Flow perfusion culture induces the osteoblastic differentiation of marrow stromal cell‐scaffold constructs in the absence of dexamethasone

Abstract

Flow perfusion culture of scaffold/cell constructs has been shown to enhance the osteoblastic differentiation of rat bone marrow stroma cells (MSCs) over static culture in the presence of osteogenic supplements including dexamethasone. Although dexamethasone is known to be a powerful induction agent of osteoblast differentiation in MSC, we hypothesied that the mechanical shear force caused by fluid flow in a flow perfusion bioreactor would be sufficient to induce osteoblast differentiation in the absence of dexamethasone. In this study, we examined the ability of MSCs seeded on titanium fiber mesh scaffolds to differentiate into osteoblasts in a flow perfusion bioreactor in both the presence and absence of dexamethasone. Scaffold/cell constructs were cultured for 8 or 16 days and osteoblastic differentiation was determined by analyzing the constructs for cellularity, alkaline phosphatase activity, and calcium content as well as media samples for osteopontin. For scaffold/cell constructs cultured under flow perfusion, there was greater scaffold cellularity, alkaline phosphatase activity, osteopontin secretion, and calcium deposition compared with static controls, even in the absence of dexamethasone. When dexamethasone was present in the cell culture medium under flow perfusion conditions, there was further enhancement of osteogenic differentiation as evidenced by lower scaffold cellularity, greater osteopontin secretion, and greater calcium deposition. These results suggest that flow perfusion culture alone induces osteogenic differentiation of rat MSCs and that there is a synergistic effect of enhanced osteogenic differentiation when both dexamethasone and flow perfusion culture are used.

Engineering anatomically shaped human bone grafts

“…The volumetric flow rate (1.8 mL/min) and the corresponding superficial velocity (an average of 0.06 cm/s) that were selected based on the demonstrated ability to sustain dense tissue growth throughout the constructs, are within the range of flow rates shown to stimulate osteogenic differentiation of hMSCs (19,20,24).…”

Influence of Shear Stress in Perfusion Bioreactor Cultures for the Development of Three-Dimensional Bone Tissue Constructs: A Review

“…3) RunX2, 98 Col1, 97 and mineralized matrix production, 43,85,87,97 can be achieved with shear stresses in the range 1Â10 À4 to 1.2 Pa. The majority of work was focused in the 1 to 5Â10 À2 Pa range, which is at least an order of magnitude below the average for 2D culture and up to two orders of magnitude lower in some cases (other groups [99][100][101] have observed osteogenic differentiation within 3D systems but shear stresses were not quantified making comparison of the data unfeasible). These values are also orders of magnitude below those expected to cause differentiation in vivo, suggesting that the influence of ECM and mineralization in vivo may reduce the sensitivity to shear stress, by reducing the levels of cell deformation, as the cell is encased; further studies will be required to determine if this is the reason.…”

Gene expression by marrow stromal cells in a porous collagen–glycosaminoglycan scaffold is affected by pore size and mechanical stimulation

“…A variety of effects have been reported for the application of mechanical stimulation, including cyclic strain, to cells cultured on two-dimensional (2D) and 3D substrates, and these effects may depend on cell and substrate type and the nature of the mechanical loading. In the mesenchymal lineage, including preparations enriched for stem cells [13], low magnitude substrate strain may induce aspects of the osteoblastic phenotype in 2D [14][15][16] and 3D [17] culture, as may fluid flow [18][19][20]. Modelling and data obtained in vivo [15] suggest that strains as high as 5% may be compatible with intramembraneous osteogenesis during fracture healing.…”