Axon repair: surgical application at a subcellular scale.

Injury to the nervous system is a common occurrence after trauma. Severe cases of injury exact a tremendous personal cost and place a significant healthcare burden on society. Unlike some tissues in the body that exhibit self healing, nerve cells that are injured, particularly those in the brain and spinal cord, are incapable of regenerating circuits by themselves to restore neurological function. In recent years, researchers have begun to explore whether micro/nanoscale tools and materials can be used to address this major challenge in neuromedicine. Efforts in this area have proceeded along two lines. One is the development of new nanoscale tissue scaffold materials to act as conduits and stimulate axon regeneration. The other is the use of novel cellular-scale surgical micro/nanodevices designed to perform surgical microsplicing and the functional repair of severed axons. We discuss results generated by these two approaches and hurdles confronting both strategies.

Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls.

As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3 microm thick and spaced 10 microm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electroporation, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.

Microtechnology and nanotechnology in nerve repair.

OBJECTIVE: This review will describe the novel contributions to the field of nerve repair from the emerging disciplines of microtechnology and nanotechnology. METHOD: This broad review will cover the advances described in the literature of the medical and biological fields and the engineering and physical sciences. The authors have also included their own work in this field. DISCUSSION: Microtechnology and nanotechnology are providing two fundamentally different pathways for pursuing nerve repair: (1) microstructured scaffolds to promote regeneration and (2) direct repair by reconnecting axons. In the first instance, many of the traditional techniques for microfabrication of microelectronics have been applied to the development of implantable tissue scaffolds with precisely formed architectures. Combined with nanotechnological capabilities to control their surface chemistries, these tissue constructs have been designed to create a microenvironment within nerve tissue to optimally promote the outgrowth of neurites. With some initial successes in animal models, these next generation tissue scaffolds may provide a marked improvement over traditional nerve grafts in the ability to overcome nerve degenerative processes and to coax nerve regeneration leading to restoration of at least some nerve function. A second, completely different repair strategy aims to directly repair nerves at the microscale by acutely reconnecting severed or damaged axons immediately after injury and potentially forestalling the usual downstream degenerative processes. This strategy will take advantage of the traditional capabilities of microfabrication to create microelectromechanical systems that will serve as ultramicrosurgical tools that can operate at the micron scale and reliably manipulate individual axons without incurring damage. To bring about some restoration of a nerve's function, axon repair will have to be performed repetitively on a large scale and soon after injury. Development work is currently underway to bring about the feasibility of this technique. CONCLUSION: With the emergence of microtechnology and nanotechnology, new methods for repairing nerves are being explored and developed. There have been two fundamental benefits from the technologies of the ultrasmall scale: (1) enhancement of regeneration using new tissue scaffold materials and architecture; (2) direct repair of nerves at the scale of single neurons and axons.

Novel high-resolution micropatterning for neuron culture using polylysine adsorption on a cell repellant, plasma-polymerized background.

The ability to organize individual neurons and their processes in culture provides important benefits to both basic neuroscience research applications and the development of biomedical microdevices. While numerous methods have been used to produce such micropatterning of neurons and cells in general, there has yet been no method to simultaneously provide high-resolution patterns with high compliance of cells to desired patterns and good manufacturability. To develop such a process, this work used a plasma polymerized, nonfouling poly ethylene oxide (PEO)-like film to provide a cell repellant substrate on which cell adhesive micropatterns can be selectively laid down. While the use of plasma polymerized, organic films have been used for cell micropatterning, this process exploits the often-overlooked tendency for the surface of this PEO-like material to adsorb polylysine from aqueous solution while remaining nonfouling with respect to other species, such as bovine serum albumin (BSA) and immunoglobulin G (IgG). When the adsorption of polylysine was enhanced by brief plasma oxidation, which slightly alters the surface chemistry of the material, simple photolithographic liftoff could be used to micropattern stable, cell adhesive areas on an otherwise cell repellant background. We showed that the application of photolithography itself on the PEO-like material did not alter its chemical properties, nor did it result in the erosion of the micropatterned polylysine on its surface. Hippocampal neurons from embryonic mice flourished on these micropatterned substrates and exhibited viability comparable to neurons cultured on polylysine coated glass. Furthermore, the compliance of cell bodies and outgrowing neurites to the micropatterns was nearly perfect. In addition to providing cell adhesive regions, the micropatterned polylysine coating also served as a template mediating the immobilization of other bioactive species such as IgG and laminin. Using this "piggybacking" of laminin on polylysine, we were also able to culture and micropattern retinal ganglion cells (RGC).

In vivo use of a nanoknife for axon microsurgery.

OBJECTIVE: Microfabricated devices with nanoscale features have been proposed as new microinstrumentation for cellular and subcellular surgical procedures, but their effectiveness in vivo has yet to be demonstrated. In this study, we examined the in vivo use of 10 to 100 microm-long nanoknives with cutting edges of 20 nm in radius of curvature during peripheral nerve surgery. METHODS: Peripheral nerves from anesthetized mice were isolated on a rudimentary microplatform with stimulation microelectrodes, and the nanoknives were positioned by a standard micromanipulator. The surgical field was viewed through a research microscope system with brightfield and fluorescence capabilities. RESULTS: Using this assembly, the nanoknife effectively made small, 50 to 100 microm-long incisions in nerve tissue in vivo. This microfabricated device was also robust enough to make repeated incisions to progressively pare down the nerve as documented visually and by the accompanying incremental diminution of evoked motor responses recorded from target muscle. Furthermore, this nanoknife also enabled the surgeon to perform procedures at an unprecedented small scale such as the cutting and isolation of a small segment from a single constituent axon in a peripheral nerve in vivo. Lastly, the nanoknife material (silicon nitride) did not elicit any acute neurotoxicity as evidenced by the robust growth of axons and neurons on this material in vitro. CONCLUSION: Together, these demonstrations support the concept that microdevices deployed in a neurosurgical environment in vivo can enable novel procedures at an unprecedented small scale. These devices are potentially the vanguard of a new family of microscale instrumentation that can extend surgical procedures down to the cellular scale and beyond.

Microtechnology in medicine: the emergence of surgical microdevices.

With the emergence of technologies to fabricate and mass-produce microscale tools and micromachines, microsurgery stands to potentially benefit through the development of a fundamentally new class of instruments. These new instruments may provide the surgeon with access to the smallest reaches of the body and perform operations that are currently not possible with manually operated tools. These new devices can be variably constructed and configured based on a wide range of design possibilities and can be built to serve many different fundamental surgical functions requiring the manipulation and handling of small tissues and structures, including grasping, cutting, and monitoring. With these functionalities also comes a high degree of integration, allowing tools and space to be used efficiently. Adapted from the techniques of the microelectronics industry, the fabrication methods and materials produce structures that are mechanically strong and easy to reproduce on a large scale. Well-developed design and physical modeling tools mean that the process of instrument development and validation can be streamlined. Along with these new instruments comes the need to provide automated interfaces to effectively translate human operator intentions into the appropriate actuation and motion of these devices. These interfaces must include the capability to scale down human motions to the range of microns. Most likely, the operation of these new microsurgical devices will resemble the control schemes developed for robotic surgery. The control schemes will provide accurate motions while minimizing the chances of damaging tools or unnecessarily injuring tissues. Naturally, these new tools and surgical schemes will require a transition from the conventional paradigm. However, with new surgical capabilities that may allow direct intervention into the inner workings of a cell, MEMS and nanotechnology-based tools may become a crucial part of the arsenal for the next generation of surgeons. Invariably, future developments of this new class of instruments will depend in large part on needs identified by the surgeon and an understanding of the enabling properties of microtechnology and nanotechnology. Thus, recognition of the vast potentials of this new technology among clinicians will greatly help to accelerate the development and integration of new microdevices and novel procedures that address disease and injury with unprecedented precision.

Isolation of neuronal substructures and precise neural microdissection using a nanocutting device.

We describe a set of microfabricated nanocutting devices with a cutting edge of less than 20 nm radius of curvature that enables high precision microdissection and subcellular isolation of neuronal structures. With these devices, it is possible to isolate functional substructures from neurons in culture such as segments of axons and dendrites, dendritic spines and Nodes of Ranvier. By fine-tuning the mechanical compliance of these devices, they can also act as alternatives to costly laser capture microdissection workstations for harvesting specific neuronal populations from tissue sections for analysis. The small size of the device (1 mm2x100 microm) allows convenient insertion into researcher specific experimental set-ups. Its ease of use and possibility for batch fabrication makes this a highly effective and versatile tool for tissue microdissection and the microanalysis of neuronal function.

Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel.

A basic step in many biological assays is separating and isolating different types of cells from raw samples. To better meet these requirements in microfluidic devices for miniature biomedical analytical systems, an alternative method for separating cells has been devised by mimicking the physiological process of leukocyte recruitment to blood vessel walls: adhesive cell rolling and transient tethering. Reproducing these interactions for cells on surfaces of microstructured fluidic channels can serve to capture and concentrate cells and even to fractionate different cell types from a continuously flowing sample. To demonstrate this principle, two designs for microstructured fluidic channels were fabricated: an array of Square pillars and another with slender, Offset pillars. These structures were coated with E-selectin IgG chimera and the interactions of HL-60 and U-937 cells with these structures were characterized. With inflow of fluidic cell suspensions, the structures were able to efficiently capture and arrest cells directly from the rapid free stream flow. After capture, cells transit through the channel in three phases: cell rolling, cell tethering, and transient re-suspension in free stream flow before re-capture. Under these interactions, captured cells were enriched several hundred-fold from the original concentration. Additionally, among collected cells, the difference in flow-driven, adhesion-mediated cell transit in the Square design suggested that the two cell types could at least be partially fractionated.