title: 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.
authors.affiliation: Lehrstuhl für Tissue Engineering und Regenerative Medizin, Universitätsklinikum Würzburg , Röntgenring 11, D-97070 Würzburg, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Tabisz
authors.forename: Werner
authors.initials: W
authors.lastname: Schmitz
authors.affiliation: Lehrstuhl für Funktionswerkstoffe der Medizin und der Zahnheilkunde, Universitätsklinikum Würzburg , Pleicherwall 2, D-97070 Würzburg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schmitz
authors.forename: Tessa
authors.initials: T
authors.lastname: Luehmann
authors.forename: Eva
authors.initials: E
authors.lastname: Heusler
authors.forename: Jens-Christoph
authors.initials: JC
authors.lastname: Rybak
authors.forename: Lorenz
authors.identifier: 0000-0002-7549-7627
authors.initials: L
authors.lastname: Meinel
authors.affiliation: Lehrstuhl für molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs Institut für Biowissenschaften, Universität Würzburg , Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
authors.forename: Juliane E
authors.initials: JE
authors.lastname: Fiebig
authors.affiliation: Lehrstuhl für molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs Institut für Biowissenschaften, Universität Würzburg , Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Mueller
authors.affiliation: Lehrstuhl für Tissue Engineering und Regenerative Medizin, Universitätsklinikum Würzburg , Röntgenring 11, D-97070 Würzburg, Germany.|Translationszentrum Würzburg "Regenerative Therapien für Krebs- und Muskuloskelettale Erkrankungen", Institutsteil Würzburg, Fraunhofer-Institut für Grenzflächen- und Bioverfahrenstechnik (IGB) , Röntgenring 11, D-97070 Würzburg, Germany.
authors.forename: Joachim
authors.identifier: 0000-0003-0154-1370
authors.initials: J
authors.lastname: Nickel
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001672:Biocompatible Materials / Q000737:chemistry; D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry / Q000235:genetics*; D001861:Bone Regeneration / Q000502:physiology; D001842:Bone and Bones / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D010012:Osteogenesis / Q000502:physiology; D011108:Polymers / Q000737:chemistry; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: C525718:BMP2 protein, human; D001672:Biocompatible Materials; D055396:Bone Morphogenetic Protein 2; D055767:Immobilized Proteins; D011108:Polymers; D000469:Alkaline Phosphatase