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Preclinical evaluation of bio-artificial conduits developed by tissue engineering for peripheral nerve regeneration

Boek - Dissertatie

Peripheral nerves are histologically complex and delicate organs that form an organized network through the body. Histologically, peripheral nerves are composed of a nerve tissue or parenchyma and a variable amount of connective tissue or stroma. The parenchyma is composed of conductive units called peripheral nerve fibers, which are myelinated or unmyelinated. The stroma is composed by three layers, the epineurium, the perineurium and the endoneurium. Peripheral nerves are responsible to establish specific motor, sensory and autonomic pathways between the central nervous system and peripheral targets. Unfortunately, their function could be affected by different pathological conditions, and there is a high incidence of traumatic injuries that could result in physical and psychological consequences for these patients. Currently, nerve transections are generally subjected to direct surgical repair with acceptable recovery. For the case of critical nerve defects, they are bridged by using grafts and conduits obtaining variable results. Nerve autograft is the gold standard technique for critical nerve defects. In the case of conduits, they are relatively efficient in the treatment of short and non-critical nerve defects and currently it is still necessary to find an efficient alternative to the use of nerve autograft in the treatment of critical nerve defects. In this regard, the general aim of this Doctoral Thesis was to generate by tissue engineering three novel 3D bio-artificial tissue-like substitutes by using fibrin-agarose hydrogels (FAH) and adipose-derived mesenchymal stem cells (ADMSCs). For the first and second engineered bio-artificial nerve substitutes, acellular FAH or FAH containing ADMSCs were used as intraluminal fillers of collagen conduits and used to repair 10 mm nerve gap in rats. Furthermore, the regeneration and functional recovery was evaluated by clinical, functional and histological techniques. In the case of the third engineered bio-artificial substitute, a nanostructured FAH bio-artificial substituted was generated and characterized in vitro by using ultrastructural, biomechanical and histological techniques. The in vivo studies revealed that the use of FAH and FAH containing ADMSCs as intraluminal fillers of collagen conduits improved the peripheral nerve regeneration profile as compared to the use of hollow conduits (controls). Clinical and functional analyses revealed that these novel strategies induced a significant reduction of neurotrophic ulcers, neurogenic muscle retraction, and superior values for pinch test of sensory recovery, toe-spread and electromyography. Histological analyses showed a significant enhance of the peripheral nerve regeneration, especially when FAH containing ADMSCs was used. The analysis of the growth associating protein 43 (GAP-43) and neurofilament revealed that both axonal markers were differentially expressed during peripheral nerve regeneration, and both proteins were significantly increased by the use of FAH and specially FAH containing cells. The in vitro studies demonstrated that it is possible to generate biomimetic and biomechanically stable bio-artificial substitutes for peripheral nerve repair by combining nanostructured FAH and human ADMSCs. Biomechanical test revealed that the nanostructuration technique significantly improves the biomechanical and structural properties of these substitutes. Histological analyses showed that the mesenchymal stem cells were able to proliferate, form cell clusters and synthetize different kind of extracellular matrix molecules over the time. Finally, the experimental results obtained in this Doctoral Thesis, demonstrated that it is possible to improve the peripheral nerve regeneration by the incorporation of FAH into collagen conduits and, even more significant results can be obtained by the use of FAH containing ADMSCs. On the other hand, in vitro studies demonstrated that it is possible to generate 3D biomimetic and biologically active nanostructured FAH bio-artificial substituted for peripheral nerve repair by using nanostructured FAH and human ADMSCs. Finally, all these results confirm the suitability of FAH in peripheral nerve repair, especially when it is combined with ADMSCs.
Jaar van publicatie:2017