Combinations of magnetic nanoparticle-labeled human mesenchymal stem cells (hMSCs) and BMP2-releasing microparticles in 2.0% collagen hydrogels compared with either nanoparticles or BMP2 alone and controls, which were hMSCs alone. All hydrogels initially contracted within 72 hours of seeding; subsequent matrix formation and proliferation of MSCs resulted in an increase in hydrogel size by day 28 of the experiment. Significant differences were seen in the total size and density of the gels, which approximately match the positions of the equivalent groups from femur-injection studies (Fig. 4) as determined by x-ray microtomography data. (A): BMP2 alone resulted in an increase in hydrogel size, whereas prelabeling hMSCs with either magnetic nanoparticle caused an increase in gel density. Combinations of both BMP2-releasing microparticles and magnetic nanoparticles resulted in an increase in both volume and density, with the greatest effect seen from the combination of BMP2 and TREK-Ab labeled hMSCs. (B): Increasing the analysis threshold for the microtomography to quantify only the mineralized portion of the hydrogel revealed that the volume of the mineralizing high-density material was similar for the control and BMP2 alone, whereas cells treated with either nanoparticle in combination with BMP2 were significantly more mineralized. Nanoparticle-only and nanoparticle + BMP combinations resulted in the formation of more numerous and thicker mineralized regions within the hydrogel than either controls or BMP2 alone when analyzed as “trabeculae.” (C): The diameter of each circle surrounding the data point reflects the average density of these regions. (D–F): These mineralized nodular regions were significantly less interconnected in the TREK-nanoparticle-containing groups compared with the control group (D) and are highlighted in green in the microtomography reconstructions, shown as cross-sections through the center of MSC-seeded hydrogels containing BMP2 particles alone (E), compared with hydrogels containing BMP2 particles plus TREK-nanoparticle-labeled hMSC (F). Error bars show standard error of the mean (n = 7). Scale bar = 1 mm. ∗, p < .05; ∗∗, p < 0.01; ∗∗∗, p < .001. Abbreviations: BMP2, bone morphogenetic protein 2; RGD, Arg-Gly-Asp tripeptide.