Biomaterials Research (2011) 15(4) : 159-167

159

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 medicalexpense in the United States.1) 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 andlose functionality. This review focuses on improving peripheral nerve regeneration with microfabrication techniquesand 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-containingpolymer) to improve nerve regeneration in the cellular and tissue levels.

Key words: peripheral nerve regeneration, micropatterns, microfluidics, gradient, polyNSAID

Peripheral Nervous System

he nervous system is a communicating network to inte-

grate and monitor the actions throughout the body. This

system is classified as the peripheral nervous system (PNS) and

central nervous system (CNS). The PNS senses a variety of

environmental reactions with sensory neurons and transmits the

information to the CNS. Central neurons in the brain and spi-

nal cord generate motor commands and peripheral neurons

execute them.2,3)

Peripheral Nervous System StructureThe nervous system has two main cell classes: neurons and

glial cells. Neurons are the functional unit of the nervous sys-

tem and glial cells support neuronal functions in numerous

ways. Neurons consist of three parts: cell body (soma), axon

(nerve fiber) and dendrites as described in Figure 1.2-4) Den-

drites receive signals from neighboring cells, whereas the neu-

ron cell body (soma) and axon send information by electrical

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

forward direction from dendrite to axon.2)

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

impulses (Figure 1).5,6) In addition, Schwann cells perform vari-

ous roles to support neurons. First, they produce biomolecules

such as laminin, cell adhesion molecules, integrins, and neu-

rotrophins, which give growth signals to provide guidance for

regenerating axons and control cellular differentiation and sur-

vival. Second, Schwann cells are involved in removing cell

debris after nerve injury.7-11)

Several nerve fibers constitute a single nerve, and each nerve

fiber is composed of Schwann cells, neurons, endoneurium,

perineurium and epineurium (Figure 2). Endoneurium consists

of single neurons and Schwann cells, perineurium includes sev-

eral endoneurium with connective tissues, and epineurium

contains multiple perineurium with fibrous connective tissue,

fibroblasts and blood vessels.12,13) The nerve injury types

related with fiber structure are described in the next section.

Peripheral Nerve Injury and Regeneration In most cases, injuries in the nervous system divide the

axons into a proximal segment that is intact with the cell body

and a distal segment that is detached from cell body.1)

T

*Corresponding author: songmj@gmail.com Figure 1. Structure of a typical neuron and Schwann cells.

160 MinJung Song

Biomaterials Research 2011

Sunderland classified nerve injury into five degrees based on

nerve histologic structure changes (Figure 3).12) First degree

injury (1) is the mildest one that involves local ion-induced

conduction blockage at the injury site with possible segmental

demyelination, a degenerative process removing the myelin

sheath that normally protects nerve fibers. Second degree injury

(2) is a complete interruption of nerve axon and surrounding

myelin, while the surrounding endoneurial sheath is preserved.

In third and fourth degree injuries (3 and 4), nerves lose the

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

structural proteins, this process is called axonal reaction.14)

Peripheral nerve regeneration processes are described in sev-

eral reviews.2,14,15) 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

the Schwann cells in the distal stump of the nerve proliferate

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

of neurons, occurs.16,17) 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

macrophages.14,18) 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 CellLevel with Micropatterned Surfaces

Micropatterning is a method to create patterns on both

organic and inorganic surfaces that can provide physical and/or

chemical cues for cellular guidance. Various cell types including

osteoblast, peripheral and central neurons, smooth muscle cells

and endothelial cells have been succesfully guided on micropat-

terned surfaces.19-23) Neural cells are particularly sensitive to their

geometrical environment, and guidance cues are really impor-

tant in neural cell growth. Therefore, micropatterned surfaces

have been extensively used in nerve regeneration studies.24-33)

Figure 2. Nerve bundle cross-section structure comprised of neu-ron, Schwann cells and endoneurium, perineurium and epineu-rium.

Figure 3. Sunderland’s five nerve injury classification from firstdegree to fifth degree injury.

Biomaterials and microfabrication for nerve regeneration 161

Vol. 15, No. 4

Micropatterning Methods Micropatterned surfaces are generated using various methods

including photolithography, microcontact printing (µCP), microf-

luidic patterning,34) micromolding in capillaries, stencil pattern-

ing and microscale plasma-initiated patterning (µPIP).35-38) Pho-

tolithography uses a projection-printing system in which light

passes through a photomask and selectively exposes a spin-

coated photoresist (photosensitive polymer). Following develop-

ment, this method generates a patterned photoresist.37,38) This

method has the advantage of generating patterns at the nanos-

cale level, whereas it has many disadvantages in its application

to biology; photolithography requires an “expensive” clean-

room facility and the processing are not necessary amenable

to biological systems.36,37,39)

Microcontact printing (µCP) (Figure 4) and plasma-initiated

patterning (µPIP) (Figure 5) methods utilize photolithography

to generate masters, but the micropatterns are then generated

with soft lithography. Microcontact printing utilizes poly

(dimethylsiloxane) (PDMS) stamp to transfer biomolecules to

the more hydrophilic surfaces (Figure 4).27,40) This method is

simple and does not require expensive or complex instruments.

The µCP method was used to generate micropatterns on

inorganic surfaces such as glass.24,41) Further studies generated

micropatterns on organic surfaces (e.g., polymers), which in-

creased applications of patterned surfaces.27,42) Generating

micropatterns on polymer surfaces is unique because polymers

can be implanted following patterning. µCP has many advan-

tages,26-28) but a few issues regarding regarding biomolecule

transferring limit further applications. Because biomolecule

transfer depends on hydrophilic differences between the

PDMS stamp and polymer surfaces, two major issues are: (i)

ink affinity to the PDMS is sometimes too strong to transfer

proteins from the PDMS stamp to the surface;43) and (ii) the

aqueous solution containing the biomolecules may dry up.39)

The µPIP method was developed in our laboratory by selec-

tively exposing surfaces to oxygen plasma to temporarily pro-

mote hydrophilicity (Figure 5).39) This method is relatively

simple and generates patterns consistently and reproducibly. In

addition, it allows complicated or gradient protein micropat-

terns to be generated. This article will describe both microcon-

tact printing and microscale plasma-initiated methods to pattern

protein on biocompatible polymer surfaces.

Micropatterning and Nerve RegenerationExtracellular matrix and oriented tissue structures influence

cell migration and neural cell guidance during nerve regenera-

tion and development,3,44,45) Micropatterned surfaces may

mimic the in vivo microenvironment system at in vitro level by

Figure 4. Microcontact printing method (µCP) to generate micro-patterns on PMMA surfaces.

Figure 6. Connecting nerve injured site with a nerve guidanceconduit.

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

162 MinJung Song

Biomaterials Research 2011

providing guidance cues (i.e., chemical, physical and biological

cues). Neurons or glial cells recognize, adhere or extend based

on these guidance cues. Micropatterned surfaces provide chem-

ical and physical cues for neural cell guidance, thus, it has

been extensively applied in nerve regeneration study.24-33)

Dorsal root ganglia (DRG) neurons and Schwann cells pref-

erentially adhered to, and subsequently aligned on, laminin

micropatterned polymer26,27,46) and glass24,31,41) surfaces. The

studies demonstrate that neural cell behavior can be controlled

with micropatterning. Aligned Schwann cell surfaces can be fur-

ther used to guide neurons as a biological cue.25,28) Protein

micropatterned surfaces are also utilized to generate central

neuron guidance, for example, brain stem neurons and hippoc-

ampal neurons form the neural network.30,32,47-49) Furthermore,

the patterned surfaces successfully guide neural progenitor cells

and show the possibility of the surface applications onto

progenitor cell differentiation.45)

Nerve Regeneration at The Cell Level with Microfluidics

Microfluidics is a newly developed technique that has many

applications in biological fields such as tissue engineering,50)

biological and cell-based assays.51) This micro- level system uti-

lizes a small amount of liquid, reagents and cells in short reac-

tion times, at low cost and power. Therefore, multiple biological

assays can be conducted, and many parallel operations are pos-

sible.51-54) Microfluidic applications have been reviewed in cel-

lular biology including immunoassay, protein and DNA

separation, cell sorting and manipulation.51,53)

Another primary advantage of microfluidics is the ability to

create gradients. Many cell types respond to a molecular or

extracellular matrix gradient, and the microfluidic system enables

the systemic analysis of cell-biomolecular gradient interac-

tions.51) Epithelial cells,55) neutrophils56-58) endothelial cell59)

and neurons51,54,60) respond to molecular or extracellular gra-

dients. As a chemical gradient can be the guidance cue to

neurons, a gradient is closely related to nerve regeneration.

Many studies have demonstrated that neuron growth cone can

be guided by gradient both in vitro44,61,62) and in vivo.63)

Gradient Generation via MicrofluidicsIn microfluidic systems, most fluids undergo laminar flow

because the channel diameter is relatively small. Fluid ten-

dency is described with Reynolds number (Re = dρυ/µ) when

d is the channel diameter and ρ, υ, µ are the density, velocity

and viscosity of fluid, respectively. Laminar flow is a fluid

stream (Re << 2000) and turbulent flow is chaotic and unpre-

dictable (Re >> 2000).36,52) In most cases, the channel diame-

ter (d) is less than 500 µm and flow rate (υ) is slow such that

the flow is typically laminar with Re values, around 0.1~ 1.53)

When two or more laminar flow streams from independent

inlets join at a single stream, the combined streams flow paral-

lel. Mixing of streams occurs only by diffusion across the inter-

face, which produces gradients in microfluidic systems.52,54,64)

Diffusion is the process in which molecules spread from areas

of high concentration to areas of low concentration to create

a concentration gradient. Therefore, gradients created by

microfluidics are well defined.52,54)

Microfluidics and Nerve RegenerationGradients of substrate-bound substances (haptotaxis), mech-

anical rigidity (durotaxis) or diffusible substances (chemotaxis)

play an important role in neuron guidance during nervous

system development.3,61-63) Axons reach their target by re-

sponding to repulsive or attractive molecular cues and follow

the molecule gradient.65,66) For example, semaphorin, netrins

and slits are neuron-attractive molecules and ephrin is a

neuron-repulsive molecule.66,67) Because the molecular gradient

is known as the main guidance cue to neurons during devel-

opment,3) a molecular gradient in nerve regeneration may be

potentially significant. Therefore, the microfluidic system is an

attractive tool for nerve regeneration. In recent studies,

microfluidic systems successfully generated diverse biomolecular

gradients and maintained the constant gradient over long

periods of time; the gradient systems were then used to

manipulate cellular microenvironments.51,54,55,58-60)

The influence of a diffusible substance gradient (i.e., chemo-

taxis) has also been extensively studied and used to elucidate

the molecular and cellular mechanisms of axon guidance.

However, neuron chemotaxis by microfluidics has not been

reported. By comparison, haptotaxis has been investigated; it is

the cells motility or outgrowth guided by a gradient of mem-

brane-bound ligand. A laminin-based protein gradient was gen-

erated with microfluidics and studied for haptotatic influence

on neuron guidance.60) In addition, axon outgrowth can be

guided by a gradient of rigidity (durotaxis).68,69) The rigid gradi-

ent is established by controlling the cross-linking degree on a

substrate. Lo et al., showed cell migration on collagen-coated

polyacrylamide substrates with rigidity gradients.70) In addition,

the rate of neurite extension was correlated to the mechanical

stiffness of agarose gels.69) No study has yet shown durotatic

generation through microfluidics. This article also focuses on

generating an adhesive peptide bound gradient in a three-

dimensional cell culture system using microfluidics.

Nerve Regeneration in Tissue byNerve Guidance Conduit

When the nerve injury become severe to the level of

extracellular component disconnection, (Sunderland’s injury

degree III, IV and V), implanting a nerve guidance conduit is

necessary to reconnect the injured site. By bridging a nerve

defect region with a guidance channel, improved nerve

Biomaterials and microfabrication for nerve regeneration 163

Vol. 15, No. 4

regeneration is expected in the peripheral nervous system

(Figure 6).14) As a nerve guidance channel, the autologous nerve

graft is considered the gold standard.71,72) 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-

resistant.14,73)

Collagen and laminin as natural-based materials, and poly

(glycolic acid), poly (D, L-lactide-co-glycolide) (PLGA) and poly

(lactide-co-caprolactone) as synthetic materials are current

examples of nerve guidance materials.74-77) Particularly, PLGA

is biodegradable and approved by a Food and Drug Adminis-

tration (FDA) for implantation. All materials demonstrated

improved nerve regeneration upon grafting, however full func-

tional recovery is limited compared to the autologous

grafting.74,78,79) One of the main reasons for limited regener-

ation is inflammation at the nerve injury site. This article will

discuss this issue and describe novel materials to address the

current inflammation issue.

Drug-based Poly(anhydride-esters)Poly (anhydride-ester) is a biodegradable polymer that has

been applied to drug delivery, tissue engineering and medical

devices.80) Previously, Erdmann et al., synthesized and charac-

terized salicylic acid-based poly (anhydride-esters) (1).81) This

polymer (1) is unique in that the drugs are incorporated into

the polymer backbone, not attached as a side group or phys-

ically admixing (Figure 7). These polymers (1) degraded into

salicylic acid (2) and sebacic acid (3) upon hydrolysis due to

the instability of the anhydride and ester bonds. Salicylic acid

(2) is generated upon hydrolysis of acetylsalicylic acid (aspirin)

and sebacic acid (3) is the linker group that connects the sali-

cylic acid unit. Other non-steroidal anti-inflammatory drugs

(NSAIDs) such as thiosalicylic acid, diflunisal and salicylsalicylic

acid were also incorporated into polymer backbone82-85) and

different types of linker groups, (e.g., diethylmalonic and adi-

pic acid), have been used to connect the drug molecules.84)

From the unique properties described above, these poly

(anhydride-esters) have many advantages. First, the polymer

contains high drug amounts (e.g., 62 wt% of polymer 1 is sali-

cylic acid). Second, polymer properties such as drug release

rate, and melting temperature can be regulated by controlling

the polymer structure.84) Third, the polymer is biocompatible

and the degradation products are nontoxic.83) These polymers

have shown reduced inflammation, inhibition of bone resorp-

tion, prevented bacterial contamination and reduced foreign-

body responses.86-88) Based upon these characteristics, the

drug-based poly(anhydride-ester) is a good candidate as a

nerve guidance material. This article will further discuss the

possibility of applying this polymer to nerve guidance conduit.

Future Work and Recommendations

Micropatterned Surfaces in Nerve RegenerationWe have developed protein micropatterning systems to

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).

Figure 8. Protein micropatterned surfaces can be rolled up intoconduits.

164 MinJung Song

Biomaterials Research 2011

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

important roles in cell differentiation.89) As micropatterned

surfaces can provide physical and biomolecular cues, studying

stem cell 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-

tion.90,91)

Microfluidic Applications in Nerve RegenerationWe have successfully generated an adhesive gradient in three

dimensions. Neuron guidance by the gradient can be assessed

after immunostaining and image analysis. Simple measurement

of neurite movement, either positive or negative to the gradi-

ent, will describe the impact of the adhesive gradient (Figure

10). As a future study, gradients of neuron-specific peptides

such as YIGSR and IKVAV can be generated with a current

microfluidic system, then their influence on neuron guidance

elucidated in vitro.

To use the biomolecular gradient collagen gel as a biomate-

rial in vivo, detaching the gel from a PDMS network remains

as an issue. To perform this function, a freeze-drying method

may be utilized. After the gradient is generated in a collagen

gel with an open microfluidic system, the gel may be frozen at

−20oC (Figure 11). After freeze-drying, the collagen gel may

be detached from the network.

Nerve Guidance ConduitsNerve guidance conduits can be fabricated as described in

Figure 12. For example, SAA polymer can be used as an outer

layer to reduce acute inflammation and an inner layer based

upon diflunisal polymer can reduce a chronic inflammation

upon implantation. Filling the current hollow conduit with col-

lagen gel may further improve nerve regeneration.92)

Iodinated salicylate-based poly(anhydride-esters) (SAA) can

be a novel material for nerve guidance conduits. Iodinated SAA

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-faces on day 3.

Figure 10. Neuron guidance analysis to a biomolecular gradient(negative or positive).

Figure 11. A gradient collagen gel detachment from the PDMSnetwork using freeze-drying method.

Figure 12. Further modified nerve guidance conduit consistingof salicylic acid- and diflunisal-based polymers.

Figure 13. Iodinated salicylate-based poly(anhydride-ester) struc-tures.93)

Biomaterials and microfabrication for nerve regeneration 165

Vol. 15, No. 4

patibility.93) If the nerve guidance conduit is fabricated with the

polymer and implanted in vivo, observing regeneration pro-

cesses can be easily monitored without sacrificing animals.

References

1. Yannas, I.V., Hill, B.J., “Selection of biomaterials for peripheralnerve regeneration using data from the nerve chamber model,”Biomaterials. 25, 1593-1600 (2004).

2. Willis, W.D., The Nervous System. in Physiology, Berne, R.M.,Levy, M.N., Koeppen, B.M. and Stanton, B.A. (ed) St. Louis: Mosby,1998, pp. 81-226.

3. Thomas M, J., Sanes., J.R., The Development of the Nervous Sys-tem. in Principles of Neural Science, Kandel, E.R., Schwartz, J.H.and Jessell, T.M.(ed). by New York: McGraw-Hill Health Profes-sions Division, 2000, pp. 1019-1161.

4. Guyton, A.C., Hall, J.E., Textbook of Medical Physiology. 10th edn(Philadelphia: Saunders, 2000), pp. xxxii, 1064.

5. Bhatheja, K., Field, J., “Schwann cells: origins and role in axonalmaintenance and regeneration,” Int J Biochem Cell Biol. 38,1995-1999 (2006).

6. Bunge, R.P., “The role of the Schwann cell in trophic support andregeneration,” J Neurol. 242, S19-21 (1994).

7. Mirsky, R., Jessen, K.R., Brennan, A., Parkinson, D., Dong, Z.,Meier, C., Parmantier, E., Lawson, D., “Schwann cells as regulatorsof nerve development,” J Physiol Paris. 96, 17-24 (2002).

8. Ard, M.D., Bunge, R.P., Bunge, M.B., “Comparison of the Schwanncell surface and Schwann cell extracellular matrix as promoters ofneurite growth,” J Neurocytol. 16, 539-555 (1987).

9. Bunge, M.B., Williams, A.K., Wood, P.M., Uitto, J., Jeffrey, J.J.,“Comparison of nerve cell and nerve cell plus Schwann cell cul-tures, with particular emphasis on basal lamina and collagen forma-tion,” J Cell Biol. 84, 184-202 (1980).

10. Jessen, K.R., Mirsky, R., “Schwann cells and their precursors emergeas major regulators of nerve development,” Trends Neurosci. 22,402-410 (1999).

11. Morrissey, T.K., Kleitman, N., Bunge, R.P., “Isolation and functionalcharacterization of Schwann cells derived from adult peripheralnerve,” J Neurosci. 11, 2433-2442 (1991).

12. Sunderland, S., “The anatomy and physiology of nerve injury,”Muscle Nerve. 13, 771-784 (1990).

13. Flores, A.J., Lavernia, C.J., Owens, P.W., “Anatomy and physiologyof peripheral nerve injury and repair,” Am J Orthop. 29, 167-173(2000).

14. Schmidt, C.E., Leach, J.B., “Neural tissue engineering: strategies forrepair and regeneration,” Annu Rev Biomed Eng. 5, 293-347(2003).

15. Makwana, M., Raivich, G., “Molecular mechanisms in successfulperipheral regeneration,” FEBS J. 272, 2628-2638 (2005).

16. Johnson, E.O., Zoubos, A.B., Soucacos, P.N., “Regeneration andrepair of peripheral nerves,” Injury. 36 Suppl 4, S24-29 (2005).

17. Richardson, P.M., Lu, X., “Inflammation and axonal regenera-tion,” J Neurol. 242, S57-60 (1994).

18. Stoll, G., Jander, S., Myers, R.R., “Degeneration and regenera-tion of the peripheral nervous system: from Augustus Waller'sobservations to neuroinflammation,” J Peripher Nerv Syst. 7, 13-27(2002).

19. Hasenbein, M.E., Andersen, T.T., Bizios, R., “Micropatterned sur-faces modified with select peptides promote exclusive interactionswith osteoblasts,” Biomaterials. 23, 3937-3942 (2002).

20. Kaji, H., Takii, Y., Nishizawa, M., Matsue, T.,“Pharmacological char-

acterization of micropatterned cardiac myocytes,” Biomaterials. 24,4239-4244 (2003).

21. Kulkarni, S.S., Orth, R., Ferrari, M., Moldovan, N.I., “Micropattern-ing of endothelial cells by guided stimulation with angiogenic fac-tors,” Biosens Bioelectron. 19, 1401-1407 (2004).

22. Thakar, R.G., Ho, F., Huang, N.F., Liepmann, D., Li, S., “Regula-tion of vascular smooth muscle cells by micropatterning,” Bio-chem. Biophys. Res. Commun. 307, 883-890 (2003).

23. Glawe, J.D., Hill, J.B., Mills, D.K., McShane, M.J., “Influence ofchannel width on alignment of smooth muscle cells by high-aspect-ratio microfabricated elastomeric cell culture scaffolds,” J.Biomed. Mater. Res. A. 75, 106-114 (2005).

24. Thompson, D.M., Buettner, H.M., “Schwann cell response tomicropatterned laminin surfaces,” Tissue Eng. 7, 247-265 (2001).

25. Thompson, D.M., Buettner, H.M., “Oriented Schwann cell mono-layers for directed neurite outgrowth,” Ann Biomed Eng. 32, 1120-1130 (2004).

26. Schmalenberg, K.E., Uhrich, K.E., “Micropatterned polymer sub-strates control alignment of proliferating Schwann cells to directneuronal regeneration,” Biomaterials. 26, 1423-1430 (2005).

27. Schmalenberg, K.E., Buettner, H.M., Uhrich, K.E., “Microcontactprinting of proteins on oxygen plasma-activated poly(methyl meth-acrylate),” Biomaterials. 25, 1851-1857 (2004).

28. Miller, C., Jeftinija, S., Mallapragada, S., “Micropatterned Schwanncell-seeded biodegradable polymer substrates significantly enhanceneurite alignment and outgrowth,” Tissue Eng. 7, 705-715 (2001).

29. Miller, C., Shanks, H., Witt, A., Rutkowski, G., Mallapragada, S.,“Oriented Schwann cell growth on micropatterned biodegrad-able polymer substrates,” Biomaterials. 22, 1263-1269 (2001).

30. Chang, J.C., Brewer, G.J., Wheeler, B.C., “A modified micros-tamping technique enhances polylysine transfer and neuronalcell patterning,” Biomaterials. 24, 2863-2870 (2003).

31. Tai, H.C., Buettner, H.M., “Neurite outgrowth and growth conemorphology on micropatterned surfaces,” Biotechnol Prog. 14,364-370 (1998).

32. Vogt, A.K., Stefani, F.D., Best, A., Nelles, G., Yasuda, A., Knoll,W., Offenhausser, A., “Impact of micropatterned surfaces on neu-ronal polarity,” J Neurosci Methods. 134, 191-198 (2004).

33. Oliva, A.A., Jr., James, C.D., Kingman, C.E., Craighead, H.G.,Banker, G.A., “Patterning axonal guidance molecules using a novelstrategy for microcontact printing,” Neurochem Res. 28, 1639-1648 (2003).

34. Tan, W., Desai, T.A., “Microfluidic patterning of cells in extracellu-lar matrix biopolymers: effects of channel size, cell type, and matrixcomposition on pattern integrity,” Tissue Eng. 9, 255-267 (2003).

35. Park, T.H., Shuler, M.L., “Integration of cell culture and microfabri-cation technology,” Biotechnol Prog. 19, 243-253 (2003).

36. Whitesides, G.M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D.E.,“Soft lithography in biology and biochemistry,” Annu Rev BiomedEng. 3, 335-373 (2001).

37. Xia, Y.W., George M., “Soft Lithography,” Angew. Chem. Int. Ed.37, 550-575 (1998).

38. Schmalenberg, K.E., Thompson, D.M., H.M., B., K.E., U., L.F.,G., “In situ stepwise surface analysis of micropatterned glass sub-strates in liquids using functional near-field scanning opticalmicroscopy,” Langmuir. 18, 8593-8600 (2002).

39. Langowski, B.A., Uhrich, K.E., “Microscale plasma-initiated pat-terning (µPIP),” Langmuir. 21, 10509-10514 (2005).

40. Langowski, B.A., Uhrich, K.E., “Oxygen plasma-treatment effectson Si transfer,” Langmuir. 21, 6366-6372 (2005).

41. Clark, P., Britland, S., Connolly, P., “Growth cone guidance andneuron morphology on micropatterned laminin surfaces,” J Cell

166 MinJung Song

Biomaterials Research 2011

Sci. 105 ( Pt 1), 203-212 (1993).42. Csucs, G., Michel, R., Lussi, J.W., Textor, M., Danuser, G., “Micro-

contact printing of novel co-polymers in combination with pro-teins for cell-biological applications,” Biomaterials. 24, 1713-1720(2003).

43. Tan, J.T., J; Chen CS, “Microcontact printing of proteins on mixedself-assembled monolayers,” Langmuir. 18, 519-523 (2002).

44. Gurdon, J.B., Bourillot, P.Y., “Morphogen gradient interpretation,”Nature. 413, 797-803 (2001).

45. Recknor, J.B., Sakaguchi, D.S., Mallapragada, S.K., “Directedgrowth and selective differentiation of neural progenitor cells onmicropatterned polymer substrates,” Biomaterials. 27, 4098-4108 (2006).

46. Song, H.K., Toste, B., Ahmann, K., Hoffman-Kim, D., Palmore,G.T., “Micropatterns of positive guidance cues anchored to poly-pyrrole doped with polyglutamic acid: a new platform for charac-terizing neurite extension in complex environments,” Biomaterials.27, 473-484 (2006).

47. Kam, L., Shain, W., Turner, J.N., Bizios, R., “Axonal outgrowth ofhippocampal neurons on micro-scale networks of polylysine-con-jugated laminin,” Biomaterials. 22, 1049-1054 (2001).

48. Saneinejad, S., Shoichet, M.S., “Patterned glass surfaces direct celladhesion and process outgrowth of primary neurons of the cen-tral nervous system,” J Biomed Mater Res. 42, 13-19 (1998).

49. Lauer, L., Vogt, A., Yeung, C.K., Knoll, W., Offenhausser, A., “Elec-trophysiological recordings of patterned rat brain stem slice neu-rons,” Biomaterials. 23, 3123-3130 (2002).

50. Khademhosseini, A., Langer, R., Borenstein, J., Vacanti, J.P.,“Microscale technologies for tissue engineering and biology,”Proc Natl Acad Sci U S A. 103, 2480-2487 (2006).

51. Sia, S.K., Whitesides, G.M., “Microfluidic devices fabricated inpoly(dimethylsiloxane) for biological studies,” Electrophoresis. 24,3563-3576 (2003).

52. Beebe, D.J., Mensing, G.A., Walker, G.M., “Physics and applica-tions of microfluidics in biology,” Annu Rev Biomed Eng. 4, 261-286 (2002).

53. Kenis, P.J., Ismagilov, R.F., Whitesides, G.M., “Microfabricationinside capillaries using multiphase laminar flow patterning,” Sci-ence. 285, 83-85 (1999).

54. Dertinger, S.K., Chiu, D.L., Li Jeon, N., Whitesides, G.M., “Gener-ation of gradients having complex shapes using microfluidic net-works,” Anal. Chem. 73, 1240-1246 (2001).

55. Gunawan, R.C., Choban, E.R., Conour, J.E., Silvestre, J., Schook,L.B., Gaskins, H.R., Leckband, D.E., Kenis, P.J., “Regiospecific con-trol of protein expression in cells cultured on two-componentcounter gradients of extracellular matrix proteins,” Langmuir. 21,3061-3068 (2005).

56. Lin, F., Nguyen, C.M., Wang, S.J., Saadi, W., Gross, S.P., Jeon, N.L.,“Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration,” Biochem Biophys Res Commun. 319, 576-581(2004).

57. Lin, F., Nguyen, C.M., Wang, S.J., Saadi, W., Gross, S.P., Jeon, N.L.,“Neutrophil migration in opposing chemoattractant gradients usingmicrofluidic chemotaxis devices,” Ann Biomed Eng. 33, 475-482(2005).

58. Li Jeon, N., Baskaran, H., Dertinger, S.K., Whitesides, G.M., Van deWater, L., Toner, M., “Neutrophil chemotaxis in linear and com-plex gradients of interleukin-8 formed in a microfabricated device,”Nat Biotechnol. 20, 826-830 (2002).

59. Burdick, J.A., Khademhosseini, A., Langer, R., “Fabrication of gradi-ent hydrogels using a microfluidics/photopolymerization process,”Langmuir. 20, 5153-5156 (2004).

60. Dertinger, S.K., Jiang, X., Li, Z., Murthy, V.N., Whitesides, G.M.,“Gradients of substrate-bound laminin orient axonal specificationof neurons,” Proc Natl Acad Sci USA. 99, 12542-12547 (2002).

61. Messersmith, E.K., Leonardo, E.D., Shatz, C.J., Tessier-Lavigne, M.,Goodman, C.S., Kolodkin, A.L., “Semaphorin III can function as aselective chemorepellent to pattern sensory projections in the spi-nal cord,” Neuron. 14, 949-959 (1995).

62. Ming, G.L., Wong, S.T., Henley, J., Yuan, X.B., Song, H.J., Spitzer,N.C., Poo, M.M., “Adaptation in the chemotactic guidance ofnerve growth cones,” Nature. 417, 411-418 (2002).

63. Isbister, C.M., Mackenzie, P.J., To, K.C., O'Connor, T.P., “Gradientsteepness influences the pathfinding decisions of neuronal growthcones in vivo,” J Neurosci. 23, 193-202 (2003).

64. Atencia, J., Beebe, D.J., “Controlled microfluidic interfaces,”Nature. 437, 648-655 (2005).

65. Song, H., Poo, M., “The cell biology of neuronal navigation,” NatCell Biol. 3, E81-88 (2001).

66. Dickson, B.J., “Molecular mechanisms of axon guidance,” Sci-ence. 298, 1959-1964 (2002).

67. von Philipsborn, A.C., Lang, S., Loeschinger, J., Bernard, A., David,C., Lehnert, D., Bonhoeffer, F., Bastmeyer, M., “Growth cone nav-igation in substrate-bound ephrin gradients,” Development. 133,2487-2495 (2006).

68. Dubey, N., Letourneau, P.C., Tranquillo, R.T., “Neuronal contactguidance in magnetically aligned fibrin gels: effect of variation ingel mechano-structural properties,” Biomaterials. 22, 1065-1075(2001).

69. Balgude, A.P., Yu, X., Szymanski, A., Bellamkonda, R.V., “Agar-ose gel stiffness determines rate of DRG neurite extension in 3Dcultures,” Biomaterials. 22, 1077-1084 (2001).

70. Lo, C.M., Wang, H.B., Dembo, M., Wang, Y.L., “Cell move-ment is guided by the rigidity of the substrate,” Biophys J. 79,144-152 (2000).

71. Brunelli, G.A., Brunelli, G.R., “Restoration of walking in paraple-gia by transferring the ulnar nerve to the hip: A report on thefirst patient,” Microsurgery. 19, 223-226 (1999).

72. Carlstedt T, G.P., Hallin RG, Noren G, “Return of function afterspinal cord implantation of avulsed spinal nerve roots,” Lancet.346, 1323-1325 (1995).

73. Kannan, R.Y., Salacinski, H.J., Butler, P.E., Seifalian, A.M., “Artificialnerve conduits in peripheral-nerve repair,” Biotechnol Appl Bio-chem. 41, 193-200 (2005).

74. Evans, G.R., Brandt, K., Katz, S., Chauvin, P., Otto, L., Bogle, M.,Wang, B., Meszlenyi, R.K., Lu, L., Mikos, A.G., Patrick, C.W., Jr.,“Bioactive poly(L-lactic acid) conduits seeded with Schwann cellsfor peripheral nerve regeneration,” Biomaterials. 23, 841-848(2002).

75. Ceballos, D., Navarro, X., Dubey, N., Wendelschafer-Crabb, G.,Kennedy, W.R., Tranquillo, R.T., “Magnetically aligned collagen gelfilling a collagen nerve guide improves peripheral nerve regenera-tion,” Exp Neurol. 158, 290-300 (1999).

76. Hadlock, T., Sundback, C., Hunter, D., Cheney, M., Vacanti, J.P.,“A polymer foam conduit seeded with Schwann cells promotesguided peripheral nerve regeneration,” Tissue Eng. 6, 119-127(2000).

77. Taras, J.S., Nanavati, V., Steelman, P., “Nerve conduits,” J HandTher. 18, 191-197 (2005).

78. Evans, G.R., Brandt, K., Niederbichler, A.D., Chauvin, P., Her-rman, S., Bogle, M., Otta, L., Wang, B., Patrick, C.W., Jr., “Clinicallong-term in vivo evaluation of poly(L-lactic acid) porous conduitsfor peripheral nerve regeneration,” J Biomater Sci Polym Ed. 11,869-878 (2000).

Biomaterials and microfabrication for nerve regeneration 167

Vol. 15, No. 4

79. Yoshii, S., Oka, M., Shima, M., Taniguchi, A., Akagi, M., “Bridginga 30-mm nerve defect using collagen filaments,” J Biomed MaterRes A. 67, 467-474 (2003).

80. Kumar, N., Langer, R.S., Domb, A.J., “Polyanhydrides: an over-view,” Adv Drug Deliv Rev. 54, 889-910 (2002).

81. Erdmann, L., Uhrich, K.E., “Synthesis and degradation characteris-tics of salicylic acid-derived poly(anhydride-esters),” Biomaterials.21, 1941-1946 (2000).

82. Schmeltzer, R.A., T.; Uhrich, K, “Optimized synthesis of salicylate-based poly(anhydride-esters),” Polymer Bulletin. 49, 441-448 (2003).

83. Schmeltzer, R.C., Schmalenberg, K.E., Uhrich, K.E., “Synthesis andcytotoxicity of salicylate-based poly(anhydride esters),” Biomacro-molecules. 6, 359-367 (2005).

84. Prudencio, A., Schmeltzer, R. C., Uhrich K. E., “Effect of linkerstructure on salicylic acid-derived poly(anhydride-esters),” Macro-molecules. 38, 6895-6901 (2005).

85. Schmeltzer, R.C., Uhrich, K.E., “Synthesis and characterization ofsalicylic acid-based poly(anhydride-ester) copolymers,” J of Bioac-tive Compatible Poly. 21, 123-133 (2006).

86. Erdmann, L., Macedo, B., Uhrich, K.E., “Degradable poly(anhy-dride ester) implants: effects of localized salicylic acid release onbone,” Biomaterials. 21, 2507-2512 (2000).

87. Bryers, J.D., Jarvis, R.A., Lebo, J., Prudencio, A., Kyriakides, T.R.,

Uhrich, K.E., “Biodegradation of poly(anhydride-esters) into non-steroidal anti-inflammatory drugs and their effect on Pseudomo-nas aeruginosa biofilms in vitro and on the foreign-body responsein vivo,” Biomaterials. 27, 5039-5048 (2006).

88. Harten, R.D., Svach, D.J., Schmeltzer, R., Uhrich, K.E., “Salicylicacid-derived poly(anhydride-esters) inhibit bone resorption andformation in vivo,” J. Biomed. Mater. Res. A. 72, 354-362 (2005).

89. Philp, D., Chen, S.S., Fitzgerald, W., Orenstein, J., Margolis, L.,Kleinman, H.K., “Complex extracellular matrices promote tissue-specific stem cell differentiation,” Stem Cells. 23, 288-296 (2005).

90. Myckatyn, T.M., Mackinnon, S.E., McDonald, J.W., “Stem celltransplantation and other novel techniques for promoting recoveryfrom spinal cord injury,” Transpl Immunol. 12, 343-358 (2004).

91. Tohill, M., Terenghi, G., “Stem-cell plasticity and therapy for inju-ries of the peripheral nervous system,” Biotechnol Appl Biochem.40, 17-24 (2004).

92. Schmidt, C.E., Leach, J.B., “Neural tissue engineering: strategies forrepair and regeneration,” Annu. Rev. Biomed. Eng. 5, 293-347(2003).

93. Carbone, A.L., Song, M.J., Uhrich, K.E., Melt-condensation andsolution polymerization of iodinated salicylate-based poly(anhy-dride-esters) as radiopaque biomaterials, Biomacromolecules.9(6),1604-12 (2008).