Tissue engineering with glial cells and a novel biodegradable matrix to promote functional repair following experimental spinal cord injuries [Elektronische Ressource] / vorgelegt Sven Möllers

Sven Stelling-Michaud

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2007
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 61 Mo

Extrait

“Tissue Engineering with Glial Cells and a Novel
Biodegradable Matrix to Promote Functional Repair
Following Experimental Spinal Cord Injuries”





Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-Westfälischen
Technischen Hochschule Aachen zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation
vorgelegt von

Diplom-Biologe Sven Möllers
aus Weert (NL)


Berichter: Herr Priv.-Doz. Ph.D. B.Sc. Gary Brook
Herr Univ.-Prof. Dr.rer.nat. Hermann Wagner


Tag der mündlichen Prüfung: 26. April 2007




Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. 2




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Table of Contents
Table of Contents ...............................................................................................................................3
1. Abstract.......................................................................................................................................7
2. Introduction..............................................................................................................................11
2.1. The spinal cord and its injuries ..........................................................................................11
2.2. Endogenous nervous tissue repair......................................................................................12
2.3. Cellular and molecular mechanism involved in preventing CNS regeneration.................13
2.4. Grafting strategies to enhance CNS tissue repair and behavioural recovery.....................15
2.4.1. Orientated biomaterials as bridging scaffolds............................................................16
2.4.2. Olfactory ensheathing cells........................................................................................17
2.5. Hypotheses tested in this thesis..........................................................................................18
3. Materials and Methods............................................................................................................21
3.1. Cell culture constituents.....................................................................................................21
3.1.1. Tissue culture media and buffers ...............................................................................21
3.1.2. Commercial media.....................................................................................................21
3.1.3. Chemicals and reagents..............................................................................................21
3.1.4. Supplements and growth factors................................................................................22
3.1.5. Kits.............................................................................................................................23
3.1.6. Tissue culture plastics ................................................................................................23
3.1.7. Animals......................................................................................................................23
3.1.8. Source of cell gifts .....................................................................................................24
3.2. Collagen-based materials...................................................................................................24
3.2.1. Non-orientated type-I collagen matrices....................................................................25
3.2.1.1. Matricel “ACI-Maix” membranes .....................................................................25
3.2.2. Orientated three dimensional collagen matrices ........................................................26
3.2.2.1. General 3D collagen matrix lyophilisation / fabrication....................................26
3.2.2.1.1. Helmholtz institute 3D matrices26
3.2.2.1.2. Matricel 3D collagen matrices ........................................................................27
3.3. Cell culture protocols.........................................................................................................28
3.3.1. Tissue culture media ..................................................................................................28
3.3.1.1. ONEC medium...................................................................................................28
3.3.1.2. SCOTTS medium...............................................................................................28
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3.3.2. Adult rat olfactory ensheathing cell culture...............................................................29
3.3.2.1. MiniMACS cell purification ..............................................................................30
3.3.3. Post-natal rat olfactory ensheathing cell culture........................................................30
3.3.4. Post-mortem human olfactor.............................................30
3.3.5. Post-natal rat cerebral astrocyte cultures ...................................................................30
3.3.6. Human astrocyte cell line cultures.............................................................................31
3.3.7. Adult rat Schwann cell cultures .................................................................................31
3.3.8. Adult rat dorsal root ganglion cultures ......................................................................31
3.3.9. Cryo-storage...............................................................................................................31
3.4. In vitro collagen scaffold investigations ............................................................................32
3.4.1. Unorientated ACI-Maix-membranes.........................................................................32
3.4.2. Orientated collagen matrices......................................................................................33
3.4.2.1. Helmholtz Institute matrices ..............................................................................33
3.4.2.2. Matricel matrices................................................................................................33
3.4.2.2.1. Rheometry.......................................................................................................34
3.5. Cytocompatibility tests of Matricel matrices.....................................................................35
3.5.1. Cell migration............................................................................................................35
3.5.2. Cell proliferation (CyQUANT assay)........................................................................35
3.5.3. Matrix - dorsal root ganglion preparation..................................................................36
3.6. Animal surgery...................................................................................................................36
3.6.1. Adult rat spinal cord laminectomy.............................................................................36
3.6.1.1. Scaffold implantation.........................................................................................38
3.6.2. Anterograde labelling.................................................................................................39
3.6.3. Perfusion of animals...................................................................................................40
3.6.4. Slide preparation........................................................................................................40
3.6.5. Immunocytochemical staining...................................................................................40
3.6.5.1. Immunofluorescence..........................................................................................40
3.6.5.2. Immunoperoxidase staining...............................................................................41
3.6.5.3. BDA staining.....................................................................................................41
3.6.5.4. Antibodies..........................................................................................................41
3.6.6. Immunohistochemistry: statistical data acquisition ...................................................42
3.6.6.1. ED1 (macrophage response) ..............................................................................42
3.6.6.2. GFAP (astrocytic process length and number) ..................................................43
3.6.6.3. Nf200 (axonal response) ....................................................................................43
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3.6.7. Scanning electron microscopy ...................................................................................44
3.7. Behavioural analyses..........................................................................................................44
3.7.1. Staircase test...............................................................................................................45
3.8. Statistical Analysis.............................................................................................................45
4. Results .......................................................................................................................................47