Current Micropatterning, Microfluidics and Nerve Guidance Conduit for Nerve Regeneration and Future Recommendations

Biomaterials Research (2011) 15(4) : 159-167 Biomaterials Research C The Korean Society for Biomaterials Current Micropatterning, Microfluidics and Nerve Guidance Conduit for Nerve Regeneration and Future Recommendations MinJung Song* Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854 (Received October 26, 2011/Acccepted November 7, 2011) Each year, approximately 200,000 patients suffer from peripheral nerve injuries, resulting in tremendous medical 1) expense in the United States. The peripheral nerves are damaged by injury, disease, trauma, or other medical dis- orders. After the injury, peripheral nerves can regenerate, but when the injury is too severe, nerves degenerate and lose functionality. This review focuses on improving peripheral nerve regeneration with microfabrication techniques and biomaterials. In this review, several concepts will be introducing relating to the peripheral nervous system, microfabrication techniques (e.g., micropatterning and microfluidics) and biomaterials (e.g., drug-containing polymer) to improve nerve regeneration in the cellular and tissue levels. Key words: peripheral nerve regeneration, micropatterns, microfluidics, gradient, polyNSAID Peripheral Nervous System ous roles to support neurons. First, they produce biomolecules such as laminin, cell adhesion molecules, integrins, and neu- he nervous system is a communicating network to inte- rotrophins, which give growth signals to provide guidance for T grate and monitor the actions throughout the body. This regenerating axons and control cellular differentiation and sur- system is classified as the peripheral nervous system (PNS) and vival. Second, Schwann cells are involved in removing cell 7-11) central nervous system (CNS). The PNS senses a variety of debris after nerve injury. environmental reactions with sensory neurons and transmits the Several nerve fibers constitute a single nerve, and each nerve information to the CNS. Central neurons in the brain and spi- fiber is composed of Schwann cells, neurons, endoneurium, nal cord generate motor commands and peripheral neurons perineurium and epineurium (Figure 2). Endoneurium consists 2,3) execute them. of single neurons and Schwann cells, perineurium includes several endoneurium with connective tissues, and epineurium Peripheral Nervous System Structure contains multiple perineurium with fibrous connective tissue, 12,13) The nervous system has two main cell classes: neurons and fibroblasts and blood vessels. The nerve injury types glial cells. Neurons are the functional unit of the nervous sys- related with fiber structure are described in the next section. tem and glial cells support neuronal functions in numerous ways. Neurons consist of three parts: cell body (soma), axon Peripheral Nerve Injury and Regeneration 2-4) (nerve fiber) and dendrites as described in Figure 1. Den- In most cases, injuries in the nervous system divide the drites receive signals from neighboring cells, whereas the neu- axons into a proximal segment that is intact with the cell body 1) ron cell body (soma) and axon send information by electrical and a distal segment that is detached from cell body. impulses. The axon terminal and dendrites form synapses to transmit impulses to another neuron, which allows the signal to continue on to another neuron. The signals pass only in the 2) forward direction from dendrite to axon. Representative glial cells in the PNS are Schwann cells. The primary function of Schwann cells is to produce a myelin which surrounds and insulates axons that conduct electrical 5,6) impulses (Figure 1). In addition, Schwann cells perform vari- *Corresponding author: [email protected] Figure 1. Structure of a typical neuron and Schwann cells. 159 160 MinJung Song continiuity of the endoneurium and perineurium, respectively, with the disruption of the axon, but with an intact epineurium. Fifth degree injury (5) is the complete loss of continuity of the epineurium. The lack of surrounding extracellular protein connections ensure that nerve regeneration will not occur. In the first and second degree injuries, if the environment allows, the neuron attempts to repair the axon by making new 14) structural proteins, this process is called axonal reaction. Peripheral nerve regeneration processes are described in sev- 2,14,15) eral reviews. In specific, nerve regeneration initiates when growth factors, adhesion molecules and structural com- ponents arrive at the injury site for axonal elongation. The proximal stump of the damaged axon develops sprouts and Figure 2. Nerve bundle cross-section structure comprised of neu- the Schwann cells in the distal stump of the nerve proliferate ron, Schwann cells and endoneurium, perineurium and epineurium. and form routes along the course previously taken by the axons. Axon sprouts elongate and grow along the path of the original nerve to the distal stump if this route is available. Growth cones of the sprouting axons find their way along the routes of Schwann cells and eventually reinnervate the original peripheral target structures. Once they return to their targets, the regenerated axons can form new functional nerve endings. At the distal segments in injured nerve. A Wallerian degeneration which is the process of degeneration at the distal part 16,17) of neurons, occurs. Axon degeneration and disintegration from calcium influx and axonal proteases initiated a Wallerian degeneration. As a response of axonal breakdown, the myelin sheath produced by Schwann cells degenerates. Degeneration products and other debris are removed by phagocytic cells and 14,18) macrophages. In the case of Sunderland’s third, fourth and fifth degree injuries (Figure 3 - 3, 4, 5), a Wallerian degeneration occurs at the distal segments. In the third degree injury, axons undergo a distal Wallerian degeneration and the endoneurium is disconnected. In the fourth degree injury, all portions of the nerve are disrupted except the epineurium and the fifth degree injury is the complete severance of the nerve trunk. In these injury degrees, a functional recovery is rarely obtained in a natural environment. This article will focus on the third, fourth and fifth degree injury types. Improving Nerve Regeneration at The Cell Figure 3. Sunderland’s five nerve injury classification from first Level with Micropatterned Surfaces degree to fifth degree injury. Micropatterning is a method to create patterns on both Sunderland classified nerve injury into five degrees based on organic and inorganic surfaces that can provide physical and/or 12) nerve histologic structure changes (Figure 3). First degree chemical cues for cellular guidance. Various cell types including injury (1) is the mildest one that involves local ion-induced osteoblast, peripheral and central neurons, smooth muscle cells conduction blockage at the injury site with possible segmental and endothelial cells have been succesfully guided on micropat- 19-23) demyelination, a degenerative process removing the myelin terned surfaces. Neural cells are particularly sensitive to their sheath that normally protects nerve fibers. Second degree injury geometrical environment, and guidance cues are really impor- (2) is a complete interruption of nerve axon and surrounding tant in neural cell growth. Therefore, micropatterned surfaces 24-33) myelin, while the surrounding endoneurial sheath is preserved. have been extensively used in nerve regeneration studies. In third and fourth degree injuries (3 and 4), nerves lose the Biomaterials Research 2011 Biomaterials and microfabrication for nerve regeneration 161 Micropatterning Methods micropatterns on polymer surfaces is unique because polymers Micropatterned surfaces are generated using various methods can be implanted following patterning. µCP has many advan- 26-28) including photolithography, microcontact printing (µCP), microf- tages, but a few issues regarding regarding biomolecule 34) luidic patterning, micromolding in capillaries, stencil pattern- transferring limit further applications. Because biomolecule 35-38) ing and microscale plasma-initiated patterning (µPIP). Pho- transfer depends on hydrophilic differences between the tolithography uses a projection-printing system in which light PDMS stamp and polymer surfaces, two major issues are: (i) passes through a photomask and selectively exposes a spin- ink affinity to the PDMS is sometimes too strong to transfer 43) coated photoresist (photosensitive polymer). Following develop- proteins from the PDMS stamp to the surface; and (ii) the 37,38) 39) ment, this method generates a patterned photoresist. This aqueous solution containing the biomolecules may dry up. method has the advantage of generating patterns at the nanos- The µPIP method was developed in our laboratory by selec- cale level, whereas it has many disadvantages in its application tively exposing surfaces to oxygen plasma to temporarily pro- 39) to biology; photolithography requires an “expensive” clean- mote hydrophilicity (Figure 5). This method is relatively room facility and the processing are not necessary amenable simple and generates patterns consistently and reproducibly. In 36,37,39) to biological systems. addition, it allows complicated or gradient protein micropat- Microcontact printing (µCP) (Figure 4) and plasma-initiated terns to be generated. This article will describe both microcon- patterning (µPIP) (Figure 5) methods utilize photolithography tact printing and microscale plasma-initiated methods to pattern to generate

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