Site-Directed Immobilization of BMP-2: Two Approaches for the Production of Innovative Osteoinductive Scaffolds

Abstract

The regenerative potential of bone is strongly impaired in pathological conditions, such as nonunion fractures. To support bone regeneration various scaffolds have been developed in the past, which have been functionalized with osteogenic growth factors such as bone morphogenetic proteins (BMPs). However, most of them required supra-physiological levels of these proteins leading to burst releases, thereby causing severe side effects. Site-specific, covalent coupling of BMP2 to implant materials might be an optimal strategy in order to overcome these problems. Therefore, we created a BMP-2 variant (BMP2-K3Plk) containing a noncanonical amino acid (propargyl-l-lysine) substitution introduced by genetic code expansion that allows for site-specific and covalent immobilization onto polymeric scaffold materials. To directly compare different coupling strategies, we also produced a BMP2 variant containing an additional cysteine residue (BMP2-A2C) allowing covalent coupling by thioether formation. The BMP2-K3Plk mutant was coupled to functionalized beads by a copper-catalyzed azide-alkyne cycloaddition (CuAAC) either directly or via a short biotin-PEG linker both with high specificity. After exposing the BMP-coated beads to C2C12 cells, ALP expression appeared locally restricted in close proximity to these beads, showing that both coupled BMP2 variants trigger cell differentiation. The advantage of our approach over non-site-directed immobilization techniques is the ability to produce fully defined osteogenic surfaces, allowing for lower BMP2 loads and concomitant higher bioactivities, for example, due to controlled orientation toward BMP2 receptors. Such products might provide superior bone healing capabilities with potential safety advantages as of homogeneous product outcome.

Bone physiology as inspiration for tissue regenerative therapies

“…Cell-interacting biomaterials containing covalently immobilized proteins and growth factors have been shown to modulate stem cells response and drive effective bone repair [299]. Indeed, the modification of biomaterials with covalently attached growth factors was suggested as a promising manner of maintaining physiological levels and prolong their life time [300, 301]. For example, methoxy poly(ethylene glycol) injectable hydrogels covalently modified with BMP-2 through a click reaction showed enhanced synergic osteogenic differentiation of human periodontal ligament cells as compared to soluble BMP-2 formulations [302].…”

Copresentation of BMP-6 and RGD Ligands Enhances Cell Adhesion and BMP-Mediated Signaling

“…Concerning the immobilization of the BMPs, several methods have been reported for their immobilization, mainly involving BMP-2 [28] and BMP-7 full proteins and short peptide sequences, on material surfaces [36,37]. More recently, site-specific modification of recombinant human BMP-2 for the covalent binding to a linker via click chemistry was immobilized to NeutrAvidin-agarose beads [38]. While our strategy does not allow a precise control over the orientation of the molecule, it however ensures the specific binding of growth factors on the surface without further modification of active residues of the protein.…”

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

“…However, these methods entail protein modifications thus can also affect the outcome of individual cellular responses. Recently, Tabisz et al [112] published a strategy for sitedirected coupling of BMP-2 to scaffolds by click-chemistry utilizing an artificial amino acid, which had been introduced during bacterial expression by amber codon suppression [113]. The constructed BMP-2 variant showed the same bioactivity compared to wildtype BMP-2 and could be coupled site-directed and biologically active coupled to solid surfaces.…”