Rest to allow the cells to enter the scaffold. With this method, the initial cell density (the number of cells which attached in 3D scaffold when tissue engineering bone were preparation and without culturing in vivo or in vitro) in the scaffold can be increased by increasing the cell concentration of the suspension within a certain range, though at the expense of seeding efficiency (i.e. the percentage of cells that entered the scaffold), but cannot be further increased beyond a plateau level [6]. In comparison, in the hydroASP015K dynamic seeding method, cells are allowed to adhere to the scaffold in a dynamicfluid flow created by a bioreactor. With this method, the cell agglomeration accelerates with the cell density in the seeding suspension, thus facilitating the adherence of cells to the scaffold, increasing the speed and density of cell seeding, and improving the spatial distribution of cells 
in the scaffold [7,8]. In addition to seeding, hydrodynamic conditions can also substantially affect the subsequent in vitro culture of cell-scaffold constructs. A dynamic fluid flow was found to positively affect the behavior of seeded cells, such as proliferation, purchase ��-Sitosterol ��-D-glucoside differentiation, and migration [4,7,9,10,11]. However, dynamic fluid flow may also result in cell detachment and shear-induced damage, and thus, loss in cell utilization [3,12]. A number of studies have separately exploited the advantages associated with a higher initial cell density or hydrodynamic culture [7,13]. Zhao et al increased the initial density of human umbilical cord mesenchymal stem seeded cells in injectable bone tissue engineering constructs by using hydrogel microbeads [13]. Ericka et al seeded chondrocytes onto polyglycolid acid scaffolds under hydrodynamic conditions, and obtained intermediate initial cell densities and sustained subsequent proliferation [7]. The optimal tissue engineering technique should combine methods to increase the initial cell density and create an appropriate hydrodynamic environment to accelerate the in vitro maturation of the cell-scaffold constructs into clinically applicable grafts. Here, we investigate whether a combination of fibrin glueassisted seeding and hydrodynamic culture in rotating wall vesselEffects of Initial Cell and Hydrodynamic Culturebioreactor can substantially improve the seeding efficiency and subsequent proliferation and osteoblastic differentiation. We further determined if these improvements translated into enhanced osteogenic activity in a nude mice subcutaneous implantation model. This study aims to understand the effects of the key factors of tissue engineering preparation methods, including initial cell density and hydrodynamic culture methods, in an attempt to provide experimental basis for improvement the osteogenesis performance of bone tissue engineering.Materials and Methods Ethics statementNude mice (6 weeks old) were purchased from the Laboratory Animal Center of our university. The animal experiment was approved by the ethics committee of Third Military Medical University and conducted in conformity 10457188 with the `Guiding Principles for Research Involving Animals and Human Beings’ as adopted by The American Physiological Society.Isolation and characterization of hMSCsHuman mesenchymal stem 
cells (hMSCs) derived from bone marrow of the iliac crests of young healthy volunteers were provided from Tissue Engineering Research and Development Center of The Third Military Medical University. hMSCs were isolated by dens.Rest to allow the cells to enter the scaffold. With this method, the initial cell density (the number of cells which attached in 3D scaffold when tissue engineering bone were preparation and without culturing in vivo or in vitro) in the scaffold can be increased by increasing the cell concentration of the suspension within a certain range, though at the expense of seeding efficiency (i.e. the percentage of cells that entered the scaffold), but cannot be further increased beyond a plateau level [6]. In comparison, in the hydrodynamic seeding method, cells are allowed to adhere to the scaffold in a dynamicfluid flow created by a bioreactor. With this method, the cell agglomeration accelerates with the cell density in the seeding suspension, thus facilitating the adherence of cells to the scaffold, increasing the speed and density of cell seeding, and improving the spatial distribution of cells in the scaffold [7,8]. In addition to seeding, hydrodynamic conditions can also substantially affect the subsequent in vitro culture of cell-scaffold constructs. A dynamic fluid flow was found to positively affect the behavior of seeded cells, such as proliferation, differentiation, and migration [4,7,9,10,11]. However, dynamic fluid flow may also result in cell detachment and shear-induced damage, and thus, loss in cell utilization [3,12]. A number of studies have separately exploited the advantages associated with a higher initial cell density or hydrodynamic culture [7,13]. Zhao et al increased the initial density of human umbilical cord mesenchymal stem seeded cells in injectable bone tissue engineering constructs by using hydrogel microbeads [13]. Ericka et al seeded chondrocytes onto polyglycolid acid scaffolds under hydrodynamic conditions, and obtained intermediate initial cell densities and sustained subsequent proliferation [7]. The optimal tissue engineering technique should combine methods to increase the initial cell density and create an appropriate hydrodynamic environment to accelerate the in vitro maturation of the cell-scaffold constructs into clinically applicable grafts. Here, we investigate whether a combination of fibrin glueassisted seeding and hydrodynamic culture in rotating wall vesselEffects of Initial Cell and Hydrodynamic Culturebioreactor can substantially improve the seeding efficiency and subsequent proliferation and osteoblastic differentiation. We further determined if these improvements translated into enhanced osteogenic activity in a nude mice subcutaneous implantation model. This study aims to understand the effects of the key factors of tissue engineering preparation methods, including initial cell density and hydrodynamic culture methods, in an attempt to provide experimental basis for improvement the osteogenesis performance of bone tissue engineering.Materials and Methods Ethics statementNude mice (6 weeks old) were purchased from the Laboratory Animal Center of our university. The animal experiment was approved by the ethics committee of Third Military Medical University and conducted in conformity 10457188 with the `Guiding Principles for Research Involving Animals and Human Beings’ as adopted by The American Physiological Society.Isolation and characterization of hMSCsHuman mesenchymal stem cells (hMSCs) derived from bone marrow of the iliac crests of young healthy volunteers were provided from Tissue Engineering Research and Development Center of The Third Military Medical University. hMSCs were isolated by dens.
