E 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 1338247-35-0 implantation in nude mice. At 22948146 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 order 69056-38-8 monomers [27]. These glue contents in mixture formed an efficient cross-linking network that could capture MSCsrapidly and promote the cell attachment and proliferation. Therefore, higher seeding efficiency was obtained in fibrin hydrogel-assisted seeding groups. We further identified the effect of hydrodynamic culture on cell proliferation and differentiation in vitro. There is still no consensus on whether tissue-engineered bone grafts need to be cultured in vitro before implantation. Many studies have suggested that in vitro culture can allow the seeded cells to stably adhere on the scaffold and, thereby, prevent their detachment, migration, or death resulting from changes of microenvironment [3,4,28]. Wang et al, however, suggested that the in vivo condition should be optimal for the growth, differentiation, and function of cells. In contrast, in vitro cultured constructs may be structurally unstable, mechanically weak, and subject to changes in tissue structure and type [29]. In an attempt to combine the advantages of pre-implantation culture and in vivo microenvironment, some studies also explored ectopic 
implantation to engineer mature, vascularized bone grafts [30]. These “in vivo engineered” grafts were found to have superior osteogenic activities, but the technique involves a long in viv.E 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 22948146 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 formed an efficient cross-linking network that could capture MSCsrapidly and promote the cell attachment and proliferation. Therefore, higher seeding efficiency was obtained in fibrin hydrogel-assisted seeding groups. We further identified the effect of hydrodynamic culture on cell proliferation and differentiation in vitro. There is still no consensus on whether tissue-engineered bone grafts need to be cultured in vitro before implantation. Many studies have suggested that in vitro culture can allow the seeded cells to stably adhere on the scaffold and, thereby, prevent their detachment, migration, or death resulting from changes of microenvironment [3,4,28]. Wang et al, however, suggested that the in vivo condition should be optimal for the growth, differentiation, and function of cells. In contrast, in vitro cultured constructs may be structurally unstable, mechanically weak, and subject to changes in tissue structure and type [29]. In an attempt to combine the advantages of pre-implantation culture and in vivo microenvironment, some studies also explored ectopic implantation to engineer mature, vascularized bone grafts [30]. These “in vivo engineered” grafts were found to have superior osteogenic activities, but the technique involves a long in viv.
