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

7.5K vistas.  National University of Singapore. 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...