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 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.
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 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.
1. Alginate Solution Preparation
2. Alginate Microbead Fabrication
3. Size Measurement of Alginate Microbeads
4. Sterilization
5. Protamine and Heparin Coating
6. Protamine Content
7. Heparin Content
8. Confocal Image of Layer-by-layer Structure
9. BMP-2 and NELL-1 Uptake and Release
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).
11. Cell Viability
12. Packaging into Scaffold and BMP-2 & NELL-1 Loading
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 (0.35 mm nozzle, flow rate of 4 ml/hr & 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.
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. Please click here to view a larger version of this figure.
Figure 2: Measuring alginate microbeads with ImageJ software. After opening the image file by clicking File → 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 µm by using formula: length of alginate/length of Scale bar x 500 µm. Please click here to view a larger version of this figure.
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). Please click here to view a larger version of this figure.
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 µm.) Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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 ± standard deviation. Please click here to view a larger version of this figure.
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 ± standard deviation. Please click here to view a larger version of this figure.
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 ± standard deviation. Please click here to view a larger version of this figure.
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 µm) packed with PEC carrier. (B) PCL-TCP scaffold. (C) High magnification: PEC maintains its shape after packing. Please click here to view a larger version of this figure.
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 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 > strontium > 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 °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 µm 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 authors have nothing to disclose.
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.
Life Science Acrodisc 25mm Syring Filter w/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 Labelled 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 labelling |
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 (100µm) | BD Science | 352360 | Hold PEC for ALP assay |
Cell Scraper 290mm Bladewide 20mm | SPL Life Science | 90030 | Detach the cell from 24 well plate |
CF 405S, Succinimidyl Ester | Sigma Aldrich | SCJ4600013 | Blue fluorescent dye for protamine labelling |
CF 594, Hydrazide | Sigma Aldrich | SCJ4600031 | Deep red fluorescent dye for heparin labelling |
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 labelling |
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 |
NELL-1 | Aragen Bioscience Morgan Hill, CA, USA | N/A | Osteogenic growth factor, keep at -80˚C |
Nisco cell encapsulator | Nisco Engineering Inc | Encapsulation unit VAR V1 | |
Fluorescent Microscope | Olympus | IX71 | |
mPCL-TCP Scaffold (Pore size is 1.3mm) | Osteopore | PCL-TCP 0/90 | Hold PEC for in vivo study |
Penicillin-Streptomycin 10,000 unit/ml, 100ml | 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 22mm 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 | ||
5ml syringe | Terumo | 140425R | Diameter of syringe affects the flow rate |
75cm2 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 |