Neural Crest-Derived Dental Pulp Stem Cells in DPSC transplants showed positive DMP1, DSP, and BSP staining in both cytoplasmic area and extracellular matrices (Figures 7J–7O). We observed elongated and polarized cells that were positive for DMP1 and DSP in the DPSC transplantations (Figures 7K, 7M, 7N insets). On the other hand, the bone-like structures created by BMSCs were negative for DSP, but positively for BSP (Figures 7Q and 7R inset). We also observed positive DMP1 staining in both DPSC-derived odontoblast-like cells and BMSC-derived osteoblast-/osteocyte-like cells (Figures 7M inset, 7P osteoblast-like cells indicated by arrowheads, osteocyte-like cells indicated by arrows). These results, based on tissue morphology and antibody staining, indicate that in vivo DPSCs differentiated into odontoblast-like cells and generated dentin-like matrix whereas BMSCs differentiated into osteoblast-like cells and formed bone condensations. Upon closer examination of the odontoblastic niche in the 12- week DPSC transplants, similar to 5-week transplants, we observed abundant microvessels in close proximity to odonto- blast-like cells with fenestrated morphology surrounded by PKH- 26+ cells, in both non clonal and clonal DPSC transplants (Figures 6E, 6F black arrowheads, S9G, 8A, and 8B). In contrast, matrices that were generated by BMSC transplants surrounded areas rich in HAp/TCP (Figures 6G black arrowheads, and 6J). Although microvessels were observed in the BMSC transplants, the BMSC mineralized areas did not show close proximity to vessels (Figure 8C black arrowheads). Interestingly, in the BMSC transplants microvessels were predominantly found in areas rich in adipocytes (Figures 6G and S9H). In both transplants, microvessels were mature as circulating red blood cells can be seen throughout (Figure 8D). Therefore, we performed a quantification of the average distance of donor nuclei in condensed matrices to nearest capillaries. Surprisingly, the distance from odontoblast nuclei to nearest capillary was very close (9 mm +/2 4.49) and very consistent across all DPSC transplants whereas the distance of osteocyte nuclei to capillaries in bone condensations were significantly farther (187 mm+/2 76.88) (Figure 8E). This capillary Figure 8. Proximity of donor cell nuclei to blood vessels in DPSC and BMSC transplantation. The average distance of donor nuclei in condensed matrices to nearest capillaries was measured using ImageJ v1.43u (Wayne Rasband, NIH; http://rsb.info.nih.gov/ij). The distances between donor nuclei to blood vessels were represented by yellow lines. (A and B) In the 12-week DPSC transplants, abundant microvessels with fenestrated morphology were seen in close proximity to odontoblast-like cells in both non clonal and clonal DPSC transplants. (C) In BMSC transplants microvessels were predominantly found in areas rich in adipocytes whereas mineralized areas formed by BMSCs were distant to microvessels. Microvessels were mature as circulating red blood cells can be seen throughout in both DPSC and BMSC transplants. (D) Abundant microvessels with fenestrated morphology containing circulating red blood cells (insets) are seen in close proximity to mineralized matrix formed by DPSCs. (E) The bar graph shows that the average distance from odontoblast nuclei to nearest capillary was significantly close (9 mm 6 4.49) and consistent across all DPSC transplants whereas the average distance of osteocyte nuclei in bone condensations to nearest microvessels were significantly farther (187 mm 6 76.88). The average distances from donor cell nuclei to blood vessels in non-clonal DPSCs (n = 5, n; numbers of measured area) or clonal DPSCs (n = 7) and BMSCs (n = 5) are statistically significant. Student’s t-test calculated * P#0.05. Error bars represent 6SEM. Scale bars indicate 25 mm. doi:10.1371/journal.pone.0027526.g008 PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27526