Integrating chemical and topographical cues to enhance Schwann cell migration in 3D Derek Hernandez July 9, 2012 I’d like to welcome everyone who made.

Integrating chemical and topographical cues to enhance Schwann cell migration in 3D
Derek Hernandez July 9, 2012 I’d like to welcome everyone who made it this morning to listen to my talk about the development and characterization of a versatile 3-dimensional (3D) scaffold to investigate the effects of chemical and topographical cues on Schwann cell (SC) migration and nerve regeneration

Motivation Designed a method to control topography and chemistry in 3D
Chemical Matrix composition Growth factors Cell behavior Migration Adhesion Differentiation Proliferation Contact Matrix stiffness Topography Compliance Cellular Junctions Paracrine signals The extracellular matrix is a complex environment that contains a plethora of signals, which are known to direct cell behaviors such as differentiation, proliferation, and migration. The signals can be separated into 3 categories: chemical cues define the composition of the matrix such as collagen for instance or soluble growth factors that may be present. Contact cues incorporate the mechanical properties of the matrix as well as the fibrillar topography, labeled pink in the image. Other cells also reside in the matrix and can impact cell behavior through direct cell-to-cell contact. We have developed a technique to control the presentation of two of these cues, topographical and bound chemical cues, on a sub-cellular scale in 3D environments and aim to use this technique to elucidate what role these cues play in cell infiltration into scaffolds. Specifically, we aim to assess the impact of these cues on Schwann cell migration, and believe the work will contribute to improve future designs of nerve injury scaffolds. Lust, JR. University of Rochester, Institute of Optics. Scale bar = 2 µm Designed a method to control topography and chemistry in 3D Improve scaffold designs for treating nerve injuries

Schwann cells Major glial cell of the peripheral nervous system
Primary function to support and protect neurons Myelinating and non-myelinating phenotype So, what are Schwann cells? Schwann cells are the major glial cell of the peripheral nervous system. In the adult mammal, two phenotypes of Schwann cells exist. Myelinating Schwann cells form the myelin sheath around axons, forming a protective layer and aiding in the conduction of action potentials. Non-myelinating Schwann cells are present to help maintain smaller axons and neuronal health. The primary function of both phenotypes is to support and protect neurons.

Aid in the repair of nerve injury
Role of Schwann cells Neural Development Post-injury in vivo Aid in the repair of nerve injury Nave, KA and Schwab, MH. 2005 Axons Schwann Cells During neural development SC progenitor cells derived from the neural crest precede the projection of axons. These cells rapidly proliferate and migrate through the ECM, laying down basement membrane and secreting soluble cues to direct and promote axon guidance. Once the axons have reached their targets, SCs either myelinate surrounding axons or remain non-myelinating. After a nerve injury, Schwann cells de-differentiate to precursor-like cells, assist in the clearance of debris, and once again lead the way for regenerating axons. This behavior was observed in vivo by Wes Thompson’s lab after nerve injury. The Schwann cells, stained green here, migrate ahead of the axons, which are stained red, and the axons follow the paths of the SCs. This provided the first evidence that regeneration speed correlated directly with migration speed of Schwann cells (20 um scalebar: cut foreign nerve, distal end transplanted on calf muscle). The question that remains is how do we recreate the developmental environment to promote faster SC migration after a nerve injury. Son, YJ and Thompson, WJ. 1995 Scale bar = 20 µm Regeneration speed correlates directly with SC migration speed How do we recapitulate the developmental environment to promote regeneration?

Desirable properties for nerve guidance channels
Li and Hoffman-Kim. Tiss Eng. Part B. 2008 Numerous biomaterials have been tested for their potential to treat nerve injuries however none have matched the success of the autograft transplant. The schematic shown here represents a collection of desirable biomaterial properties for nerve regeneration scaffolds. These include many of the signals which are found in the ECM such as topographical cues, chemical cues, and cellular cues. All of these cues have been shown to independently and in some cases collaboratively promote cell migration and/or axon extension in vitro but in many cases, the promising results have not translated to in vivo success. For my research I aim to independently control two of these signals, chemical and topographical cues, and assess how they can be utilized to improve SC migration.

Chemical cues Chemotaxis -directed cellular behavior in response to chemical gradients Gradients play a major role in neural development Adams, DN. et al. J Neurobio Scale bar = 50 µm Chemical cues are important because of the their ability to direct cell behaviors such as migration and axon extension through a process known as chemotaxis. An example of this phenomena was displayed by the presentation of immobilized gradients of the laminin derived peptide IKVAV to DRGs. Neurites from the DRG were introduced near the center of the gradient and preferentially extended processes toward the higher concentration of IKVAV. Additionally, Chemotaxis is known to play a critical role in the development of the neural crest and the regionalization of the brain.

Topographical cues Topographical cues in vivo
Topography impacts cell alignment and motility in vitro Mitchell, JA. et. al. PloS ONE. 2011 Evidence of topographical cues in vivo exist, such as the migration of cortical neurons along the tracts of radial glia. Topographical cues have been used extensively with SCs and other cell types to control cell alignment and motility. The majority of topographies investigated are iterations on aligned fibers or grooved substrates, such as the one shown here, which can be easily and rapidly produced using photolithographic methods. The specific substrate for the experiment presented here was laminin-coated, PDMS and the groove width was either 30 or 60 microns. When SCs were seeded on the ridged substrates, migration velocity was significantly increased parallel to the ridges, and most significantly in the grooves. Other studies have shown that feature sizes above 75 microns have little impact on cell behavior. Alternatively though, features on the sub-micron scale can significantly dictate cell behavior.

Bridging the gap Translate research to controllable 3D environments
Decouple the effects of chemical and topographical features Lay the groundwork for future designs of nerve regeneration scaffolds Although it is known SCs display strong responses to chemical and contact cues, limited studies have been conducted in 3D matrices. That is because many of the techniques used to modify materials such as soft lithography and microcontact printing are limited to 2D substrates. Additionally, very few systems are capable of and none have looked specifically at the collaborative effects of chemical and topographical cues on SC behavior in 3D environments. The work I am proposing will control each of these factors independently to truly dissociate their impact on SC behavior. I believe this will be a valuable tool to improve our understanding of Schwann cell behavior and enlighten future designs for nerve regeneration scaffolds.

Research objectives Aim 1:
Develop a benzophenone-based multiphoton immobilization chemistry Aim 2: Create a 3D construct to investigate the effect of gradients and topographies on SC migration Aim 3: Explore the relationship between SCs and neurons My first aim is to develop and characterize a chemistry that allows for the 3D functionalization of crosslinked protein structures without altering their mechanical properties. Protein structures will then be fabricated inside of a hydrogel matrix with various topographies and the chemistry from Aim 1 will be used to immobilize chemical gradients on the structures. These cues will be used both independently and collaboratively to enhance Schwann cell migration and alignment into 3D scaffolds. Finally in Aim 3, co-cultures of Schwann cells and neurons will be seeded on the scaffolds to study the relationship between SC migration and axon extension.

Aim 1: Goals Develop a multiphoton immobilization chemistry to generate gradients of bound chemical cues in user-defined, 3-dimensional patterns Characterize and optimize benzophenone-biotin chemistry Assess the mechanical changes due to benzophenone-biotin immobilization Render protein microstructures biofunctional for SC culture More specifically in Aim 1, we will characterize and optimize a novel benzophenone-based immobilization chemistry to produce gradients of bound substrates on the surface on crosslinked protein structures. As I mentioned previously, the goal is to create these gradients without altering the physical properties of the protein structure, so I will assess the impact of the chemistry on the mechanical properties using atomic force microscopy. Finally, I aim to show we are indeed rendering the crosslinked protein structures biofunctional by using the chemistry to immobilize adhesive peptides to pattern schwann cells or localize adhesion on the structure.

Chemical modification techniques
Method Advantages Disadvantages Soft lithography low cost, simple, 10 nm resolution, rapid 2D patterns Microfluidics (absorption) Cheap, reproducible diffusion limited patterns, large solution volumes required Photolithography Parallel processing, 100 nm resolution expensive, 2D surfaces, unable to control surface chemistry 3D printing 3D low resolution, limited materials, large shear forces Multiphoton lithography sub-micron resolution, 3D time intensive, expensive There are a number of techniques others have used to impart biomaterials with chemical gradients and topographies. Shown here is a brief list of some of these techniques along with their advantages and disadvantages. Soft lithography, microfluidics, and traditional photolithography are mainly limited to modification of surfaces only. 3D printing has expanded rapidly over the last few years but the technique is not yet able to reach the desired microscale resolution necessary and is limited in the type of polymers that can be used. I elected to use multiphoton lithography because it was already a well established technique in Jason’s lab and offers precise control over both the surface chemistry and topography of 3D architectures.

Introduction to multiphoton lithography
Near simultaneous absorption of 2 or more photons Kaehr, B. 2007 Multiphoton lithography is a photolithographic method that requires the simultaneous absorption of 2 or more photons from a focused, usually pulsed laser to an electron which is then excited into a higher energy state. Since this is an extremely low probability event, the excitation is limited to the focal volume of the laser. This differs from single photon excitation where molecules throughout the entire illumination path are excited and provides true 3D control of the excitation region. Courtesy of Brad Amos MRC, Cambridge

Dynamic-mask multiphoton lithography
Ti:S Scan mirror Sample DMD Nielson, R. et al. Small Scale bar = 10 µm Digital micromirror device Coupling multiphoton excitation with a dynamic masking technique we are able to rapidly and reproducibly fabricate 3D protein architectures. On the left is a simplified representation of the optical configuration required. A Ti:S laser, where the central position is represented by the solid red line, is reflected off of a two-axis galvonometer driven scan mirror which rapidly raster scans the beam. The scan position extrema are represented by the dotted lines. The beam is focused and scanned over the face of a digital micromirror device which is an array of reflective mirrors which are either off or on based on an input from an electronic, binary mask. The mirrors in the on position reflect the beam to the back aperture of an objective and onto the sample, which is in a conjugate plane. Therefore the image projected on the DMD gets directly translated to the focal plane of the objective. Rex Nielson used this technique to fabricate the monkey skulls shown here and other complex 3D architectures. nimblegen.com Reproducible and rapid fabrication of 3D protein structures

Protein substrate Bovine serum albumin Gelatin, avidin, lysozyme
66 kDa protein pI = 4.7 Biocompatible Low immunogenic response Gelatin, avidin, lysozyme Kaehr, B. et al. PNAS. 2004 Scale bar = 1 µm The substrate fabricated using multiphoton lithography is a densely crosslinked protein network. The protein I have used for the much of the data that will be presented is BSA. BSA is harvested cows and is water soluble and biocompatible. Crosslinked BSA structures have been used previously in cell experiments with DRGs and NG-108s. It is important to note that we are not limited to using BSA and that other proteins such as gelatin and avidin have been used.

Protocol to immobilize cues on protein structures
1) Fabricate protein structure Concentrated protein solution Photosensitizer High laser intensity 2) Immobilize BP-biotin 2 mg/mL BP-biotin solution Reduced laser intensity Remove fabrication solution First the protein structure is fabricated on the surface of a glass slide by scanning a concentrated solution of protein and photosensitizer with a high laser intensity. The residual fabrication solution is then washed out and replaced with an aqueous solution of benzophenone-biotin. The protein structure is then scanned a second time, at a reduced laser intensity, which covalently bonds the benzophenone to the matrix. By doing the fabrication and functionalization with separate scans, I am able to control the architecture of the protein structure independently of the immobilization. In addition, the immobilized concentration can be tuned without affecting the protein structure as it was initially fabricated because this step is accomplished at a reduced intensity. Once benzophenone-biotin is bonded to the matrix, we can couple a peptide of interest using avidin-biotin binding chemistry. 3) Bind peptide using neutravidin-biotin chemistry Benzophenone-biotin Neutravidin Biotinylated peptide with PEG linker Protein structure Remove BP-biotin solution

Benzophenone immobilization chemistry
Benzophenone-DPEG-Biotin λ = nm First time benzophenone reacted with multiphoton excitation The immobilization chemistry relies on the multiphoton excitation of a benzophenone molecule. Upon exposure to light at a wavelength between nm, a radical is generated at the benzene ring. This radical then inserts into weak carbon-hydrogen sigma bonds in the protein matrix, forming a covalent bond with the matrix (benzene, aliphatic chains, but not extremely specific). Once the benzophenone-biotin is immobilized, neutravidin-biotin binding chemistry can be used to conjugate the peptide or protein of interest as mentioned the last slide. Benzophenone has been used previously with single photon excitation but has never been used in conjunction with multiphoton excitation. Since this immobilization can be achieved at a much lower laser intensity than fabrication, I can tune the concentration of BP-biotin without affecting the mechanical properties of the substrate. Reaction occurs at a lower laser intensity than fabrication

Controlling the degree of immobilization
Benzophenone concentration dependent on laser fluence Laser power (1 scan/plane) Scan number (17 mW) [mW] 7 10 13 16 1 2 4 6 [scans/plane] The concentration of immobilized benzophenone can be tuned by changing the laser fluence, which is an energy per unit area. This can effectively be altered by changing the laser power or increasing the number of laser scans in a single plane. To display this I immobilized step gradients of neutravidin-tmr on the surface of a BSA pad changing the two variables mentioned. In the left image I held the number of scans/plane constant and tuned the power. In the right image, the laser power was held constant and the number of scans/plane varied with each step. The plots below the fluorescence images represent the average pixel intensity and show that the degree of immobilization can be in fact be tuned using these two methods.

Continuous gradients using a Pockel’s cell
Triangle function Sine function Power Range: mW The ultimate goal was to generate continuous gradients, which required the incorporation of a Pockel’s cell into the optical alignment. A Pockel’s cell works by altering the output polarization, and thus the output power, based on a voltage input function. Numerous variables of the input function can be tuned such as waveform, frequency, and amplitude. Here I show the effect of both a triangle and sine function voltage input to the Pockel’s cell on the immobilization. The resultant fluorescence intensities displayed on the plots show nearly a 5X intensity change over 20 microns. The pockel’s cell provides an automated and reproducible way to tune laser fluence and easily translates to producing gradients in 3D. Automated and reproducible modulation of laser fluence

Immobilized gradients on BSA ramps
Front view Isometric view A B B To show that I can in fact use this chemistry to immobilize gradients on 3D substrates I fabricated BSA ramps approximately 10 microns tall and scanned a central region of the ramps for the immobilization. A sawtooth voltage function was used as the pockel’s cell input to immobilize gradients both down and up the ramps. A constant power was also used to show that immobilization is not dependent on ramp height at this scale. An example of each concentration profile is shown in the 3D projections generated from confocal stacks on the left. Over a 20 micron lateral distance and 10 micron height, fluorescence intensity was tunable over a wide range. C C BSA – Blue Scale bars = 10 µm Fluorescent NA - White

Assess the impact of immobilization on the structure
Immobilization does not alter the mechanical properties of the substrate Atomic Force Microscopy (AFM) Surface roughness Elastic modulus The next step was to ensure that the topographical and mechanical changes were not induced by the immobilization scan. To test this, I will analyze the surface roughness and the elastic modulus of structures which have and have not been functionalized using atomic force microscopy.

No effect of immobilization on surface roughness
All structures identically fabricated Performed immobilization at 85% of fabrication power To analyze the surface roughness, I acquired topographical scans of functionalized and non-functionalized structures and calculated the root-mean-squared roughness of each structure. Immobilization was performed at 85% of fabrication power and the number of scans/plane either 0, 2, 4, 6. I’ve already shown that increasing the number of scans/plane increases the immobilization, shown here again on the left, but what was most important here is that significant differences were not observed in surface roughness. *Error bars represent the standard deviation (n=5)

Force mapping to determine elastic modulus
Hertz model I want to do a similarly designed experiment to ensure that the elastic modulus is not being altered during the immobilization. To do this I will acquire 16 stress-strain curves on a single structure using a beaded cantilever. Taking the output force v. extension data, I can calculate the elastic modulus from the Hertz model. I have done preliminary experiments and am trying to identify the most appropriate cantilever to consistently probe the modulus of these structures. Force (N) F = force (N) Rc = radius of bead (m) E = elastic modulus (Pa) δ = indentation (m) v = poisson’s ratio Extension (µm)

Count cells/substrate
2D SC adhesion study Positive control PLL coated coverslip Negative controls Cues: RGD, IKVAV Medium: DMEM, High glucose, 1% FBS Seed SCs Benzophenone Biotin Neutravidin Cue with PEG linker Protein structure As a final touch to Aim 1, I would like to confirm that I am rendering the protein structures biofunctional. I plan to immobilize either the laminin-derived IKVAV peptide or the collagen derived RGD adhesive peptide on the surface of the protein structure to improve SC adhesion or pattern cells on the substrate. The number of cells adhered to functionalized and non-functionalized substrates after 6 hours will be counted and compared. If we have immobilized an adequate concentration of the peptide we should see an increase in cell adhesion on these substrates. 6 - 8 hrs Fix and Image Count cells/substrate Scrambled Peptide

Aim 1 summary Achieved a range of concentrations without altering substrate roughness Applied chemistry to functionalize of patterns on 3D substrates Still need to assess: Elastic modulus SC adhesion To summarize aim 1, I have shown that I can immobilize a range of concentrations without altering the underlying substrate roughness. In addition, I have used this chemistry to immobilize gradients on 3D architectures, such as the BSA ramps. I still need to confirm that there are no changes in modulus and that I can direct SC adhesion with this chemistry.

Aim 2: Goals Develop a 3D construct to study the effects of immobilized chemical gradients and topographies on SC adhesion and migration Incorporate topographical cues and chemical gradients in HA based hydrogels Optimize SC adhesion to protein structures by controlling geometry Investigate SC migration speed and alignment in response to various chemical and topographical cues In Aim 2 I want to use this technology to incorporate topographical and chemical gradients into hydrogels to study Schwann cell migration. Step 1 will be to extend the fabrication and immobilization into a hydrogel environment. Then I plan to optimize the structure geometry for SC adhesion independent of additional cues. Once SC adhesion has been optimized, cues will be incorporated to investigate their effects on SC migration and alignment.

Previous work IKVAV functionalized BSA structures in hydrogels support DRG cell adhesion and migration Limitations Unable to incorporate chemical gradients Structure height limited to ~30 µm Previous experiments by Seidlits have shown that dissociated DRGs adhere to IKVAV functionalized structures in HA gels and migrate into the matrix along the fabricated tracks. A 3D reconstruction of the protein structure is shown in A and cells, fixed after 6 days in culture, migrated down the spiral staircase as shown in the slices in B and D (700 nm increments). I want to add to these promising results by incorporating gradients using the chemistry described in aim 1 and topographies on the protein structure to enhance migration into the hydrogels. (were fixed after 4-7 days in culture. 30% adhesion efficiency without IKVAV) Seidlits, SK. et al. AFM Scale bar = 50 µm

Hyaluronic Acid Natural material Chemically modifiable
Controllable material properties Biocompatible Non cell-adhesive Enzymatically degradable (e.g. hyaluronidase) The base hydrogel we will use for these studies is composed of a hyaluronic acid. Hyaluronic acid is a natural glycosaminoglycan which is extracted from the vitreous humer of the cow’s eye. It is chemically modifiable, such as the methacrylation reaction shown here to produce GMHA, which can be photocrosslinked. Material properties such as crosslinked density and stiffness can be controlled. It is biocompatible and mostly non-cell adhesive, which for the proposed research is a desirable property. Leach, JB. et al. Biotech Bioeng

Fabrication and functionalization in HA gels
1-2% GMHA, 1% I2959 8 hr buffer rinse 2 - 4 min UV exposure To fabricate the hydrogels, first a viscous 1-2 % solution of GMHA and Irgacure is injected into a mold and photocrosslinked using UV light. Unreacted materials are rinsed out and the gel is soaked in a concentrated protein solution for 30 minutes prior to fabrication. Fabrication and functionalization proceed as described in aim 1, however the solution exchanges take longer since we are operating in a hydrogel. 30 min BP-biotin incubation 30 min. in protein solution Buffer wash

Fabrication in HA gels Influenced by basal lamina tubes of native nerve tissue Fabricated BSA tubes 100 µm long Fabrication time = 20 minutes Hudson, TW et al Scale bar = 10 µm In preliminary experiments I have successfully fabricated 10 micron diameter tubes up to 100 microns deep into a hydrogel. This tube geometry was inspired by the basal lamina tubes present in native nerve tissue. Confocal reconstructions of the tubes are shown in A and B from 2 different viewpoints. These tubes are continuous and take approximately 20 minutes to fabricate. Major gridlines = 10 µm

Proposed inner wall topographies
BSA tubes on glass 4 Ridge 8 Ridge Spiral Ridge dimensions: 2 µm tall 1 µm thick Ridge dimensions: 1 µm tall 1 µm thick Spiral dimensions: Extends 1 µm from wall 1 full turn in 15 µm Initially, I am proposing to incorporate 4 ridge, 8 ridge, and spiral topographies inside of these tubes. 3D mask recreations of each topography are shown with relevant dimensions below. A confocal projection of BSA tubes with each of these topographies fabricated on glass is shown on the right. Topographies have been fabricated on these walls with dimensions as small as 1 micron. It is important to note that these dimensions are not fixed and are subject to change depending on the results of the tube geometry experiment. Dimensions are adjustable Scale bar = 10 µm

Aim 2 experimental summary
Topographical Cues Topographical + Chemical Tube geometry Chemical cues First we will take a look at how tube geometry in the absence of additional cues affects cellular interaction with the structures to ensure that we get adequate cell adhesion to perform migration studies. Once the best tube geometry has been established, the impact of chemical and topographical cues on migration and alignment will be assessed independently. Here we will assess what impacts these cues have on SC migration and alignment. We will eliminate the variables that have the least impact on cell migration and alignment and use the best performing variables to conduct a combined cue study. Although there is crosstalk between the pathways that govern topographical and chemical cue integration, there is no reason to believe these cues will not coordinate. Operating under this assumption, I believe it is credible to first look at these two variables independently then combine the cues in a second experiment with a smaller set of variables. Tyrosine phosphorylation signal transduction pathway and rho and rac signaling pathways for topography. Ras and rho/rac pathways control chemotactic signaling. (dynamic formation of adhesion complexes) Adhesion Migration distance Migration distance Cell alignment Migration distance Cell alignment

Optimizing tube geometry for cell adhesion and migration
Variables Inner tube diameter (d): 10 – 30 µm Wall thickness (t): 1 – 10 µm Interstitial spacing (m): 1 – 5 µm Cell density: 30, ,000 cells/gel Criteria for success > 80% of structures with cells d t m First I plan to optimize the tube geometry to promote efficient cell adhesion to the protein tubes. Variables that will be adjusted are the diameter of the tubes, the wall thickness, and the interstitial spacing between the tubes. Arrays of tubes will be fabricated within a single gel and cells will be cultured on the gels for 24 hours. Tube success will be quantified by the # structures in an array which have at least a single cell adhered. Ideally we would like to get cell adhesion on 8 out of 10 structures. Previously reported 30% adhesion efficiency of neurons and SCs to non-modified, linear BSA structures on the surface of GMHA gels. 2-3 fold increase in adhesion to BSA structures when modified with IKVAV.

Independent cue experimental outline
Variable Characteristics (n) Topography (non-functionalized) none (8), 4 ridge (8), 8 ridge (8), spiral (8) Chemical cue IKVAV or RGD Gradient slope (no topography) constant(8), low(8), steep(8) Seed Cell-tracker stained SCs For the second experiment, arrays of tubes will be fabricated inside of a hydrogel possessing either topographical cues or chemical cues. The specific variables that will be tested are outlined in the table with the proposed sample number. Schwann cells will be stained with cell-tracker orange for visualization prior to culturing. Cultures will be fixed at time points of 4, 12 and 24 hours and migration distance will be quantified by the nuclear position of the cells. An average migration speed will be calculated as well. Cell alignment will also be quantified based on the end-to-end angle of the cell relative to the z-axis. Fix at 4, 12, and 24 hours Assess: Migration speed v. controls Cell alignment (end-end angle) DAPI Stain Protein tube Functionalized protein tube Schwann cell Legend Confocal Microscopy

Combined cue experimental outline
Take the two best performing cues from each group and combine (4 combinations) Seed Cell-tracker stained SCs Assess: Migration speed v. controls Cell alignment (end-end angle) Compare to individual cue results For the combined cue experiment I will take the 2 best performing cues from each group and pair them within single tubes. Once again I will take a look at migration speed and cell alignment and compare the results to those of the independent cue experiment. The goal is to assess whether a synergistic effect is indeed observed and if cell infiltration can be further enhanced by combining these cues. Fix at 4, 12, and 24 hours DAPI Stain Protein tube Functionalized protein tube Schwann cell Legend Confocal Microscopy

Aim 2 summary Developed a dual-scaffold system to incorporate chemical and topographical cues into hydrogels Employ scaffolds to thoroughly investigate SC migration and alignment In summary of aim 2, I have shown that we have developed a dual-scaffold system by fabricating protein structures with micron scale features inside of HA gels and plan to employ these scaffolds to study SC migration and alignment in 3D environments.

Aim 3: Goals Study the relationship between SC migration and neurite extension by seeding dissociated DRGs onto scaffolds Determine if SC migration speed directly correlates to neurite extension Determine if scaffolds pre-seeded with SCs improve neurite extension rates My third aim is to seed dissociated DRG’s on these scaffolds to determine if increased migration speed will indeed increase axon extension rates. I also want to use this system to test whether a pre-seeded scaffold improves axon extension rates.

Crosstalk between SCs and neurons
SCs promote neurite extension by secreting diffusible signals Nerve growth factor, brain derived neurotrophic factor, glial derived neurotrophic factor, neurotrophic factor-3 SC alignment promotes neurite alignment and extension SC incorporation into scaffolds to treat nerve injury There is ample evidence to support communication between SCs and neurons. Some of this communication occurs through diffusible signals released by SCs, such as growth factors, to promote axon extension. SC alignment has also been shown to promote neurite alignment and extension. Because of these findings, many researchers are beginning to use SCs and other cell types to treat nerve injuries. Cells have been delivered both in a scaffold and by injection at the site of injury. Many questions remain regarding what role these cells play in treating nerve injuries and what the appropriate delivery time may be.

Dissociated dorsal root ganglia
Contain neurons and glia Model for peripheral nerve repair Rapidly extend neurites in vitro To explore the relationship between SCs and neurons I plan to use dissociated DRGs, which are located along the vertebral column of the spine and contain a combination of neuronal and glial cells. They are commonly used as a model for peripheral nerve repair and rapidly extend neurites in culture. For example, on 2D substrates they can extend processes as quickly as 30 microns per hour and in 3D collagen gels have extended processes up to 150 microns over a 3 day period. At 3 days in a collagen matrix neurites extended up to 150 microns (hoffman-kim). With laminin gradients on 2D substrates can extend up to 30 microns/hour (bellamkonda).

Neurite extension protocol
Use best performing tube/gradient combinations from Aim 2 Quantify neurite extension and alignment Compare SCs response to Aim 2 Seed dissociated DRGs The protocol for here is schematically identical to the combined cue SC migration protocol except I will be using dissociated DRGs and a different staining method to identify neurites and Schwann cells. The time points at which I will fix are highly dependent on the results of aim 2 but based on neurite extension rates published in 3D I have recommended 12 and 24 hours here. For the analysis I would like to quantify neurite extension and alignment in modified tubes versus unmodified tubes. I will also determine if the presence of neurons have an effect on the migratory behavior of the Schwann cells. Fix at time = 12 and 24 hours Protein tube Functionalized protein tube Schwann cell Legend Neuron Stain (DAPI, Neurofilament, S100) Confocal Microscopy

Do pre-seeded SC scaffolds further enhance neurite extension
Allow SCs to infiltrate matrix 4 and 24 hours prior to seeding DRGs Compare neurite extension rates to scaffolds that are not pre-seeded Seed Cell-tracker stained SCs Seed dissociated DRGs Additionally, I would like to test the effect of pre-seeding scaffolds with SCs on the rates of neurite extension by allowing SCs to infiltrate the matrix 4 and 24 hours prior to seeding the DRGs. The pre-seeded SCs in this case will be distinguishable from the SCs of the DRG because they will be stained with cell tracker. Allowing the pre-seeded SCs to infiltrate the matrix for either a short or long period of time before presenting the neurons could have a significant effect on neurite extension. Comparing the results here to the previous experiment, the control, should also provide valuable insight regarding the use of SCs for the treatment of nerve injury. Protein tube Functionalized protein tube Schwann cell Legend Neuron Stain (DAPI, Neurofilament, S100)

Proposed timeline I want to conclude with my proposed experimental timeline. I am wrapping up aim 1 and ramping up to continue aim 2 currently. I aim to begin work on the third aim in the middle of next year and defend by the end of 2013.

Acknowledgements Advisors: Committee: Dr. Christine Schmidt
Dr. Jason Shear Committee: Dr. Lydia Contreras Dr. Chris Ellison Dr. Wesley Thompson I would like to thank my advisors, Christine Schmidt and Jason Shear, as well as my committee members and coworkers from both labs.

Multiphoton reaction details
Triplet state of photosensitizer produces singlet oxygen Singlet oxygen is a highly reactive species Aromatics – tyrosine, tryptophan Thiols - cysteine Amines - lysine, arginine Alkenes

Competing multiphoton immobilization chemistries
Mono-acrylated-PEG PEG-DA hydrogel N-vinyl pyrrolidone 2,2-dimethoxy-2-phenylacetophenone Coumarin-maleimide Coumarin modified agarose gels Fluorescein-biotin Mono-acrylated-PEG modified glass Hoffman, JC et al. Soft Matter. 2010 Wylie, RG. et al. Nature Materials. 2011 Scott, MA. et al. Lab on a Chip. 2012

Experimental questions
Topography: Which topography best promotes migration? Do topographies dictate cell alignment? Chemical cue: Which cue best promotes migration? Do gradients increase migration speed? Do gradients contribute to cell alignment? Combinatorial studies: Do topographical and chemical cues have a synergistic effect?

Crosstalk between SCs and neurons
SCs promote axon extension by secreting diffusible signals Aligned SCs promote neurite alignment and extension Armstrong, SJ. et al. Tissue Eng. 2007 Seggio, AM. et al. Journal of Neural Eng. 2010

Current peripheral repair strategies
Size of nerve gap: < 1 mm 5-7 cm > 7 cm Not necessary Leach, JB. And Schmidt, CE. Ann Rev Biomed Eng Need to develop biomaterial scaffolds to improve functional nerve regeneration over larger gap distances

Integrating chemical and topographical cues to enhance Schwann cell migration in 3D Derek Hernandez July 9, 2012 I’d like to welcome everyone who made.

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