Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Summary

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Abstract

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

Introduction

Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor (TGF-β) family and acts as inducer of de novo bone formation as well as regulator of several tissues during embryonic development and adult homeostasis^1-3. Each monomer of the biologically active homodimeric BMP-2 protein contains a "cysteine knot" motif, which is highly conserved in all BMPs⁴. Six of the seven cysteine residues form intramolecular disulfide bonds that stabilize each monomer, whereas the seventh cysteine is involved in dimerization, forming an intermolecular bond between the two monomers^5,6. This highly c....

Protocol

1. Synthesis of 11-Mercaptoundecanoyl-N-hydroxysuccinimide Ester (MU-NHS)

1. Add dropwise a solution of 500 mg N-hydroxysuccinimide and 30 mg 4-(dimethylamino)pyridine in 10 ml acetone (p.a.) to 1 g 11-mercaptoundecanoic acid in 40 ml dichloromethane (p.a.) at room temperature (RT).
2. Cool the reaction to 0 °C and add dropwise 1.1 g N,N'-dicyclohexylcarbodiimide in 10 ml dichloromethane (under nitrogen atmosphere). Keep the reaction at low temperature for 1 hr and then stir at RT overnight.
3. Filter the precipitate and dry it under reduced pressure. Purify the product by flash chromatography with petroleum b....

Results

In our setup, gold was chosen as excipient since it provides a biologically unspecific but chemically tunable system. Furthermore, the application of self-assembling monolayers entails many benefits: SAMs spontaneously adsorb via their "head-groups" on metals and form monolayers with few defects, while their functional end-groups can be further modified. Thus they provide a platform to tailor the properties of the interface in a controlled yet highly adaptable way²⁰.

For the immobil.......

Discussion

In this protocol we describe the preparation of surfaces functionalized with bioactive rhBMP-2. This approach comprises two steps: 1) the initial formation of a self-assembling monolayer (SAM) of a bifunctional linker on the gold surface; 2) covalent immobilization of the rhBMP-2 protein. In previous work, we validated the effective binding of the bifunctional linker and the growth factor, and demonstrated that surface-immobilized rhBMP-2 maintains its biological activity¹⁹. The bioactivity of growth factors p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
N-hydroxysuccinimide	Sigma-Aldrich	130672
4-(dimethylamino)pyridin	Sigma-Aldrich	522805
Acetone	AppliChem	A2282
11-mercaptoundecanoic acid	Sigma-Aldrich	674427
Dichlormethane	Merck	106050
N,N'-dicyclohexylcarbodiimide	Sigma-Aldrich	D80002
Petroleum benzene	Merck
Glass coverslips	Carl Roth	M 875
Ethylacetate	AppliChem	A3550
Methanol	Carl Roth	4627
N,N-dimethylformamide	Carl Roth	T921
rhBMP-2	R&D Systems	355-BM	Carrier-free; expressed in E.coli
PBS	PAA	H15-002
NaCl	Carl Roth	HN00.2
Poly(dimethyl siloxane) (PDMS)	Dow Corning
Sylgard 184 silicone elastomer kit	Dow Corning
Anti-rhBMP-2	Sigma	B9553
Goat anti-mouse IgG-HRP	Santa Cruz	sc-2005	Secondary antibody
Ampliflu Red assay	Sigma	90101
Dulbecco's Modified Eagle Medium (DMEM) (1x), liquid	Gibco	41966	High glucose
Fetal Bovine Serum (FBS)	Sigma	F7524	Sterile filtered, cell culture tested
Pen/Strep	Gibco	15140
Trypsin 0.05% (1x) with EDTA 4Na	Gibco	25300
Glycine (0.1 M)	Riedel-de Haën	33226
IGEPAL CA-630 (1%)	Sigma	I8896	Lysis buffer (ALP assay)19
Magnesium chloride (MgCl2)(1 mM)	Carl Roth	HNO3.2
Zinc chloride (ZnCl2) (1 mM)	Carl Roth	3533.1
p-nitrophenylphosphate (pNPP)	Sigma	S0942	Phosphatase substrate
Anti-mysin heavy chain (MHC)	Developmental Studies Hybridoma Bank, University of Iowa	MF20	Monoclonal antibody
Alexa Fluor 488 Goat anti-mouse IgG	Invitrogen	A11001
DAPI	Sigma	D9542
Equipment
Ultrsonic bath (Sonorex Super RK 102H), Frequency 35 kHz	BANDELIN electronic GmbH Co. KG
MED 020 Sputtercoating system	BAL-TEC AG		Coating conditions Cr: 120 mA, 1.3 x 10-2 mbar, 30 sec Au: 60 mA, 5.0 x 10-2 mbar, 45 sec
Tecan Infinite M200 Plate reader	Tecan