA and protein contents), mineralization (calcium accumulation), and cell-normalized oxidative metabolism (basal respiration, maximal respiration, ATP turnover, spare capacity, proton leak, and non-mitochondrial respiration rates) were measured over a 28 day time course in cultures grown in differentiating (ascorbic acid, insulin-transferrin-selenium, and β-glycerol phosphate) conditions in 1mM phosphate. These studies found that when the cells were induced to differentiate, there was a measurable increase in protein content while DNA content decreased by 30%, indicating a fraction of the cells underwent cell death. Differentiation was further associated with an overall two-fold increase in oxidative respiration. Next we assessed how differentiation, the promotion of matrix mineralization, and inorganic phosphate availability affected oxidative respiration. When differentiation was not induced with ascorbic acid and β-glycerol phosphate, there was no over growth in the cultures nor any change in total extracellular matrix mineralization or oxidative respiration. In the absence only of β-glycerol phosphate, differentiation proceeded but matrix mineralization did not occur. However, overall protein content and oxidative respiration were statistically two- and 1.5-fold higher, respectively, independent of the inorganic phosphate contents of the growth media. These results suggest that both differentiation and overall protein accumulation are strongly associated with increased oxidative metabolism while mineralization of the matrix decreased oxidative function. Only at the lowest phosphate levels were changes in basal oxidative function observed. These results are consistent with previous in vivo findings suggesting that diminished expression of mitochondrial associated genes in callus tissues from hypophosphatemic mice were associated with an overall decrease in chondrocyte differentiation.

The first objective of my work was to use the ank/ank (progressive ankylosis) mutant mice, which have a deficiency in inorganic pyrophosphate transport, to address the role of Ank in joint ankylosis. I observed the presence of hypertrophic chondrocytes in the uncalcified ank/ank mice articular cartilage. This novel phenotype is likely due to a dysregulation of chondrocyte maturation as these chondrocytes expressed hypertrophic chondrocyte markers (collagen type X and tissue non-specific alkaline phosphatase). I also showed by immunohistochemical staining that beta-catenin expression was upregulated and localized in the nuclei of articular ank/ank chondrocytes, suggesting activation of Wnt/beta-catenin signaling in these chondrocytes. The second objective was to use ank/ank mice as an informative model for understanding ankylosis mechanisms in human ankylosing spondylitis (AS) patients, as WNT/beta-catenin signaling plays an important role in ankylosis in AS patients. We attempted rescue of joint ankylosis in ank/ank mice by gene transfer of noggin, an antagonist of BMP signaling. Paradoxically, noggin-treated ank/ank mice had accelerated ankylosis, as evidenced by joint pathology and IHC staining of beta-catenin showed more intense signals in the spinal chondrocytes of the treated mice. As noggin and sclerostin (an antagonist of beta-catenin signaling) form a mutually inhibitory complex, we hypothesize that the formation of this complex results in relieving suppression of both beta-catenin and BMP signaling, leading to more severe ankylosis in ank/ank mice. By quantitative molecular imaging, I have demonstrated that ankylosis in these mutant mice developed simultaneously in distal and axial joint, instead of being a centripetal process. In summary, I have made three original observations in the ank/ank mice: the hypertrophic chondrocyte phenotype; activation of beta-catenin signaling and the simultaneous development of ankylosis in distal and axial joints. These mutant mice serve as valuable model for pre-clinical studies which enable modeling and testing of novel anti-ankylosis treatments.