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 ....

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....

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.......

Materials

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