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 conserved cysteine knot defines the three-dimensional structure of the BMP-2 protein and determines its unique properties, such as resistance against heat, denaturants and acidic pH^7-9. BMP-2 binds to serine/threonine kinase transmembrane receptors, thereby inducing signal transduction^10-12. Depending on the mode of receptor oligomerization, different signaling pathways are activated: a Smad-independent signaling cascade leads to alkaline phosphatase induction via p38 signaling, whereas a Smad-dependent pathway activated by receptor phosphorylation results in Smad complex nuclear translocation and activation of transcription of specific target genes, such as the inhibitor of differentiation (Id)^12-14.

In bone, BMP-2 induces the differentiation of mesenchymal stem cells into osteoblasts, thus stimulating the healing and de novo formation of bone. Currently, recombinantly expressed BMP-2 is applied clinically to enhance the healing of fractured sites. A common strategy in bone tissue engineering is the use of injectable growth factors, which is less invasive compared to local delivery systems. However, in vivo studies and clinical applications have shown that the short biological half-life, unspecific localization and rapid local clearance of BMP-2 may lead to several local, ectopic and systemic problems¹⁵. Hence, to obtain an effective presentation, the entrapment or immobilization of BMP-2 within or onto materials is necessary for its local and sustained delivery at the target site. Sustained delivery can be achieved with non-covalent retention approaches, such as physical entrapment, adsorption or ion complexation¹⁶. However, it is known that unspecific adsorption of proteins to surfaces may results in denaturation of the molecules¹⁷. For the covalent binding of growth factors, different types of supports have been developed over the last decade. The use of bifunctional linking molecules that target amino or carboxyl groups of the protein for example, is one type of approach that does not necessarily require protein modification to achieve its immobilization. In fact, while protein modification offers the advantage of controlling protein orientation, the introduction of artificial domains, peptide tags and site-specific chains may alter the biological activity of growth factors¹⁷. Thus, to circumvent denaturation due to interaction with the supporting material, surfaces can be functionalized beforehand, for example, with a self-assembled monolayer (SAM) of a linking molecule, followed by coupling of the desired factor¹⁸. We have used a SAM-based approach to covalently immobilize BMP-2 onto a surface by targeting its free amine residues and have shown that the immobilized protein retains both its short- and long-term biological activity¹⁹. This protocol provides a simple and efficient way to deliver BMP-2 to cells for in vitro studies on the mechanisms which occur at the cell membrane and regulate intracellular signaling responsible for osteogenic signaling.

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 benzene and ethyl acetate (p.a.) in a ratio of 1:1.

2. Preparation of Homogeneous Gold Layers

1. Clean glass coverslips with precision wipes and sonicate them for 5 min in a solution containing a 1:1 mixture of ethyl acetate (p.a.) and methanol (p.a.). Rinse substrates with methanol and dry off under nitrogen flow.
2. Place the clean substrates in the sputter coating device. Evacuate the chamber. Remove the uppermost chromium oxide layer from chromium target by sputtering for 60 sec. Place the samples underneath the metal beam and coat them with a 10 nm chromium layer, followed by coating with a 40 nm gold layer.

3. Surface Immobilization of BMP-2

1. Dissolve MU-NHS in N,N-dimethlyformamide (DMF) to a final concentration of approximately 1 mM and incubate gold substrates in the MU-NHS solution at RT for 4 hr under nitrogen atmosphere.
2. Sonicate surfaces in DMF for 2 min, rinse with DMF and MeOH and dry off under nitrogen flow.
3. Prepare a stock solution of rhBMP-2 in sterile 4 mM HCl with a concentration of 100 μg/ml (store aliquots at -80 °C). For working dilutions (3.5 μg/ml), dilute the stock solution in PBS/NaCl (PBS containing 1 M NaCl) and adjust to pH 8.5 immediately prior to use.
4. Incubate surfaces functionalized MU-NHS in the rhBMP-2 working solution at 4 °C overnight. Remove incubation supernatant. Sonicate surfaces in PBS/NaCl for 2 min and wash 3x with sterile PBS/NaCl.

4. Surface Characterization

1. To block unspecific adsorption of antibody, incubate surfaces with a 5% BSA (w/v) in PBS solution for 1 hr at RT, followed by incubation with an anti-BMP-2 antibody (1:100 in 1% (w/v) BSA/PBS solution) for 1 hr at RT. Wash surfaces twice with PBS and sonicate for 30 sec.
2. Incubate substrates with HRP-conjugated secondary antibody (1:1,000 in 1% (w/v) BSA/PBS solution) for 30 min at RT, then wash twice with PBS.
3. Use Ampliflu Red assay to measure HRP enzymatic activity at 570 nm with a plate reader.

5. Analysis of Biological Activity

1. Seed 1 x 10⁵ mouse C2C12 myoblasts per well in 6-well plates in growth medium, consisting of a high glucose DMEM containing pyruvate supplemented with 10% FBS, 1% penicillin/streptomycin and incubate them for 24 hr at 37 °C/5% CO_2.
2. Starve cells in serum-free DMEM for 3-5 hr prior to seeding onto the surfaces decorated with immobilized BMP-2.
3. For the investigation of short-term signaling induction, replace the medium by 200 μl fresh serum-free DMEM and place the immobilized BMP-2 surfaces above the cells. The surface should be handled with fine tip tweezers to avoid scratching.
4. Remove gently surfaces and aspirate the medium with a pipette, wash the cells twice with PBS. Proceed to analysis of cell responses.
5. For the analysis of long-term biological activity, plate cells onto surfaces and culture them at 37 °C/5% CO₂ in low serum conditions (2% FBS) for six days.

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