Laced at the right caudal position. Table 2. Implant groups for subcutaneous implantation.Statistical analysesData were expressed as mean 6 standard deviation. Data were analyzed by one-way analyses of variance (ANOVA) and Student?Newman euls (SNK) post hoc tests using SPSS 12.0 (SPSS, Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.Results Cell culture and characterizationThe hMSCs tended to form calcium nodus after 12 d conditional culture (Fig. 1A). The hMSCs also stained immunohistochemically positive for ALP (Fig. 1B and C), osteocalcin (Fig. 1E and F) and collagen type I (Fig. 1H and I) after 12-day osteogenic induction. The ALP activity of hMSCs (Fig. 1D) and osteocalcin concentration (Fig. 1G) in the culture Title Loaded From File medium were significantly higher in induced group than that in control group (P,0. 01).Group I II III IVScaffold DBM DBM DBM DBMSeeding condision no Hydrogel-assisted Hydrogel-assisted Hydrogel-assistedCell 114311-32-9 biological activity number per scaffold no 1.06106 1.06107 1.Culture time no dynamic culture 12 d no static flask culture 12 dPlace in nude mouse left rostral right rostral left caudal right caudaldoi:10.1371/journal.pone.0053697.tEffects of Initial Cell and Hydrodynamic CultureFigure 1. The culture and characterization of hMSCs. The hMSCs formed calcium nodus after 12 day culture (A). The hMSCs (2006) stained immunohistochemically positive for 26001275 ALP (B), osteocalcin (E) and collagen type I (H) compared to non-induced cells (C, F, and I). The ALP activity (D) of hMSCs and osteocalcin concentration (G) in the culture medium were significant higher in induced group than that in control group (non-induced cells). *p , 0. 01, compared with control group. doi:10.1371/journal.pone.0053697.gMicroscopy of cell-scaffold constructsIn group A (dynamic seeding followed by dynamic culture), after culture for 8 h, a small number of cells were observed on the surface of the pores in the scaffolds. Then, the cells gradually increased in number and became uniformly distributed on the surface of the pores. After culturing for 5 days, the cells started to produce ECM, the cells and ECM were both uniformly distributed (Fig. 2 A). In group B (hydrogel-assisted seeding followed by static culture), the cell-laden gel filled most pores in scaffold after seeding immediately and the resulting constructs remained almost unchanged during subsequent culture (Fig. 2 B). In group C (static seeding followed by static culture, control group), the seeding suspension rapidly penetrated the DBM scaffolds after seeding and reached the bottom of the wells. Two hours after seeding, a large number of spindle-like cells appeared at the well bottom. After 5 days of subsequent culture, the cells in the scaffolds started to produce extracellular matrix (ECM) resembling spider webs. The cells and ECM were distributed non-uniformly (Fig. 2 C) at this time point. In group D (hydrogel-assisted seeding followed by dynamic culture), the seeded cells were carried by the fibrin gel and filled most pores in the scaffolds after seeding. The cells were identified by their low refractivity (Fig. 2 D). A small amount of fibrin gel was found on the bottom of the wells. The cell number increased with culture time, and was higher than group C at the same time points.The number of attached cells and density of ECM fibers in the interior of the scaffold after 14-day culture are significantly different among four groups. The number of attached cells and density of E.Laced at the right caudal position. Table 2. Implant groups for subcutaneous implantation.Statistical analysesData were expressed as mean 6 standard deviation. Data were analyzed by one-way analyses of variance (ANOVA) and Student?Newman euls (SNK) post hoc tests using SPSS 12.0 (SPSS, Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.Results Cell culture and characterizationThe hMSCs tended to form calcium nodus after 12 d conditional culture (Fig. 1A). The hMSCs also stained immunohistochemically positive for ALP (Fig. 1B and C), osteocalcin (Fig. 1E and F) and collagen type I (Fig. 1H and I) after 12-day osteogenic induction. The ALP activity of hMSCs (Fig. 1D) and osteocalcin concentration (Fig. 1G) in the culture medium were significantly higher in induced group than that in control group (P,0. 01).Group I II III IVScaffold DBM DBM DBM DBMSeeding condision no Hydrogel-assisted Hydrogel-assisted Hydrogel-assistedCell number per scaffold no 1.06106 1.06107 1.Culture time no dynamic culture 12 d no static flask culture 12 dPlace in nude mouse left rostral right rostral left caudal right caudaldoi:10.1371/journal.pone.0053697.tEffects of Initial Cell and Hydrodynamic CultureFigure 1. The culture and characterization of hMSCs. The hMSCs formed calcium nodus after 12 day culture (A). The hMSCs (2006) stained immunohistochemically positive for 26001275 ALP (B), osteocalcin (E) and collagen type I (H) compared to non-induced cells (C, F, and I). The ALP activity (D) of hMSCs and osteocalcin concentration (G) in the culture medium were significant higher in induced group than that in control group (non-induced cells). *p , 0. 01, compared with control group. doi:10.1371/journal.pone.0053697.gMicroscopy of cell-scaffold constructsIn group A (dynamic seeding followed by dynamic culture), after culture for 8 h, a small number of cells were observed on the surface of the pores in the scaffolds. Then, the cells gradually increased in number and became uniformly distributed on the surface of the pores. After culturing for 5 days, the cells started to produce ECM, the cells and ECM were both uniformly distributed (Fig. 2 A). In group B (hydrogel-assisted seeding followed by static culture), the cell-laden gel filled most pores in scaffold after seeding immediately and the resulting constructs remained almost unchanged during subsequent culture (Fig. 2 B). In group C (static seeding followed by static culture, control group), the seeding suspension rapidly penetrated the DBM scaffolds after seeding and reached the bottom of the wells. Two hours after seeding, a large number of spindle-like cells appeared at the well bottom. After 5 days of subsequent culture, the cells in the scaffolds started to produce extracellular matrix (ECM) resembling spider webs. The cells and ECM were distributed non-uniformly (Fig. 2 C) at this time point. In group D (hydrogel-assisted seeding followed by dynamic culture), the seeded cells were carried by the fibrin gel and filled most pores in the scaffolds after seeding. The cells were identified by their low refractivity (Fig. 2 D). A small amount of fibrin gel was found on the bottom of the wells. The cell number increased with culture time, and was higher than group C at the same time points.The number of attached cells and density of ECM fibers in the interior of the scaffold after 14-day culture are significantly different among four groups. The number of attached cells and density of E.
