Development of tissue engineering strategies for supporting regeneration after injuries of the nervous system

This thesis deals with the preparation of artificial guiding structures for supporting regeneration after nerve injuries. Injuries of the nervous system can have serious consequences such as the loss of motoric and sensoric function. As existing treatment methods for the peripheral nervous system include several unsatisfying deficits and due to the absence of efficient treatment for injuries of the central nervous system, the development of an artificial scaffold for supporting nerve regeneration is highly desirable. Chemical composition, mechanical properties and oriented structures for aligned guidance of recovering nerves are important issues for such scaffolds. The method of electrospinning was applied to produce highly oriented fibres from different materials with low densities to achieve oriented cell growth and aligned extensions from a variety of neuronal and non-neuronal cells. For in vitro experiments fibres were collected onto glass cover slips which were surface functionalised with a thin hydrogel layer based on star-shaped poly(ethylene glycol)-stat-poly(propylene glycol) (sPEG). This layer demonstrated non-adhesive properties towards cells and proteins which enabled controlled investigation of the effects actuated by the fibres. Three different polymeric materials were developed and electrospun into fibres: Firstly, blends of poly(caprolactone) (PCL) and the extracellular matrix protein collagen with different ratios were prepared. High quality oriented suspended fibres were obtained at a PCL-collagen ratio of 3:1. Fibre diameters were between 0.4-2.4 µm. Several analytical methods demonstrated the existence of collagen at the fibre surface. Experiments with cells important in the nervous system including neurons, Schwann cells, astrocytes, oligodendroyctes and olfactory ensheathing cells showed an affinity of the cells to the collagen containing fibres. Neurites were longer and cells migrated further on these fibres, in contrast to pure PCL fibres. Secondly, amphiphilic poly(ethylene oxide)-b-poly(caprolactone) (PEO-b-PCL) block copolymers with molecular weights between 34000 to 76000 g/mol and different block length ratios were prepared. High quality oriented fibres could easily be obtained with high molecular weight polymers. Surface analysis demonstrated a hydrophilic character and reduced protein adsorption. Additionally, a GRGDS-functionalised block copolymer was prepared. Analytics verified the success of each preparation step. The functionalisation with the peptide sequence should allow cellular adhesion and guidance of neuronal cells while simultaneously inhibiting undesired protein adsorption. This makes the polymers suitable as implantable biomaterials with controlled material/body interactions. However, impurities and degradation processes during the synthesis prevented oriented electrospinning and therefore cell experiments were not performed. Thirdly, blends of sPEG, PCL and poly(caprolactone-diol) (PCL-diol) were prepared and functionalised either with the peptide sequence GRGDS or the protein fibronectin. Oriented suspended fibres could be electrospun. A reduced protein affinity the blends demonstrated the existence of sPEG at the fibre surface. Unfunctionalised PCL/sPEG fibres showed minimal cellular migration and axonal outgrowth while in contrast, the biologically functionalised fibres increased cellular response significantly. Oriented suspended and biological functionalised electrospun fibres encouraged cell adhesion and migration and induced alignment of cells and extensions according to the fibre direction. Thus, the fibres developed in this thesis are a promising tool for the preparation of scaffolds for neuronal regeneration after nerve injuries