This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1. Water/Oil/Water Double Emulsion Microsphere Production
<ol>
	<li>First prepare 2% and 0.5% w/v solutions of polyvinyl alcohol (PVA) in deionized water. Stir the solutions at 50 &#176;C until clear, this may take several hours. Prepare a solution of 2% v/v Isopropyl Alcohol in deionized water.</li>
	<li>Prepare an aqueous solution of desired hydrophilic protein. The table below provides example formulations.</li>
	</ol>
	<img alt="Table 1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> 
	Table 1: Example Protein Solutions. The following protein solutions have been successfully encapsulated and electrospun using this protocol. Other hydrophilic protein solutions can be used as needed.
	
	<ol start="3">
	<li>Place 40 ml of the 0.5% PVA solution into a 50 ml centrifuge tube and set aside.</li>
	<li>In a 15 ml centrifuge tube dissolve 300 mg of 65:35 poly(lactic-co-glycolic acid) (PLGA) in 3 ml of Dichloromethane. A vortex mixer can be used to accelerate PLGA dissolution.</li>
	<li>Combine 200 &#181;l of protein solution and 4 &#181;l of 2% PVA solution. Pour the protein mixture into the PLGA solution (step 1.4). The solutions will remain mostly separate.</li>
	<li>Place the tube into a beaker of ice water. Using a wand sonicator at ~10 Watts (RMS), agitate the solution for a few seconds (5-10) until a uniform creamy white emulsion is created.&#160;</li>
	<li>Pour the emulsion into the 50 ml tube containing 0.5% PVA (step 1.3). Mix the solution at high speed on a vortex mixer for ~20 sec. The solution will develop a cloudy appearance.</li>
	<li>Transfer the emulsion to a 200 ml beaker and place on a stir plate at 350 rpm for 2 min. Add 50 ml of 2% isopropyl alcohol to the beaker on the stir plate. Allow the mixture to continue stirring for a minimum of 1 hr to allow the DCM to evaporate and the PLGA to harden.</li>
	<li>Transfer the microsphere solution into centrifuge tubes.&#160;</li>
	<li>Centrifuge at 425 x g for 3 min. The microspheres will collect at the bottom of the tube and appear white. Carefully remove the supernatant from the tube, above the microspheres, and store in a 500 ml bottle.&#160;</li>
	<li>Rinse the microspheres with deionized water by filling the tube three quarters full and shaking it to redistribute the microspheres in the liquid.&#160;</li>
	<li>Repeat steps 1.10 &#38; 1.11 four times.</li>
	<li>Following the final rinse, remove the supernatant again and place in the 500 ml bottle with the other samples. Freeze the microspheres collected in the centrifuge tube, then lyophilize for at least 24 hr.&#160;</li>
	<li>Visualize the microspheres under a light microscope or with a Scanning Electron Microscope. Microspheres no larger than 60 &#181;m are for effective electrospinning. If microspheres are too large, longer sonicating or vortexing times may be required in step 1.6 or 1.7.</li>
	<li>Store the dried microspheres in a -20 &#176;C freezer.</li>
	<li>Optional: Use a protein assay, per manufacturer instructions, to test the amount of protein in the 500 ml bottle from step 1.1030. This is used to calculate the percent of protein encapsulated in the microspheres, by subtracting the amount in the solution from what was used in the production process.</li>
</ol>
Note: To visualize the protein location in the microsphere add Rhodamine 2 &#181;g/ml to the PLGA solution31 and encapsulate a FITC conjugated protein. Figure 1 shows an example.
2. Electrospinning with Microspheres
<ol>
	<li>Prior to preparing electrospinning solution, create a 0.5% w/v solution of photoinitiator, in deionized water by dissolving at 37 &#176;C. This process can take several hours.</li>
	<li>Create a 2% w/v methacrylated hyaluronic acid (MeHA) (see Burdick et al. for synthesis)29, 3% w/v 900 kD poly(ethylene oxide) (PEO) and 0.05% w/v photo initiator solution in deionized water.&#160;
	<ol>
		<li>Calculate the correct amount of MeHA and PEO for the desired volume. For example, 10 ml of electrospinning solution requires 200 mg of MeHA and 300 mg of PEO.</li>
		<li>Dissolve the PEO in deionized water at 90% of the final volume desired (9 ml for this example). This may take several hours, a heated stir plate or water bath at 37 &#176;C can be used to accelerate the process.</li>
		<li>Next add the MeHA and use a vortex mixer to stir the solution until clear. This will only take a few minutes.</li>
		<li>Finally add the 0.5% photo initiator solution to fill the remaining 10% volume (1 ml for this example).&#160;</li>
	</ol>
	</li>
	<li>Add microspheres at the desired concentration up to 400 mg/ml. Mix the solution on a vortex mixer until the microspheres are evenly distributed in the solution.</li>
	<li>Transfer the solution to a syringe and attach a 6 inch 18 gauge blunt tip needle.</li>
	<li>Place the syringe in a syringe pump and set it to dispense at 1.2 ml/hr.&#160;</li>
	<li>Tape a layer of aluminum foil on the collection plate or mandrel. This allows for easy clean up and storage of the finished scaffold. A rotating mandrel is used to create aligned fibers. A flat plate or stationary mandrel will result in randomly arranged fibers.</li>
	<li>Connect the ground wire from a high voltage power source to the collection apparatus. Connect the positive lead to the needle.</li>
	<li>Adjust the syringe pump and collection surface so that there is 15 cm between the needle tip and collection surface.</li>
	<li>Start the polymer pumping, when the solution is visible at the end of the syringe, turn on the voltage source and set the voltage to 24 kV. CAUTION: Once the voltage is turned on do not touch any metal part of the system.&#160; Charge may also jump short distances from electrified parts to skin.</li>
	<li>Run the solution until desired scaffold thickness is achieved. When complete turn off voltage source and syringe pump.&#160;</li>
	<li>Remove foil with scaffold attached. Completed scaffolds containing protein are stored in a -20 &#176;C freezer.</li>
</ol>
3. Protein Bioactivity Testing
<ol>
	<li>Prepare cell culture media. Add 10% v/v Fetal Bovine Serum, 1% v/v L-Glutamine, and 1% v/v Penicillin-Streptomycin to Dulbecco&#8217;s Modified Eagle&#8217;s Medium.</li>
	<li>Select glass coverslips that fit completely into a well plate.</li>
	<li>Use 3-(Trimethoxysilyl)propyl methacrylate to treat the coverslips as described by the manufacturer. Methacrylation enhances scaffold adherence to the coverslips.&#160;</li>
	<li>Attach methacrylated coverslips to the collection area of the electrospinner with removable double sided tape before electrospinning. Spinning onto the coverslips eases handling and viewing.</li>
	<li>Electrospin to desired thickness as described above.&#160;</li>
	<li>After electrospinning carefully remove coverslips from mandrel. Place the scaffold coated coverslips into a clear nitrogen chamber and ensure that all oxygen is purged.&#160;</li>
	<li>Place the chamber and scaffold under a 10 mW/cm2 365 nm light for 15 min. After crosslinking place into appropriately sized well plate. Ensure that the scaffold side is facing up.&#160;</li>
	<li>Place scaffolds under a germicidal lamp for 30 min to sterilize. If desired fibronectin or other protein is used as a coating to enhance cell adhesion. Follow manufacturer&#8217;s instructions to coat scaffolds.</li>
	<li>Harvest Dorsal Root Ganglia (DRG) as previously described by Hollenbeck32. One DRG will be needed for each scaffold covered coverslip tested.&#160;</li>
	<li>Place 100-200 &#181;l of media on each scaffold in the well plate. Carefully place one DRG on each scaffold in the media droplet. For thick scaffold more media may be needed; DRG need to be fully submerged and not floating.&#160;</li>
	<li>Incubate the scaffold and DRG at 37 &#176;C for 4 hr to allow the cell to adhere to the scaffold.&#160;</li>
	<li>Fill the media to the appropriate level for the well and place back into the incubator. Continue incubating for 4-6 days.</li>
	<li>After the incubation period carefully remove the media from each well and gently wash once with PBS. Fix cells for 30 min using 4% w/v paraformaldehyde.</li>
	<li>Stain cells using an antibody stain for neurofilament. This will allow visualizing of neurite outgrowth for quantification. DAPI can also be used to view nuclei of the cells. An example staining protocol was described by Sundararaghavan and colleagues14.</li>
	<li>Visualize the cells using a fluorescent microscope.
	<ol>
		<li>Place well plate on the stage of the microscope.</li>
		<li>Locate the cell mass using the filter and excitation settings for DAPI.&#160;</li>
		<li>Once the cell is switch the filter to FITC to visualize the extended neurites. Using the stitch function on the microscope collect and combine as many images as necessary to see the entire structure. Repeat for DAPI, FITC and bright field.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wrobel, M. R., Sundararaghavan, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+in+neural+tissue+engineering.">Directed migration in neural tissue engineering.</a> Tissue Eng Part B Rev. , (2013).</li><li>Schmidt, C. E., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering:+strategies+for+repair+and+regeneration.">Neural tissue engineering: strategies for repair and regeneration.</a> Annual Review of Biomedical Engineering. 5, 293-347 (2003).</li><li>Madduri, S., di Summa, P., Papaloizos, M., Kalbermatten, D., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Madduri, S., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+delivery+systems+and+repair+strategies+for+damaged+peripheral+nerves.">Growth factor delivery systems and repair strategies for damaged peripheral nerves.</a> J Control Release. 161, 274-282 (2012).</li><li>Madigan, N. N., McMahon, S., O'Brien, T., Yaszemski, M. J., Windebank, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+tissue+engineering+and+novel+therapeutic+approaches+to+axonal+regeneration+following+spinal+cord+injury+using+polymer+scaffolds.">Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds.</a> Respir Physiol Neurobiol. 169, 183-199 (2009).</li><li>Sundararaghavan, H. G., Monteiro, G. A., Firestein, B. L., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+growth+in+3D+collagen+gels+with+gradients+of+mechanical+properties.">Neurite growth in 3D collagen gels with gradients of mechanical properties.</a> Biotechnol. Bioeng. 102, 632-643 (2009).</li><li>Hudson, T. W., Evans, G. R., Schmidt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+strategies+for+peripheral+nerve+repair.">Engineering strategies for peripheral nerve repair.</a> Clin Plast Surg. 26, 617-628 (1999).</li><li>Kokai, L. E., Bourbeau, D., Weber, D., McAtee, J., Marra, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+growth+factor+delivery+promotes+axonal+regeneration+in+long+gap+peripheral+nerve+repair.">Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair.</a> Tissue Eng Part A. 17, 1263-1275 (2011).</li><li>Bronzino, J. D., Peterson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biomedical Engineering Handbook, Third Edition - 3 Volume Set: Tissue Engineering and Artificial Organs. , (2006).</li><li>Bell, J. H. A., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+nerve+guides:+materials,+fabrication,+growth+factors,+and+cell+delivery.">Next generation nerve guides: materials, fabrication, growth factors, and cell delivery.</a> Tissue Eng Part B Rev. 18, 116-128 (2012).</li><li>Ruiter, G. C. W., Malessy, M. J. A., Yaszemski, M. J., Windebank, A. J., Spinner, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ideal+conduits+for+peripheral+nerve+repair.">Designing ideal conduits for peripheral nerve repair.</a> Neurosurgical focus. 26, (2009).</li><li>Olakowska, E., Woszczycka-Korczy&#324;ska, I., J&#281;drzejowska-Szypu&#322;ka, H., Lewin-Kowalik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nanotubes+and+nanofibres+in+nerve+repair.+A+review.">Application of nanotubes and nanofibres in nerve repair. A review.</a> Folia Neuropathol. 48, 231-237 (2010).</li><li>Gunn, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyblend+nanofibers+for+biomedical+applications:+perspectives+and+challenges.">Polyblend nanofibers for biomedical applications: perspectives and challenges.</a> Trends Biotechnol. 28, 189-197 (2010).</li><li>Sundararaghavan, H. G., Masand, S. N., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+generation+of+haptotactic+gradients+through+3D+collagen+gels+for+enhanced+neurite+growth.">Microfluidic generation of haptotactic gradients through 3D collagen gels for enhanced neurite growth.</a> Journal of Neurotrauma. 28, 2377-2387 (2011).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multiscale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multiscale and photopatterned porosity.</a> Macromol Biosci. 10, 265-270 (2010).</li><li>Edalat, F., Sheu, I., Manoucheri, S., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+strategies+for+creating+artificial+cell-instructive+niches.">Material strategies for creating artificial cell-instructive niches.</a> Current Opinion in Biotechnology. 23, 820-825 (2012).</li><li>Annabi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+highly+porous+crosslinked+elastin+hydrogels+and+their+interaction+with+fibroblasts+in+vitro.">Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro.</a> Biomaterials. 30, 4550-4557 (2009).</li><li>Casta&#241;o, O., Eltohamy, M., Kim, H. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+technology+in+tissue+regeneration.">Electrospinning technology in tissue regeneration.</a> Methods Mol. Biol. 811, 127-140 (2012).</li><li>Chew, S. Y., Wen, J., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+release+of+proteins+from+electrospun+biodegradable+fibers.">Sustained release of proteins from electrospun biodegradable fibers.</a> Biomacromolecules. 6, 2017-2024 (2005).</li><li>Han, D., Gouma, P. -. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+bioscaffolds+that+mimic+the+topology+of+extracellular+matrix.">Electrospun bioscaffolds that mimic the topology of extracellular matrix.</a> Nanomedicine. 2, 37-41 (2006).</li><li>Prabhakaran, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+biocomposite+nanofibrous+scaffolds+for+neural+tissue+engineering.">Electrospun biocomposite nanofibrous scaffolds for neural tissue engineering.</a> Tissue Eng Part A. 14, 1787-1797 (2008).</li><li>Xie, J., MacEwan, M. R., Schwartz, A. G., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibers+for+neural+tissue+engineering.">Electrospun nanofibers for neural tissue engineering.</a> Nanoscale. 2, 35-44 (2010).</li><li>Yao, L., O'Brien, N., Windebank, A., Pandit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orienting+neurite+growth+in+electrospun+fibrous+neural+conduits.">Orienting neurite growth in electrospun fibrous neural conduits.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 90, 483-491 (2009).</li><li>Sill, T. J., von Recum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Ifkovits, J. L., Sundararaghavan, H. G., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+fibrous+polymer+scaffolds+for+tissue+engineering+and+cell+culture.">Electrospinning fibrous polymer scaffolds for tissue engineering and cell culture.</a> Journal of Visualized Experiments: JoVE. , (2009).</li><li>Sundararaghavan, H. G., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+with+depth+in+electrospun+fibrous+scaffolds+for+directed+cell+behavior.">Gradients with depth in electrospun fibrous scaffolds for directed cell behavior.</a> Biomacromolecules. 12, 2344-2350 (2011).</li><li>Meinel, A. J., Germershaus, O., Luhmann, T., Merkle, H. P., Meinel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+matrices+for+localized+drug+delivery:+current+technologies+and+selected+biomedical+applications.">Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications.</a> Eur J Pharm Biopharm. 81, 1-13 (2012).</li><li>Ji, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+electrospun+scaffolds+delivering+growth+factors+and+genes+for+tissue+engineering+applications.">Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications.</a> Pharm. Res. 28, 1259-1272 (2011).</li><li>Burdick, J. A., Chung, C., Jia, X., Randolph, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>P&#233;an, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+HSA+and+NGF+encapsulation+yields+in+PLGA+microparticles.">Optimization of HSA and NGF encapsulation yields in PLGA microparticles.</a> International Journal of Pharmaceutics. 166, 105-115 (1998).</li><li>Cartiera, M. S., Johnson, K. M., Rajendran, V., Caplan, M. J., Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Uptake+and+Intracellular+Fate+of+PLGA+Nanoparticles+in+Epithelial+Cells.">The Uptake and Intracellular Fate of PLGA Nanoparticles in Epithelial Cells.</a> Biomaterials. 30, 2790-2798 (2009).</li><li>Hollenbeck, P. J., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurons: Methods and Applications for the Cell Biologist. , (2003).</li><li>Boer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+sciatic+nerve+repair+with+a+poly-lactic-co-glycolic+acid+scaffold+and+nerve+growth+factor+releasing+microspheres.">Rat sciatic nerve repair with a poly-lactic-co-glycolic acid scaffold and nerve growth factor releasing microspheres.</a> Microsurgery. 31, 293-302 (2011).</li><li>Pujic, Z., Goodhill, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+compartment+diffusion+chamber+for+studying+axonal+chemotaxis+in+3D+collagen.">A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen.</a> Journal of Neuroscience Methods. 215, 53-59 (2013).</li><li>Madduri, S., di Summa, P., Papalo&#239;zos, M., Kalbermatten, D., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphosphoester+microspheres+for+sustained+release+of+biologically+active+nerve+growth+factor.">Polyphosphoester microspheres for sustained release of biologically active nerve growth factor.</a> Biomaterials. 23, 3765-3772 (2002).</li><li>Yan, Q., Yin, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+new+PLGL-RGD-NGF+nerve+conduits+for+promoting+peripheral+nerve+regeneration.">Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration.</a> Biomed Eng Online. 11, (2012).</li><li>Gungor-Ozkerim, P. S., Balkan, T., Kose, G. T., Sarac, A. S., Kok, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+growth+factor+loaded+microspheres+into+polymeric+electrospun+nanofibers+for+tissue+engineering+applications.">Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.</a> J Biomed Mater Res A. , (2013).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+proteins+in+poly(L-lactide-co-caprolactone)+fibers+by+emulsion+electrospinning.">Encapsulation of proteins in poly(L-lactide-co-caprolactone) fibers by emulsion electrospinning.</a> Colloids Surf B Biointerfaces. 75, 418-424 (2010).</li><li>Wang, C. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+aligned+core-shell+nanofibres+delivering+NGF+on+the+promotion+of+sciatic+nerve+regeneration.">The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration.</a> J Biomater Sci Polym Ed. 23, 167-184 (2012).</li><li>Liu, J. -. J., Wang, C. -. Y., Wang, J. -. G., Ruan, H. -. J., Fan, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+regeneration+using+composite+poly(lactic+acid-caprolactone)/nerve+growth+factor+conduits+prepared+by+coaxial+electrospinning.">Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning.</a> J Biomed Mater Res A. 96, 13-20 (2011).</li></ol>

The authors have nothing to disclose.

Many studies have shown that nerve cells can be directed by topographical cues (fiber alignment) and chemical cues (growth factors)1,2,10,11,35. Electrospinning is a facile method to create aligned fibers. Growth factors encourage nerve growth but in order to include them into nerve conduits (NC), a method for sustained release is required. To create a more robust system with both cues, these two signals should be combined. Several methods have been previously studied to provide extended release of protein wit...

One of the ongoing challenges in neural tissue engineering is creating a nerve conduit (NC) that mimics the extra cellular matrix, where nerves grow naturally. Research has shown that cells respond to several factors in their environment including mechanical, topographical, adhesive and chemical signals1-3. One of the primary challenges in this field is determining the appropriate combination of signals and fabricating a system that can maintain cues for an extended period to support cell growth4. Peripheral neurons are known to prefer a soft substrate, be directed by aligned fibers, and respond to nerve growth factor (NGF)5-7. NCs that can provide chemical cues for weeks have been shown to provide improved functional recovery closer to that of allografts, the current gold standard for nerve repair8,9.
Various materials and production methods can be used to produce mechanical and topographical cues10-13. Mechanical cues are inherent to the material chosen, making selection of the appropriate material for the application critical1,13. Production methods to control topographical cues include phase separation, self-assembly and electrospinning1,13. For microscale applications, microfluidics, photopatterning, etching, salt leaches, or gas foams can also be used14-17. Electrospinning has emerged as the most popular way to engineer fibrous substrates for tissue culture due to its flexibility and ease of production13,18-23. Electrospun nanofibers are fabricated by applying a high voltage to a polymer solution causing it to repel itself and stretch across a short gap to discharge24. An aligned scaffold can be created by collecting the fibers on a grounded rotating mandrel and nonaligned scaffolds are collected on a stationary plate25. Adhesion signaling can be achieved by coating the fibrous scaffold with fibronectin or conjugating an adhesion peptide, such as RGD, to the HA before electrospinning26.
Chemical signals, such as growth factors, are the most difficult to maintain over extended periods because they need a source for controlled release. Many systems have been attempted to add controlled release to electrospun fibrous networks with varying levels of success. These methods include blend electrospinning, emulsion electrospinning, core shell electrospinning and protein conjugation27. Additionally, electrospinning is traditionally done in a volatile solvent, which can affect the viability of the protein28, therefore maintaining bioactivity of the protein must be considered.
This approach specifically addresses combining mechanical, topographical, chemical and adhesive signals to create a tunable scaffold for peripheral nerve growth. Scaffold mechanics is precisely controlled by synthesizing methacrylated Hyaluronic Acid (HA). The methacrylation sites are used to attach photo reactive crosslinkers. The crosslinked material is no longer water soluble and is exclusively broken down by enzymes29. The amount of crosslinking changes the degradation rate, mechanics and other physical properties of the material. Using HA with 30% methacrylation, which has a tensile modulus of ~500 Pa, creates a soft substrate that is close to the native mechanics of neural tissue and is typically preferred by neurons26,29. Electrospinning on a rotating mandrel is used to create aligned fibers for a topographical cue. Using electrospinning along with microspheres provides chemical signals within the scaffold over extended periods. To support neurite growth microspheres containing NGF are used to create the chemical signal. Unlike most electrospun materials HA is soluble in water so the NGF does not encounter harsh solvents during production. To add an adhesive signal, the scaffold is coated with fibronectin. The completed system contains all four types of signals described above: soft (mechanical) aligned (topographical) fibers with NGF releasing microspheres (chemical) coated with fibronectin (adhesive). Production and testing of this system is described in this protocol.
The process begins with the production of the microspheres with a Water-in-Oil-in-Water Double Emulsion. The emulsion is stabilized with a surfactant, Polyvinyl Alcohol (PVA). The inner water phase contains the protein. As it is added to the oil phase, containing the PLGA shell material dissolved in Dichloromethane (DCM), the surfactant creates a barrier between the phases protecting the protein from the DCM. This emulsion is than dispersed in another water phase containing PVA to create the outer surface of the microspheres. The stable emulsion is stirred to allow the DCM to evaporate. After rinsing and lyophilizing you are left with the dry microspheres containing the protein.
After the microspheres are completed they are ready to be electrospun into scaffolds. First you prepare the electrospinning solution. The viscosity of the solution is critical to proper fiber formation. Solutions of pure HA do not meet this requirement; PEO is added as a carrier polymer to allow for electrospinning. The microspheres are added to the solution and electrospun resulting in a fibrous scaffold with microspheres distributed throughout.
Once the production is complete, the protein should be tested to verify its viability. To do this, a primary cell that responds to NGF can be used. This protocol uses Dorsal Root Ganglia (DRG) from 8-10 day old chicken embryos. The cell bundles are seeded onto scaffolds containing microspheres filled with NGF or ones that are empty. If the NGF is still viable you should see enhanced neurite growth on the NGF containing scaffolds. If the NGF is no longer viable it will not promote neurites to extend and should appear similar to the control.
The exact procedure described herein is focused on neural support, however, with simple modifications to the material, electrospinning method, and proteins the system can be optimized for various cell and tissue types.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DAPI</td>
			<td style="width:208px;">Invitrogen</td>
			<td style="width:139px;">D1306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Irgacure 2959</td>
			<td style="width:208px;">BASF</td>
			<td style="width:139px;">24650-42-8</td>
			<td style="width:291px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEO 900 kDa</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">189456</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Methacryloxethyl thiocarbamoyl rhodamine B</td>
			<td style="width:208px;">Polysciences, Inc.</td>
			<td style="width:139px;">23591-100</td>
			<td style="width:291px;">Prepare stock solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe Pump</td>
			<td style="width:208px;">KD Scientific</td>
			<td style="width:139px;">KDS100</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Power Source</td>
			<td style="width:208px;">Gamma High Voltage</td>
			<td style="width:139px;">ES30P-5W</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motor</td>
			<td style="width:208px;">Triem Electric Motors, Inc</td>
			<td style="width:139px;">0132022-15</td>
			<td style="width:291px;">Must attach to a custom built mandrel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tachometer</td>
			<td style="width:208px;">Network Tool Warehouse</td>
			<td style="width:139px;">ESI-330</td>
			<td style="width:291px;">Use to monitor mandrel speed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Omnicure UV Spot Cure System with collimating adapter</td>
			<td style="width:208px;">EXFO</td>
			<td style="width:139px;">S1000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Needles</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">14-825-16H</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coverslips</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">12-545-81</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Isoporopyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">I9030-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">BP9703-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">BSA-FITC</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">080M7400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">&#946;-Nerve Growth Factor (NGF)</td>
			<td style="width:208px;">R&#38;D Systems</td>
			<td style="width:139px;">1156-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001554270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dichloromethane</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">34856-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td align="right" style="width:139px;">1856209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001558456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fibronectin</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:139px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:208px;">Atlanta Biologicals</td>
			<td style="width:139px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:208px;">Hyclone</td>
			<td style="width:139px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glutamine</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pen-Strep</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td style="width:208px;">Alfa Aesar</td>
			<td style="width:139px;">A11313&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrospinning growth factor releasing microspheres into fibrous scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

Microspheres 50&#177;14 &#181;m in diameter with an over 85% protein encapsulation have been consistently produced and electrospun into scaffolds. Size was determined by imaging samples of microspheres from three separate production batches. The images where captured on an optical microscope and lengths where measured using commercial lab software. Figure 1 shows a histogram of the size distribution. Encapsulation rate was also tested from three separate microsphere batches, by quantifying the protein th...

Watch this Scientific Journal Video about Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds at JoVE.com

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

Research

JoVE Journal

Bioengineering

12.2K Views.  Wayne State University. This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Video: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...