Moreover, immunofluorescence staining data confirmed that the application of G protein as a linker for anti-BMP-2 to ACS scaffold leads to increased expression and/or accumulation of BMP-2, BMP-4, and BMP-7 ligands within reconstructed tissues, while specimens immobilized with anti-BMP2 mAb alone showed significantly less amounts of positive staining. The present study utilized Protein G to control the orientation of binding of anti-BMP-2 mAb to scaffold. G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (< 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies < 0.05). Altogether, our results demonstrated that application of Protein G as a linker to adsorb anti-BMP-2 monoclonal antibodies onto the scaffold was accompanied by increased binding of the anti-BMP-2 mAb/BMP immune complex to BMP-receptor positive cell, as well as increased volume and strength of bone formation capturing of endogenous BMP-2, -4 and -7 by anti-BMP-2 mAb, as well as bone formation.15C17 This approach was termed antibody-mediated osseous regeneration (AMOR). Our previous studies have demonstrated ability of both murine-derived,15C17 as well as chimeric anti-BMP2 monoclonal antibodies to be effective in AMOR.18 Stork et al. in their studies reported that fusing a single-chain diabody to an albumin-binding domain from streptococcal Protein G improved the circulation time by a factor of 6.19 Therefore, we have hypothesized that anti-BMP-2 mAb captures BMPs, which are then presented to their cellular receptors, triggering their osteogenic differentiation. This will require availability of the antigen-binding region of antibody to bind to BMPs in domain(s), which do PQBP3 not interfere with interactions with their cellular receptors. To begin to further test this hypothesis, it was sought to determine whether binding of anti-BMP-2 mAb to the scaffold through its Fc region may be a more effective strategy, since this is likely to leave antigen-binding sites available to binding BMP ligands. To that end, Protein G, which is a bacterial cell wall protein with specific affinity for immunoglobulin (IgG) was utilized. If confirmed, this information will have utility in optimizing AMOR for translational applications. Materials and methods Antibodies and Protein G We generated and used a chimeric anti-BMP2 IgG2 mAb according to the method previously reported.18 An isotype-matched mAb (Iso mAb) with no specificity for BMP2 was utilized as the negative control. The rec-Protein G (Recombinant Protein G from < 0.01, and ***< 0.001. In vitro binding and release characteristics of anti-mAb Protein G complex To examine potential differences in the binding and release profile of the chimeric mAb or chimeric mAb Protein G complex on ACS scaffold, an binding and release kinetics study was performed. Results demonstrated sustained release of anti-BMP-2 mAb or Protein G/anti-BMP-2 mAb immune complex for up to 14 days (Figure 3(a)). Additionally, no statistically significant difference was found in the levels of the mAb detected on ACS scaffold after 14 days (Figure 3(b)). These results confirmed that when Protein G (either recombinant or Protein G coupled to microbeads) is used as linker for binding of anti-BMP-2 mAb to ACS, release of the mAb from the ACS scaffold is not inhibited. Open in a separate window Figure 3 Characterization of the release profile and binding of chimeric mAb and chimeric mAb Protein G complex-loaded scaffolds. (a) The release of mAb was calculated by measuring mAb concentrations in solution at various time points. (b) Fluorescence microscopic analysis demonstrating binding of anti-BMP-2 mAb on Ixabepilone ACS scaffold detected by FITC-conjugated goat anti-human secondary antibody at different time intervals. *< 0.05. In vivo osteogenic properties of Protein G/anti-BMP2 mAb complex To determine the effects of orientation of binding of anti-BMP-2 to scaffold, Protein G-coupled microbeads were first incubated with ACS, followed by incubation with anti-BMP-2 mAbs. The ACS/Protein G/anti-BMP-2 mAb or ACS/Protein G/isotypic mAb, ACS/anti-BMP-2 mAb or ACS/isotypic mAb were each implanted into critical size rat calvarial defects. After eight weeks, healing of calvarial defects was studied by micro-CT and histology. Micro-CT analysis (Figure 4(a)) showed increased volume of bone formation within calvarial defects implanted with ACS/Protein G/anti-BMP2 mAb in comparison to the defects implanted with anti-BMP2 mAb adsorbed directly on ACS (< Ixabepilone 0.05) (Figure 4(b)). Substitution of anti-BMP-2 mAb with isotype control mAb with or without Protein G was not associated with any significant bone formation. Open in a separate window Figure 4 (a) Representative 3D reconstruction of micro-CT images of bone volume within rat calvaria. Anti-BMP-2 mAb immobilized on ACS with or without Protein Ixabepilone G-coupled microbeads linker implanted.