| Summary: |
Recent advances in nerve tissue engineering have greatly promoted the generation of nerve conduits, which may be implanted empty, or may be filled with growth factors, cells or fibres. A multidisciplinary team, including veterinaries, medical doctors like neurologists and surgeons through Experimental Surgery, has a crucial role in the development of these biomaterials and in testing the surgical techniques that involve their application, always considering animal welfare. The purpose of this study is testing two different biomaterials developed by this research group, used for tube-guides, PLGA with a novel proportion (90:10) of the two polymers, poly(L-lactide):poly(glycolide) and chitosan, in promoting nerve regeneration of the rat sciatic nerve across a 10 mm-gap (neurotmesis) or in a 3 mm axonotmesis lesion. Both type of tube-guides will be tested in vivo, also covered by a cellular system obtained from autologous in vitro differentiated stem cells collected by bone marrow aspiration (Kramer et al., 1999; Archer et al., 1981). This neural cells differentiated from rMSCs, are able to produce growth factors in the local of the nerve injury, during the necessary healing period. Since this process of differentiation implies the inhibition of the cell growth and mitosis, the transferred cells must be able to survive during the nerve's healing period. The best period during the in vitro processing, of the neural cells, what concerns survival and grade of differentiation, will be correlated with the [Ca2+]i, measured by epifluorescence technique, using the fluorescence probe Fura-2-AM ester (Rodrigues et al., 2005a; Rodrigues et al., 2005b). Ca2+ serves as an important intracellular signal for cellular processes such as growth and differentiation. It is also known to be toxic to cells and is involved in the triggering of events leading to excitotoxic cell death in neurons. The emitted fluorescence intensities at 510 nm are recorded at 340 / 380 nm excitation  |
Summary
Recent advances in nerve tissue engineering have greatly promoted the generation of nerve conduits, which may be implanted empty, or may be filled with growth factors, cells or fibres. A multidisciplinary team, including veterinaries, medical doctors like neurologists and surgeons through Experimental Surgery, has a crucial role in the development of these biomaterials and in testing the surgical techniques that involve their application, always considering animal welfare. The purpose of this study is testing two different biomaterials developed by this research group, used for tube-guides, PLGA with a novel proportion (90:10) of the two polymers, poly(L-lactide):poly(glycolide) and chitosan, in promoting nerve regeneration of the rat sciatic nerve across a 10 mm-gap (neurotmesis) or in a 3 mm axonotmesis lesion. Both type of tube-guides will be tested in vivo, also covered by a cellular system obtained from autologous in vitro differentiated stem cells collected by bone marrow aspiration (Kramer et al., 1999; Archer et al., 1981). This neural cells differentiated from rMSCs, are able to produce growth factors in the local of the nerve injury, during the necessary healing period. Since this process of differentiation implies the inhibition of the cell growth and mitosis, the transferred cells must be able to survive during the nerve's healing period. The best period during the in vitro processing, of the neural cells, what concerns survival and grade of differentiation, will be correlated with the [Ca2+]i, measured by epifluorescence technique, using the fluorescence probe Fura-2-AM ester (Rodrigues et al., 2005a; Rodrigues et al., 2005b). Ca2+ serves as an important intracellular signal for cellular processes such as growth and differentiation. It is also known to be toxic to cells and is involved in the triggering of events leading to excitotoxic cell death in neurons. The emitted fluorescence intensities at 510 nm are recorded at 340 / 380 nm excitation wavelengths. [Ca2+]i is estimated from the ratio equation described by Grynkiewiez et al. (1985). Groups of 6 adult male Sasco Sprague rats weighing 300-350 g will be used. Under general anaesthesia the sciatic nerve unilaterally is exposed. After nerve mobilization it is performed a transaction injury, just above the terminal nerve ramification (neurotmesis). For the crush injury, a non-serrated clamp exerting a force of 54 N, is used for a period of 30 seconds to create a 3 mm long crush injury, 10 mm above the bifurcation (Varejão et al., 2003). For reconstruction, the two types of biodegradable tubes will be used, for a nerve gap of 10 mm or to involve the axonotmesis lesion area. Opposite leg and sciatic nerve is not operated upon and serves as control. The aim is to move even further, considering that the success in the repair of peripheral nerve insults depends not just in bridging the sectioned ends of the nerve but also in optimizing the reinnervation of the target tissues. In this respect, new surgical techniques will be combined with innovative rehabilitation strategies aimed at shortening the time necessary for the healing nerves to bridge the gap and also to improve the reinnervation of affected nerve target areas. One such strategy is walking/running in motorized treadmills imposing a diary exercise in 6 groups of animals. Motor functional recovery after the sciatic nerve reconstruction will be assessed serially using video recording of the gait for biomechanical analysis, by measuring extensor postural thrust (EPT), sciatic functional index (SFI) and sciatic functional index under static conditions (SSI), every 2 weeks. The sensitive recovery will be tested by the withdrawal reflex latency (WRL). The repaired nerves are processed for light microscope analysis, imunohistochemistry and confocal microscopy and histomosphometric studies. Electrophysiological measurements will be performed under general anaesthesia just before the euthanasia of the animals. |