Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering. Bone is a complex, highly organized tissue consisting of a structured extracellular matrix composed of inorganic and organic elements containing a conglomeration of cell types responsible for its metabolism and upkeep that are responsive to a variety of signals (1). As would be expected from the skeleton's central role in support and structural integrity, bone cells cultured in vitro respond to a variety of different mechanical signals including fluid flow, hydrostatic pressure, and substrate deformation.Current studies indicate that fluid flow is a potentially stronger stimulus for bone cell behavior than either hydrostatic compression (2) or substrate deformation (3, 4). The in vitro mechanical stimulation of bone cells by fluid flow has been reported to impact the levels of many biochemical factors including intracellular calcium (5, 6), nitric oxide (4, 7-9), prostaglandin E 2 (3, 4, 7, 8), the expression of the genes for osteopontin, cyclooxygenase-2, and c-Fos (6, 10-12) as well as other intracellular messengers and transcription factors (6,(13)(14)(15). This mechanostimulation of bone cells in vitro by fluid flow mimics the physiological response of bone cells in vivo where pressure gradients from mechanical loading of locomotion and other stressors deform the mineralized bone matrix and move extracellular fluid radially outward toward the cortex through the lacunocanalicular network (16)(17)(18)(19). Mechanical loading of bone plays an important role because it can both increase bone formation and decrease bone resorption (1). Indeed, its absence can lead to lower bone matrix protein production, mineral content, and bone formation plus incr...

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...