Although neurotransplantation of primary fetal cells into the striatum of patients with Parkinson’s disease (PD) has been reported to be effective, poor clinical outcome and severe side effects lower clinical long-term results. A major drawback of cell replacement therapies in PD is the low cell survival and lacking regeneration of the neuronal circuitries due to the ectopic transplantation of cells into the host striatum. More anatomic and functional integration could potentially be reached by an orthotopic cell transplantation into their natural position within the rostral mesencephalon at the site of the Substantia nigra, where dopaminergic cells get lost in PD. The aim of the thesis was to provide the scientific basis for the use of injectable bioscaffols containing chemo-attractants promoting cell survival, differentiation and axo-dendritic outgrowth of dopaminergic cells. With the so called “bridging” transplantation technology an artificial axon pathway between the substantia nigra and the striatum with targeted nigro-striatal re-innervation should be generated. Thereby, the central dopaminergic nigro-striatal pathway would be reconstructed enabling a fully integration of grafted neurons into the basal ganglia circuitries.
The main focus of the thesis was to explore the influence of bioscaffolds on cell survival and morphology of dopaminergic neurons in vitro. The investigations included isolation of primary fetal mesencephalic cells and fetal mesencephalic neural stem cells (NSCs) from embryonic (E14) mouse brain and their culture on ECM compounds and starPEG-heparin hydrogels. Initial characterizations of the gels showed separate as well as simultaneous immobilization and release of growth factors demonstrating that hydrogels could serve as an efficient storage and delivery system for growth factors. The axo-dendritic outgrowth of dopaminergic cells including primary branching, total branching and neurite elongation; cell survival studies; cell type analysis and cell migration were analyzed by immunostaining.
Both cell sources showed distinct growth properties depending on the stiffness of the gel material and the presence of biomolecules with increased cell survival by the presence of RGD and FGF-2 in the hydrogel independent of network characteristic. Moreover, the presence of RGD on hydrogels was found to initiate differentiation of NSCs, whereas FGF-2 bound to hydrogels was shown to promote the viability of undifferentiated cells. Additionally, survival and axo-dendritic outgrowth of dopaminergic cells were observed to be affected by the gel properties: RGD or FGF-2 modification of hydrogels with intermediate network density showed the best results for dopaminergic growth. With the addition of GDNF to hydrogels the total amount of cells decreased strongly by an equal quantity of dead cells compared to FGF-2 bound hydrogels. Furthermore, differential effects were found for the survival of different brain cells depending on the growth factor which is loaded. GDNF was found to increase the survival of astrocytes, whereas FGF-2 bound to gels stimulated the viability of oligodendrocyte precursor cells. No differential effects were found for the survival of NSCs and mature neuronal cells on GDNF or FGF-2 bound gels. By showing the penetration of primary fetal mesencephalic cells expressing MMPs as endogenous endopeptidases into MMP-cleavable hydrogels, the potential biodegradability of the starPEG-heparin hydrogels was demonstrated.
Together the findings provide the in vitro proof-of-principle data for combining dopaminergic neurons or predopaminergic NSCs with biomaterials for reconstructing the central dopaminergic nigro-striatal pathway by the “bridging” transplantation strategy as an alternative transplantation approach in PD. Further studies should focus on three-dimensional cell culture studies using starPEG-heparin hydrogels with cleavable peptide sequences and their functionalization with gradients of axon guidance molecules to selectively promote dopaminergic outgrowth.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:14-qucosa-112900 |
| Date | 14 May 2013 |
| Creators | Schurig, Katja |
| Contributors | Technische Universität Dresden, Fakultät Mathematik und Naturwissenschaften, Prof. Dr. Carsten Werner, Prof. Dr. Alexander Storch, Prof. Dr. Carsten Werner, Prof. Dr. Alexander Storch |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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