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 , 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 and control and sur- system is classified as the peripheral nervous system (PNS) and vival. Second, Schwann cells are involved in removing cell 7-11) (CNS). The PNS senses a variety of debris after . 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 sev- eral 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 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), 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 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.

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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 epineu- rium. 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 degen- eration 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 . 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 , 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 masters, but the micropatterns are then generated protein on biocompatible polymer surfaces. with soft lithography. Microcontact printing utilizes poly (dimethylsiloxane) (PDMS) stamp to transfer biomolecules to Micropatterning and Nerve Regeneration 27,40) the more hydrophilic surfaces (Figure 4). This method is and oriented tissue structures influence simple and does not require expensive or complex instruments. cell migration and neural cell guidance during nerve regenera- 3,44,45) The µCP method was used to generate micropatterns on tion and development, Micropatterned surfaces may 24,41) inorganic surfaces such as glass. Further studies generated mimic the in vivo microenvironment system at in vitro level by micropatterns on organic surfaces (e.g., polymers), which in- 27,42) creased applications of patterned surfaces. Generating

Figure 5. Microscale plasma initiated patterning (µPIP) method to generate micropatterns on PMMA surfaces.

Figure 4. Microcontact printing method (µCP) to generate micro- Figure 6. Connecting nerve injured site with a nerve guidance patterns on PMMA surfaces. conduit.

Vol. 15, No. 4 162 MinJung Song providing guidance cues (i.e., chemical, physical and biological inlets join at a single stream, the combined streams flow paral- cues). Neurons or glial cells recognize, adhere or extend based lel. Mixing of streams occurs only by diffusion across the inter- 52,54,64) on these guidance cues. Micropatterned surfaces provide chem- face, which produces gradients in microfluidic systems. ical and physical cues for neural cell guidance, thus, it has Diffusion is the process in which molecules spread from areas 24-33) been extensively applied in nerve regeneration study. of high concentration to areas of low concentration to create Dorsal root ganglia (DRG) neurons and Schwann cells pref- a concentration gradient. Therefore, gradients created by 52,54) erentially adhered to, and subsequently aligned on, laminin microfluidics are well defined. 26,27,46) 24,31,41) micropatterned polymer and glass surfaces. The studies demonstrate that neural cell behavior can be controlled Microfluidics and Nerve Regeneration with micropatterning. Aligned surfaces can be fur- Gradients of substrate-bound substances (haptotaxis), mech- 25,28) ther used to guide neurons as a biological cue. Protein anical rigidity (durotaxis) or diffusible substances (chemotaxis) micropatterned surfaces are also utilized to generate central play an important role in neuron guidance during nervous 3,61-63) neuron guidance, for example, brain stem neurons and hippoc- system development. Axons reach their target by re- 30,32,47-49) ampal neurons form the neural network. Furthermore, sponding to repulsive or attractive molecular cues and follow 65,66) the patterned surfaces successfully guide neural progenitor cells the molecule gradient. For example, semaphorin, netrins and show the possibility of the surface applications onto and slits are neuron-attractive molecules and ephrin is a 45) 66,67) progenitor cell differentiation. neuron-repulsive molecule. Because the molecular gradient is known as the main guidance cue to neurons during devel- 3) Nerve Regeneration at The Cell Level with opment, a molecular gradient in nerve regeneration may be Microfluidics potentially significant. Therefore, the microfluidic system is an attractive tool for nerve regeneration. In recent studies, Microfluidics is a newly developed technique that has many microfluidic systems successfully generated diverse biomolecular 50) applications in biological fields such as , gradients and maintained the constant gradient over long 51) biological and cell-based assays. This micro- level system uti- periods of time; the gradient systems were then used to 51,54,55,58-60) lizes a small amount of liquid, reagents and cells in short reac- manipulate cellular microenvironments. tion times, at low cost and power. Therefore, multiple biological The influence of a diffusible substance gradient (i.e., chemo- assays can be conducted, and many parallel operations are pos- taxis) has also been extensively studied and used to elucidate 51-54) sible. Microfluidic applications have been reviewed in cel- the molecular and cellular mechanisms of axon guidance. lular biology including immunoassay, protein and DNA However, neuron chemotaxis by microfluidics has not been 51,53) separation, cell sorting and manipulation. reported. By comparison, haptotaxis has been investigated; it is Another primary advantage of microfluidics is the ability to the cells motility or outgrowth guided by a gradient of mem- create gradients. Many cell types respond to a molecular or brane-bound ligand. A laminin-based protein gradient was gen- extracellular matrix gradient, and the microfluidic system enables erated with microfluidics and studied for haptotatic influence 60) the systemic analysis of cell-biomolecular gradient interac- on neuron guidance. In addition, axon outgrowth can be 51) 55) 56-58) 59) 68,69) tions. Epithelial cells, neutrophils endothelial cell guided by a gradient of rigidity (durotaxis). The rigid gradi- 51,54,60) and neurons respond to molecular or extracellular gra- ent is established by controlling the cross-linking degree on a dients. As a chemical gradient can be the guidance cue to substrate. Lo et al., showed cell migration on -coated 70) neurons, a gradient is closely related to nerve regeneration. polyacrylamide substrates with rigidity gradients. In addition, Many studies have demonstrated that neuron growth cone can the rate of extension was correlated to the mechanical 44,61,62) 63) 69) be guided by gradient both in vitro and in vivo. stiffness of agarose . No study has yet shown durotatic generation through microfluidics. This article also focuses on Gradient Generation via Microfluidics generating an adhesive peptide bound gradient in a three- In microfluidic systems, most fluids undergo laminar flow dimensional cell culture system using microfluidics. because the channel diameter is relatively small. Fluid ten- dency is described with Reynolds number (Re = dρυ/µ) when Nerve Regeneration in Tissue by d is the channel diameter and ρ, υ, µ are the density, velocity Nerve Guidance Conduit and viscosity of fluid, respectively. Laminar flow is a fluid stream (Re << 2000) and turbulent flow is chaotic and unpre- When the nerve injury become severe to the level of 36,52) dictable (Re >> 2000). In most cases, the channel diame- extracellular component disconnection, (Sunderland’s injury ter (d) is less than 500 µm and flow rate (υ) is slow such that degree III, IV and V), implanting a nerve guidance conduit is 53) the flow is typically laminar with Re values, around 0.1~ 1. necessary to reconnect the injured site. By bridging a nerve When two or more laminar flow streams from independent defect region with a guidance channel, improved nerve

Biomaterials Research 2011 Biomaterials and microfabrication for nerve regeneration 163 regeneration is expected in the peripheral nervous system 14) (Figure 6). As a nerve guidance channel, the autologous nerve 71,72) graft is considered the gold standard. The autologous nerve tissue contains growth factors and cytokines for regeneration, without a foreign body response. However, autologous nerve graft supply is a significant problem; because a healthy nerve is utilized, a second surgery is required and causes additional injury. Various natural-based and synthetic materials have been synthesized or modified as alternative nerve guidance systems. To be a nerve guidance conduit, materials should possess specific properties: (i) biocompatible; (ii) biodegradable, is other- wise, the remaining graft must be removed from the injury site; (iii) retain proper mechanical properties, they must strong enough to resist collapse during implantation and regeneration yet be flexible enough to handle; (iv) easily fabricated and modified with desired dimensions; and (v) sterilizable and tear- Figure 8. Protein micropatterned surfaces can be rolled up into 14,73) conduits. resistant. Collagen and laminin as natural-based materials, and poly (), poly (D, L-lactide-co-glycolide) (PLGA) and poly cylic acid unit. Other non-steroidal anti-inflammatory drugs (lactide-co-caprolactone) as synthetic materials are current (NSAIDs) such as thiosalicylic acid, diflunisal and salicylsalicylic 74-77) 82-85) examples of nerve guidance materials. Particularly, PLGA acid were also incorporated into polymer backbone and is biodegradable and approved by a Food and Drug Adminis- different types of linker groups, (e.g., diethylmalonic and adi- 84) tration (FDA) for implantation. All materials demonstrated pic acid), have been used to connect the drug molecules. improved nerve regeneration upon grafting, however full func- From the unique properties described above, these poly tional recovery is limited compared to the autologous (anhydride-esters) have many advantages. First, the polymer 74,78,79) grafting. One of the main reasons for limited regener- contains high drug amounts (e.g., 62 wt% of polymer 1 is sali- ation is inflammation at the nerve injury site. This article will cylic acid). Second, polymer properties such as drug release discuss this issue and describe novel materials to address the rate, and melting temperature can be regulated by controlling 84) current inflammation issue. the polymer structure. Third, the polymer is biocompatible 83) and the degradation products are nontoxic. These polymers Drug-based Poly(anhydride-esters) have shown reduced inflammation, inhibition of resorp- Poly (anhydride-ester) is a biodegradable polymer that has tion, prevented bacterial contamination and reduced foreign- 86-88) been applied to drug delivery, tissue engineering and medical body responses. Based upon these characteristics, the 80) devices. Previously, Erdmann et al., synthesized and charac- drug-based poly(anhydride-ester) is a good candidate as a 81) terized salicylic acid-based poly (anhydride-esters) (1). This nerve guidance material. This article will further discuss the polymer (1) is unique in that the drugs are incorporated into possibility of applying this polymer to nerve guidance conduit. the polymer backbone, not attached as a side group or phys- ically admixing (Figure 7). These polymers (1) degraded into Future Work and Recommendations salicylic acid (2) and sebacic acid (3) upon hydrolysis due to the instability of the anhydride and ester bonds. Salicylic acid Micropatterned Surfaces in Nerve Regeneration (2) is generated upon hydrolysis of acetylsalicylic acid (aspirin) We have developed protein micropatterning systems to and sebacic acid (3) is the linker group that connects the sali- guide neural cells on biocompatible polymer substrates. For in

Figure 7. Hydrolytic degradation of poly(anhydride ester) (1) into salicylic acid (2) and sebacic acid (3).

Vol. 15, No. 4 164 MinJung Song vivo use, generating micropatterns and guiding cells on biode- gradable polymer drug substrates such as salicylic acid-based poly(anhydride-esters) should be attempted. Micropatterned surfaces in biodegradable polymer can be rolled up as a con- duit shape to implant in animals (Figure 8). As this conduit contains an inner surface with laminin micropatterns, improved nerve regeneration is expected. In stem cells, physical cues as well as biomolecular cues play 89) important roles in cell differentiation. As micropatterned surfaces can provide physical and biomolecular cues, studying differentiation with these cues will be an interesting topic. Figure 6.2 shows embryonic stem cells on laminin micro- patterned surfaces; the cells recognized and aligned on patterns, indicating that a patterned surface may influence a stem cell differentiation (Figure 9). Differentiated stem cells will bring various potential therapies methods for nerve regenera- Figure 11. A gradient collagen detachment from the PDMS 90,91) tion. network using freeze-drying method.

Microfluidic Applications in Nerve Regeneration gel with an open microfluidic system, the gel may be frozen at o We have successfully generated an adhesive gradient in three −20 C (Figure 11). After freeze-drying, the collagen gel may dimensions. Neuron guidance by the gradient can be assessed be detached from the network. after immunostaining and image analysis. Simple measurement of neurite movement, either positive or negative to the gradi- Nerve Guidance Conduits ent, will describe the impact of the adhesive gradient (Figure Nerve guidance conduits can be fabricated as described in 10). As a future study, gradients of neuron-specific peptides Figure 12. For example, SAA polymer can be used as an outer such as YIGSR and IKVAV can be generated with a current layer to reduce acute inflammation and an inner layer based microfluidic system, then their influence on neuron guidance upon diflunisal polymer can reduce a chronic inflammation elucidated in vitro. upon implantation. Filling the current hollow conduit with col- 92) To use the biomolecular gradient collagen gel as a biomate- lagen gel may further improve nerve regeneration. rial in vivo, detaching the gel from a PDMS network remains Iodinated salicylate-based poly(anhydride-esters) (SAA) can as an issue. To perform this function, a freeze-drying method be a novel material for nerve guidance conduits. Iodinated SAA may be utilized. After the gradient is generated in a collagen was synthesized in our laboratory (Figure 13), and showed X- ray opacity in clinical X-ray techniques with good biocom-

Figure 9. Embryonic stem cell guidance on micropatterned sur- Figure 12. Further modified nerve guidance conduit consisting faces on day 3. of salicylic acid- and diflunisal-based polymers.

Figure 10. Neuron guidance analysis to a biomolecular gradient Figure 13. Iodinated salicylate-based poly(anhydride-ester) struc- 93) (negative or positive). tures.

Biomaterials Research 2011 Biomaterials and microfabrication for nerve regeneration 165

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