Effect of Low-Frequency Pulsatile Flow on Expression of Osteoblastic Genes by Bone Marrow Stromal Cells

Abstract

Perfusion culture of osteoprogenitor cells is a promising means to form a bone-like extracellular matrix for tissue engineering applications, but the mechanism by which hydrodynamic shear stimulates expression of bone extracellular matrix (ECM) proteins is not understood. Osteoblasts are mechanosensitive and respond differently to steady and pulsatile flow. Therefore, to probe the effect of flow, bone marrow stromal cells (BMSCs)--cultured under osteogenic conditions--were exposed to steady or pulsatile flow at frequencies of 0.015, 0.044, or 0.074 Hz. Following 24 h of stimulus, cells were cultured statically for an additional 13 days and then analyzed for the expression of bone ECM proteins collagen 1alpha1 (Col1alpha1), osteopontin, osteocalcin (OC), and bone sialoprotein (BSP). All mRNA levels were elevated by flow, but OC and BSP were enhanced modestly with pulsatile flow. To determine if these effects were related to gene induction during flow, BMSCs were again exposed to steady or pulsatile flow for 24 h, but then analyzed immediately for expression of growth and differentiation factors bone morphogenetic proteins (BMP)-2, -4, and -7, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor-A. All growth and differentiation factors were significantly elevated by flow, except BMP-4 which was suppressed. In addition, expression of BMP-2 and -7 were enhanced and TGF-beta1 suppressed by pulsatile flow relative to steady flow. These results demonstrate that pulsatile flow modulates expression of BMP-2, -7, and TGF-beta1 and suggest that enhanced expression of bone ECM proteins by pulsatile flow may be mediated through the induction of BMP-2 and -7.

Fluid Flow-induced Soluble Vascular Endothelial Growth Factor Isoforms Regulate Actin Adaptation in Osteoblasts

“…On the other hand, the PFSS-induced up-regulation of Vegf-a gene expression (ϳ6-fold in our previous study (2)) and the increased VEGF protein level within MC3T3-E1 cells in the current study clearly support a role for VEGF in osteoblast mechanotransduction. Similar findings have been reported in a murine embryonic mesenchymal progenitor cell line and in bone marrow stromal cells that showed significantly increased VEGF mRNA expression in response to laminar or pulsatile fluid shear stress (28,29). Likewise, up-regulation of VEGF expression under cyclic strain has been shown in various cell types (30,31), including osteoblasts (14,32).…”

Mechanical Stimulation of Bone Marrow In Situ Induces Bone Formation in Trabecular Explants

“…Shear stress is known to regulate cell differentiation and is capable of stimulating an osteogenic response in vitro 1,3,11,53 . The magnitude of the calculated shear stress (mean shear stress ranges from 0.575 -0.702 Pa across the Vibrated group) in the explants was at a level consistent with osteogenic response by MSCs in 2D parallel plate flow chambers 35,53 and 3D scaffold fluid flow experiments 11 .…”

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

“…When physiologically relevant flow patterns have been explored, comparisons are often difficult as single flow types with respect to no flow controls are studied in individual systems. In instances where flow types have been compared side by side, the consensus is mixed; data from Sharp et al 77 suggest that pulsatile flow is best for the upregulation of bone sialoprotein (BSP), osteocalcin (OC), and bone morphogenetic proteins 2 and 7 (BMP2/7), and the downregulation of transforming growth factor-b 1 ; Jaasma and O'Brien 74 highlighted that pulsatile flow is best for the upregulation of cyclooxygenase-2 (COX-2), whereas oscillatory flow produces the greatest increase in prostaglandin E 2 (PGE 2 ). There is some evidence to suggest that constant stimulation of cells from biomechanical stimuli, irrespective of flow type, leads to quiescence of the cellular response: cellular desensitization.…”

“…A wealth of literature exists for nonhuman 70 70 and peroxisome proliferator activated receptor gamma (PPARg) 70 showed upregulation of gene or protein expression as a consequence of shear stress application, whereas its influence was negative or even suppressive for expression of BMP4, 77 COX1, 90 and transforming growth factor-b 1 . 77 The variation in cell types used, culture conditions, and experimental platforms has increased the complexity of prizing out the importance of the role of fluid shear stress; nevertheless, it appears that a range of 0.1-0.5 Pa is most successful, and in some instances, values as high as 2 Pa have yielded positive responses, showing a good correlation with the levels of shear stress expected to occur in vivo (0.8-3 Pa).…”

“…77 The variation in cell types used, culture conditions, and experimental platforms has increased the complexity of prizing out the importance of the role of fluid shear stress; nevertheless, it appears that a range of 0.1-0.5 Pa is most successful, and in some instances, values as high as 2 Pa have yielded positive responses, showing a good correlation with the levels of shear stress expected to occur in vivo (0.8-3 Pa).…”