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G Ciofani1, L Ricotti1, S Danti2,3, S Moscato4, C Nesti2, D D’Alessandro2,4, D Dinucci5, F Chiellini5, A Pietrabissa3, M Petrini2,3, A Menciassi1,61Scuola Superiore Sant’Anna, Pisa, Italy; 2CUCCS-RRMR, Center for the Clinical Use of Stem Cells – Regional Network of Regenerative Medicine, 3Department of Oncology, Transplants and Advanced Technologies, 4Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy; 5Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab), UdR INSTM, Department of Chemistry and Industrial Chemistry, University of Pisa, San Piero a Grado, Italy; 6Italian Institute of Technology, Genova, ItalyAbstract: Boron nitride nanotubes (BNNTs) have generated considerable interest within the scientific community by virtue of their unique physical properties, which can be exploited in the biomedical field. In the present in vitro study, we investigated the interactions of poly-L-lysine-coated BNNTs with C2C12 cells, as a model of muscle cells, in terms of cytocompatibility and BNNT internalization. The latter was performed using both confocal and transmission electron microscopy. Finally, we investigated myoblast differentiation in the presence of BNNTs, evaluating the protein synthesis of differentiating cells, myotube formation, and expression of some constitutive myoblastic markers, such as MyoD and Cx43, by reverse transcription – polymerase chain reaction and Western blot analysis. We demonstrated that BNNTs are highly internalized by C2C12 cells, with neither adversely affecting C2C12 myoblast viability nor significantly interfering with myotube formation.Keywords: boron nitride nanotubes, C2C12 cells, cytocompatibility, up-take, differentiation, MyoD, connexin 43

Mixed cryoglobulinaemia (MC) is a systemic disorder characterized clinically by the typical triad of purpura, arthritis and weakness, and serologically by the presence of cryoprecipitating complexes, formed by a monoclonal or polyclonal rheumatoid factor and polyclonal immunoglobulins. The presence of anti-HCV antibodies and viral mRNA in the serum of MC patients has shed new light on the pathogenesis of the disease. However, the mechanisms leading to the different manifestations of MC are still unknown.

Osteogenic growth peptide (OGP) is an endogenous tetradecapeptide present in micromolar concentrations in mammalian serum; its carboxy-terminal pentapeptide, OGP(10-14), represents its physiologically active fragment. OGP(10-14) induces proliferation and differentiation in fibroblast and osteoblast cell lines, and it enhances hematopoiesis in vitro and in vivo. The signaling pathways triggered by OGP(10-14) are not yet fully known. In the present report, we evaluated the effect of OGP(10-14) on differentiation of a cancer megakaryoblast cell line and its involvement on RhoA and Src family kinases signaling pathway.Cell proliferation of the Mo-7e line was evaluated using the MTT test. Mo-7e differentiation was evaluated by microscopic observation of cell morphology and by expression of the factor VIII-related antigen. Involvement of RhoA and Src kinases on signaling pathways triggered by OGP(10-14) was analyzed using RhoA and Src family kinase (SFK) inhibitors (C3 and PP2) and an immunoperoxidase technique.OGP(10-14) induces expression of the factor VIII-related antigen, morphologic changes indicative of megakaryocytic differentiation, and a down-regulation of the Fyn Src kinase. These OGP(10-14) effects were prevented by C3 and enhanced by PP2.The anti-proliferative and pro-differentiating activities of OGP(10-14) on thrombopoietin (TPO)-primed Mo-7e cells are mediated by RhoA and Src kinase pathways as demonstrated by the use of C3 and PP2.

The tympanic membrane (TM) is an anatomical structure with unique histological and physiological features playing a fundamental role in sound transmission. In particular, the middle layer of the pars tensa, which represents the widest and thickest surface portion of the TM, consists of connective tissue mainly composed of collagen types II and III fibers, while collagen type I is present at a lesser extent [1]. Several pathologies affect the TM, including otitis media, tympanosclerosis, cholesteatoma and perforation that require reconstructive surgery depending on the lesion extent [2]. To this purpose, the temporalis fascia is currently considered as the gold standard material. However, due to limited graft availability, fully synthetic substitutes are also applied, with poorly satisfactory outcomes. For these reasons new strategies for TM replacement are still needed. In this study, we employed a tissue engineering (TE) approach for the regeneration of TM substitutes selecting some biocompatible and bioresorbable polymeric matrices to be cultured with human bone marrow-derived mesenchymal stem cells (MSCs). We set up a cell differentiation protocol using an appropriate mix of growing factors to obtain the in vitro differentiation of MSCs into TM fibroblasts. Furthermore, because of the role played by mechanical forces in TM motion, these engineered substitutes underwent mechanical stress during the culture. The obtained biohybrid constructs were characterized about cellular viability assays, gene expression quantification as well as histochemical and immunohistochemical analyses. Moreover, native TMs from cadavers were investigated for assessment and optimization of the engineered constructs. Our results showed that MSCs were able to grow and differentiate properly on the selected biomaterials and to synthesize appropriate extracellular matrix molecules. Moreover, the applied mechanical forces seem to promote TM-fibroblastic differentiation, increasing the production of collagen type II, that is a peculiarity of TM structure., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012