Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Summary

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.

Abstract

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.

Introduction

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 used^4,5.

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 immobilization^7,8. The alginate microbead core was fabricated by crosslinking α-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.

Protocol

1. Alginate Solution Preparation

1. 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 °C overnight. Filter the alginate solution with a sterile 0.2 µm syringe filter before alginate microbead fabrication.

2. Alginate Microbead Fabrication

1. Disinfect the electrostatic bead generator and syringe pump with 70% ethanol and place them in a Class II Biological Safety Cabinet (Figure 1).
2. Place a glass basin with a magnetic stir bar inside the bead generator.
3. Set the arm electrode of the bead generator 9 cm above the basin.
4. 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.
5. Load 5 ml of 0.2 µm 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.
6. 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.
7. 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.
8. Store the microbeads in 20 ml of 0.2 M strontium chloride solution at 4 °C overnight to complete cross linking and stabilize the gel.

3. Size Measurement of Alginate Microbeads

1. 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 µm) in TIFF format at resolution 2,048 x 1,536.
2. 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.
   1. Click on the line tools and draw a line across the middle of the alginate bead.
   2. Click "Analyze" on the menu bar and select "Measure". A pop-up window will appear.
   3. Repeat steps 3.2.1-3.2.2 to measure all alginate beads within the image. Measure the scale bar on the image.
   4. 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 µm. For example, 1.420 (diameter measured by ImageJ) / 2.657 (scale bar length measured by ImageJ) * 500 µm = 267 µm.
3. Consider the average size of 100 microbeads (mean ± standard deviation) as the representative size of each batch of microbeads.

4. Sterilization

1. Collect the microbeads using a 100 µm nylon strainer and wash the beads with double distilled water.
2. 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.
3. Finally, sterilize the microbeads by autoclaving using liquid mode (115 °C, 15 min) or in accordance with manufacturer's specifications. Add 1.5 L of distilled water to the chamber to prevent beads from drying.

5. Protamine and Heparin Coating

1. Inside the BSL-2 hood, incubate the sterile microbeads with 1 ml of 2 mg/ml protamine solution (sterilized using a 0.2 µm syringe filter) for 1 hr at room temperature.
2. After incubating the microbeads for 1 hr (Step 5.1), collect 150 µl of the protamine solution for the micro bicinchoninic acid (microBCA) test (section 6).
3. 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.
4. Incubate protamine coated microbeads with 1 ml of 0.5 mg/ml heparin solution (sterilized using a 0.2 µm syringe filter) for 30 min to create polyelectrolyte complex (PEC).
5. After incubating the protamine coated microbeads for 30 min (Step 5.4), collect 400 µl of the heparin solution to determine heparin content (section 7).
6. After incubation, wash off the unbound heparin from the PECs by washing twice with double distilled water.

6. Protamine Content

1. Perform the microBCA test according to manufacturer instructions. Briefly, add 150 µl protamine solution (collected before and after incubation with microbeads) into a 96 well plate. Add 150 µl microBCA working solution.
2. Use albumin solution (0, 0.5, 1, 2, 5, 10, 20, 40 and 200 µg/ml) as calibration standards.
3. Incubate the mixture for 60 min at 60 °C. Measure the absorbance with a spectrophotometer at 562 nm.
4. Use the standard curve to determine the protamine concentration of each unknown sample according to manufacturer's instructions.
5. 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).

7. Heparin Content

1. Prepare the 10 ml of working solution by dissolving 4 mg toluidine blue and 20 mg sodium chloride in 0.01 N hydrochloric acid.
2. Add 400 µl of sample (from step 5.5) to the working solution at a ratio of 2:3 and vortex for 30 sec.
3. Add 600 µl of n-hexane (equivalent volume to the working reagent solution) and vortex the mixture to extract the toluidine blue heparin complex.
4. Aspirate 200 µl of the aqueous phase by syringe after phase separation.
5. Measure the amount of un-extracted toluidine blue contained in the aqueous phase using a spectrophotometer at 631 nm.
6. Prepare heparin standard solutions of 0-20 µg/ml.
7. Plot the 631 nm reading of each heparin standard vs. heparin concentration in µg/ml. Use the standard curve to determine the heparin concentration of each sample.

8. Confocal Image of Layer-by-layer Structure

1. 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's technical datasheet.
2. Coat 100 µg microbeads with 300 µl 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.
3. Observe the layer-by-layer structure (Figure 3) by using a confocal microscope at 10X magnification.⁷

9. BMP-2 and NELL-1 Uptake and Release

1. Load 13.3 µl of 1.5 mg/ml of BMP-2 or NELL-1 solution on 100 µg of PEC. Incubate PEC at 4 °C under 30 rpm shaking for 10 hr.
2. Immerse the microbeads in 1 ml of phosphate buffered saline (PBS) at 37 °C with constant shaking (30 rpm).
3. Collect 1 ml of the supernatant and replace it with 1 ml PBS after 1, 3, 6, 10 and 14 days.
4. Evaluate the uptake and release efficiency of BMP-2 using the ELISA method according to the manufacturer'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's protocol.
5. Determine cumulative release at time (t):
   Cumulative release at time (t) = Release at time (t) + Previous release at time (t-1).
6. Plot cumulative release of BMP-2 and NELL-1 against time.

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).

1. Seed 20,000 rBMSCs per well in a 24-well plate and allow them to grow for one day with 1 ml of Dulbecco's Modified Eagle's Medium (DMEM) + 10% Fetal bovine serum (FBS) at 37 °C and 5% CO₂.
2. After 24 hr, replace the medium with 1 ml of an osteogenic medium (DMEM supplemented with 10% FBS, 2% penicillin streptomycin, 50 µg/ml ascorbic acid, 10 mmol/L beta-glycerophosphate, and 10^-8 mol/L dexamethasone) for 7 days at 37 °C and 5% CO₂.
3. Place 300 µg 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.
4. 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.
5. After 7 and 14 days of incubation, determine ALP activity with an ALP assay kit in accordance with the kit manufacturer's protocol.
   1. Lyse cells with assay buffer containing 0.1% TritonX-100 at 4 °C for 10 min. Scrape off adhered cells using a cell scraper. Incubate cell suspension at 4 °C under agitation for at least 60 min.
   2. Centrifuge the cell suspension at 2,500 x g at 4 °C for 10 min. Collect the supernatant for the ALP assay.
   3. Add 50 µl 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.
   4. Add the supernatant from step 10.5.2 (50 µl/well) and dilute with dilution buffer.
   5. Add 50 µl 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.
   6. Calculate ALP activity using the calibration curve.
6. Determine protein content using the microBCA protein assay kit according to manufacturer's instructions. Normalize the ALP activity by dividing ALP activity by protein content.

11. Cell Viability

1. Incubate 200 mg of PEC-NELL-1 with 1 ml of DMEM +10% FBS at 37 °C for 24 hr for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
2. Seed 2,000 rBMSCs per well (in 100 µl of DMEM with 10% FBS) in a 96 well plate and incubate for 1 day at 37 °C, 5% CO₂.
3. Replace DMEM + 10% FBS with 100 µl of PEC-NELL-1/PEC extract and incubate at 37 °C, 5% CO₂.
4. After 1 day or 3 days of incubation, add 10 µl of 5 mg/ml MTT solution and further incubate at 37 °C, 5% CO₂ for 4 hr in darkness.
5. Add 100 µl DMSO solution to each well to dissolve formazan crystals.
6. Determine the absorbance at 570 nm using a microplate reader.
7. Calculate the relative growth rate:
   

12. Packaging into Scaffold and BMP-2 & NELL-1 Loading

1. 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.
2. 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 °C.

Results

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 ± 14 µm. (0.35 mm nozzle, flow rate of 5 ml/hr & 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 ± 30 µm (0.3...

Discussion

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 ± 2.7%, BMP-2: 70.5 ± 3.1%. The PEC carrier has a better modulation of NELL-1 (20%) release compared to a pure surface ad...

Materials

List of materials used in this article

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