Peripheral nerve regeneration monitoring using multilayer microchannel scaffolds

Over 200,000 Americans have peripheral nerve injuries annually that result in a loss of function and a compromised quality of life. Of these, a significant percent involves unsuccessful repair of peripheral nerve gaps that occur due to traumatic limb injury or collateral damage to peripheral nerves during tumor resection. The clinical gold standard to repair a nerve gap is to use sural nerve autografts. However, autografts are not ideal because of the need for secondary surgery to source the nerve, loss of function at the donor site, lack of source nerve in the event of diabetic neuropathies, neuroma formation, and the need for multiple grafts to bridge nerves. An alternative to autografting that has proved to have significantly less risks and sacrifices is a nerve conduit. While there are some nerve conduits approved for clinical applications (Pabari et al., 2010; Giusti et al., 2012), commercial nerve conduits for nerve repair are usually composed of type I collagen or biodegradable polymers, such that the conduit will degrade once the nerve has healed. Although possible complication from foreign materials is not negligible, nerve conduits have had success in bridging nerve gaps and restoring functionality to limbs. Unlike autografting, it does not require the sacrifice of the donor sural nerve. In instances where nerve injury takes the form of long nerve gaps, the nerve regeneration, even with conduit supports, is insufficient to connect the proximal nerve ending to the distal stump. In order to reliably provide more physical support for cellular substrate formation, several types of microchannel scaffolds, occasionally combined with neurotrophic factors, have been developed to physically connect the proximal and distal nerve ends. Microchannel scaffolds have been developed to artificially provide the necessary physical support and direction to Schwann cell migration by further constricting the direction of outgrowth and increasing the surface area available for support. As opposed to nerve conduits that allow axon outgrowth within the guide itself to be relatively disorganized, microchannel scaffolds arrange axon outgrowth into a series of linear arrays, each one with the physical restraints necessary for reattachment to the distal nerve stump within the three dimensional in vivo environment. Although microchannel scaffolds have been used successfully in several nerve regeneration studies on rats and other small mammals (Lacour et al., 2009; Billiar et al., 2010), the details of the biological events during axonal regeneration have not been reported. In order to understand the process of peripheral nerve regeneration within microchannel scaffolds, longitudinal observation of axon outgrowth via the microchannel is crucial. Analyzing individual axonal growth patterns and tracing the gradual progress of nerve regeneration inside microchannel scaffolds may be required to further investigate and confirm microchannel functionality. For these functional and investigational purposes, multilayer microchannel scaffolds were developed to visualize and monitor the progress of axonal regeneration (Hossain et al., 2015; Kim et al., 2015). During the fabrication process, no special micromachining equipment was required and commercially available microwires were efficiently used to implement the microchannel structures. The multilayer PDMS microchannel scaffold consisting of individual layers of microchannels were manually stacked up to eight layers to form the required implant size to match the approximate size of the rat sciatic nerve model (1.5 mm diameter). A schematic view of the multilayer microchannel scaffold is shown in Figure 1A, B. This approach allows a flexibility of sample size because the microchannels can be cut to any length from the initial length, which only depends upon the size of the molding structure and microwires. In this case, 100 mm long microchannel layers were developed and cut for the each designed 3 mm long microchannel scaffolds. These layers were not secured together using an adhesive, but were wrapped with a PDMS thin film which was anchored to itself with a small portion of PDMS as a glue, which made feasible to disassemble individual PDMS microchannel layers after explant from nerve tissue. The stacked microchannel layers could be extracted and separated after nerve regeneration without damaging the harvested regenerated nerve tissue as shown in Figure 1B. A systematic study of the peripheral nerve regeneration through microchannel scaffolds has been performed using multilayer PDMS microchannel scaffolds in rat sciatic nerve model. One of the standard analysis techniques for nerve regeneration through an artificial conduit is immunohistochemistry using specific biomarkers, such as neurofilament (NF160, N5264, Sigma, St Louis, MO) (Gökbuget et al., 2015). NF160 is specific to the neurofilament which is a major component of the neuronal cytoskeletal structure and provide structural support for the axon and to regulate axon diameter. NF160 has been used as a major antibody to investigate nerve regeneration due to its strong specificity to the neurofilament. The red stained NF 160 lines in Figure 1C, D show neurofilament structures inside axons. The dash blue lines represent the PDMS microchannel walls and red colored regenerating axons are shown inside microchannels. Surprising results were achieved after nerve regeneration using the multilayer PDMS microchannel scaffolds. We were able to trace growth cones from the regenerating nerves and observed axonal branching in the individual microchannels. Two major cellular responses within damaged nerves (transected in animal models) are growth cone migration and axonal branching. The former term refers to how a severed nerve navigates to the disconnected target muscles, and the latter term describes how an axon actively searches for local guidance cues. Due to high interest in regenerative medicine and neurodegenerative disease, studies of growth cone motility and axonal branching from the transected nerve have been recently emphasized.