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 ...

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