Gh convenient, this method can attain only limited cell density. Under the action of gravity force, the seeded cells are Ergocalciferol easily detached from the scaffold and became concentrated at its bottom side, thus resulting in loss of cells. VariousFigure 6. Nude mice subcutaneous implantation model for the evaluation of osteogenic activity; (A) a photograph showing a nude mouse with four implants; (B) a radiograph 4 weeks after implantation; (C) a radiograph 8 weeks after implantation; (D) a radiograph 12 weeks after implantation. The radiographic densities of the implants increased from week 4 to week 12. The osteogenesis of implants was not clear at weeks 4 and 8 postoperative. It was not until 12 weeks postoperative that the imagings of implants in the radiographs were clearly observed. At week 12, implant II clearly showed increased density indicating calcification. doi:10.1371/journal.pone.0053697.gEffects of Initial Cell and Hydrodynamic CultureFigure 8. HE staining of ectopic bone formation in nude mice at 12 weeks (6100), Implant I can be seen partially degraded DBM stand, surrounded by fibrous connective tissue replaced; Implant II showed more mature 25331948 bone structure of a small beam than other groups; both Implant III and IV showed small beam structure of bone with some cartilage-like structure partially visible, bone formation maturity lower than Implant II. doi:10.1371/journal.pone.0053697.gFigure 7. Wet weight and bone mineral density of implants after subcutaneous implantation in nude mice. At 12 weeks postoperative, implant in group II showed higher wet weight (A) and bone mineral density (B) than that in other groups(p,0.05). *indicates a statistically significantly lower value compared with other implants; # indicates a statistically higher value compared with other implants. doi:10.1371/journal.pone.0053697.gbecause of their poor strength and limited bone conductivity. Despite this major disadvantage, hydrogels may improve the adhesion between seeded cells and the scaffold [13]. In this study, we compared the seeding efficiency and initial cell density resulting from three seeding methods: fibrin hydrogelassisted seeding, hydrodynamic seeding (simulated microgravity in RWVB), and the simple static infiltration. Microscopy, cell counting, and viability assays showed that fibrin hydrogel-assisted seeding generated a significantly higher seeding efficiency and initial cell density than the other two methods. The improvement can increase the utilization of seeded cells and is expected to increase the osteogenic activity of the resulting grafts. Fibrin glue has been clinically confirmed to be safe, biocompatible, and fully absorbable within two weeks [23]. A recent clinical study used fibrin as a carrier for chondrocytes to treat cartilage defects and obtained Eledoisin positive results [24]. The fibrin glue used in this study was a mixture of fibrinogen, thrombin, factor XIII, and calcium salt. Fibrinogen is a major plasma protein (350 kDa) that stimulates proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing [25]. Thrombin is an enzyme that converts soluble fibrinogen into insoluble fibrin between 10 and 60 seconds and acts as a tissue adhesive [26]. Factor XIII, which exists in the fibrinogen component of the glue, cross links and stabilises the clot’s fibrin monomers [27]. These glue contents in mixture forme.Gh convenient, this method can attain only limited cell density. Under the action of gravity force, the seeded cells are easily detached from the scaffold and became concentrated at its bottom side, thus resulting in loss of cells. VariousFigure 6. Nude mice subcutaneous implantation model for the evaluation of osteogenic activity; (A) a photograph showing a nude mouse with four implants; (B) a radiograph 4 weeks after implantation; (C) a radiograph 8 weeks after implantation; (D) a radiograph 12 weeks after implantation. The radiographic densities of the implants increased from week 4 to week 12. The osteogenesis of implants was not clear at weeks 4 and 8 postoperative. It was not until 12 weeks postoperative that the imagings of implants in the radiographs were clearly observed. At week 12, implant II clearly showed increased density indicating calcification. doi:10.1371/journal.pone.0053697.gEffects of Initial Cell and Hydrodynamic CultureFigure 8. HE staining of ectopic bone formation in nude mice at 12 weeks (6100), Implant I can be seen partially degraded DBM stand, surrounded by fibrous connective tissue replaced; Implant II showed more mature 25331948 bone structure of a small beam than other groups; both Implant III and IV showed small beam structure of bone with some cartilage-like structure partially visible, bone formation maturity lower than Implant II. doi:10.1371/journal.pone.0053697.gFigure 7. Wet weight and bone mineral density of implants after subcutaneous implantation in nude mice. At 12 weeks postoperative, implant in group II showed higher wet weight (A) and bone mineral density (B) than that in other groups(p,0.05). *indicates a statistically significantly lower value compared with other implants; # indicates a statistically higher value compared with other implants. doi:10.1371/journal.pone.0053697.gbecause of their poor strength and limited bone conductivity. Despite this major disadvantage, hydrogels may improve the adhesion between seeded cells and the scaffold [13]. In this study, we compared the seeding efficiency and initial cell density resulting from three seeding methods: fibrin hydrogelassisted seeding, hydrodynamic seeding (simulated microgravity in RWVB), and the simple static infiltration. Microscopy, cell counting, and viability assays showed that fibrin hydrogel-assisted seeding generated a significantly higher seeding efficiency and initial cell density than the other two methods. The improvement can increase the utilization of seeded cells and is expected to increase the osteogenic activity of the resulting grafts. Fibrin glue has been clinically confirmed to be safe, biocompatible, and fully absorbable within two weeks [23]. A recent clinical study used fibrin as a carrier for chondrocytes to treat cartilage defects and obtained positive results [24]. The fibrin glue used in this study was a mixture of fibrinogen, thrombin, factor XIII, and calcium salt. Fibrinogen is a major plasma protein (350 kDa) that stimulates proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing [25]. Thrombin is an enzyme that converts soluble fibrinogen into insoluble fibrin between 10 and 60 seconds and acts as a tissue adhesive [26]. Factor XIII, which exists in the fibrinogen component of the glue, cross links and stabilises the clot’s fibrin monomers [27]. These glue contents in mixture forme.