Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation

Pohl, Theresa L. M.; Boergermann, Jan H.; Schwaerzer, Gerburg K.; Knaus, Petra; Cavalcanti‐Adam, Elisabetta Ada

doi:10.1016/j.actbio.2011.10.019

Cited by 74 publications

(80 citation statements)

References 43 publications

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“…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 activity 19 . The bioactivity of growth factors presented in an immobilized form has also been shown in other studies, indicating that the release of the proteins and their subsequent internalization are not essential for cell stimulation 24,25 .…”

Section: Discussionmentioning

confidence: 70%

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

Pohl

Schwab

Cavalcanti‐Adam

2013

JoVE

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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. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 70%

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

Pohl

Schwab

Cavalcanti‐Adam

2013

JoVE

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“…The observed time scale of internalization does not correlate with the activation of downstream Smad1/5/8 signaling. In fact, recent experiments with immobilized BMP2 clearly show that binding of BMP2 is sufficient to trigger Smad1/5/8 phosphorylation (Pohl et al, 2012). Thus, ligand internalization and its modulation by physiological BMP antagonists, acts at a different level and provides two modes of action: inhibition of BMP2 binding (Chordin); or enhanced removal of BMP2 from the cellular environment (Noggin, Gremlin).…”

Section: Discussionmentioning

confidence: 99%

Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists

Alborzinia

Schmidt-Glenewinkel

Ilkavets

et al. 2013

Journal of Cell Science

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SummaryBone morphogenetic proteins (BMPs) are members of the TGFb family of signaling proteins and play an important role during development and in tissue formation. BMP signaling is a well-studied process, which is initiated through binding of cognate receptors and processed through activation of Smad downstream mediators. A hallmark of BMP signaling is its modulation at the extracellular level through specific antagonists. Although it had been shown that BMP and TGFb receptors are internalized following activation, little is known about the fate of BMP ligands. We prepared biologically active fluorescently labeled BMP2 and quantitatively analyzed its binding and uptake in cells using flow cytometry and confocal microscopy. Exogenous BMP2 was rapidly bound to the cell surface and subsequently internalized in a time-dependent manner and accumulated in the cell center. Although binding to the cell surface was limited by binding sites at the beginning, internalization continously increased with time, after a short delay. Using different inhibitors we found that internalization of BMP2 through endosomal particles occurred in a clathrin-dependent pathway. Furthermore, uptake of BMP2 was modulated in strikingly different ways by BMP2 antagonists. Although Noggin and Gremlin increased BMP2 uptake, Chordin blocked BMP2 uptake, which was concentration dependent in both cases. In conclusion, our findings present interesting mechanisms for the modulation of BMP signaling by concentration gradients of BMP ligands and antagonists in a dose-and timedependent manner, which can provide an explanation of some properties of the BMP regulatory network.

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“…For tracking purpose, BMP-2 has been iodinated with Na 125 I prior to coupling. The activated surfaces were used in cell based assays using C2C12 cells [114]. Also here, the covalently immobilized BMP-2 activated BMP-dependent signal transduction, thus resulting in the expected cellular responses like suppression of myotube formation and upregulated ALP expression.…”

Section: Delivery Strategies Using Covalently Coupled Recombinant Bmp-2mentioning

confidence: 99%

How to use BMP-2 for clinical applications? A review on pros and cons of existing delivery strategies

Mumcuoglu¹,

Siverino²,

Tabisz³

et al. 2017

J Transl Sci

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Surface immobilization of bone morphogenetic protein 2 via a self-assembled monolayer formation induces cell differentiation

Cited by 74 publications

References 43 publications

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

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

Quantitative kinetics analysis of BMP2 uptake into cells and its modulation by BMP antagonists

How to use BMP-2 for clinical applications? A review on pros and cons of existing delivery strategies

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