Is most, then group D (Fig. 5 D), followed by group A (Fig. 5A) and group C (Fig. 5 C).X-ray radiographyThe radiographic densities of all implants increased from week 4 to week 12 (Fig. 6). Implant II(hydrogel-assisted seeding of 26107/ ml hMSCs, followed by dynamic culture for 12 d) showed substantially higher 79831-76-8 density than the other implants, and implant I (cell-free DBM scaffold) had the lowest densities at both time points. At week 12, implant I showed a slightly higher density compared with the host soft tissue, while implant II clearly showed increased density indicating calcification. The implants III (hydrogel-assisted seeding of 16108/ml hMSCs without further in vitro culture) and IV (hydrogel-assisted seeding 26107/ml MSCs followed by SPDB custom synthesis static culture for 12 d) also showed signs of calcification, but substantially weaker than that in implant II.Figure 2. Phase-contrast photomicrographs (6100) of cellscaffold constructs after in vitro culture for 12 d; (A) group A (dynamic seeding and dynamic culture), (B) group B (hydrogelassisted seeding and static flask culture, (C) group C (static seeding and static flask culture, control group), and (D) group D (hydrogel-assisted seeding and dynamic culture). Bar lengths are 100 um. doi:10.1371/journal.pone.0053697.gWet weight and bone mineral densityTwelve weeks after implantation, implant I showed a significantly lower wet weight compared with other implants (all p,0.01). Moreover, the wet weight of implant II was statistically higher than the implants III (p = 0.008) and D (p = 0.004). Implants III and IV were similar (p = 0.770) (Fig. 7A). Twelve weeks after 
implantation, implant II showed a significantly higher bone mineral density than all other implants (all p,0.01). The bone mineral density of implant I was significantly lower than the other implants (all p,0.01). Implants III and IV were similar (p = 0.741) (Fig. 7B).In comparison, the cell number in group D decreased slightly between 8?4 h in culture, then decreased more rapidly between days 1?, and remained stable thereafter. The ALP activities in all groups increased from day 2 to day 6 (Fig. 4B). The activities in groups A and B remained stable thereafter. 
In comparison, the activities in groups C and D continued to increase, although at lower levels and slopes.Histology of retrieved implantsTwelve weeks after implantation, implant I (Fig. 8A) showed partial degradation of DBM scaffold and replacement by fibrousFigure 3. Photomicrographs (6100, 15755315 methyl violet staining) of cell-scaffold constructs after in vitro culture for 12 d. The number of attached cells and density of extracellular matrix (ECM) fibers in the interior of the scaffold are obvious different among four groups, with group B (B) . group D (D) . group A (A) . group C (C). Bar lengths are 100 um. doi:10.1371/journal.pone.0053697.gEffects of Initial Cell and Hydrodynamic CultureFigure 5. Scanning electron micrographs of cell-scaffold constructs after in vitro culture for 12 days. The attached cells and extracellular matrix (ECM) fibers presented on the scaffolds in group B (B) and group D (D) are significantly outnumber those in group A (A) as well as group C (C).Bar lengths are 100 um. The black arrows indicate cells and the blue arrows indicate ECM fibers. doi:10.1371/journal.pone.0053697.gFigure 4. Proliferation of seeded cells in cell-scaffold constructs was detected by cell counting kit-8 (A) and osteoblastic differentiation of seeded cells in cell-scaffold const.Is most, then group D (Fig. 5 D), followed by group A (Fig. 5A) and group C (Fig. 5 C).X-ray radiographyThe radiographic densities of all implants increased from week 4 to week 12 (Fig. 6). Implant II(hydrogel-assisted seeding of 26107/ ml hMSCs, followed by dynamic culture for 12 d) showed substantially higher density than the other implants, and implant I (cell-free DBM scaffold) had the lowest densities at both time points. At week 12, implant I showed a slightly higher density compared with the host soft tissue, while implant II clearly showed increased density indicating calcification. The implants III (hydrogel-assisted seeding of 16108/ml hMSCs without further in vitro culture) and IV (hydrogel-assisted seeding 26107/ml MSCs followed by static culture for 12 d) also showed signs of calcification, but substantially weaker than that in implant II.Figure 2. Phase-contrast photomicrographs (6100) of cellscaffold constructs after in vitro culture for 12 d; (A) group A (dynamic seeding and dynamic culture), (B) group B (hydrogelassisted seeding and static flask culture, (C) group C (static seeding and static flask culture, control group), and (D) group D (hydrogel-assisted seeding and dynamic culture). Bar lengths are 100 um. doi:10.1371/journal.pone.0053697.gWet weight and bone mineral densityTwelve weeks after implantation, implant I showed a significantly lower wet weight compared with other implants (all p,0.01). Moreover, the wet weight of implant II was statistically higher than the implants III (p = 0.008) and D (p = 0.004). Implants III and IV were similar (p = 0.770) (Fig. 7A). Twelve weeks after implantation, implant II showed a significantly higher bone mineral density than all other implants (all p,0.01). The bone mineral density of implant I was significantly lower than the other implants (all p,0.01). Implants III and IV were similar (p = 0.741) (Fig. 7B).In comparison, the cell number in group D decreased slightly between 8?4 h in culture, then decreased more rapidly between days 1?, and remained stable thereafter. The ALP activities in all groups increased from day 2 to day 6 (Fig. 4B). The activities in groups A and B remained stable thereafter. In comparison, the activities in groups C and D continued to increase, although at lower levels and slopes.Histology of retrieved implantsTwelve weeks after implantation, implant I (Fig. 8A) showed partial degradation of DBM scaffold and replacement by fibrousFigure 3. Photomicrographs (6100, 15755315 methyl violet staining) of cell-scaffold constructs after in vitro culture for 12 d. The number of attached cells and density of extracellular matrix (ECM) fibers in the interior of the scaffold are obvious different among four groups, with group B (B) . group D (D) . group A (A) . group C (C). Bar lengths are 100 um. doi:10.1371/journal.pone.0053697.gEffects of Initial Cell and Hydrodynamic CultureFigure 5. Scanning electron micrographs of cell-scaffold constructs after in vitro culture for 12 days. The attached cells and extracellular matrix (ECM) fibers presented on the scaffolds in group B (B) and group D (D) are significantly outnumber those in group A (A) as well as group C (C).Bar lengths are 100 um. The black arrows indicate cells and the blue arrows indicate ECM fibers. doi:10.1371/journal.pone.0053697.gFigure 4. Proliferation of seeded cells in cell-scaffold constructs was detected by cell counting kit-8 (A) and osteoblastic differentiation of seeded cells in cell-scaffold const.
