RESUMO
Due to pronounced species differences, hepatotoxicity of new drugs often cannot be detected in animal studies. Alternatively, human hepatocytes could be used, but there are some limitations. The cells are not always available on demand or in sufficient amounts, so far there has been only limited success to allow the transport of freshly isolated hepatocytes without massive loss of function or their cultivation for a long time. Since it is well accepted that the cultivation of hepatocytes in 3D is related to an improved function, we here tested the Optimaix-3D Scaffold from Matricel for the transport and cultivation of hepatocytes. After characterization of the scaffold, we shipped cells on the scaffold and/or cultivated them over 10 days. With the evaluation of hepatocyte functions such as urea production, albumin synthesis, and CYP activity, we showed that the metabolic activity of the cells on the scaffold remained nearly constant over the culture time whereas a significant decrease in metabolic activity occurred in 2D cultures. In addition, we demonstrated that significantly fewer cells were lost during transport. In summary, the collagen-based scaffold allows the transport and cultivation of hepatocytes without loss of function over 10 days.
RESUMO
The formation of cystic cavitation following severe spinal cord injury (SCI) constitutes one of the major barriers to successful axonal regeneration and tissue repair. The development of bioengineered scaffolds that assist in the bridging of such lesion-induced gaps may contribute to the formulation of combination strategies aimed at promoting functional tissue repair. Our previous in vitro investigations have demonstrated the directed axon regeneration and glial migration supporting properties of microstructured collagen scaffold that had been engineered to possess mechanical properties similar to those of spinal cord tissues. Here, the effect of implanting the longitudinally orientated scaffold into unilateral resection injuries (2mm long) of the mid-cervical lateral funiculus of adult rats has been investigated using behavioural and correlative morphological techniques. The resection injuries caused an immediate and long lasting (up to 12 weeks post injury) deficit of food pellet retrieval by the ipsilateral forepaw. Implantation of the orientated collagen scaffold promoted a significant improvement in pellet retrieval by the ipsilateral forepaw at 6 weeks which continued to improve up to 12 weeks post injury. In contrast, implantation of a non-orientated gelatine scaffold did not result in significant functional improvement. Surprisingly, the improved motor performance was not correlated with the regeneration of lesioned axons through the implanted scaffold. This observation supports the notion that biomaterials may support functional recovery by mechanisms other than simple bridging of the lesion site, such as the local sprouting of injured, or even non-injured fibres.
Assuntos
Regeneração Tecidual Guiada , Traumatismos da Medula Espinal/terapia , Alicerces Teciduais , Animais , Axônios/patologia , Colágeno Tipo I/uso terapêutico , Feminino , Atividade Motora , Ratos , Ratos Endogâmicos Lew , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/cirurgia , Regeneração da Medula EspinalRESUMO
The use of bioengineered nerve guides as alternatives for autologous nerve transplantation (ANT) is a promising strategy for the repair of peripheral nerve defects. In the present investigation, we present a collagen-based micro-structured nerve guide (Perimaix) for the repair of 2 cm rat sciatic nerve defects. Perimaix is an open-porous biodegradable nerve guide containing continuous, longitudinally orientated channels for orientated nerve growth. The effects of these nerve guides on axon regeneration by six weeks after implantation have been compared with those of ANT. Investigation of the regenerated sciatic nerve indicated that Perimaix strongly supported directed axon regeneration. When seeded with cultivated rat Schwann cells (SC), the Perimaix nerve guide was found to be almost as supportive of axon regeneration as ANT. The use of SC from transgenic green-fluorescent-protein (GFP) rats allowed us to detect the viability of donor SC at 1 week and 6 weeks after transplantation. The GFP-positive SC were aligned in a columnar fashion within the longitudinally orientated micro-channels. This cellular arrangement was not only observed prior to implantation, but also at one week and 6 weeks after implantation. It may be concluded that Perimaix nerve guides hold great promise for the repair of peripheral nerve defects.
Assuntos
Axônios/efeitos dos fármacos , Colágeno/farmacologia , Regeneração Tecidual Guiada/métodos , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/patologia , Alicerces Teciduais/química , Animais , Axônios/patologia , Axônios/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Porosidade/efeitos dos fármacos , Implantação de Prótese , Ratos , Ratos Endogâmicos Lew , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/transplanteRESUMO
Traumatic injury to the nervous system induces functional deficits as a result of axonal destruction and the formation of scar tissue, cystic cavitation, and physical gaps. Bioengineering bridging materials should ideally act as cell carriers for the implantation of axon growth-promoting glia, as well as supporting integration with host cell types. Here, we describe the cytocompatibility of a novel, micro-structured porcine collagen scaffold containing densely packed and highly orientated channels that, in three-dimensional (3D) tissue culture, supports attachment, proliferation, aligned process extension, and directed migration by populations of glial cells (olfactory nerve ensheathing cells and astrocytes) and orientated axonal growth by neurons (differentiated human SH-SY5Y neuroblastoma cell line). The seeded glia required several weeks to penetrate deeply into the highly porous scaffold, where they adopted an orientated morphology similar to that displayed in simple 2D cultures. The direct interaction between SH-SY5Y-derived nerve fibers and the collagen scaffold also resulted in highly orientated axonal growth. It is likely that biocompatible scaffolds that are capable of promoting glial cell attachment, migration, and highly orientated process outgrowth will be important for future repair strategies for traumatically injured nervous tissues.
Assuntos
Materiais Biocompatíveis/farmacologia , Colágeno/farmacologia , Teste de Materiais , Tecido Nervoso/citologia , Tecido Nervoso/efeitos dos fármacos , Alicerces Teciduais , Cicatrização/efeitos dos fármacos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Colágeno/ultraestrutura , Reagentes de Ligações Cruzadas/farmacologia , Imunofluorescência , Indóis , Neurônios/citologia , Neurônios/ultraestrutura , Peroxidase/metabolismo , Ratos , Esterilização , Sus scrofaRESUMO
Therapeutic benefits of autologous nerve grafting in repair of peripheral nerve lesions have not been reached using any alternative nerve guide. Nevertheless, issues of co-morbidity and limited availability of donor nerves urgently ask for a need of bioartificial nerve guides which could either replace or complement autologous nerve grafts. It is increasingly appreciated that optimal nerve guides comprise both physical and molecular cues in support of peripheral axon regeneration. Now, we present a collagen-based microstructured 3D nerve guide containing numerous longitudinal guidance channels with dimensions resembling natural endoneurial tubes. Moreover, these nerve guides could be functionalized by Schwann cell (SC) seeding. Viable SCs did not only adhere to the nerve guide, but also migrated throughout the guidance channels. Of particular importance was the observation that SCs within the guidance channels formed cellular columns reminiscent of "Bands of Büngner", which are crucial structures in the natural process of peripheral nerve regeneration during the Wallerian degeneration. We, therefore, conclude that our orientated 3D nerve guides (decorated with SCs) with their physical and molecular properties may hold great promise in the repair of peripheral nerve lesion and serve as a basis for future experimental regeneration studies.
Assuntos
Regeneração Tecidual Guiada/métodos , Regeneração Nervosa/fisiologia , Nervos Periféricos/fisiologia , Células de Schwann/fisiologia , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Colágeno/química , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Imuno-Histoquímica , Microscopia de Força Atômica , Proteínas de Neurofilamentos/análise , Ratos , Células de Schwann/citologia , Suínos , Alicerces Teciduais , Vimentina/análiseRESUMO
Complete nerve transection (neurotmesis) of the rat sciatic nerve is a well-established animal model. The most frequently used behavioural for evaluation of neurotmesis-induced deficits is the walking track analysis with calculation of the sciatic functional index (SFI). More recently, the static sciatic index (SSI) has been developed, which shows a good correlation with the SFI. However, despite all advantages (high accessibility, easy handling, high accuracy, cost-effectiveness), the SSI is still not widely used. We, therefore, developed a novel programme ("Visual-SSI"), which will be made freely available for the assessment of the SSI. As gold-standard for the treatment of neurotmesis-induced nerve gaps, autologous nerve transplantation studies in the rat sciatic nerve model (n=16 [6 weeks], n=8 [12 weeks]) were carried out to test the effectiveness and feasibility of the Visual-SSI software. We observed a significant recovery starting from the pre-operative condition over the 3rd, 6th, 9th weeks until the 12th week after surgery (p<0.05). Theoretically, the SSI can be calculated from both rearing and normal standing position of the rats and we investigated whether the SSI is affected differentially by these positions. We observed no significant differences between animals in a rearing and normal standing stance (p>0.05). The present method combines efficiency (simplicity of use, rapid and economical setup) with accurate and precise quantification of the functional regeneration in the sciatic nerve lesion model of the rat.
Assuntos
Algoritmos , Regeneração Nervosa/fisiologia , Nervo Isquiático/fisiologia , Nervo Isquiático/transplante , Animais , Interpretação Estatística de Dados , Membro Posterior/inervação , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/lesões , SoftwareRESUMO
The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel, 3-dimensional (3D), highly oriented, cross-linked porcine collagen scaffold to promote directed axonal growth has been studied. Collagen nerve guides with longitudinal guidance channels were manufactured using a series of chemical and mechanical treatments with a patented unidirectional freezing process, followed by freeze-drying (pore sizes 20-50 microm). Hemisected rat dorsal root ganglia (DRG) were positioned such that neural and non-neural elements could migrate into the collagen scaffold. After 21 days, S100-positive Schwann cells (SCs) migrated into the scaffold and aligned within the guidance channels in a columnar fashion, resembling "Bands of Büngner." Neurofilament-positive axons (mean length +/- SD 756 microm +/- 318 microm, maximum 1496 microm) from DRG neurons entered the scaffold where the growth within the guidance channels was closely associated with the oriented SCs. This study confirmed the importance of SCs in the regeneration process (neurotrophic theory). The alignment of SCs within the guidance channels supported directional axonal growth (contact guidance theory). The microstructural properties of the scaffold (open, porous, longitudinal pore channels) and the in vitro data after DRG loading (axonal regeneration along migrated and columnar-aligned SCs resembling "Band of Büngner") suggest that this novel oriented 3D collagen scaffold serves as a basis for future experimental regeneration studies in the PNS.
Assuntos
Axônios/ultraestrutura , Colágeno/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/transplante , Regeneração Tecidual Guiada/métodos , Regeneração Nervosa/fisiologia , Engenharia Tecidual/métodos , Animais , Axônios/fisiologia , Técnicas de Cultura de Células/métodos , Crescimento Celular , Células Cultivadas , Gânglios Espinais/fisiologia , Regeneração Tecidual Guiada/instrumentação , Ratos , Ratos Endogâmicos LewRESUMO
Artificial organs within the blood stream are generally associated with flow-induced blood damage, particularly hemolysis of red blood cells. These damaging effects are known to be dependent on shear forces and exposure times. The determination of a correlation between these flow-dependent properties and actual hemolysis is the subject of this study. For this purpose, a Couette device has been developed. A fluid seal based on fluorocarbon is used to separate blood from secondary external damage effects. The shear rate within the gap is controlled by the rotational speed of the inner cylinder, and the exposure time by the amount of blood that is axially pumped through the device per given time. Blood damage is quantified by the index of hemolysis (IH), which is calculated from photometric plasma hemoglobin measurements. Experiments are conducted at exposure times from texp=25 - 1250 ms and shear rates ranging from tau=30 up to 450 Pa ensuring Taylor-vortex free flow characteristics. Blood damage is remarkably low over a broad range of shear rates and exposure times. However, a significant increase in blood damage can be observed for shear stresses of tau>or= 425 Pa and exposure times of texp>or= 620 ms. Maximum hemolysis within the investigated range is IH=3.5%. The results indicate generally lower blood damage than reported in earlier studies with comparable devices, and the measurements clearly indicate a rather abrupt (i.e., critical levels of shear stresses and exposure times) than gradual increase in hemolysis, at least for the investigated range of shear rates and exposure times.