Nerve Tissue Bioreactor

Design of a Nerve Tissue Bioreactor

Nerve injuries complicate successful rehabilitation more than any other form of trauma because of loss of protective sensibility and tactile discrimination, denervation atrophy of muscles, and pain syndromes. In many cases, a patient can recover from these injuries by the regeneration of their own nervous system. Several techniques have been developed to enhance the regenerative properties of the peripheral nervous system, the system that sends allows communication between ones brain and spinal column (the central nervous system) and their environment. Currently techniques include physical reattachment of the two ends of the severed nerve or bridging the gap with donor nerve tissue or with a biodegradable tubular conduit.

These techniques are very promising and can be applied to injuries in the central nervous system also where regeneration is more difficult. For surgical repair of nerve tissue, the two ends of the damaged nerve are typically sutured together. For more sever injuries, the ends cannot be connected without stretching the nerve tissue and causing further damage. In these cases, nerve tissue from a donor or another part of the patient is used to bridge the gap. The primary drawbacks are the reliance on suitable donors and the denervation of part of the patient's own tissue. To overcome these problems, a novel bioreactor is being developed to provide an ongoing source of nerve tissue for surgical repair (Figure 1). This bioreactor utilizes microfabrication techniques to create and environment that closely mimics what exists during embryonic development of the nervous system. Microfabrication techniques will be used to create a two-dimensional patterned surface containing "islands" of Schwann cells (Figure 2). Neurons will then be co-cultured with the Schwann cells to evaluate pattern parameters. A diffusion model is also being developed to describe the concentration profiles of growth factors in this environment. Growth factors will form concentration gradients as they diffuse away from the Schwann cells. The neurons react to this chemotactic signal to by extending their axon toward the Schwann cells. Experiments are planned to study the effect of the steepness of these gradients as well as the total concentration of growth factors.

Figure 1: Schematic of nerve tissue bioreactor.A nerve cell source is placed at one end of the Microfabricated insert held inside the bioreactor. The nerve tissue will extend across the length of the bioreactor. Electrodes spaced along the length of the reactor will be used to record electrical signal as well as provide stimulus.

Other tissues (muscle or skin) can be placed at the other end of the bioreactor to provide a target for the growing axons. This patterned system will be placed into the nerve tissue bioreactor and tissue will be grown for an extended period. The tissue that is grown within the bioreactor will be tested for structural integrity, rate of growth, number of axons, fasciculation (bundling of axon fibers), and the degree of myelination. Comparisons will be made with native nerve tissue. Eventually, the tissue will be examined for its ability to assist in the regeneration of nerve tissue. Besides, nerve repair, the bioreactor can also be used as an in vitro model for nerve regeneration as well as a system for nerve/muscle and nerve/skin combination tissues.

Figure 2: Micropatterning of Schwann cells. Photolithographic techniques are used to modify the surface of the glass substrate in order to create regions that the Schwann cells will preferentially adhere. The size and spacing of these "islands" will be optimized in order to promote axon growth while controlling branching. The pattern will guide the regenerating neurons to the target tissue.

Other experimental work is planned to further enhance the extension rate of the axons. Because the growth factors will be stimulating the increasing the extension rate of the axons, bottlenecks may form in other parts of the mechanism. Because neurons are very large cells, they need to produce enough cell membrane to allow the axon to extend. If cell membrane production becomes a limiting factor, the axon may shrink in diameter and become more fragile. This shrinkage has been observed in several experiments using bioartificial nerve grafts to enhance regeneration. By providing, lipids and other cell membrane precursors this problem bottleneck can be eliminated. Another project involves the effect of fluid shear stress on the alignment and extension of axons. Fluid flow can lead to an increase in shear stresses that may encourage the axons to extend in the direction of flow. Modeling of the flow characteristics will help in establishing the magnitude of these forces.

Specific Projects

• Construct the basic bioreactor system which can be adapted to include the microelectrodes, and microfabricated inserts

• Determine the impact of various lipids and cell membrane precursors under high nerve growth conditions

• Determine the effect of concentration gradients of various growth factors of the direction and speed of axon extension

• Evaluate the effect of shear stresses on the direction and growth of the axons.