Nerve guidance conduit | EPFL Graph Search

A nerve guidance conduit (also referred to as an artificial nerve conduit or artificial nerve graft, as opposed to an autograft) is an artificial means of guiding axonal regrowth to facilitate nerve regeneration and is one of several clinical treatments for nerve injuries. When direct suturing of the two stumps of a severed nerve cannot be accomplished without tension, the standard clinical treatment for peripheral nerve injuries is autologous nerve grafting. Due to the limited availability of donor tissue and functional recovery in autologous nerve grafting, neural tissue engineering research has focused on the development of bioartificial nerve guidance conduits as an alternative treatment, especially for large defects. Similar techniques are also being explored for nerve repair in the spinal cord but nerve regeneration in the central nervous system poses a greater challenge because its axons do not regenerate appreciably in their native environment. The creation of artificial cond

Interactions of Peripheral Nerve Cells with Soft Micropillar Interfaces

Cédric Xavier Paulou

Regenerative peripheral nerve interfaces are particularly invasive implant devices. They rely on the growth of axons from transected nerve stumps through electrode-bearing structures in order to establish highly efficient communication channels with the nervous system. The functioning and lifetime of these devices is challenged by the host response from the nervous tissue, which complicates their use in long-term applications. The elicited foreign body reaction is characterized by chronic inflammation and excessive deposition of collagen. The difference in mechanical properties between implant material and nervous tissue, which are among the softest in the body, is thought to be an important contributor to these adverse effects. With the motivation of improving the bio-integration of implanted nerve interfaces, this thesis evaluate micropillar arrays as a texture to modulate the behaviour of peripheral nerve cells on polydimethylsiloxane (PDMS) silicone surfaces. Thanks to their flexibility, micropillars can reduce the effective stiffness perceived by cells, while providing topographic cues at the microscopic level. Importantly, these mechanical and topographic cues can be easily tailored by modification of their geometrical dimensions. Micropillar arrays with varying dimensions were fabricated by soft lithography process. Their mechanical behaviour under bending was simulated by finite element method (FEM) analysis to approximate their spring constants. The softest micropillar configuration had an effective surface stiffness of 0.7 kPa, representing a reduction of 3 orders of magnitude compared to bulk PDMS. The effect of micropillar diameter (from 1.2 to 4.2 um), modulating the surface stiffness, and the effect of interpillar spacing (from 1.0 to 7.6 um), modifying micropillars density, were investigated in vitro. Dissociated cell cultures from dorsal root ganglion (DRG), comprising neurons and glial cells, were probed for different parameters after 7 days of incubation. While neurons spread and established neurite networks on all configurations, fewer glial cells were found on the softest and lowest density of micropillars. Neurites explored this pseudo-3D environment and preferentially anchored to the top of pillar shafts, well above the bottom surface. The matrix of pillars provided physical cues for growing neurites, which strongly aligned with the densest arrays. The morphology of neuronal bodies was affected by the most flexible pillars, which induced smaller and rounder somas. Macrophages and adipose-derived stem cells showed significantly increased attachment to micropillar topographies compared to flat surface. The effect of micropillars was investigated on nerve regeneration in vivo. Nerve guidance conduits patterned with micropillars on their lumen surface were sutured to the transected sciatic nerve of rats. While the axonal regeneration was slightly affected by the micropillar texture, macrophages were massively recruited to the micropillar surface in comparison to the flat control. Their phenotype was also affected, with predominantly classically activated macrophage in contact with the micropillars. Altogether, these results demonstrate the possibility to modulate the behaviour of a variety of cell types in vitro and in vivo, through the patterning of micropillars on the surface. This approach is amenable to implementation on silicone neural implants for mitigating tissue-material interactions.

EPFL2015

Micro-engineering the Cerebral Cortical Cell Niche

Anja Kunze

A major problem in traditional cell culture methods, such as Petri dishes and culture flasks, is the very simplified artificial environment around the cells. Traditional cell culture methods lack features of the native cell niche, such as gradients and cell organization. This lack probably explains why pharmaceutics against the neurodegenerative Alzheimer's disease successfully stop the propagation of the disease in the Petri dish, but fail so far in clinical trials. This thesis intends to improve cell culture methods for neuroscience research related to neural developmental questions and neurodegenerative diseases. As the cortex is the main part in our brain, related to memory, emotions and perception, this thesis does focus on cell culture tools and protocols for primary cortical neurons. Currently, dissociated neurons are cultured in pure or co-culture of neural and non-neural cells, but structuring elements and controlled gradient formation is missing. The first part of this thesis discusses different studies that implicate environmental components for neural cells in their native neural cell niche. We will establish a generic neural cell niche, which consists of different neural and non-neural cells, a structured 3D environment, molecular gradients and oriented neurite networks, in a nutshell. Additionally, a simplified model of the generic neural cell niche is introduced that is implemented in a microfluidic base cell culture tool. This novel artificial neural cell niche will provide cell layer structure in 3D and local control of molecular gradients at the microscale. We will use microfluidic technology to integrate missing features in cell culture techniques for primary cortical neurons. The microfluidic device will consist of three parts: (1) a main cell culture channel that is used to organize neural cells in 3D hydrogel layers, side-by-side; (2) parallel perfusion channels to mimic nutrient supply and to control stable gradient formation, (3) interconnecting microchannels, called junction channels, that separate perfusion driven molecular transport from diffusive molecular transport. The perfusion channels are connected to device-incorporated reservoirs that allow maintenance of stable molecular gradients based on perfusion and diffusion without the use of peristaltic or syringe pumps. By injecting cortical neurons entrapped in an agarose-alginate solution in the microfluidic device, we generated 3D micropatterned neural cell layers with stable gradients perpendicular to the layer orientation. We demonstrated neurite outgrowth behavior until three weeks in culture. The application of different cell organization patterns revealed an influence of the pattern on the cell culture response. Using neurotrophic gradients of nerve growth factor (NGF) and nutrient supplements (B27), we showed that neurite guidance and synapse formation followed synergistic NGF/B27 gradients. We found that the gradient induced effects are very sensitive to changes in the environmental structure, such as the cell layer organization. Using a gradient of a phosphatase inhibitor, okadaic acid, we generated locally diseased states of the protein Tau in both 2D and 3D micropatterned neural cell cultures. The diseased form of Tau, hyper-phosphorylated Tau, is a major hallmark of Alzheimer's disease. For the first time, local formation of hyper-phosphorylated Tau was demonstrated to affect a defined cell population in a compartmentalized 2D or 3D neural cell culture. Our results revealed that the propagation mechanism of this Alzheimer's diseased lesion is determined through the formation of the 2D or 3D environment. We think that this new neural cell culture tool has the potential to answer biological questions related to environmental and structural parameters involved in the formation of the cerebral cortex and in the propagation of neurodegenerative diseases. Future studies in modern neuroscience research can now better investigate the effect of gradient parameters such as the slope, average concentration or gradient profile on cell culture and disease propagation in a controlled manner. Furthermore, the influence of cell patterns in 2D and 3D is addressed with the ability to modify cell position, density and type at the microscale. The local formation of neurodegenerative disease lesions in a micropatterned neural cell culture is generic, which makes the integration of other neurodegenerative disease models, such as Parkinson's disease, Amyotrophic lateral sclerosis or Huntington's disease, possible.

EPFL2012