S in groups C and D continued to increase, although at lower levels and slopes.Histology of retrieved implantsTwelve weeks after order PHCCC implantation, implant I (Fig. 8A) showed partial degradation of DBM scaffold and replacement by fibrousFigure 3. Photomicrographs (6100, 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 constructs was evaluated by ALP activities (B). The number of cells was increased with culture time except group C. The dynamic culture 26001275 (groups A and B) showed an obvious ability of promoting proliferation of cells. The ALP activities in all groups increased from day 2 to day 14 (B). The ALP activities in groups A, B, D were statistically higher than that in groups C(p,0.05) from day 4 to day 14. indicates a statistically higher value compared with group C(p,0.05). doi:10.1371/journal.pone.0053697.gmethods have been used to promote cell penetration and minimize cell detachment [20,21], such as the use of negative pressure and magnetic field. Although effective to varying degrees, these methods cannot substantially increase the initial cell density in the scaffold. Recent studies found that RWVBs can produce a simulated microgravity environment to allow cells to diffuse and become uniformly distributed in the interior of scaffolds [9,22]. Hydrogels have been combined with seeded cells to construct grafts for the repair of cartilage as well as bone [13]. Hydorgels alone, however, are not satisfactory for constructing bone graftsconnective tissues around the periphery. Implant II (Fig. 8B) showed relatively mature bone trabeculae but no chondroid tissues. Implant III (Fig. 8C) showed less mature bone trabeculae than implant II, in addition to chondroid structures in a few locations. Implant IV (Fig. 8D) showed new bone trabeculae that were less mature than those formed in implants II and III; transformation of chondroid tissue to immature bony tissue was also locally observed.DiscussionIn the present study, we evaluated the effects of seeding methods on seeding efficiency and initial cell density for constructing tissueengineered bone. Compared with other synthetic bone substitutes, tissue-engineered grafts generally have superior osteogenic activities because of the incorporation of seeded cells. Various factors can influence the osteoblastic differentiation of marrow stromal cells in tissue engineering scaffolds during cultivation, including the density and SMER 28 custom synthesis spatial distribution of the seeded cells in the scaffolds [1,2,4]. Seeded cells are commonly seeded in scaffolds by static infiltration. Althou.S 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, 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 constructs was evaluated by ALP activities (B). The number of cells was increased with culture time except group C. The dynamic culture 26001275 (groups A and B) showed an obvious ability of promoting proliferation of cells. The ALP activities in all groups increased from day 2 to day 14 (B). The ALP activities in groups A, B, D were statistically higher than that in groups C(p,0.05) from day 4 to day 14. indicates a statistically higher value compared with group C(p,0.05). doi:10.1371/journal.pone.0053697.gmethods have been used to promote cell penetration and minimize cell detachment [20,21], such as the use of negative pressure and magnetic field. Although effective to varying degrees, these methods cannot substantially increase the initial cell density in the scaffold. Recent studies found that RWVBs can produce a simulated microgravity environment to allow cells to diffuse and become uniformly distributed in the interior of scaffolds [9,22]. Hydrogels have been combined with seeded cells to construct grafts for the repair of cartilage as well as bone [13]. Hydorgels alone, however, are not satisfactory for constructing bone graftsconnective tissues around the periphery. Implant II (Fig. 8B) showed relatively mature bone trabeculae but no chondroid tissues. Implant III (Fig. 8C) showed less mature bone trabeculae than implant II, in addition to chondroid structures in a few locations. Implant IV (Fig. 8D) showed new bone trabeculae that were less mature than those formed in implants II and III; transformation of chondroid tissue to immature bony tissue was also locally observed.DiscussionIn the present study, we evaluated the effects of seeding methods on seeding efficiency and initial cell density for constructing tissueengineered bone. Compared with other synthetic bone substitutes, tissue-engineered grafts generally have superior osteogenic activities because of the incorporation of seeded cells. Various factors can influence the osteoblastic differentiation of marrow stromal cells in tissue engineering scaffolds during cultivation, including the density and spatial distribution of the seeded cells in the scaffolds [1,2,4]. Seeded cells are commonly seeded in scaffolds by static infiltration. Althou.
