In this study we report on direct involvement of fluid shear stresses on the osteoblastic differentiation of marrow stromal cells. Rat bone marrow stromal cells were seeded in 3D porous titanium fiber mesh scaffolds and cultured for 16 days in a flow perfusion bioreactor with perfusing culture media of different viscosities while maintaining the fluid flow rate constant. This methodology allowed exposure of the cultured cells to increasing levels of mechanical stimulation, in the form of fluid shear stress, whereas chemotransport conditions for nutrient delivery and waste removal remained essentially constant. Under similar chemotransport for the cultured cells in the 3D porous scaffolds, increasing fluid shear forces led to increased mineral deposition, suggesting that the mechanical stimulation provided by fluid shear forces in 3D flow perfusion culture can indeed enhance the expression of the osteoblastic phenotype. Increased fluid shear forces also resulted in the generation of a better spatially distributed extracellular matrix inside the porosity of the 3D titanium fiber mesh scaffolds. The combined effect of fluid shear forces on the mineralized extracellular matrix production and distribution emphasizes the importance of mechanosensation on osteoblastic cell function in a 3D environment. O ut of all of the myriad of tissue types present in the body, bone is probably the type most associated with all things mechanical. Although the bones of the skeletal system serve other functions in diverse areas such as calcium metabolism and hematopoiesis, it is their role in skeletal integrity, support, and locomotion that is most prominent. Because of this role, bone, in addition to its structured extracellular matrix of inorganic and organic elements, contains a conglomeration of cell types that continually monitor and modify the bony structure in response to the ever-changing mechanical stressors (1). As would be expected, these same bone cells when cultured in vitro respond to a variety of mechanical signals including fluid flow, hydrostatic pressure, and substrate deformation.Of these mechanical stressors, fluid flow has emerged as one of the strongest stimuli of bone cell behavior (2-5). The in vitro mechanical stimulation of bone cells by fluid flow has been implicated in the alteration of a variety of biochemical factors in cell behavior. Short-term exposure of osteoblastic cells to fluid shear induces a rapid increase in intracellular calcium (6, 7), a response that resembles the effect of parathyroid hormone on osteoblastic cells. Fluid flow-induced shear stress applied to osteoblastic cells for several hours has been shown to stimulate the release of nitric oxide (5, 8, 9), a short-lived radical and messenger implicated in several cellular functions, and prostaglandin (4,5,8,10,11) that may have an autocrine effect on osteoblastic cells. Fluid shear has been shown to up-regulate a variety of genes, including those of osteopontin and cyclooxygenase-2, and several other transcription factors and intracellula...

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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.