Die Rolle von GABA- und Glyzinrezeptoren w��hrend der radialen Migration kortikaler Neurone in der pr��natalen Maus

F��r die Entwicklung des zerebralen Kortex ist die radiale Migration von Neuronen von elementarer Bedeutung. F��r diese radiale Migration sind extrazellul��re Signale, die mit den Neuronen interagieren und eine Umgestaltung des Zytoskeletts vermitteln, notwendig. Zu den extrazellul��ren Signalen geh��rt auch der Neurotransmitter GABA, der ��ber Depolarisation der Neurone einen Ca2+-Einstrom vermittelt und dadurch die Modulation der Migration ��ber Ca2+-abh��ngige Signalwege erm��glicht. Auch von Taurin ist bekannt, dass es die neuronale Migration beeinflusst. Fr��here Studien zeigten, dass die Depolarisation von GABAA-Rezeptoren durch GABA zu einem Migrationsstop f��hrt, wohingegen Picrotoxin-sensitive Rezeptoren die Migration von der Ventrikul��ren Zone in die Intermedi��re Zone des pr��natalen Kortex vermitteln. Obwohl zu den Picrotoxin-sensitiven Rezeptoren GABAA-, GABAC- und bestimmte Glyzinrezeptoren geh��ren, wurde die Rolle von GABAC- und Glyzinrezeptoren w��hrend der radialen Migration nie ��berpr��ft. Ziel dieser Dissertation war deshalb, den Einfluss von GABAC- und Glyzinrezeptoren auf die radiale Migration zu untersuchen. Unter Verwendung von Migrationsanalysen, Fluoreszenzmessungen, molekularbiologischen und histologischen Methoden wurde gezeigt, dass GABAC-Rezeptoren im unteren Bereiche des pr��frontalen Kortex exprimiert werden, ihre Aktivierung durch GABA in der Intermedi��ren Zone zu einer Depolarisation f��hrt, dass GABAC-Rezeptoren die Migration f��rdern und dieser Effekt ��ber den migrationsstoppenden Effekt der GABAA-Rezeptoren dominiert. Durch Aktivierung der Glyzinrezeptoren f��rdert Taurin die Migration.

Differentiation and extracellular matrix interactions of chondrocytes in response to signaling molecules and biomaterials for cartilage repair = Differenzierung und Interaktionen von Chondrozyten mit der extrazellul��ren Matrix als Reaktion auf Signalmolek��le und Biomaterialien f��r die Wiederinstandsetzung von Knorpel

Chondrocytes live isolated in the voluminous extracellular matrix of cartilage, which they secrete and is neither vascularized nor innervated. Nutrient and waste exchanges occur through diffusion leading to low oxygen tension around the cells. Consequently even normal cartilage under normal physiological conditions suffers from a poor reparative potential that predisposes to degenerative conditions, such as osteoarthritis of the joints, with significant clinical effects.rnOne of the key challenges in medicine is the structural and functional replacement of lost or damaged tissues. Current therapeutical approaches are to transplant cells, implant bioartificial tissues, and chemically induce regeneration at the site of the injury. None of them reproduces well the biological and biomechanical properties of hyaline cartilage.rnThis thesis investigates the re-differentiation of chondrocytes and the repair of cartilage mediated by signaling molecules, biomaterials, and factors provided in mixed cellular cultures (co-culture systems). As signaling molecules we have applied prostaglandin E2 (PGE2) and bone morphogenetic protein 1 (BMP-1) and we have transfected chondrocytes with BMP-1 expressing vectors. Our biomaterials have been hydrogels of type-I collagen and gelatin-based scaffolds designed to mimic the architecture and biochemistry of native cartilage and provide a suitable three-dimensional environment for the cells. We have brought chondrocytes to interact with osteosarcoma Cal 72 cells or with murine preosteoblastic KS483 cells, either in a cell-to-cell or in a paracrine manner.rnExogenous stimulation with PGE2 or BMP-1 did not improve the differentiation or the proliferation of human articular chondrocytes. BMP-1 induced chondrocytic de-differentiation in a dose-dependent manner. Prostaglandin stimulation from gelatin-based scaffolds (three-dimensional culture) showed a certain degree of chondrocyte re-differentiaton. Murine preosteoblastic KS483 cells had no beneficial effect on human articular chondrocytes jointly cultivated with them in hydrogels of type I collagen. Although the hydrogels provided the chondrocytes with a proper matrix in which the cells adopted their native morphology; additionally, the expression of chondrocytic proteoglycan increased in the co-cultures after two weeks. The co-culture of chondrocytes with osteoblast-like cells (in transwell systems) resulted in suppression of the regular de-differentiation program that passaged chondrocytes undergo when cultured in monolayers. Under these conditions, the extracellular matrix of the chondrocytes, rich in type-II collagen and aggrecan, was not transformed into the extracellular matrix characteristic of de-differentiated human articular chondrocytes, which is rich in type-I collagen and versican.rnThis thesis suggests novel strategies of tissue engineering for clinical attempts to improve cartilage repair. Since implants are prepared in vitro (ex-vivo) by expanding human articular chondrocytes (autologous or allogeneic), we conclude that it will be convenient to provide a proper three-dimensional support to the chondrocytes in culture, to supplement the culture medium with PGE2, and to stimulate chondrocytes with osteoblastic factors by cultivating them with osteoblasts.rn

Monitoring the extracellular matrix mineralization processes in biological scaffolds using bioreactors, x-ray ��ct technology and 3-d modeling methods (monitoraggio dei processi di mineralizzazione della matrice extracellulare in scaffolds biologici, utilizzando bioreattore, tecnologia ��ct a raggi x e metodi di modellizzazione 3d)

Al fine di migliorare le tecniche di coltura cellulare in vitro, sistemi a bioreattore sono sempre maggiormente utilizzati, e.g. ingegnerizzazione del tessuto osseo. Spinner Flasks, bioreattori rotanti e sistemi a perfusione di flusso sono oggi utilizzati e ogni sistema ha vantaggi e svantaggi. Questo lavoro descrive lo sviluppo di un semplice bioreattore a perfusione ed i risultati della metodologia di valutazione impiegata, basata su analisi ��CT a raggi-X e tecniche di modellizzazione 3D. Un semplice bioreattore con generatore di flusso ad elica �� stato progettato e costruito con l'obiettivo di migliorare la differenziazione di cellule staminali mesenchimali, provenienti da embrioni umani (HES-MP); le cellule sono state seminate su scaffold porosi di titanio che garantiscono una migliore adesione della matrice mineralizzata. Attraverso un microcontrollore e un'interfaccia grafica, il bioreattore genera tre tipi di flusso: in avanti (senso orario), indietro (senso antiorario) e una modalit�� a impulsi (avanti e indietro). Un semplice modello �� stato realizzato per stimare la pressione generata dal flusso negli scaffolds (3���10-2 Pa). Sono stati comparati tre scaffolds in coltura statica e tre all���interno del bioreattore. Questi sono stati incubati per 21 giorni, fissati in paraformaldehyde (4% w/v) e sono stati soggetti ad acquisizione attraverso ��CT a raggi-X. Le immagini ottenute sono state poi elaborate mediante un software di imaging 3D; �� stato effettuato un sezionamento ���virtuale��� degli scaffolds, al fine di ottenere la distribuzione del gradiente dei valori di grigio di campioni estratti dalla superficie e dall���interno di essi. Tale distribuzione serve per distinguere le varie componenti presenti nelle immagini; in questo caso gli scaffolds dall���ipotetica matrice cellulare. I risultati mostrano che sia sulla superficie che internamente agli scaffolds, mantenuti nel bioreattore, �� presente una maggiore densit�� dei gradienti dei valori di grigio ci�� suggerisce un migliore deposito della matrice mineralizzata. Gli insegnamenti provenienti dalla realizzazione di questo bioreattore saranno utilizzati per progettare una nuova versione che render�� possibile l���analisi di pi�� di 20 scaffolds contemporaneamente, permettendo un���ulteriore analisi della qualit�� della differenziazione usando metodologie molecolari ed istochimiche.

Extracellular glutamate-GABA balance in the developing neocortex

It has been shown in the study that glutamate transporters (EAAT) are capable to modulate GABA transports (GAT). Here we also report that DL-TBOA, a non-transportable glutamate uptake blocker, eliminates GAT-mediated GABA release, while D-aspartate, an EAAT substrate, does not block the latter. The strength or even the operating mode of GABA uptake/release could be influenced by the work of EAATs. Considering the interaction between EAATs and GATs we can conclude that ambient glutamate and GABA levels are mutually dependent. The EAAT-GAT crosstalk observed in this work is mediated by EAAT1 and GAT-2/3. Since both transporters are Na+ dependent and mainly glial, next we investigated the role of [Na+]i in astrocytic-mediated glutamate uptake. We tested whether [Na+]i changes affect paired-pulse plasticity of STCs recorded from cortical layer 2/3 astrocytes. We report that an elevation of [Na+]i induced either by using a high [Na+]i intrapipette solution or by application of GABA slows STCs kinetics and decrease paired-pulse facilitation (PPF) of STCs at short inter-stimulus intervals. Moreover, GAT inhibitors decrease PPF of STCs under control conditions, suggesting that endogenous GABA operating via GATs influences EAAT-mediated transport

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.