Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Abstract

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...

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor

“…Differences in geometrical configurations of perfusion bioreactor systems and the complex internal architecture of scaffolds (Bancroft et al, 2002;Bonvin et al, 2010;Wendt et al, 2003;Zhao and Ma, 2005) makes correlating observed biological outcomes on the basis of inlet fluid flow-rate very difficult. Calculation of the  within systems allows comparisons to be made on a single representative scale.…”

“…However, static culturing regimes neglect the important role of biomechanical stimuli in the normal formation, development and homeostasis of bone tissue: the effects of which are clearly observed when normal, physiologically relevant levels of mechanical stimulation are removed (Morey and Baylink, 1978;Zerwekh et al, 1998). Therefore, in-vitro bone tissue cultivation strategies have sought to enhance the osteogenic differentiation of cell-seeded constructs by employing mechanical stimulation via flow-perfusion (Bancroft et al, 2002;Cartmell, S. et al, 2003;Keogh et al, 2011;Sikavitsas et al, 2003;Sikavitsas et al, 2005;Vance et al, 2005). We have successfully employed an in-house designed perfusion bioreactor to examine the effects of mechanical stimulation on gene expression levels of key osteogenic markers for MC3T3 (pre-osteoblasts) cell-seeded collagen-GAG (CG) scaffolds subjected to a range of fluid flow regimes and for exposure times of 1h to 14 days Partap et al, 2009;Plunkett et al, 2010).…”

Mathematical modelling of fibre-enhanced perfusion inside a tissue-engineering bioreactor

“…Abousleiman and Sikavitsas, 2006;Cimetta et al, 2007;Kim et al, 2007). For small tissue-engineered constructs these methods have been shown to be successful in comparison to static culture (Glowacki et al, 1998;Goldstein et al, 2001;Bancroft et al, 2002;Cartmell et al, 2003). However, problems arise when the tissue size is scaled up.…”

Evaluation of bioreactor-cultivated bone by magnetic resonance microscopy and FTIR microspectroscopy

“…Typically, constructs are grown under perfusion conditions either in a columnar (3) or rotating bioreactor (4) to promote the formation of bone throughout the scaffold. A better understanding of the regulatory role of various endogenous growth factors and mechanical loading conditions could greatly improve the quality of bioreactor-derived bone (5).…”

“…In addition, the continuous perfusion of the cells attached to the fiber surface will promote the appositional growth of bone in the radial direction, resulting in more mineralized tissue compared to monolayer cultures or cells seeded onto highly porous polymer meshes cultured under static conditions (3,7). Lastly, bone cells are expected to thrive within the HFBR system because the cells experience fluid shear stresses (3,(7)(8)(9)(10)(11). The drawback of this HFBR system is the inability to sample the tissue as it forms in the reactor.…”