Marco Vincenzo Patruno

MUSCLE GROWTH IN RESPONSE TO CHANGING DEMANDS OF FUNCTIONS IN THE TELEOST SPARUS AURATA (L.) DURING DEVELOPMENT FROM HATCHING TO JUVENILE

Abstractu2002Growth of laterarl muscle in the teleost fish Sparus aurata (L.) was examined from hatching to juvenile by a basic morphofunctional approach that takes into account structural and ecophysiological aspects and combines in vivo observations and LM and TEM microscopic analysis. As shown in most teleost fishes, muscle growth proceeds by a double mechanism of hyperplasia and hypertrophy that contribute differentially to the overall development of the lateral muscle, giving rise in each myomere to a typical pattern of structurally and functionally different fibre types (slow-red and fast-white fibres, plus pink intermediate fibres) in a nerve-dependent process. During larval life the muscle growth takes place mainly due to hyperplastic growth at the level of specific proliferative zones of the myomeres, from which slow, pink and white muscle fibres are derived. In those species that reach a large adult size a new typical hyperplastic process disseminated throughout the fast white muscle layer takes place during post-larval life. In contrast, hypertrophic growth occurs in all stages, but is the dominant mechanism of muscle growth only in juvenile and adult. The suitable recruitment of the different fibre types enables the fish to optimize its performances according to specific functional and metabolic requirements related to the swimming behaviour and hydrodynamic regimes. The different mechanisms of growth are here analysed in their detailed structural and ultrastructural aspects in order to interpret their adaptive significance in the light of the fish life cycle, with particular reference to locomotion and feeding behaviour.

Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence.

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor–β (TGF–β) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF–β–like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF–β–like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non–neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF–β–like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.

Scleraxis and MyoD fused with the TAT polypeptide may induce equine adult stem cells towards tenogenic and myogenic fates

B design and 3-D printing of scaffold with heterogeneous internal geometry is essential for cell distribution, blood vessel in growth and biomaterial degradation in bone tissue engineering. This study was designed to demonstrate the heterogeneous pores and channels in 3-D printed scaffolds for bone tissue engineering. Scaffolds were prepared using ceramic particles through 3-D printing. Pores and connecting channels with diameters of 200mm-500mm were designed for facilitating cell seeding and cell distribution. Internal pores of 50mm-200mm were designed for bone regeneration. Nano-sized surface topography was designed for enhanced degradation of scaffold. The fabricated scaffolds were evaluated using scanning electronic microscopy. SEM of fabricated scaffolds revealed that 400mm500mm inter-connecting channels crossed over the entire scaffold, that ~200 mm internal pores were scattered over the scaffold and connected to each other and to the interconnecting channels, and that ~200 nm pores showed on the surfaces of inter-connecting channels and internal pores, which would play an important role in increasing the surface ratio of materials and facilitating material degradation. A heterogeneous profile of connecting channels and internal pores was evident in these 3-D printed biomimetic scaffolds. As a conclusion, the biomimetic design and fabrication of scaffolds for bone tissue engineering can be fulfilled by a 3-D printing process. Heterogeneous profiles of inter-connecting channels, internal pores, and nano-sized surface topography can be generated to provide a biomimetic environment suitable for bone tissue engineering.Background: Myocardial infarction (MI) was the leading cause of death in worldwide. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of biological processes, including cell viability, proliferation, development and differentiation. The purpose of this study was to investigate difference of miRNA profiles between infarct zone and border zone in post-MI remodeling using the second generation sequencing.Neia Naldaiz-Gastesi1, Patricia Garcia-Parra1, Maria Goicoechea1, Sonia Alonso-Martin2, Ana Aiastui1, Macarena Lopez-Mayorga3, Paula Garcia-Belda4, Jaione Lacalle1,5, Veronique Le Berre6, Ander Matheu1, Jose Manuel Garcia-Verdugo4, Jaime J. Carvajal3, Frederic Relaix2, Adolfo Lopez de Munain1 and Ander Izeta1 1Instituto Biodonostia, Spain 2Myology Research Center, France 3Centro Andaluz de Biologia del Desarrollo, Spain 4Instituto Cavanilles, Universidad de Valencia, Spain 5University of the Basque Country (UPV-EHU), Spain 6UMR INSA, FranceOur knowledge of the regenerative ability of the auditory system is still inadequate. Moreover, new treatment techniques for hearing impairment using cochlear implant and tissue engineering, call for further investigations. Tissue engineering and regenerative strategies have many applications ranging from studies of cell behavior to tissue replacement and recently there have been significant advances in the biotechnological tools followed by development of new interventions, including molecules, cells, and even biodegradable biomaterials. This thesis presents results of tissue engineering approaches used in vitro with the long-term aim of facilitating auditory nerve and spiral ganglion regeneration. The first part describes the use of neurotrophic factors and neurosteroids for promoting survival and growth of nerve cells and the second part describes the effective usage of a biotechnology method, microcontact imprinting technique, to control key cellular parameters modifying chemical cues on the surface. The failure of the spiral ganglion neurons to regenerate was postulated to be due to the limited capacity of neurons to re-grow axons to their target. In paper I, we focused our studies on the role of GDNF in promoting spiral ganglion neuron outgrowth. The effect of three neurotrophins, among them GDNF, on spiral ganglion neurons in vitro was evaluated. The neuronal outgrowth was characterized by light microscopy and immunohistochemistry. The results speak in favor of GDNF, which promoted neuronal growth and branching, and Schwann cell alignment along the neurons in culture. The study support the role of GDNF as a potent factor, exerted neurogenic effects on cochlear cells in a degree dependent on the concentration used, confirming the hypothesis of GDNF being an oto-protector for chemicaland noiseinduced hearing loss and potential drug candidate for the inner ear. This might be relevant for future regenerative therapies and could have implications for tissue engineering techniques. In the second study, paper II, the objective was similarly to evaluate the efficacy of dendrogenin, a neurosteroid analogue, which can be applied to the cochlea. Dendrogenin was also tested in the presence and absence of other growth factors and the effect on adult neural stem cells was investigated. The study showed that neural stem cells exhibited proliferation/differentiation responses. Based on fluorescent labeling and a sphere-formation assay, we observed that adult neural stem cells induced proliferation. We asked whether the stem cells would differentiate into the major cell types of the nervous system and mainly neurons. Thus, neurotrophic supplement was added to the culture medium and was shown to have a selective effect on outgrowth of neuronal population. β3-tubulin positive neurons with BrdU positive nuclei were found and similar to other studies, we observed that the rate of differentiation increased with declining of BrdU expression. We found that despite the ongoing neuronal differentiation, there was an apparent difference of the neuronal outgrowth among the spheres treated with dendrogenin. The newly formed neurons were not found to send long projections into the local circuitry and the total cell number and length remained limited. Taken together, the protocols described inhere provide a robust tool to expand the biological role of dendrogenin that was in favor of differentiation when added to neuronal cell lines. The results of this study add new knowledge and better understanding of the possible action of dendrogenin in regenerative therapy. In paper III a strategy to guide spiral ganglion neurons was developed using a microcontact technique. The surface for neuronal guidance was designed with favorable extracellular proteins to promote the neurite outgrowth. Micro-contact imprinting provided a versatile and useful technique for patterning the guidance surface. Imprinting generated a patterned surface in a controllable, predictable, and quantifiable manner. A range of events followed the patterning including alignment, polarity and directionality was reported and observed by microscopic description. The dynamic microenvironment that resulted from the synergistic combination of extracellular guidance cues and Schwann cells selectively instructed and directed the terminal extension of neurons into unior bi-polar fate. In summary, applying new factors such as molecules, cells and surfaces provides unique possibilities to recruit spiral ganglion neurons into their regenerative ability. Additionally, creating an environment that incorporates multiple molecular and cellular cues will offer exciting opportunities for elucidating the mechanisms behind nerve regeneration and highlight specific considerations for the future tissue engineering. LIST OF PUBLICATIONS This thesis is based on the following original papers, which will be referred to in the text by their Roman numerals. I. Marja Bostrom, Shaden Khalifa, Henrik Bostrom, Wei Liu, Ulla Friberg, Helge Rask-Andersen. Effects of neurotrophic factors on growth and glial cell alignment of cultured adult spiral ganglion cells. Audiology Neurootology 2010; 15(3): 175-186. II. Shaden AM Khalifa, de Medina Philippe, Sandrine Silvente-Poirot, Anna Erlandsson, Hesham ElSeedi and Marc Poirot. The novel steroidal alkaloids dendrogenin A and B promote proliferation of adult neural stem cells. Under revision in Biochemical and Biophysical Research Communications. III. Shaden AM Khalifa, Per Bjork, Christian Vieider, Mats Ulfendahl, and Eric Scarfone. Neuronal Polarity Mediated by Micro-scale Protein Patterns and Schwann Cells in vitro. Tissue Engineering and Regenerative Medicine 2013; 10(5): 266-272. CONTENTS Abstract List of publications Abbreviations Chapter 1: Introduction 1 1.1 Ear anatomy 1 1.2 Hearing physiology 6 1.3 Hearing Loss 8 1.4 Cochlear implant 9 1.5 Tissue engineering strategies 11 Aims 18 Chapter 2: Materials and methods 19 2.1 Ethical permission and animal care 19 2.2 Tissue dissection 19 2.3 Micro-pattern fabrication 20 2.4 Culture procedure 22 2.5 Immunohistochemistry 24 2.6 Imaging 25 2.7 Time Lapse Video 26 2.8 Imaging analysis 26 2.9 Axon analyzer software 27 2.10 Statistical Analysis 27 Chapter 3: Results 28 3.1 Neurotrophins stimulate neuronal regeneration in vitro 28 3.2 Dendrogenin activity on adult neural stem cells 29 3.3 Protein patterning 30 3.4 Neuronal polarity 30 3.5 Cells in culture 31 Chapter 4: Discussion 33 4.1 GDNF effects on spiral ganglion cells in vitro 33 4.2 Dendrogenin effects on adult neural stem cells in vitro 34 4.3 Patterning proteins 35 4.4 Neuronal guidance and polarity 35 Chapter 5: Conclusions and future prospective 37 5.1 Conclusions 37 5.2 Prospective 38 Acknowledgments 40 References 43 LIST OF ABBREVIATIONSS cells have been recognized as a potential tool to restore cells damaged by cerebral ischemic injury. Key functions such as the replacement of neural cells have been recently challenged by intrinsic bystander capacities of undifferentiated donor cells. One of opportunity for neurological disorder treatment is the transplantation of mesenchymal stem cells (MSCs) which have neuroprotective, neuroregenerative and anti-inflamatory properties. However, a comprehensive knowledge how transplanted MSCs exert their therapeutic achievements is still lacking. The aim of the project was to analyze the presence, distribution and quantity of human bone marrow mesenchymal stem cells (hBM-MSCs) transplanted into focal brain ischemic rats. The experiments were performed in adult male Wistar rats withbrain focal ischemiainduced with 1μl/50nmol ouabain(sodium-potassium pump inhibitor) injection into right stratium. Then 5x105 hBM-MSC (Lonza) stained with iron nanoparticles and rhodamine (Molday, BioPAL) were transplanted into internal carotid artery, 48 hours after brain insult. At 1, 3, 7 and 14 days rat brains were removed. Immunocytochemical analysis of human markers using different antibodies anti: CD44, STEM121and Ku80 were performed. The preliminary results showed that after intra-arterially injection of hBM-MSC, the donor cells were present in the ipsilateral rat hemisphere between cortical cortex and stratium near the ischemic lesion. The positive staining for Molday particles and human antigens were observed at 1, 2, 3 and 7 days after hBM-MSC transplantation. The further studies relating to the function of transplanted cells are in progress.M stem cells (MSCs) represent a population of multipotent stem cells with immunomodulatory, antiapoptotic and cytoprotective capabilities and thus hold a great promise for treatment of many inflammatory diseases and for use in a regenerative medicine. Numerous studies have shown that the administration of MSCs in combination with an immunosuppressive drug prolongs allograft survival in comparison with use of MSCs or the drug alone. However, the exact mechanism of such synergism has not yet been described.

Comparison in vivo applications between peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) in injured tendons of sheep

T properties of DLC films, which are similar to those of diamond, are attractive because of their extreme mechanical hardness, high electrical resistivity, low friction, stability against acids, uniformly flat surface and optical transparency over a wide area. DLC films are deposited on several kinds of substrates at room temperature. These advantages of the DLC films lead to many application in mechanical, electrical and medical fields. In this study, the cytocompatibility of amorphous hydrogenated carbon-a-C:H:N films on polymeric fibrous materials has been investigated to apply to the biodevices. The a-C:H:N films was deposited on fibrous scaffolds by decomposed source methane gas in addition with N2 gas as dopant. The deposited a-C:H:N films were characterized for their chemical, optical, structural and electrical properties using X-ray photoelectron spectroscopy, contact angle measurement, Raman spectroscopy, and cellular affinity. We have found that the cell culture of a-C:H:N films increased monotonously with the increase of nitrogen concentration up to 40% of N2 concentration. The introduction of nitrogen gas leaded the addition of nitrogen atoms into the films. The cytocompatibility of a-C:H films was improved by the formation of C=N arrangement on the surface.