Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy in man with stromal fibrosis as one of the main features. Carcinoma cells synthesized no or little collagen I protein. Pro-α1(I) collagen mRNA was expressed by stromal cells throughout the tumor, but expression of procollagen type I protein was restricted to stromal cells situated close to nests of carcinoma cells. These data suggest that the carcinoma cells stimulated collagen type I deposition by increasing pro-α1(1) collagen mRNA translation. Cocultures, of the human ATC cell line KAT-4, with fibroblasts under conditions that allow the study of stimulatory factors on collagen mRNA translation, showed that the KAT-4 cells stimulated collagen type I protein synthesis in fibroblasts. Specific inhibitors of PDGF and TGF-β1 and -β3 were able to inhibit this carcinoma cell-induced stimulation of collagen type I synthesis. These findings suggest that tumor cells were able to stimulate collagen mRNA translation in stromal fibroblasts by, at least in part, transferring PDGF and/or TGF-β1 and -β3. Xenograft transplantation of different ATC cell lines into athymic mice demonstrated that the low collagen producing carcinoma cell lines were less tumorigenic compared to non-collagen producing carcinoma cell lines. The morphology of tumors derived from non-collagen producing ATC cell lines showed a well demarked stroma surrounding carcinoma cell nests. TGF-β1 and -β3 were found to play a role in generating a high tumor interstitial fluid pressure (TIPF) in experimental KAT-4 tumors. A specific inhibitor of TGF-β1 and -β3 was able to lower TIPF and reduce tumor growth after a prolonged period of treatment, suggesting that TGF-β1 and -β3 have a role in maintaining a stroma that support tumor growth.

Avaliamos os efeitos da glicação do colágeno (CG) e da glicose elevada sobre a migração de fibroblastos. Utilizamos células de ratos controle e diabéticos (D) e células NIH-3T3, cultivadas em glicose 5 mM ou 30 mM (HG). Para glicação utilizou-se ácido glioxílico. O CG apresentou menor resistência à tração e elasticidade. Fibroblastos migraram menos sob HG e sobre o CG. As células D no CG não se deslocaram, apresentaram menos integrina β141 e expressaram mais α-actina de músculo liso. A viscoelasticidade do citoesqueleto foi menor em células D, especialmente sobre o CG. Sobre fibronectina, células NIH-3T3 em HG apresentaram menos fibras de estresse e deficiência na retração da parte traseira. A expressão de miosinas IIA (MIIA), IIB (MIIB) e MRLC não foi alterada, mas a fosforilação de MII diminuiu. A distribuição de MIIB ficou mais difusa, enquanto MIIA não mudou. Células HG exerceram menor força sobre o substrato. A migração de fibroblastos em ambiente hiperglicêmico é deficiente, especialmente frente ao CG, em parte devido a uma redução da contratilidade celular. / We evaluated the effects of collagen glycation (GC) and high glucose concentrations on fibroblasts migration. Fibroblasts derived from control and diabetic rats (D) and NIH-3T3 cells were cultured under 5 mM or 30 mM glucose (HG). For glycation, glyoxylic acid was used. The GC showed lower tensile strength and elasticity. Fibroblasts migrated less in HG and over the GC. D cells did not move on GC, showed less β141 integrin and a higher expression of smooth muscle α-actin. The viscoelasticity of the cytoskeleton was lower in D cells, especially on the GC. On fibronectin, NIH-3T3 cells under HG had fewer stress fibers and showed impaired contraction at the rear, presenting long tails. The expression of myosin IIA (MIIA), IIB (MIIB) and MRLC has not changed, but the phosphorylation of MII decreased. The distribution of MIIB became more diffuse, while MIIA has not changed. Cells under HG exerted less force on the substrate. The migration of fibroblasts in hyperglycemic environment is impaired, especially on GC, partly due to a reduction of cell contractility.

Les ligaments jouent un rôle important dans le mouvement et la stabilité des articulations. Les accidents et la fatigue chronique sont les principales raisons des ruptures de ligaments qui n’ont généralement pas de capacité de guérison, ce qui conduit à de graves dysfonctionnements du ligament et des articulations. La construction des ligaments en bioingénierie donne un nouvel espoir thérapeutique. Pour construire un tel tissu, les cellules sont très importantes dans la mise en oeuvre de la construction d’un biotissu ayant de bonnes propriétés tant biologiques que mécaniques. Quelle source cellulaire et quel microenvironnement doivent être utilisés pour la reconstruction des ligaments ? L’objectif de ce travail était d’étudier la différenciation des cellules souches mésenchymateuses médullaires (CSMM) en fibroblaste. Pour ce faire, d’une part, nous avons co-cultivé des CSMM du rat sans contact avec des fibroblastes ligamentaires, et d’autre part, nous avons stimulé mécaniquement les CSMM. Un suivi des ARNm et des protéines associées caractéristiques des ligaments (collagènes I et III et ténascine-C) a été analysés. Nos résultats expérimentaux ont montré que la culture des CSMM dans un microenvironnement fibroblastique de ligaments ou l’étirement favorisent les synthèses de collagènes I et III et de ténascine-C dans les proportions proches des ligaments. L’ensemble de cette étude suggère qu’il est envisageable d’utiliser les CSMM comme source cellulaire, pour une application clinique, en ingénierie tissulaire du ligaments / Ligaments play an important role in the movement and stability of joints. Accidents and chronic fatigues are the main reasons for ligament lesion which usually is difficult for self healing and leads to serious dysfunction of ligaments and joints. The construction of bioengineering ligaments gives a new way to overcome this problem. Cells are very important in the construction of a biotissue with appropriate biological as well as mechanical properties. Which cellular source and microenvironment should be used for the reconstruction of ligaments? The objective of this work is to study the differentiation of bone marrow mesenchymal stem cell (BMSC) into fibroblast. We co-cultured indirectly rat BMSC with ligament fibroblasts or stimulated them by mechanical stretching. After that, the expressions of characteristics mRNA and protein of ligaments (collagen I, III and tenascin-C) have been analyzed. Our experimental results showed that the culture of BMSC in a microenvironment of ligament fibroblast or under stretching favored the syntheses of collagen I, III and tenascin-C in the proportions close to ligaments. In summary, these studies suggest it is feasible to use BMSC as cellular source for a clinical application in tissue engineering of ligaments

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Le basculement de paradigme apporté par l'ingénierie tissulaire et de la médecine régénérative par rapport à l'approche thérapeutique utilisant les biomatériaux, questionne aujourd'hui les méthodes d'évaluation de ces thérapies avancées en histopathologie. Les outils d'évaluation actuellement disponibles en histopathologie ne sont pas pleinement satisfaisants pour l'évaluation locale de ces thérapies avancées, notamment en matière d'évaluation de leur performance. Nous avons développé une nouvelle méthode quantitative numérique, simple, peu coûteuse fournissant des indicateurs clés pour la caractérisation structurelle et compositionnelle des tissus régénérés. Cet indicateur mesure le taux de croissance tissulaire (TIR) en intégrant deux autres indicateurs, le taux de croissance cellulaire (CIR) et le contenu total en collagène (TCC). Il se traduit par l'équation suivante TIR (%) = CIR (%) + TCC (%). D'autre part, un sous-ensemble d'indicateurs quantitatifs décrivant l'organisation directionnelle du collagène (relation entre structure et propriétés mécaniques des tissus), le ratio collagène I / collagène III (qualité du remodelage) et la propriété anisotropique optique du collagène (indicateur de maturité), a également été produit automatiquement. A l'aide d'un analyseur d'images assisté par ordinateur tous les indicateurs sont extraits uniquement à partir de deux lames sériées colorées soit avec du Feulgen & Rossenbeck (spécificité cellulaire) ou à l'aide de la coloration au rouge picrosirius F3BA (spécifique du collagène). Pour valider cette nouvelle approche, des échafaudages 3D identiques ont été implantés en site intrapéritonéal chez un groupe de rats sains et chez un groupe de rats diabétiques. L'hypothèse émise était que quantitativement la régénération tissulaire serait significativement retardée et défectueuse chez les rats diabétiques par rapport aux rats sains. De plus, un échafaudage 3D chimiquement modifié a été similairement implanté chez un troisième groupe de rats sains avec l'hypothèse qu'une modulation de la croissance tissulaire serait mise en évidence quantitativement par rapport au groupe de rats sains portant l'échafaudage 3D non-modifié. Après 21 jours d'implantation, les deux hypothèses ont été vérifiées, validant cette nouvelle approche d'analyse quantitative computationnelle. Les résultats quantitatifs ont révélé des différences tissulaires fines qui n'ont pas été détectées à l'évaluation semi-quantitative conduite en parallèle. Cette méthode automatisée et supervisée réduit la dépendance à l'opérateur à un minimum et s'est montrée sensible, simple, peu coûteuse et permet de gagner du temps. Elle offre le double avantage d'objectiver les comparaisons thérapeutiques et de comprendre la régénération des tissus fonctionnels localement et dans le temps / The paradigm shift brought about by the expansion of tissue engineering and regenerative medicine away from the use of biomaterials, currently questions the value of histopathologic methods in the evaluation of biological changes. To date, the available tools of evaluation are not fully consistent and satisfactory for these advanced therapies. We have developed a new, simple and inexpensive quantitative digital approach that provides key metrics for structural and compositional characterization of the regenerated tissues. For example, metrics provide the tissue ingrowth rate (TIR) which integrates two separate indicators; the cell ingrowth rate (CIR) and the total collagen content (TCC) as featured in the equation, TIR%=CIR%+TCC%. Moreover a subset of quantitative indicators describing the directional organization of the collagen (relating structure and mechanical function of tissues), the ratio of collagen I to collagen III and the optical anisotropy property of the collagen (maturity indicator) was automatically produced as well. Using an image analyzer, all metrics were extracted from only two serial sections stained with either Feulgen & Rossenbeck (cell specific) or Picrosirius Red F3BA (collagen specific). To validate this new procedure, 3D scaffolds were intraperitoneally implanted in healthy and diabetic rats. It was hypothesized that quantitatively; the healing tissue would be significantly delayed and of poor quality in diabetic rats in comparison to healthy rats. In addition, a chemically modified 3D scaffold was similarly implanted in a third group of healthy rats with the assumption that modulation of the ingrown tissue would be quantitatively present in comparison to the 3D scaffold-healthy group. After 21 days of implantation, both hypotheses were verified by use of this novel computerized approach. When the two methods were run in parallel, the quantitative results revealed fine details and differences not detected by the semi-quantitative assessment, demonstrating the importance of quantitative analysis in the performance evaluation of soft tissue healing. This automated and supervised method reduced operator dependency to a minimum and proved to be simple, sensitive, cost-effective, time-effective, a way of doing objective therapeutic comparisons and a way to elucidate regeneration and the dynamics of a functional tissue