Étude de l'angiogenèse et de la distribution des cellules souches mésenchymateuses dans des défauts ostéochondraux chez le lapin

Les défis majeurs de la recherche orthopédique actuelle sont la restauration, le maintien et l’amélioration des tissus du système musculo-squelettique, afin de remédier aux déficiences causées par des maladies, telles que l’arthrite rhumatoïde et l’arthrose. En 2011, plus de 16 % de la population canadienne recensée souffrait de ces maladies, ayant donc à endurer des douleurs articulaires et une mobilité réduite des articulations affectées. À cause de l’absence de vaisseaux sanguins dans le cartilage et la faible densité cellulaire, le processus de réparation tissulaire n’est amorcé que difficilement au niveau de lésions du cartilage. Diverses techniques chirurgicales ont donc été développées pour stimuler la réparation ou la régénération du cartilage, incluant par exemple les transplantations de chondrocytes autologues et les techniques de stimulation de la moelle osseuse. Ces dernières sont parmi les plus utilisées et la stimulation de la moelle osseuse définit des techniques chirurgicales basées sur la microfracture ou la microperforation du cartilage et de l’os sous-jacent. Dans ce cas, la réparation est possible grâce à la création de canaux, qui permet la formation d’un caillot sanguin et l’infiltration des cellules souches mésenchymateuses, à partir des dommages causés au système vasculaire de l’os sous-chondral. Cependant, de nombreuses études ont démontré que le cartilage résultant était un mélange de cartilage hyalin et de cartilage fibreux, dont les propriétés mécaniques sont insuffisantes pour restaurer la mobilité initiale de l’articulation. Comme montrée dans de précédents travaux entrepris au laboratoire, la combinaison des techniques de microperforation avec un implant composé de chitosan, de glycérolphosphate et de sang autologue permet d’améliorer la qualité du cartilage réparé. Ceci est possible grâce au recrutement de plusieurs populations cellulaire au site de la lésion, comme les neutrophiles et les macrophages, et à travers divers mécanismes biologiques, incluant l’angiogenèse et la chondrogenèse. Il a été démontré que la solidification in situ de l’implant pouvait être accélérée par l’ajout de facteurs de coagulation. Les facteurs testés sont la thrombine (IIa), le facteur VIIa recombiné humain (rhFVIIa) et le facteur tissulaire (TF). Ils ont été ajoutés à l’implant chitosan-GP/sang, afin d’accélérer la solidification in situ au cours de l’application clinique , qui pourrait rendre possible la livraison de l’implant par arthroscopie. Dans un modèle lapin de microperforation, l’implant chitosan-GP/sang solidifié in situ par la thrombine permet d’améliorer la qualité du cartilage réparé vis-avis la thrombine seule (Marchand et al., 2012). Cependant, l’effet précis des facteurs de coagulation sur l’angiogenèse et les mécanismes de réparation tissulaire doivent encore être étudiés. Dans cette étude, les deux hypothèses suivantes sont posées : - L’ajout de facteurs de coagulation, notamment la thrombine (IIa), le facteur VIIa recombiné humain (rhFVIIa) et le facteur tissulaire (TF) + rhFVIIa, à la solution de chitosan, de glycérolphosphate et de sang autologue, engendre plus d’angiogenèse dans l’os sous-chondral des microperforations en réparation par rapport à la microperforation seule, à 3 semaines postchirurgie dans un modèle lapin de la stimulation de la moelle. - L’utilisation de l’implant chitosan-GP/sang, avec les techniques de stimulation de la moelle osseuse, attire un plus grand nombre de cellules souches mésenchymateuses (MSC) aux lésions microperforées, comparativement à l’utilisation des techniques de stimulation de la moelle osseuse seules. Pour répondre à ces hypothèses, les objectifs de cette étude sont les suivants : - Évaluer l’effet de l’implant chitosan-GP/sang coagulé dans la lésion avec la thrombine (IIa), du facteur VIIa recombiné humain (rhFVIIa) et du facteur tissulaire (TF) + rhFVIIa sur la réparation du cartilage induite par la microperforation, par rapport à la microperforation seule, à partir de la quantification stéréographique des vaisseaux sanguins et des données histomorphométriques de la matrice extracellulaire du tissu de granulation; - Proposer des anticorps qui se lient aux cellules souches humaines et qui peuvent être utilisés en immunohistochimie pour détecter des MSC dans les tissus de lapin NZW et dans des coupes osteochondrales; - Localiser les MSC dans les lésions microperforées dans le cartilage, pour une meilleure compréhension du processus de réparation du cartilage. Dans la première partie de ce projet de mémoire, l’effet de l’implant chitosan-GP/sang avec ou sans un facteur de coagulation a été étudié à travers une méthode stéréographique. Cette dernière permet de récolter des données quantitatives reproductibles, qui peuvent être comparées aux données d’études passées et d’études futures. L’augmentation de la densité de surface et de volume des vaisseaux sanguins pour des implants avec IIa ou rhFVIIa ajouté montrent que ces facteurs de coagulation peuvent stimuler plus d’angiogenèse dans l’os sous-chondral au cours de la réparation induite par microperforation. Il a également été démontré que l’implant chitosan-GP/sang retarde le dépôt des fibres de collagènes (de type I et de type II) dans la matrice extracellulaire. Dans la deuxième partie de ce mémoire, les anticorps contre la β-catenin, la nestin et l’actine αSMA ont été utilisés pour des tests d’immunohistochimie, afin de détecter les cellules souches mésenchymateuses dans les tissus d’études animales in vivo. Malgré l’absence de signal positif avec les anticorps anti-β-catenin et anti-nestin, l’actine αSMA a été détectée dans divers tissus du lapin NZW, incluant les tissus fœtaux, les tissus adultes du foie, du poumon, de la peau, du placenta, de la trochlée intacte ou en réparation. Les résultats obtenus dans les tissus de trochlée en réparation montrent que les cellules exprimant l’actine αSMA sont localisées sur les bords de la microperforation, ainsi qu’à la surface du tissu de granulation. Bien que des tests supplémentaires soient nécessaires pour mieux comprendre les mécanismes impliquant les MSC, les colorations IHC effectuées indiquent que les cellules αSMA-positives sont des « bone lining cells » et qu’elles sont impliquées dans le processus de remodelage et réparation de l’os, et dans les « pre-chondrogenic foci » survenant à l’intersection du tissu de granulation et de l’os nouvellement formé. Les pre-chondrogenic foci, qui sont formés à la surface des plaques sous-chondrales, sont les structures précoces du cartilage articulaire régénéré. Dans son ensemble, cette étude a permis des informations supplémentaires sur l’implication de l’angiogenèse et des cellules souches mésenchymateuses lors de la réparation du cartilage et de l’os sous-chondral. Elle bénéficie aux progrès de la recherche dans ce domaine en démontrant que l’angiogenèse et les MSC sont des éléments détectable et quantifiable dans le tissu de granulation des lésions microperforées du cartilage, avant l’émergence des « chondrogenic foci ». Des études futures devraient être en mesure d’optimiser l’implant chitosan-GP/sang par la sélection d’additifs, comme les facteurs de coagulation, qui peuvent améliorer l’efficacité et la durabilité de la réparation du cartilage. L’un des buts majeurs est de pouvoir engendrer la formation d’un cartilage « purement » hyalin à travers la promotion de l’angiogenèse et du recrutement des MSC pour restituer le cartilage à son état sain initial. ---------- The major challenges of current orthopedic research are the restitution, preservation and improvement of the musculoskeletal system, in treating degenerative diseases such as osteoarthritis and rheumatoid arthritis. In 2011, these diseases affected more than 16% of the population in Canada, leading patients to suffer articular pain and reduced mobility of the joint. The absence of repair in focal lesions of articular cartilage can be explained by the avascular nature of the cartilage tissue and its low cellular density. Hence various surgical techniques were developed to trigger the repair of the cartilage and the subchondral bone, as for example autologous chondrocyte transplantation or bone marrow stimulation. The latter surgical technique uses microfracture or microdrilling in the cartilage and bone tissues and is one of the most commonly used. It creates access channels to the subchondral bone to allow the formation in situ of a blood clot and the migration of mesenchymal stem cells, through the rupture of subchondral vasculature. But studies have shown that the produced cartilage is a mixture of fibrous and hyaline cartilage, which cannot sustain the same loads as intact hyaline cartilage in the joint. Thus a chitosan-based implant was developed to induce more hyaline cartilage repair and ensure the residency of the otherwise fragile blood clot in the cartilage defect. Previous work in our laboratory has shown that combining bone marrow stimulation techniques with a solution of chitosan, glycerol phosphate and autologous blood enhances the cartilage quality through mechanisms involving various cellular populations, such as neutrophils and macrophages, and different repair processes, such as angiogenesis and chondroinduction. It was shown that the in situ solidification of the chitosan-GP/blood implant can be accelerated by the addition of the coagulation factors, such as thrombin (IIa), tissue factor (TF) and recombinant human Factor VIIa (rhFVIIa). These coagulation factors were added to the chitosan-based implant to accelerate in situ solidification of the implant for clinical application, which could potentially allow implant delivery by arthroscopic injection. In a rabbit microdrill model, thrombin-solidified implant improved cartilage repair over thrombin-treatment alone. However, the precise effect of coagulation factors on angiogenesis and the repair process has yet to be elucidated. Thus, the purpose of this study was to test the following two hypotheses: - The addition of coagulation factors, including thrombin (IIa), tissue factor (TF) and recombinant human Factor VIIa (rhFVIIa), to the chitosan-GP/blood implant enhances subchondral angiogenesis in repairing microdrilled defects, compared to microdrilling alone, at 3 weeks post-operative in a rabbit model of cartilage repair. - The use of a chitosan-GP/blood implant with microdrilling attracts more mesenchymal stem cells to the microdrilled cartilage defect, in comparison to bone marrow stimulation techniques alone. To test these hypotheses, the study’s principal aims were: - To evaluate the effect of the chitosan-GP/blood implant coagulated in the microdrilled lesion with coagulation factors, including thrombin (IIa), tissue factor (TF) + rhFVIIa and recombinant human Factor VIIa (rhFVIIa), on cartilage repair compared to microdrilling-alone, through blood vessel stereology and extracellular matrix histomorphometry data; - To identify the antibodies that recognize human stem cell markers that can be used for immunohistochemistry in NZW rabbit tissues and osteochondral samples; - To locate the MSC in the microdrilled defects and to better understand the cartilage repair process. In the first part of this study, the effect of the chitosan-GP/blood implant with or without added coagulation factor was investigated through a stereological method. It results in quantitative reproducible data, which allows comparison to data from previous and future studies. The observed increase in blood vessel surface and volume density for implant with either thrombin or recombinant factor rhFVIIa, indicates that these coagulation factors enhance subchondral angiogenesis in the chitosan-induced cartilage repair. It is also shown that the chitosan-GP/blood implant delays collagen (type I and type II) deposition in the extracellular matrix. In the second part of this study, antibodies for β-catenin, nestin and αSMA were used in immunohistochemistry tests in rabbit tissues in order to detect mesenchymal stem cells in tissues collected from in vivo studies. Although positive signals for β-catenin and nestin were not obtained, αSMA was detected in various tissues, including foetal tissue, adult lung, skin, liver, placenta, osteochondral repairing and intact tissue from NZW rabbit animal models. Results in repairing osteochondral tissue also demonstrated the presence of αSMA expressing cells at the edges of the microperforated subchondral bone defect and at the surface of the granulation tissue in NZW rabbit osteochondral sections. Further tests would be necessary to full understand the mechanisms implicating mesenchymal stem cells, but the IHC results do show that αSMA expressing cells are bone lining cells and that they are implicated in the bone remodelling and repair processes, and are present in “pre-chondrogenic foci” structures occurring at the interface between the granulation tissue and the newly formed bone. Chondrogenic foci that form along the repairing subchondral bone plate are the precursor structures that regenerate hyaline articular cartilage. Altogether, this study allowed a better understanding of the implication of angiogenesis and mesenchymal stem cells in subchondral bone and cartilage repair. It benefits research in the field, as it shows that angiogenesis and mesenchymal stem cells are important process that can be located and quantified in the drilled defect granulation tissue, before the emergence of chondrogenic foci. Future studies should be able to determine which additive, such as coagulation factor, can enhance the chitosan-GP/blood implant efficiency and generation “pure” articular cartilage through the promotion of subchondral angiogenesis and mesenchymal stem cell recruitment in repairing drilled defects

Bone-repair properties of biodegradable hydroxyapatite nano-rod superstructures

Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material´s surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants.Fil: D'elía, Noelia Laura. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Química del Sur; ArgentinaFil: Mathieu, Colleen. École Polytechnique. Institute of Biomedical Engineering; CanadáFil: Hoemann, Caroline D.. École Polytechnique. Institute of Biomedical Engineering; Canadá. Groupe de Recherche en Sciences et Technologies Biomédicales; Canadá. École Polytechnique. Department of Chemical Engineering; CanadáFil: Laiuppa, Juan Andrés. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Santillan, Graciela Edith. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Messina, Paula Veronica. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Química del Sur; Argentin

Identification of genes expressed by immune cells of the colon that are regulated by colorectal cancer-associated variants.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

How to build science-action partnerships for local land-use planning and management: Lessons from Durban, South Africa

The gap between scientific knowledge and implementation in the fields of biodiversity conservation, environmental management, and climate change adaptation has resulted in many calls from practitioners and academics to provide practical solutions responding effectively to the risks and opportunities of global environmental change, e.g., Future Earth. We present a framework to guide the implementation of science-action partnerships based on a real-world case study of a partnership between a local municipality and an academic institution to bridge the science-action gap in the eThekwini Municipal Area, South Africa. This partnership aims to inform the implementation of sustainable land-use planning, biodiversity conservation, environmental management, and climate change adaptation practice and contributes to the development of human capacity in these areas of expertise. Using a transdisciplinary approach, implementation-driven research is being conducted to develop several decision-making products to better inform land-use planning and management. Lessons learned through this partnership are synthesized and presented as a framework of enabling actions operating at different levels, from the individual to the interorganizational. Enabling actions include putting in place enabling organizational preconditions, assembling a functional well-structured team, and actively building interpersonal and individual collaborative capacity. Lessons learned in the case study emphasize the importance of building collaborative capacity and social capital, and paying attention to the process of transdisciplinary research to achieve more tangible science, management, and policy objectives in science-action partnerships. By documenting and reflecting on the process, this case study provides conceptual and practical guidance on bridging the science-action gap through partnerships

Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action

A workshop sponsored by the Human Toxicology Project Consortium (HTPC), “Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action” brought together experts from a wide range of perspectives to inform the process of pathway development and to advance two prototype pathways initially developed by the European Commission Joint Research Center (JRC): liver-specific fibrosis and steatosis. The first half of the workshop focused on the theory and practice of pathway development; the second on liver disease and the two prototype pathways. Participants agreed pathway development is extremely useful for organizing information and found that focusing the theoretical discussion on a specific AOP is helpful. It is important to include several perspectives during pathway development, including information specialists, pathologists, human health and environmental risk assessors, and chemical and product manufacturers, to ensure the biology is well captured and end use is considered