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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 ....

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 electrostati.......

Representative 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 &#.......

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.......

Disclosures

We have no conflict of interest.

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Life Science Acrodisc 25mm Syring Filter w/0.2 µm Supor  MembranePALL PN4612Sterile protamine,
 heparin solution by ultrafiltration
24 well plateCell Star 662160
96 well plate Nuclon Delta SurfaceThermo Fisher Scientific167008
(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), MTTSigma AldrichM5655Measure cytotoxicity of PEC-NELL-1
AcetoneFisher ScientificA/0600/17Precipitate CF-405
Labelled protamine 
Alamar BlueInvitrogen, Life TechnologiesDAL 1025Measure cytotoxicity of PEC-BMP-2
Alkaline Phosphatase Assay (ALP) assay kitAnaspecAS-72146
Ammonium ChlorideMerckArt 1145Stop reagent in FITC labelling
Anhydrous Dimethyl Sulfoxide (DMSO)Invitrogen, Life TechnologiesD12345Solvent for fluorescent isothiocyanate I
Dimethyl Sulfoxide (DMSO)Sigma AldrichDissolve  formazan 
AutoclaveHirayamaHU-110Sterilize alginate beads by steam
Beta-glycerophosphateSigma AldrichG9422
BMP-2 (Infuse Bone Graft Large II Kit) Medtronic Sofarmor Danek, Memphis TN, USA7510800Osteogenic Growth  Factor,
 dialysis is needed to remove stabilizer component that interferes with FITC coupling
Carboxybenzoyl quinoline-2-Carboxaldehyde (CBQCA) Thermo Fisher ScientificA-6222To quantify NELL-1 protein
Cell Strainer (100µm)BD Science352360Hold PEC for ALP assay
Cell Scraper 290mm Bladewide 20mmSPL Life Science 90030Detach the cell from 24 well plate 
CF 405S, Succinimidyl EsterSigma AldrichSCJ4600013Blue fluorescent dye for protamine labelling
CF 594, HydrazideSigma AldrichSCJ4600031Deep red fluorescent dye for heparin labelling 
CentrifugeBeckman CoulterMicrofuge 22R
Confocal MicroscopeOlympus FV1000
DexamethasoneSigma AldrichD4902Component of osteogenic growth medium
Dextran Desalting ColumnsPierce (Thermo Scientific) 43230
DMEMGibco 12320
BMP-2 Quantikine ELISA KitR&D SystemDBP200Determine BMP-2 release
Fetal Bovine Serum FBSHycloneSV30160.03
Fluoescein Isothiocyananate, Isomer ISigma AldrichF7250Green fluorescent dye for NELL-1 and BMP-2 labelling
ThinCert Cell Culture Inserts,
For 24 Well plates, Sterile
Greiner 662630Prevents PEC wash out when
 changing osteogenic medium
Havard Appartus Syringe Pump (11 plus)Havard Apparatus70-2208
n-Hexane (>99%)Sigma Aldrich139386
HeparinSigma AldrichH3149Binds with osteogenic
growth factor with heparin binding domain
Hydrochloric acid (37%)Merck100317Highly Corrosive
IncubatorBinderC8150
MicroBCA Protein Assay kitThermoscientific23235
Microplate ReaderTecanInfinite M200For ALP and microBCA assays
NELL-1Aragen Bioscience Morgan Hill, CA, USAN/AOsteogenic growth factor, keep at -80˚C
Nisco cell encapsulatorNisco Engineering IncEncapsulation unit VAR V1
Fluorescent MicroscopeOlympusIX71
mPCL-TCP Scaffold (Pore size is 1.3mm)OsteoporePCL-TCP 0/90Hold PEC for in vivo study
Penicillin-Streptomycin 10,000 unit/ml, 100mlHyclone Cell CultureSV30010Antibiotic
10X Phosphate Buffered Saline (PBS) VivantisPB0344-1L10x Solution, Ultra Pure Grade
Poly-L-Lysine MW 15,000-30,000Sigma AldrichP2568Polycation
Protamine Sulfate salt, from SalmonSigma AldrichP4020Polycation
ShakerLabnetS2025
Snakeskin Dialysis Tubing 3,500 MWCO 22mm x 35 feetThermo Fisher Scientific68035Remove unreacted FITC by dialysis
Sodium ChlorideMerck1.06404.1000
Sodium HydroxideQrecS5158
Sodium BicarbonateUS BiologicalS4000Buffer
Sodium carbonateSigma AldrichS7795-500GBuffer
Strontium Chloride HexahydrateSigma Aldrich255521Crosslinker for alginate
Spatula3dia
5ml syringeTerumo140425RDiameter of syringe
affects the flow rate 
75cm2 Cell Culture Flask Canted NeckCorning730720
Toluidine Blue Sigma Aldrich52040Heparin assay
Trypsin 1XHyclone Cell CultureSH30042.01
Sodium alginateNovamatrix (FMC Biopolymer, Princeton, NJ)Pronova UPMVGCore material of microbeads

References

  1. Yuan, W., et al. NELL-1 based demineralized bone graft promotes rat spine fusion as compared to commercially available BMP-2 product. Orthop Sci. 18, 646-657 (2013).
  2. Anderson, C. L., Whitaker, M. C.

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