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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

Published: August 22nd, 2016

DOI:

10.3791/54202

1Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 2Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, 3Department of Bioengineering, Faculty of Engineering, National University of Singapore, 4Tissue Engineering Program, National University of Singapore, 5Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles

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 ....

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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.......

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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 &#.......

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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.......

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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.

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Name Company Catalog Number Comments
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

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