Name: polyelectrolyte complex for heparin binding domain osteogenic growth factor delivery
Uploaded: 2016-08-22T14:00:00
Description: Watch this Scientific Journal Video about Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery at JoVE.com

These studies were funded by National Medical Research Council Clinician Scientist - Individual Research Grant (CS-IRG) NMRC/CIRG/1372/2013 and NMRC EDG/0022/2008.

1. Alginate Solution Preparation
<ol>
	<li>Dissolve 200 mg of sodium alginate (non-irradiated) or 400 mg of 8 MRad irradiated sodium alginate in 10 ml double distilled water and shake for 1 hr for non-radiated alginate and 15 min for irradiated alginate. Store the alginate solution at 4 &#176;C overnight. Filter the alginate solution with a sterile 0.2 &#181;m syringe filter before alginate microbead fabrication.</li>
</ol>
2. Alginate Microbead Fabrication
<ol>
	<li>Disinfect the electrostati...

<ol>
	<li>Yuan, 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=NELL-1+based+demineralized+bone+graft+promotes+rat+spine+fusion+as+compared+to+commercially+available+BMP-2+product.">NELL-1 based demineralized bone graft promotes rat spine fusion as compared to commercially available BMP-2 product.</a> Orthop Sci. 18, 646-657 (2013).</li><li>Anderson, C. L., Whitaker, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+associated+with+recombinant+human+bone+morphogenetic+protein-2+(infuse)+in+posterolateral+lumbar+spine+fusion:+a+case+report.">Heterotopic ossification associated with recombinant human bone morphogenetic protein-2 (infuse) in posterolateral lumbar spine fusion: a case report.</a> Spine. 37, 502-506 (2012).</li><li>Glassman, S. D., 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=RhBMP-2+versus+iliac+crest+bone+graft+for+lumbar+spine+fusion:+a+randomized,+controlled+trial+in+patients+over+sixty+years+of+age.">RhBMP-2 versus iliac crest bone graft for lumbar spine fusion: a randomized, controlled trial in patients over sixty years of age.</a> Spine. 33, 2843-2849 (2008).</li><li>Tannoury, C. A., An, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+with+the+use+of+bone+morphogenetic+protein+2+(BMP-2)+in+spine+surgery.">Complications with the use of bone morphogenetic protein 2 (BMP-2) in spine surgery.</a> Spine J. 14, 552-559 (2014).</li><li>Carragee, E. J., Hurwitz, E. L., Weiner, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+recombinant+human+bone+morphogenetic+protein-2+trials+in+spinal+surgery:+emerging+safety+concerns+and+lessons+learned.">A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned.</a> Spine J. 11, 471-491 (2011).</li><li>Abbah, S. A., Lam, C. X., Hutmacher, D. W., Goh, J. C., Wong, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+performance+of+a+polycaprolactone-based+scaffold+used+as+fusion+cage+device+in+a+large+animal+model+of+spinal+reconstructive+surgery.">Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery.</a> Biomaterials. 30, 5086-5093 (2009).</li><li>Abbah, S. A., Liu, J., Lam, R. W., Goh, J. C., Wong, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioactivity+of+rhBMP-2+delivered+with+novel+polyelectrolyte+complexation+shells+assembled+on+an+alginate+microbead+core+template.">In vivo bioactivity of rhBMP-2 delivered with novel polyelectrolyte complexation shells assembled on an alginate microbead core template.</a> J. Control. Release. 162, 364-372 (2012).</li><li>Wang, 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=Polyelectrolyte+Complex+Carrier+Enhances+Therapeutic+Efficiency+and+Safety+Profile+of+Bone+Morphogenetic+Protein-2+in+Porcine+Lumbar+Interbody+Fusion+Model.">Polyelectrolyte Complex Carrier Enhances Therapeutic Efficiency and Safety Profile of Bone Morphogenetic Protein-2 in Porcine Lumbar Interbody Fusion Model.</a> Spine. 40, 964-973 (2015).</li><li>Abbah, S. A., Lam, W. M., Hu, T., Goh, J., Wong, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestration+of+rhBMP-2+into+self-assembled+polyelectrolyte+complexes+promotes+anatomic+localization+of+new+bone+in+a+porcine+model+of+spinal+reconstructive+surgery.">Sequestration of rhBMP-2 into self-assembled polyelectrolyte complexes promotes anatomic localization of new bone in a porcine model of spinal reconstructive surgery.</a> Tissue Eng. Part A. 20, 1679-1688 (2014).</li><li>Hu, T., 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=Novel+Protamine-Based+Polyelectrolyte+Carrier+Enhances+Low-Dose+rhBMP-2+in+Posterolateral+Spinal+Fusion.">Novel Protamine-Based Polyelectrolyte Carrier Enhances Low-Dose rhBMP-2 in Posterolateral Spinal Fusion.</a> Spine. 40, 613-621 (2015).</li><li>Hu, J., Hou, Y., Park, H., Lee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-tricalcium+phosphate+particles+as+a+controlled+release+carrier+of+osteogenic+proteins+for+bone+tissue+engineering.">Beta-tricalcium phosphate particles as a controlled release carrier of osteogenic proteins for bone tissue engineering.</a> J Biomed Mater Res A. 100, 1680-1686 (2012).</li><li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L-Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Li, X., Min, S., Zhao, X., Lu, Z., Jin, A. <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+entrapping+conditions+to+improve+the+release+of+BMP-2+from+PELA+carriers+by+response+surface+methodology.">Optimization of entrapping conditions to improve the release of BMP-2 from PELA carriers by response surface methodology.</a> Biomed Mater. 10, 015002 (2015).</li></ol>

This protocol presents a method for the preparation of PECs through layer-by-layer self-assembly. The layer-by-layer structure is visualized using fluorescent analogues of protamine, heparin, BMP-2 and NELL-1 and confocal microscopy. Uptake and release tests show that heparin on PEC mediates osteogenic growth factor uptake and release. The uptake efficiency of the PEC method is: NELL-1: 86.7 &#177; 2.7%, BMP-2: 70.5 &#177; 3.1%. The PEC carrier has a better modulation of NELL-1 (20%) release compared to a pure surface ad...

The incidence of pseudoarthrosis has been reported to be as high as 10 to 45% in degenerative spinal fusion and revision spinal surgeries1. To reduce the rate of pseudarthrosis during spine fusion and other reconstructive bone surgeries, osteogenic growth factors such as BMP-2, Nell-11 and platelet derived growth factor (PDGF) have been introduced to promote de novo osteogenesis. Among these, BMP-2 is a popular choice for spinal fusion2. Although the potency of BMP-2 in inducing and facilitating new bone formation has been well established3; clinically significant complications such as heterotopic bone formation, seroma and hematoma formation, inflammatory response, radiculitis, vertebral body osteolysis, and retrograde ejaculation continue to be issues of concern due to the supraphysiological amounts used4,5.
Therefore, lowering the dose of BMP-2 remains a relevant strategy in attempts to minimize side effects. Besides, efficient carrier systems are required to suppress the initial burst release of BMP-2 observed in contemporary collagen sponge carrier systems and further enhance prolonged and localized delivery of this potent cytokine. The layer-by-layer self-assembly of alternating cationic and anionic polyelectrolytes can be employed as a tunable method to build up polyelectrolyte complexes on the surface of scaffold matrices or implantable materials6. In this respect, heparin (known for having the highest negative charge density of all biological agents) has been recognized to avidly bind with a variety of growth factors via electrostatic and heparin binding domains. Indeed, heparin has been shown to prolong the half-life and thus potentiate the bioactivity of several growth factors.
Based on this, our group adapted a layer-by-layer self-assembly protocol to fabricate a heparin-based polyelectrolyte complex (PEC) that loads and preserves the bioactivities of osteogenic growth factors during immobilization7,8. The alginate microbead core was fabricated by crosslinking &#945;-L-guluronate (G) residues of alginate with divalent cation calcium or strontium ions. The alginate core is a biodegradable scaffold matrix; which after implantation, it is resorbed in the fusion bed providing room for bony ingrowth. Poly-L-lysine (PLL) or protamine is used as the cationic layer to interlace with both the scaffold matrix (in this case, the alginate microbead carrier core) and the negatively charged heparin; while the anionic heparin layer functions to stabilize and localize loaded growth factors. The triple layer PEC has been shown to increase growth factor loading capacity in a porcine model9. Recently, PEC carriers have been shown to successfully reduce the effective dose of BMP-2 by at least 20-fold in rat10 and porcine models of spinal fusion8.
Here, we report the methods of fabricating PECs for enhanced growth factor delivery in spinal fusion and the other reconstructive bone surgeries using BMP-2 as a model osteogenic growth factor.

<table border="0" cellpadding="0" cellspacing="0" width="981">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Life Science Acrodisc 25mm Syring Filter w/0.2 &#181;m Supor&#160; Membrane</td>
			<td style="width:167px;">PALL&#160;</td>
			<td style="width:79px;">PN4612</td>
			<td style="width:311px;">Sterile protamine, 
			&#160;heparin solution by ultrafiltration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">24 well plate</td>
			<td style="width:167px;">Cell Star&#160;</td>
			<td style="width:79px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">96 well plate Nuclon Delta Surface</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">167008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), MTT</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">M5655</td>
			<td>Measure cytotoxicity of PEC-NELL-1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Acetone</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:79px;">A/0600/17</td>
			<td style="width:311px;">Precipitate CF-405 
			Labelled protamine&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Alamar Blue</td>
			<td style="width:167px;">Invitrogen, Life Technologies</td>
			<td style="width:79px;">DAL 1025</td>
			<td>Measure cytotoxicity of PEC-BMP-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Alkaline Phosphatase Assay (ALP) assay kit</td>
			<td style="width:167px;">Anaspec</td>
			<td style="width:79px;">AS-72146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Ammonium Chloride</td>
			<td style="width:167px;">Merck</td>
			<td style="width:79px;">Art 1145</td>
			<td>Stop reagent in FITC labelling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Anhydrous Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:167px;">Invitrogen, Life Technologies</td>
			<td style="width:79px;">D12345</td>
			<td style="width:311px;">Solvent for fluorescent isothiocyanate I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;"></td>
			<td style="width:311px;">Dissolve&#160; formazan&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Autoclave</td>
			<td style="width:167px;">Hirayama</td>
			<td style="width:79px;">HU-110</td>
			<td>Sterilize alginate beads by steam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beta-glycerophosphate</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">G9422</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">BMP-2 (Infuse Bone Graft Large II Kit)&#160;</td>
			<td style="width:167px;">Medtronic Sofarmor Danek, Memphis TN, USA</td>
			<td style="width:79px;">7510800</td>
			<td style="width:311px;">Osteogenic Growth&#160; Factor, 
			&#160;dialysis is needed to remove stabilizer component that interferes with FITC coupling</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:425px;">Carboxybenzoyl quinoline-2-Carboxaldehyde (CBQCA)&#160;</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">A-6222</td>
			<td>To quantify NELL-1 protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Cell Strainer (100&#181;m)</td>
			<td style="width:167px;">BD Science</td>
			<td style="width:79px;">352360</td>
			<td>Hold PEC for ALP assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Cell Scraper 290mm Bladewide 20mm</td>
			<td style="width:167px;">SPL Life Science&#160;</td>
			<td style="width:79px;">90030</td>
			<td>Detach the cell from 24 well plate&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">CF 405S, Succinimidyl Ester</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">SCJ4600013</td>
			<td>Blue fluorescent dye for protamine labelling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">CF 594, Hydrazide</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">SCJ4600031</td>
			<td>Deep red fluorescent dye for heparin labelling&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Centrifuge</td>
			<td style="width:167px;">Beckman Coulter</td>
			<td style="width:79px;">Microfuge 22R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Confocal Microscope</td>
			<td style="width:167px;">Olympus&#160;</td>
			<td style="width:79px;">FV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Dexamethasone</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">D4902</td>
			<td>Component of osteogenic growth medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Dextran Desalting Columns</td>
			<td style="width:167px;">Pierce (Thermo Scientific)&#160;</td>
			<td style="width:79px;">43230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">DMEM</td>
			<td style="width:167px;">Gibco&#160;</td>
			<td style="width:79px;">12320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">BMP-2 Quantikine ELISA Kit</td>
			<td style="width:167px;">R&#38;D System</td>
			<td style="width:79px;">DBP200</td>
			<td>Determine BMP-2 release</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Fetal Bovine Serum FBS</td>
			<td style="width:167px;">Hyclone</td>
			<td style="width:79px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Fluoescein Isothiocyananate, Isomer I</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">F7250</td>
			<td>Green fluorescent dye for NELL-1 and BMP-2 labelling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">ThinCert Cell Culture Inserts, 
			For 24 Well plates, Sterile</td>
			<td style="width:167px;">Greiner&#160;</td>
			<td style="width:79px;">662630</td>
			<td style="width:311px;">Prevents PEC wash out when 
			&#160;changing osteogenic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Havard Appartus Syringe Pump (11 plus)</td>
			<td style="width:167px;">Havard Apparatus</td>
			<td style="width:79px;">70-2208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">n-Hexane (&#62;99%)</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">139386</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Heparin</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">H3149</td>
			<td style="width:311px;">Binds with osteogenic 
			growth factor with heparin binding domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Hydrochloric acid (37%)</td>
			<td style="width:167px;">Merck</td>
			<td style="width:79px;">100317</td>
			<td>Highly Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Incubator</td>
			<td style="width:167px;">Binder</td>
			<td style="width:79px;">C8150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">MicroBCA Protein Assay kit</td>
			<td style="width:167px;">Thermoscientific</td>
			<td style="width:79px;">23235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Microplate Reader</td>
			<td style="width:167px;">Tecan</td>
			<td style="width:79px;">Infinite M200</td>
			<td>For ALP and microBCA assays</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">NELL-1</td>
			<td style="width:167px;">Aragen Bioscience Morgan Hill, CA, USA</td>
			<td style="width:79px;">N/A</td>
			<td>Osteogenic growth factor, keep at -80&#730;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">Nisco cell encapsulator</td>
			<td style="width:167px;">Nisco Engineering Inc</td>
			<td style="width:79px;">Encapsulation unit VAR V1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Fluorescent Microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:79px;">IX71</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">mPCL-TCP Scaffold (Pore size is 1.3mm)</td>
			<td style="width:167px;">Osteopore</td>
			<td style="width:79px;">PCL-TCP 0/90</td>
			<td>Hold PEC for in vivo study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Penicillin-Streptomycin 10,000 unit/ml, 100ml</td>
			<td style="width:167px;">Hyclone Cell Culture</td>
			<td style="width:79px;">SV30010</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">10X Phosphate Buffered Saline (PBS)&#160;</td>
			<td style="width:167px;">Vivantis</td>
			<td style="width:79px;">PB0344-1L</td>
			<td>10x Solution, Ultra Pure Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Poly-L-Lysine MW 15,000-30,000</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">P2568</td>
			<td>Polycation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Protamine Sulfate salt, from Salmon</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">P4020</td>
			<td>Polycation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Shaker</td>
			<td style="width:167px;">Labnet</td>
			<td style="width:79px;">S2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Snakeskin Dialysis Tubing 3,500 MWCO 22mm x 35 feet</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:79px;">68035</td>
			<td>Remove unreacted FITC by dialysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Sodium Chloride</td>
			<td style="width:167px;">Merck</td>
			<td style="width:79px;">1.06404.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Sodium Hydroxide</td>
			<td style="width:167px;">Qrec</td>
			<td style="width:79px;">S5158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Sodium Bicarbonate</td>
			<td style="width:167px;">US Biological</td>
			<td style="width:79px;">S4000</td>
			<td>Buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Sodium carbonate</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">S7795-500G</td>
			<td>Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Strontium Chloride Hexahydrate</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">255521</td>
			<td>Crosslinker for alginate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Spatula</td>
			<td style="width:167px;">3dia</td>
			<td style="width:79px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">5ml syringe</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:79px;">140425R</td>
			<td style="width:311px;">Diameter of syringe 
			affects the flow rate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">75cm2 Cell Culture Flask Canted Neck</td>
			<td style="width:167px;">Corning</td>
			<td style="width:79px;">730720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:425px;">Toluidine Blue&#160;</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:79px;">52040</td>
			<td>Heparin assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:425px;">Trypsin 1X</td>
			<td style="width:167px;">Hyclone Cell Culture</td>
			<td style="width:79px;">SH30042.01</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:425px;">Sodium alginate</td>
			<td style="width:167px;">Novamatrix (FMC Biopolymer, Princeton, NJ)</td>
			<td style="width:79px;">Pronova UPMVG</td>
			<td>Core material of microbeads</td>
		</tr>
	</tbody>
</table>

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.

In our carrier, protamine was chosen as a substitute of poly-L-lysine as it has similar chemical properties and it is FDA approved as an antidote of heparin. Optical microscope results showed that the non-irradiated microbeads were spherical in shape with a diameter of 267 &#177; 14 &#181;m. (0.35 mm nozzle, flow rate of 5 ml/hr &#38; 5.8 kV). The majority of the irradiated microbeads are of teardrop shape. The diameter measured on the round portion of the irradiated microbeads was 212 &#...

Watch this Scientific Journal Video about Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery at JoVE.com

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

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 ...

The overall goal of this procedure is to fabricate a polyelectrolyte complexation shell. A carrier that can entrap and regulate the release of a wide range of growth factors with a heparin binding domain. This method can answer key questions in orthopedic tissue engineering field for efficient entrapment and delivery dedicate growth factors.The main advantages of this technique is it does not require usage of organics often. Which is known to affect the bio activities of growth factors. Dissolve 200 milligrams of non irradiated sodium alginate or 400 milligrams of eight megarad irradiated sodium alginate in 10 milliliters of double distilled water.And shake for one hour. Store the alginate solution at four degrees celsius overnight. Filter the alginate solution with a sterile 0.2 micron syringe filter before alginate microbead fabrication.Disinfect the electrostatic BE generator and syringe pump with 70%ethanol and place them in a class two biological safety cabinet. Then place a glass basin with a magnetic stir bar inside the BE generator. Set the arm electrode of the BE generator nine centimeters above the basin.Connect the electrode cable of the BE generator to the neuro screw two of the arm electrode. And pour 80 milliliters of strontium chloride solution into the basin. Next, load five milliliters of 0.2 micron filtered alginate solution into the syringe in rubber tube.After connecting the rubber tube to the arm electrode, switch on the syringe pump at five milliliters per hour. For two minutes. To expel the air inside the tubing and deliver the alginate solution to the tip of the nozzle.Then, turn off the syringe pump. To commence microbead generation, set the encapsulator to 5.8 kilovolts. Followed by the syringe pump at an alginate flow rate of five milliliters per hour.Discard the microbeads generated during the first two minutes as these microbeads tend to be irregularly sized and shaped. Collect subsequent microbeads in the 0.2 molar strontium chloride solution. Turn off the syringe pump followed by the encapsulator.After pumping the preplanned volume of alginate solution. Repeat this for subsequent batches of microbead fabrication. Store the microbeads in 20 milliliters of 0.2 molar strontium chloride solution at four degrees celsius overnight to complete cross linking and stabilize the gel.Collect 0.5 milliliters of alginate microbeads with a plastic pipette. And place it on a glass slide. View the microbeads under an optical microscope at 10x magnification.Take 10 images of the microbeads with a microscope CCD camera. Save the images with a 500 micron scale bar in TIFF format at a resolution of 2048 by 1536. Using the length tools in image J, measure the size of the microbeads.And the scale bar. Then convert the microbead length from pixels to micrometers. Click on the line tools and draw a line across the middle of the alginate bead.Click analyze on the menu bar and select measure. A pop up window will appear. Repeat these steps to measure all alginate beads within the image.Measure the scale bar on the image. Collect the microbeads using the 100 micron nylon strainer. And wash the beads with double distilled water.Using a spatula, transfer all the microbeads made from 0.1 milliliters of the alginate solution into a two milliliter microcentrifuge tube and cover with gauze to prevent drying. Finally, sterilize the microbeads by auto cleaving using liquid mode or in accordance with manufacturer's specifications. Add 1.5 liters of distilled water to the chamber to prevent beads from drying.Inside the BSL2 hood, incubate the sterile microbeads with one milliliter of sterilized two milligrams per milliliter protamine solution for one hour at room temperature. Wash the protamine coated microbeads twice with double distilled water. Then spin down, using a benchtop centrifuge at 200 times G for three minutes at room temperature.After centrifugation, aspirate the water using a syringe. Incubate protamine coated microbeads with one milliliter of sterilized 0.5 milligrams per milliliter heparin solution for 30 minutes. This creates a polyelectrolyte complexation shell with avid affinity for heparin binding growth factors.After incubation, aspirate away the heprain by using a syringe. And wash off the unbound heparin from the polyelectrolyte complexes by washing twice with double distilled water. Load 13.3 microliters of 1.5 milligram per milliliter bone morphogenetic protein 2 or NELL like molecule 1 solution onto 100 micrograms of the polyelectrolyte complex.Incubate the polyelectrolyte complex at four degrees celsius under 30 RPM, shaking for 10 hours. Next, immerse the microbeads in one milliliter of phosphate buffered saline, or PBS, at 37 degrees celsius with constant shaking. Collect one milliliter of the supernatant and replace it one milliliter of PBS after one, three, six, 10 and 14 days.Evaluate the uptake and release efficiency of NELL like molecule one using the CBQCA protein assay method according to the manufacturer's protocol. Alternatively, perform elisa. The bioactivity of NELL like molecule one released from the polyelectrolyte complex is assessed by measuring its ability to increase the expression of alkaline phosphatase in rabbit bone marrow stem cells.Seed 20, 000 rabbit bone marrow stem cells per well in a 24 well plate. And allow them to grow for one day with one milliliter of dulbecco's modified eagle's medium plus 10%fetal bovine serum at 37 degrees celsius and 5%CO2. After 24 hours, replace the medium with one milliliter of an osteogenic medium for seven days at 37 degrees celsius.And 5%CO2. Place 300 micrograms of polyelectrolyte complex and polyelectrolyte complex NELL like molecule one inside cell culture inserts to keep the complexes separate from the cells. Place the insert into the 24 well plate to avoid the wash out of polyelectrolyte complex microbeads during the osteogenic medium change.Once every three days, aspirate one milliliter of the osteogenic medium by placing a needle outside the insert. And replace with one milliliter of fresh osteogenic medium. After seven and 14 days of incubation, determine alkaline phosphatase activity with an alkaline phosphatase assay kit.In accordance with the kit manufacturer's protocol. Package the polyelectrolyte complexes into the pores of a bioresorbable medical grade polycaprolactone tri calcium phosphate scaffold. Using a sterilized spatula inside a BSL2 chamber.Add a 1.5 milligram or milliliter solution of bone morphogenetic protein two or NELL like molecule one on to the scaffold packed with polyelectrolyte complex. And incubate overnight at 4 degrees celsius. Alginate microbeads are inspected under a microscope to monitor their size and morphology.Non irradiated alginate microbeads are spherical and the irradiated counterpart is teardrop shaped. Size distribution is an important factor of growth factor uptake. And reduction in size increases the surface to area ratio.When measuring alginate microbeads with image J software, an average of 100 microbeads provides a good estimation of the size distribution in each match of microbeads. Since growth factor uptake depends on proper coating technique, the layer thickness of each coating is observed by confocal microsopy. Here, confocal laser scanning microsopy images of alginate microbeads incublated with fluorescent analogs of protamine, heparin, NELL like molecule one, and bone morphogenetic protein two are shown.Alkaline phosphatase activity is used to measure bioactivity of released growth factor. A 2.2 fold increase in alkaline phosphatase activity was observed after incubation with polyelectrolyte complex NELL like molecule one on day 14. Once mastered, this technique can be done within five hours if it is performed properly.While performing this procedure, it is important to remember that alginate concentrations affect microbead stability. For irradiated alginates, we required four way percent. For non irradiated counterparts, we required two way percent.Following this procedures, we can further increase growth factor loading by multi layer coating. This technique allows researchers in tissue engineering field to explore growth factor delivery in other disease models. After watching this video, you are able to fabricate polyelectrolyte complex with narrow size distribution.

polyelectrolyte-complex-for-heparin-binding-domain-osteogenic-growth

Research

JoVE Journal

Bioengineering

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. ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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.

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, ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

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 ...

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 ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...