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Nuclear factor of activated T cells (NFAT)-c1 is known as a key regulator in osteoclast differentiation and immune response. This study is a follow-up to our previous study showing the antiresorptive activity of VIVIT, a peptide type NFATc1 inhibitor, using absorbable collagen sponge (ACS). This study aimed to investigate the effective concentration range of local VIVIT that suppresses early excessive osteoclast activation and inflammation induced by high-dose recombinant human bone morphogenetic protein (rhBMP)-2 and concomitantly enhances bone healing in a rat critical-sized calvaria defect model. High-dose rhBMP-2 (40 μg/defect) alone significantly increased in vivo osteoclast activation and expression of the inflammatory cytokines interleukin-1β and transforming necrosis factor-α on the scaffold at 7 days after surgery. However, rhBMP-2 had no direct effect on osteoclast activation in vitro. Osteoclast activation by rhBMP-2 was significantly suppressed by combined treatment with VIVIT at concentrations of 75 and 150 μM, but not at 15 μM, whereas suppression of inflammation occurred at all doses of VIVIT. Microcomputed tomography at 4 and 8 weeks after implantation revealed that the combination of rhBMP-2 and VIVIT at 75 μM VIVIT led to a greater bone fraction at the initial defect area, compared with rhBMP-2 alone. These findings revealed that local administration of VIVIT at certain concentrations has multiple positive effects that weaken early excessive osteoimmunological responses and enhance bone healing after rhBMP-2 administration. VIVIT has the potential to expand the therapeutic area of high-dose rhBMP-2 therapy to inflammatory bone loss. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1299-1310, 2018.

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Protein microarrays are miniaturized two-dimensional arrays, incorporating thousands of immobilized proteins, typically printed in minute amounts on functionalized solid substrates, which can be analyzed in a high-throughput fashion. Irreproducibility of the printing techniques adopted, resulting in inconsistently and nonuniformly deposited microscopic spots, nonuniform signal intensities from the printed microspots, and significantly high background noise are some of the critical issues that affect protein analysis using traditional protein microarrays. To overcome such issues, in this study, we introduced a novel gold grid pattern-based protein microarray. The grid patterns incorporated in our microarray are equivalent to the spots used for protein analysis in conventional protein microarrays. We utilized the signal intensities from the grid patterns acting as spots for quantifying the protein concentration levels. To demonstrate the utility of our novel design concept, we quantified as low as 66.7 ng/mL of bovine serum albumin using our gold grid pattern-based protein microarray. Our grid pattern-based design concept for protein quantification overcame the signal nonuniformity issues and ensured that the dominance of any distorted signal from a single spot did not affect the overall protein quantification results as encountered in conventional protein microarrays.