D 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 More emphasis on pathways whose member genes show fold changes that 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 vivo culture and additional damage to the patient. Recent development of bioreactor techniques has made it possible to better simulate 26001275 the in vivo microenvironment, promote mass exchange, and create appropriate mechanical stimuli. These improvements may be used to produce more mature and bioactive tissue-engineered grafts [31]. In tissue engineering of grafts, the supply of nutrients and removal of metabolic wastes is more difficult than in conventional cell culture. The mass transport in the common static culture method relies on the concentration gradient and is thus inefficient [32]. As a result, cells typically do not survive well in the center of the graft and in some cases even undergo necrosis to form voids [33]. This has severely limited the size of grafts that can be obtained by tissue engineering [34]. An appropriately designed bioreactor may provide hydrodynamic conditions to promote mass transfer, stimulate stem cells to differentiate into osteoblasts, and thus overcome this disadvantage. In this study, we found that when comparing static and hydrogel-assisted seeding, the statically cultured cell-scaffold constructs achieved lower plateau values. In comparison, regardless of the initial cell densities, the dynamically cultured constructs showed continued increase in cell density and became approximately two times higher than the statically cultured grafts.Effects of Initial Cell and Hydrodynamic CultureFurthermore, with a higher seeding efficiency and cell density by the hydrogel-assisted seeding, group B achieved plateau earlier than the group A. The ALP activities of the constructs (Fig. 3A) followed the order of: group B.group A.group D.group C, consistent with the trend of cell number between days 6?4 (Fig. 3B). These findings suggest that hydrogel-assisted seeding followed by hydrodynamic culture can substantially increase the initial seed cell density in constructs, achieve a higher cell density earlier than static culture, and is the optimal one among the four methods studied here. The favourable effect of hydrodynamic culture may be attributed to three factors. First, the Title Loaded From File vortex in the bioreactor generated fluid flow in the construct, which enhanced mass transfer and improve.D 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 vivo culture and additional damage to the patient. Recent development of bioreactor techniques has made it possible to better simulate 26001275 the in vivo microenvironment, promote mass exchange, and create appropriate mechanical stimuli. These improvements may be used to produce more mature and bioactive tissue-engineered grafts [31]. In tissue engineering of grafts, the supply of nutrients and removal of metabolic wastes is more difficult than in conventional cell culture. The mass transport in the common static culture method relies on the concentration gradient and is thus inefficient [32]. As a result, cells typically do not survive well in the center of the graft and in some cases even undergo necrosis to form voids [33]. This has severely limited the size of grafts that can be obtained by tissue engineering [34]. An appropriately designed bioreactor may provide hydrodynamic conditions to promote mass transfer, stimulate stem cells to differentiate into osteoblasts, and thus overcome this disadvantage. In this study, we found that when comparing static and hydrogel-assisted seeding, the statically cultured cell-scaffold constructs achieved lower plateau values. In comparison, regardless of the initial cell densities, the dynamically cultured constructs showed continued increase in cell density and became approximately two times higher than the statically cultured grafts.Effects of Initial Cell and Hydrodynamic CultureFurthermore, with a higher seeding efficiency and cell density by the hydrogel-assisted seeding, group B achieved plateau earlier than the group A. The ALP activities of the constructs (Fig. 3A) followed the order of: group B.group A.group D.group C, consistent with the trend of cell number between days 6?4 (Fig. 3B). These findings suggest that hydrogel-assisted seeding followed by hydrodynamic culture can substantially increase the initial seed cell density in constructs, achieve a higher cell density earlier than static culture, and is the optimal one among the four methods studied here. The favourable effect of hydrodynamic culture may be attributed to three factors. First, the vortex in the bioreactor generated fluid flow in the construct, which enhanced mass transfer and improve.
