Name: polyelectrolyte complex for heparin binding domain osteogenic growth factor delivery
Uploaded: 2016-08-22T14:00:00.000+00:00
Duration: 12 min 27 s
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 electrostatic bead generator and syringe pump with 70% ethanol and place them in a Class II Biological Safety Cabinet (Figure 1).</li>
	<li>Place a glass basin with a magnetic stir bar inside the bead generator.</li>
	<li>Set the arm electrode of the bead generator 9 cm above the basin.</li>
	<li>Connect the electrode cable of the bead generator to the knurled screw 2 of the arm electrode, and pour 80 ml of SrCl2 solution into the basin.</li>
	<li>Load 5 ml of 0.2 &#181;m filtered alginate solution into the syringe and rubber tube. After connecting the rubber tube to the arm electrode, switch on the syringe pump at 5 ml/hr for 2 min to expel the air inside the tubing and deliver the alginate solution to the tip of the nozzle. Turn off the syringe pump.</li>
	<li>Next, switch on the encapsulator and then, the syringe pump to commence microbead generation. Set the alginate flow rate at 5 ml/hr and the voltage at 5.8 kV on the encapsulator. Discard the microbeads generated during the first two minutes (or the initial 0.5 ml alginate solution pumped out of the syringe), as these microbeads tend to be irregularly sized and shaped.</li>
	<li>Collect subsequent microbeads in 0.2 M strontium chloride solution. Turn off both the syringe pump and the encapsulator (in that order) after pumping the pre-planned volume of alginate solution. Repeat this for subsequent batches of microbead fabrication. Upon completion, turn off the syringe pump first, followed by the encapsulator.</li>
	<li>Store the microbeads in 20 ml of 0.2 M strontium chloride solution at 4 &#176;C overnight to complete cross linking and stabilize the gel.</li>
</ol>
3. Size Measurement of Alginate Microbeads
<ol>
	<li>Collect 0.5 ml 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 ten images of the microbeads with a microscope CCD camera. Save the images with scale bar (500 &#181;m) in TIFF format at resolution 2,048 x 1,536.</li>
	<li>Using the length tools in ImageJ, measure the size of the microbeads and scale bar (Figure 2). Convert the microbeads length from pixel to micrometer.
	<ol>
		<li>Click on the line tools and draw a line across the middle of the alginate bead.</li>
		<li>Click &#34;Analyze&#34; on the menu bar and select &#34;Measure&#34;. A pop-up window will appear.</li>
		<li>Repeat steps 3.2.1-3.2.2 to measure all alginate beads within the image. Measure the scale bar on the image.</li>
		<li>Convert the diameter of the alginate bead to the actual length by using the formula:length of alginate bead/length of scale bar x 500 &#181;m. For example, 1.420 (diameter measured by ImageJ) / 2.657 (scale bar length measured by ImageJ) * 500 &#181;m = 267 &#181;m.</li>
	</ol>
	</li>
	<li>Consider the average size of 100 microbeads (mean &#177; standard deviation) as the representative size of each batch of microbeads.</li>
</ol>
4. Sterilization
<ol>
	<li>Collect the microbeads using a 100 &#181;m nylon strainer and wash the beads with double distilled water.</li>
	<li>Using a spatula, transfer all the microbeads made from 0.1 ml alginate solution into a 2 ml microcentrifuge tube and cover with gauze to prevent drying.</li>
	<li>Finally, sterilize the microbeads by autoclaving using liquid mode (115 &#176;C, 15 min) or in accordance with manufacturer&#39;s specifications. Add 1.5 L of distilled water to the chamber to prevent beads from drying.</li>
</ol>
5. Protamine and Heparin Coating
<ol>
	<li>Inside the BSL-2 hood, incubate the sterile microbeads with 1 ml of 2 mg/ml protamine solution (sterilized using a 0.2 &#181;m syringe filter) for 1 hr at room temperature.</li>
	<li>After incubating the microbeads for 1 hr (Step 5.1), collect 150 &#181;l of the protamine solution for the micro bicinchoninic acid (microBCA) test (section 6).</li>
	<li>Wash the protamine coated microbeads twice with double distilled water. Spin down using a bench top centrifuge at 200 x g for 3 min at room temperature. After centrifugation, aspirate the water using a syringe.</li>
	<li>Incubate protamine coated microbeads with 1 ml of 0.5 mg/ml heparin solution (sterilized using a 0.2 &#181;m syringe filter) for 30 min to create polyelectrolyte complex (PEC).</li>
	<li>After incubating the protamine coated microbeads for 30 min (Step 5.4), collect 400 &#181;l of the heparin solution to determine heparin content (section 7).</li>
	<li>After incubation, wash off the unbound heparin from the PECs by washing twice with double distilled water.</li>
</ol>
6. Protamine Content
<ol>
	<li>Perform the microBCA test according to manufacturer instructions. Briefly, add 150 &#181;l protamine solution (collected before and after incubation with microbeads) into a 96 well plate. Add 150 &#181;l microBCA working solution.</li>
	<li>Use albumin solution (0, 0.5, 1, 2, 5, 10, 20, 40 and 200 &#181;g/ml) as calibration standards.</li>
	<li>Incubate the mixture for 60 min at 60 &#176;C. Measure the absorbance with a spectrophotometer at 562 nm.</li>
	<li>Use the standard curve to determine the protamine concentration of each unknown sample according to manufacturer&#39;s instructions.</li>
	<li>Determine the protamine content of the microbeads by subtracting the total amount of protamine in the coating solution (before incubation with microbeads) from the amount of protamine remaining in the coating solution (after incubation with microbeads).</li>
</ol>
7. Heparin Content
<ol>
	<li>Prepare the 10 ml of working solution by dissolving 4 mg toluidine blue and 20 mg sodium chloride in 0.01 N hydrochloric acid.</li>
	<li>Add 400 &#181;l of sample (from step 5.5) to the working solution at a ratio of 2:3 and vortex for 30 sec.</li>
	<li>Add 600 &#181;l of n-hexane (equivalent volume to the working reagent solution) and vortex the mixture to extract the toluidine blue heparin complex.</li>
	<li>Aspirate 200 &#181;l of the aqueous phase by syringe after phase separation.</li>
	<li>Measure the amount of un-extracted toluidine blue contained in the aqueous phase using a spectrophotometer at 631 nm.</li>
	<li>Prepare heparin standard solutions of 0-20 &#181;g/ml.</li>
	<li>Plot the 631 nm reading of each heparin standard vs. heparin concentration in &#181;g/ml. Use the standard curve to determine the heparin concentration of each sample.</li>
</ol>
8. Confocal Image of Layer-by-layer Structure
<ol>
	<li>Fabricate protamine, heparin and NELL-1/BMP-2 fluorescent analog CF-405 protamine (blue), CF 594 heparin (red) and FITC labeled NELL-1/FITC labeled (green) BMP-2 + heparin + protamine according to manufacturer&#39;s technical datasheet.</li>
	<li>Coat 100 &#181;g microbeads with 300 &#181;l of fluorescent analog (coating method as described in 5.3-5.6) CF-405 protamine (blue) (2 mg/ml, 1 hr incubation), CF 594 heparin (red) (0.5 mg/ml, 30 min) and FITC labeled NELL-1/FITC labeled (green) BMP-2 (1.5 mg/ml, overnight). Wash the microbeads twice with distilled water to eliminate the unbound fluorescent protamine, heparin and NELL-1/BMP-2.</li>
	<li>Observe the layer-by-layer structure (Figure 3) by using a confocal microscope at 10X magnification.7</li>
</ol>
9. BMP-2 and NELL-1 Uptake and Release
<ol>
	<li>Load 13.3 &#181;l of 1.5 mg/ml of BMP-2 or NELL-1 solution on 100 &#181;g of PEC. Incubate PEC at 4 &#176;C under 30 rpm shaking for 10 hr.</li>
	<li>Immerse the microbeads in 1 ml of phosphate buffered saline (PBS) at 37 &#176;C with constant shaking (30 rpm).</li>
	<li>Collect 1 ml of the supernatant and replace it with 1 ml PBS after 1, 3, 6, 10 and 14 days.</li>
	<li>Evaluate the uptake and release efficiency of BMP-2 using the ELISA method according to the manufacturer&#39;s protocol. Evaluate the uptake and release efficiency of NELL-1 using the carboxybenzoyl quinoline-2-carboxaldehyde (CBQCA) protein assay method according to the manufacturer&#39;s protocol.</li>
	<li>Determine cumulative release at time (t): 
	Cumulative release at time (t) = Release at time (t) + Previous release at time (t-1).</li>
	<li>Plot cumulative release of BMP-2 and NELL-1 against time.</li>
</ol>
10. In Vitro Bioactivity of NELL-1
Note: The bioactivity of NELL-1 released from PEC was assessed by measuring its ability to increase the expression of alkaline phosphatase (ALP) in rabbit bone marrow stem cells (rBMSC).
<ol>
	<li>Seed 20,000 rBMSCs per well in a 24-well plate and allow them to grow for one day with 1 ml of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) + 10% Fetal bovine serum (FBS) at 37 &#176;C and 5% CO2.</li>
	<li>After 24 hr, replace the medium with 1 ml of an osteogenic medium (DMEM supplemented with 10% FBS, 2% penicillin streptomycin, 50 &#181;g/ml ascorbic acid, 10 mmol/L beta-glycerophosphate, and 10-8 mol/L dexamethasone) for 7 days at 37 &#176;C and 5% CO2.</li>
	<li>Place 300 &#181;g PEC-NELL-1 (from step 8.2) and PEC inside cell culture inserts (TC insert) to keep the PECs separate from the cells (this avoids the washout of PEC microbeads during the osteogenic medium change). Place TC insert into the 24 well plate for 14 days.</li>
	<li>Once every three days, aspirate 1 ml of the osteogenic medium by placing a needle outside the TC insert, and replace with 1 ml of fresh osteogenic medium.</li>
	<li>After 7 and 14 days of incubation, determine ALP activity with an ALP assay kit in accordance with the kit manufacturer&#39;s protocol.
	<ol>
		<li>Lyse cells with assay buffer containing 0.1% TritonX-100 at 4 &#176;C for 10 min. Scrape off adhered cells using a cell scraper. Incubate cell suspension at 4 &#176;C under agitation for at least 60 min.</li>
		<li>Centrifuge the cell suspension at 2,500 x g at 4 &#176;C for 10 min. Collect the supernatant for the ALP assay.</li>
		<li>Add 50 &#181;l of serially diluted alkaline phosphatase standard solution from 200 to 0 ng/ml to the wells of a 96 well plate. The final amounts of alkaline phosphatase standard are 10, 5, 2.5, 1.2, 0.6, 0.3, 0.15, and 0 ng/well.</li>
		<li>Add the supernatant from step 10.5.2 (50 &#181;l/well) and dilute with dilution buffer.</li>
		<li>Add 50 &#181;l of p-nitrophenyl phosphate (pNPP) substrate solution into each well. Mix the reagents by gently shaking the plate for 30 sec. Incubate the mixture for 30 min in the dark. Measure the absorbance at 405 nm by plate reader.</li>
		<li>Calculate ALP activity using the calibration curve.</li>
	</ol>
	</li>
	<li>Determine protein content using the microBCA protein assay kit according to manufacturer&#39;s instructions. Normalize the ALP activity by dividing ALP activity by protein content.</li>
</ol>
11. Cell Viability
<ol>
	<li>Incubate 200 mg of PEC-NELL-1 with 1 ml of DMEM +10% FBS at 37 &#176;C for 24 hr for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.</li>
	<li>Seed 2,000 rBMSCs per well (in 100 &#181;l of DMEM with 10% FBS) in a 96 well plate and incubate for 1 day at 37 &#176;C, 5% CO2.</li>
	<li>Replace DMEM + 10% FBS with 100 &#181;l of PEC-NELL-1/PEC extract and incubate at 37 &#176;C, 5% CO2.</li>
	<li>After 1 day or 3 days of incubation, add 10 &#181;l of 5 mg/ml MTT solution and further incubate at 37 &#176;C, 5% CO2 for 4 hr in darkness.</li>
	<li>Add 100 &#181;l DMSO solution to each well to dissolve formazan crystals.</li>
	<li>Determine the absorbance at 570 nm using a microplate reader.</li>
	<li>Calculate the relative growth rate: 
	<img alt="Equation 1" src="/files/ftp_upload/54202/54202eq1.jpg" /></li>
</ol>
12. Packaging into Scaffold and BMP-2 &#38; NELL-1 Loading
<ol>
	<li>Pack the PECs into the pores of a bioresorbable medical grade Polycaprolactone - tri-calcium phosphate (mPCL-TCP) scaffold using a sterilized spatula inside a BSL-2 chamber.</li>
	<li>Add 1.5 mg/ml solution of BMP-2 or NELL-1 onto the mPCL-TCP scaffold packed with PEC and incubate overnight at 4 &#176;C.</li>
</ol>

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	<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. 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We have no conflict of interest.

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 adsorption carrier such as calcium apatite particles (40-80%)11.
Besides modulating release, heparin neutralizes the excessive positive charge of polycations such as protamine to avoid unwanted cytotoxicity related issues12. The PECs do not show any signs of cytotoxicity, as determined by the MTT assay and the Alamar Blue assay7. The ALP assay shows the PEC carrier can maintain the bioactivity of both NELL-1 and BMP-2. Although developed for osteogenic growth factor BMP-2 therapy in spinal fusion surgeries, PEC can also take up other growth factors with heparin binding domains such as NELL-1 and PDGF-BB. Compared to other delivery methods such as encapsulation of growth factors in polyglycolic acid microspheres, PECs do not require organic solvents that tend to inactivate growth factors13.
A number of factors in the PEC fabrication procedure may affect carrier performance. Firstly, microbead size affects the surface area/volume ratio. Higher osteogenic growth factor loading can be achieved with smaller microbeads. Secondly, the alginate concentration should be sufficient to maintain the stability of the microbead structure. Microbead stability depends on alginate type, chain length (affected by gamma irradiation) and the divalent ion used (barium &#62; strontium &#62; calcium). While 2% alginate solution is sufficient to manufacture PECs with stable microbead structures, 4% alginate is required following 8 MRad gamma irradiation to compensate for the effects of alginate chain shortening during irradiation. Thirdly, the rate of in vivo degradation of alginate microbeads is strongly influenced by alginate chain length. Based on our experience from rat and porcine models, PEC fabricated using 8 MRad irradiated alginate shows rapid (28 days) and complete degradation of the alginate core (unpublished data). Degradation of the alginate core provides room essential for bony ingrowth. Fourthly, the overnight incubation of BMP-2 and NELL-1 at 4 &#176;C with constant shaking (e.g., 30 rpm) can improve uptake efficiency. Lastly, the protamine coating thickness is time dependent. Since the extent of the protamine-heparin interaction determines the release of osteogenic growth factors such as BMP-2 or NELL-1, 1 hr of protamine incubation is adopted to improve the stability of the PEC structure.
The use of heparin in this technique is critical in stabilizing the delicate growth factors and thus important for prolonging in vivo bioactivity. Given the very limited amount of heparin involved and coupled with the choice of the antidote, i.e., protamine (a highly effective drug in neutralizing the anti-coagulant activities of heparin), prolonged bleeding time in decorticated bone is largely theoretical and practically inconsequential.
Loading the PECs into the PCL-TCP scaffold enhances localization of beads at implant sites. Scaffolds provide necessary mechanical support that is crucial for spine fusion. In the present studies, we used PCL-TCP scaffolds with 1,300 &#181;m pores to facilitate proper packing (Figure 9). Although the current illustration shows PEC osteogenic growth factor delivery with the PCL-TCP scaffold, our group has also evaluated carrier performance with a polyetherketoneketone (PEKK) bone chamber in one rabbit study with similar efficacy.
In this study, the lack of comparison with other previously evaluated carriers of rhBMP-2 and NELL-1 could represent a limitation.
In conclusion, the presented procedure provides a useful carrier to control release of osteogenic growth factors with heparin domains such as BMP-2 and NELL-1. The described strategy combines many advantages: it is not restricted to BMP-2 and applicable to other growth factors with heparin binding domains such as NELL-1. Dose reduction on osteogenic growth factor can reduce undesirable side effects such as seroma, heterotrophic bone formation and lower the overall cost of treatment.

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><tbody><tr><td>Life Science Acrodisc 25&amp;nbsp;mm Syring Filter with 0.2&amp;nbsp;&amp;micro;m Supor Membrane</td><td>PALL&amp;nbsp;</td><td>PN4612</td><td>Sterile protamine, heparin solution by ultrafiltration</td></tr><tr><td>24 well plate</td><td>Cell Star&amp;nbsp;</td><td>662160</td><td></td></tr><tr><td>96 well plate Nuclon Delta Surface</td><td>Thermo Fisher Scientific</td><td>167008</td><td></td></tr><tr><td>(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), MTT</td><td>Sigma Aldrich</td><td>M5655</td><td>Measure cytotoxicity of PEC-NELL-1</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A/0600/17</td><td>Precipitate CF-405&lt;br/&gt; Labeled protamine</td></tr><tr><td>Alamar Blue</td><td>Invitrogen, Life Technologies</td><td>DAL 1025</td><td>Measure cytotoxicity of PEC-BMP-2</td></tr><tr><td>Alkaline Phosphatase Assay (ALP) assay kit</td><td>Anaspec</td><td>AS-72146</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Merck</td><td>Art 1145</td><td>Stop reagent in FITC labeling</td></tr><tr><td>Anhydrous Dimethyl Sulfoxide (DMSO)</td><td>Invitrogen, Life Technologies</td><td>D12345</td><td>Solvent for fluorescent isothiocyanate I</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td></td><td>Dissolve&amp;nbsp; formazan&amp;nbsp;</td></tr><tr><td>Autoclave</td><td>Hirayama</td><td>HU-110</td><td>Sterilize alginate beads by steam</td></tr><tr><td>Beta-glycerophosphate</td><td>Sigma Aldrich</td><td>G9422</td><td></td></tr><tr><td>BMP-2 (Infuse Bone Graft Large II Kit)&amp;nbsp;</td><td>Medtronic Sofarmor Danek, Memphis TN, USA</td><td>7510800</td><td>Osteogenic Growth Factor, dialysis is needed to remove stabilizer component that interferes with FITC coupling</td></tr><tr><td>Carboxybenzoyl quinoline-2-Carboxaldehyde (CBQCA)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A-6222</td><td>To quantify NELL-1 protein</td></tr><tr><td>Cell Strainer (100&amp;nbsp;&amp;micro;m)</td><td>BD Science</td><td>352360</td><td>Hold PEC for ALP assay</td></tr><tr><td>Cell Scraper 290&amp;nbsp;mm Bladewide 20&amp;nbsp;mm</td><td>SPL Life Science</td><td>90030</td><td>Detach the cell from 24 well plate</td></tr><tr><td>CF 405S, Succinimidyl Ester</td><td>Sigma Aldrich</td><td>SCJ4600013</td><td>Blue fluorescent dye for protamine labeling</td></tr><tr><td>CF 594, Hydrazide</td><td>Sigma Aldrich</td><td>SCJ4600031</td><td>Deep red fluorescent dye for heparin labeling</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Microfuge 22R</td><td></td></tr><tr><td>Confocal Microscope</td><td>Olympus&amp;nbsp;</td><td>FV1000</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma Aldrich</td><td>D4902</td><td>Component of osteogenic growth medium</td></tr><tr><td>Dextran Desalting Columns</td><td>Pierce (Thermo Scientific)&amp;nbsp;</td><td>43230</td><td></td></tr><tr><td>DMEM</td><td>Gibco&amp;nbsp;</td><td>12320</td><td></td></tr><tr><td>BMP-2 Quantikine ELISA Kit</td><td>R&amp;amp;D System</td><td>DBP200</td><td>Determine BMP-2 release</td></tr><tr><td>Fetal Bovine Serum FBS</td><td>Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>Fluoescein Isothiocyananate, Isomer I</td><td>Sigma Aldrich</td><td>F7250</td><td>Green fluorescent dye for NELL-1 and BMP-2 labeling</td></tr><tr><td>ThinCert Cell Culture Inserts,&lt;br/&gt; For 24 Well plates, Sterile</td><td>Greiner&amp;nbsp;</td><td>662630</td><td>Prevents PEC wash out when changing osteogenic medium</td></tr><tr><td>Havard Appartus Syringe Pump (11 plus)</td><td>Havard Apparatus</td><td>70-2208</td><td></td></tr><tr><td>n-Hexane (&gt;99%)</td><td>Sigma Aldrich</td><td>139386</td><td></td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H3149</td><td>Binds with osteogenic&lt;br/&gt; growth factor with heparin binding domain</td></tr><tr><td>Hydrochloric acid (37%)</td><td>Merck</td><td>100317</td><td>Highly Corrosive</td></tr><tr><td>Incubator</td><td>Binder</td><td>C8150</td><td></td></tr><tr><td>MicroBCA Protein Assay kit</td><td>Thermoscientific</td><td>23235</td><td></td></tr><tr><td>Microplate Reader</td><td>Tecan</td><td>Infinite M200</td><td>For ALP and microBCA assays</td></tr><tr><td>Nisco cell encapsulator</td><td>Nisco Engineering Inc</td><td>Encapsulation unit VAR V1</td><td></td></tr><tr><td>Fluorescent Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>mPCL-TCP Scaffold (Pore size is 1.3&amp;nbsp;mm)</td><td>Osteopore</td><td>PCL-TCP 0/90</td><td>Hold PEC for &lt;em&gt;in vivo&lt;/em&gt; study</td></tr><tr><td>Penicillin-Streptomycin 10,000 unit/ml, 100&amp;nbsp;ml</td><td>Hyclone Cell Culture</td><td>SV30010</td><td>Antibiotic</td></tr><tr><td>10x Phosphate Buffered Saline (PBS)</td><td>Vivantis</td><td>PB0344-1L</td><td>10x Solution, Ultra Pure Grade</td></tr><tr><td>Poly-L-Lysine MW 15,000-30,000</td><td>Sigma Aldrich</td><td>P2568</td><td>Polycation</td></tr><tr><td>Protamine Sulfate salt, from Salmon</td><td>Sigma Aldrich</td><td>P4020</td><td>Polycation</td></tr><tr><td>Shaker</td><td>Labnet</td><td>S2025</td><td></td></tr><tr><td>Snakeskin Dialysis Tubing 3,500 MWCO 22&amp;nbsp;mm x 35 feet</td><td>Thermo Fisher Scientific</td><td>68035</td><td>Remove unreacted FITC by dialysis</td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>1.06404.1000</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Qrec</td><td>S5158</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>US Biological</td><td>S4000</td><td>Buffer</td></tr><tr><td>Sodium carbonate</td><td>Sigma Aldrich</td><td>S7795-500G</td><td>Buffer</td></tr><tr><td>Strontium Chloride Hexahydrate</td><td>Sigma Aldrich</td><td>255521</td><td>Crosslinker for alginate</td></tr><tr><td>Spatula</td><td>3dia</td><td></td><td></td></tr><tr><td>5 ml syringe</td><td>Terumo</td><td>140425R</td><td>Diameter of syringe affects the flow rate</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Cell Culture Flask Canted Neck</td><td>Corning</td><td>730720</td><td></td></tr><tr><td>Toluidine Blue</td><td>Sigma Aldrich</td><td>52040</td><td>Heparin assay</td></tr><tr><td>Trypsin 1x</td><td>Hyclone Cell Culture</td><td>SH30042.01</td><td></td></tr><tr><td>Sodium alginate</td><td>Novamatrix (FMC Biopolymer, Princeton, NJ)</td><td>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 &#177; 30 &#181;m (0.35 mm nozzle, flow rate of 4 ml/hr &#38; 6 kV). (Figure 4).
Confocal images of the PEC microbeads reveal layer-by-layer coating of CF-405 labeled protamine (blue), CF594-labeled heparin (red) and FITC-BMP-2/FITC-NELL-1 (green). The results indicate that the PECs can bind with positively charged BMP-2 and negatively charged NELL-1 via the heparin binding domain (Figure 5). This suggests that the interaction between the PECs and the osteogenic growth factors is not charge dependent.
To prove that the PECs can uptake and release BMP-2, we used an ELISA assay to determine the amount of BMP-2 remaining after incubation and the amount of BMP-2 in the PBS on Day 1, 3, 6, 10 and 14. However, a similar approach does not work well with the NELL-1 protein, since heparin blocks the antibody binding site, significantly reducing the signal. Therefore, the CBQCA protein assay was used to determine the difference between PEC-NELL-1 and PEC. From the cumulative release curve, PECs not only show a higher NELL-1 uptake efficiency compared to BMP-2 but also release it much slower than BMP-2 (NELL-1: 20% vs. BMP-2: 25%) (Figure 6). This suggests that PECs bind more tightly with NELL-1 than BMP-2.
From the MTT assay, PEC-NELL-1 is not cytotoxic (Figure 7). The result matches with the Alamar Blue assay result in a previous study7. Heparin neutralizes the positive charge of protamine which plays an important role in maintaining the biocompatibility of PEC.
To determine whether NELL-1 release from PEC affects long-term osteogenic differentiation, the expression level of an osteogenic marker, alkaline phosphatase (ALP), was investigated by a colorimetric assay. NELL-1 release from PEC increases the ALP activity of rabbit bone marrow stem cells by 2.2 fold at day 14 compared to the PEC control group (Figure 8). BMP-2 shows maximum increase of ALP activity (3.75 fold) on day 7. Both PEC-BMP-2 and PEC-BMP-2 + extra BMP-2 show decrease of activity on day 9.
ALP activity with BMP-2 in the medium without PEC is shown in Figure 8C. Although 70% of the growth factor remains on the PEC, the ALP activity of PEC BMP-2 group is equivalent to the free BMP-group. In vivo, growth factor must be delivered by the carrier to avoid washout. From our rodent and porcine model, we can lower the dose of BMP-2 by 20-fold and 6-fold, respectively. The reduction of does not only reduce unwanted side effects but also cuts down the cost of growth factor usage.
<img alt="Figure 1" src="/files/ftp_upload/54202/54202fig1.jpg" /> 
Figure 1: Electrostatic bead generator and syringe pump set up in BSL-2 biosafety cabinet. (A) Nozzle secured on nozzle holder. (B) Big basin for strontium chloride solution. (C) Electrode cable. (D) Hose delivers alginate solution. (E) Nozzle holder and arm. (F) Electrodes supply the potential difference to regulate the bead size. (G) Magnetic stirrer. (H) 5 ml syringe. (I) Syringe pump to regulate alginate flow. (J) Agitator control knob to control stirring speed. (K) Voltage control knob to regulate the potential difference between 0-10 kV. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54202/54202fig2.jpg" /> 
Figure 2: Measuring alginate microbeads with ImageJ software. After opening the image file by clicking File &#8594; Open, follow Step 1: click the line tool and draw a line on the alginate microbeads. In Step 2, click Analyze on the menu bar and a pop-up window will appear. Repeat Step 1 and Step 2 until all microbeads are measured. In Step 3, click the line tool, draw a line across the scale bar and measure the length of the scale bar. Convert the microbeads length to &#181;m by using formula: length of alginate/length of Scale bar x 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54202/54202fig3.jpg" /> 
Figure 3: Schematic representation of Polyelectrolyte Complex. Alginate core (dark green) positive charge protamine layer (pale green), negative charge heparin layer (red), osteogenic growth factor, e.g., BMP-2/NELL-1 on outermost layer (pale blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54202/54202fig4.jpg" /> 
Figure 4: Bright field images of alginate microbeads. (A) Non-irradiated. (B) 8M Rad irradiated. Non-irradiated alginate microbeads are spherical and the 8M Rad irradiated counterpart is teardrop shaped. Magnification 100X. (Scale bar = 500 &#181;m.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54202/54202fig5.jpg" /> 
Figure 5: Confocal laser scanning microscopy images of alginate microbeads incubated with fluorescent analogues of protamine, heparin, NELL-1 and BMP-2. (A) CF 405 Protamine (blue), (B) CF 594 Heparin (red), (C) FITC NELL-1 (green), and (D) FITC BMP-2 (green). Microbeads remain spherical even after incubation with fluorescent analogues. Scale bar = 250 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54202/54202fig6.jpg" /> 
Figure 6: Uptake and release of BMP-2 and NELL-1. (A) Uptake of NELL-1 protein (black) BMP-2 (red), (B) Cumulative release curve of BMP-2 (red) and NELL-1 (black) from PEC carrier. Results are presented as mean &#177; standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54202/54202fig7.jpg" /> 
Figure 7: Cytotoxicity Assay. (A) MTT assay of rabbit bone marrow stems cell with protamine based PEC NELL-1 200 mg/ml extract (black), PEC-NELL-1 100 mg/ml extract (white) at Day 1 and 3. (B) Alamar Blue assay of rabbit bone marrow stems cell with PEC BMP-2 (blue), PEC (green) Experiments were performed in triplicate and results are presented as mean &#177; standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54202/54202fig8.jpg" /> 
Figure 8: Bioactivity Assay for BMP-2 and NELL-1 release from PEC carrier. (A) ALP activity of rabbit bone marrow stem cell incubated with PEC-NELL-1 (black), PEC (red). ALP activity was measured as absorbance at 405 nm. 2.2 fold increase in ALP activity was observed after incubation with PEC-NELL-1 at Day 14. (B) ALP activity of rabbit bone marrow stem cell incubated with PEC-BMP-2 (red) PEC-BMP-2 + addition of BMP-2 (green) and PEC (blue). Increase in ALP activity after incubation with PEC-BMP-2 and PEC-BMP-2 + addition of BMP-2 at Day 7, ALP activity drops on Day 9. (C) ALP activity of rabbit bone marrow stem cell incubated with PEC-BMP-2 (red) BMP-2 (blue) and Medium (green). Results are presented as mean &#177; standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54202/54202fig9.jpg" /> 
Figure 9: Polycaprolactone Tri-calcium phosphate (PCL-TCP) scaffold packed with PEC carrier. (A) Polycaprolactone Tri-calcium phosphate (PCL-TCP) scaffold (pore size 1,300 &#181;m) packed with PEC carrier. (B) PCL-TCP scaffold. (C) High magnification: PEC maintains its shape after packing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54202/54202fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.6K Views.  National University of Singapore. 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.

Video: Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Chemistry

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicine

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, ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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 ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Immunology and Infection

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

3D Co-Culture Assay: Vascularized Osteogenic Niches for Cancer Drug Screening

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Author Spotlight: Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(Ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

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