%0 Journal Article %1 siverino2018sitedirected %A Siverino, C. %A Tabisz, B. %A Luhmann, T. %A Meinel, L. %A Muller, T. %A Walles, H. %A Nickel, J. %D 2018 %J J Vis Exp %K Animals myOwn uni_network %N 133 %R 10.3791/56616 %T Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry %U https://www.ncbi.nlm.nih.gov/pubmed/29658921https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933278/pdf/jove-133-56616.pdf %X Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

@article{siverino2018sitedirected, abstract = {Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.}, added-at = {2024-02-15T15:08:22.000+0100}, author = {Siverino, C. and Tabisz, B. and Luhmann, T. and Meinel, L. and Muller, T. and Walles, H. and Nickel, J.}, biburl = {https://www.bibsonomy.org/bibtex/2f09c9f4c4056c4e3fe2d40aadbff8c9d/jvsi_all}, doi = {10.3791/56616}, interhash = {b653600889098f0d6f41cc0282002dcf}, intrahash = {f09c9f4c4056c4e3fe2d40aadbff8c9d}, issn = {1940-087X (Electronic) 1940-087X (Linking)}, journal = {J Vis Exp}, keywords = {Animals myOwn uni_network}, note = {Siverino, Claudia Tabisz, Barbara Luhmann, Tessa Meinel, Lorenz Muller, Thomas Walles, Heike Nickel, Joachim eng Video-Audio Media 2018/04/17 J Vis Exp. 2018 Mar 29;(133):56616. doi: 10.3791/56616.}, number = 133, timestamp = {2024-02-15T15:11:55.000+0100}, title = {Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry}, type = {Journal Article}, url = {https://www.ncbi.nlm.nih.gov/pubmed/29658921https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933278/pdf/jove-133-56616.pdf}, year = 2018 }