The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent

Loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. However, the nature of the stimulus that activates the cell is debated. Candidate stimuli include wall shear stress, streaming potentials, and chemotransport. We have addressed the nature of the flow-derived cell stimulus by comparing variations in fluid transport with variations in wall shear stress, using nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production as a parameter of bone cell activation. Adult mouse long bone cell cultures were treated for 15min with or without pulsating fluid flow using the following regimes: Low PFF, mean flow rate 0.20 cm(3)/s, 3 Hz, shear stress 0.4+/-0.12 Pa; Medium PFF, 0.33 cm(3)/s, 5 Hz, 0.6+/-0.27 Pa; and High PFF, 0.63 cm(3)/s, 9Hz, 1.2+/-0.37 Pa. In some Low PFF experiments, 2.8% neutral dextran (mol. wt. 4.98x10(4)) was added to the flow medium to increase the viscosity, thereby increasing the wall shear stress 3-fold to a level similar of the High PFF stimulus, but without affecting streaming potentials or chemotransport. NO and PGE(2) production were stimulated by Low, Medium, and High PFF in a dose-dependent manner. Application of Low PFF using dextran-supplemented medium, enhanced both the NO and PGE(2) response by 3-fold, to a level mimicking the response to High PFF at normal viscosity. These results show that the production of NO and PGE(2) by bone cells can be enhanced in a dose-dependent manner by fluid flow of increasing wall shear stress. Therefore, the stimulus leading to NO and PGE(2) production is the flow-derived shear stress, and not streaming potentials or chemotransport.     

Mechanical stress induces COX-2 mRNA expression in bone cells from elderly women

Mechanical loading-induced fluid flow in the lacuno-canalicular network is a possible signal for bone cell adaptive responses. In an earlier study we found that pulsating fluid flow (PFF, 0.7+/-0.02 Pa, 5 Hz, 0.4 Pa/s) stimulates the production of prostaglandins by neonatal mouse calvarial cells. In addition, mRNA expression of the inducible form of cyclooxygenase (COX-2), but not the constitutive form (COX-1), the major enzymes in prostaglandin production, was increased by PFF. The present study was performed to determine whether human primary bone cells from the iliac crest, respond to mechanical stress in a similar way as neonatal mouse calvarial cells. We subjected bone cells originating from the iliac crest of nine elderly women, between 56 and 80 yr of age, for 1 h to PFF and measured prostaglandin production and COX-1 and COX-2 mRNA expression. One hour PFF treatment stimulated the release of PGE2 by 3.5 fold and PGI2 by 2.2 fold. PFF also increased the expression of COX-2 mRNA by 2.9 fold, but did not change COX-1 mRNA. No correlation was found between donor age and PFF effect, neither on prostaglandin production nor on COX-2 mRNA expression. This study shows that bone cells from the iliac crest of elderly women react to PFF treatment in a similar way as neonatal mouse calvarial cells, namely with increased production of prostaglandins and upregulation of COX-2 mRNA expression. These results suggest that human bone cells from the iliac crest and neonatal mouse calvarial cells share a similar mechanotransduction pathway.     

Human dental pulp cells exhibit bone cell-like responsiveness to fluid shear stress

Background aimsFor engineering bone tissue to restore, for example, maxillofacial defects, mechanosensitive cells are needed that are able to conduct bone cell-specific functions, such as bone remodelling. Mechanical loading affects local bone mass and architecture in vivo by initiating a cellular response via loading-induced flow of interstitial fluid. After surgical removal of ectopically impacted third molars, human dental pulp tissue is an easily accessible and interesting source of cells for mineralized tissue engineering. The aim of this study was to determine whether human dental pulp-derived cells (DPC) are responsive to mechanical loading by pulsating fluid flow (PFF) upon stimulation of mineralization in vitro.MethodsHuman DPC were incubated with or without mineralization medium containing differentiation factors for 3 weeks. Cells were subjected to 1-h PFF (0.7 ± 0.3Pa, 5Hz) and the response was quantified by measuring nitric oxide (NO) and prostaglandin E₂ (PGE₂) production, and gene expression of cyclooxygenase (COX)-1 and COX-2.ResultsWe found that DPC are intrinsically mechanosensitive and, like osteogenic cells, respond to PFF-induced fluid shear stress. PFF stimulated NO and PGE₂ production, and up-regulated COX-2 but not COX-1 gene expression. In DPC cultured under mineralizing conditions, the PFF-induced NO, but not PGE₂, production was significantly enhanced.ConclusionsThese data suggest that human DPC, like osteogenic cells, acquire responsiveness to pulsating fluid shear stress in mineralizing conditions. Thus DPC might be able to perform bone-like functions during mineralized tissue remodeling in vivo, and therefore provide a promising new tool for mineralized tissue engineering to restore, for example, maxillofacial defects.     

Dynamic shear stress in parallel-plate flow chambers

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.     

Shear stress inhibits while disuse promotes osteocyte apoptosis

Cell apoptosis operates as an organizing mechanism in biology in addition to removing effete cells. We have recently proposed that during bone remodeling, osteocyte apoptosis steers osteonal alignment in relation to mechanical loading of the whole bone [J. Biomech. 36 (2003) 1453]. Here we present evidence that osteocyte apoptosis in cell culture is modulated by shear stress. Under static culture conditions, serum starved osteocytes exposed phosphatidylserine (PS) on their cell membrane 6x more often than periosteal fibroblasts and 3x more often than osteoblasts. Treatment with shear stress reduced the number of osteocytes that exposed PS by 90%, but did not affect the other cell types. Fluid shear stress of increasing magnitude, dose-dependently stimulated Bcl-2 mRNA expression in human bone cells, while shear stress did not change Bax expression. These data suggest that disuse promotes osteocyte apoptosis, while mechanical stimulation by fluid shear stress promotes osteocyte survival, by modulating the Bcl-2/Bax expression ratio.     

Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain chondrogenesis and osteogenesis. We hypothesize that BMP-2 induces an osteogenic, and BMP-7 a chondrogenic phenotype in adipose tissue-derived mesenchymal stem cells (AT-MSCs). We compared the effects of a short 15min BMP-2 or BMP-7 (10ng/ml) treatment on osteogenic and chondrogenic differentiation of AT-MSCs. Gene expression was studied 4 and 14 days after BMP-treatment. At day 4 BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression, and at day 14 BMP-7 down-regulated expression of these genes. At day 4 BMP-2 and BMP-7 stimulated biglycan gene expression, which was down-regulated by BMP-7 at day 14. BMP-7 stimulated aggrecan gene expression at day 14. Our data indicate that BMP-2 treatment for 15min induces osteogenic differentiation, whereas BMP-7 stimulates a chondrogenic phenotype of AT-MSCs. Therefore, AT-MSCs triggered for only 15min with BMP-2 or BMP-7 provide a feasible tool for bone and cartilage tissue engineering.     

Mechanical stimulation of osteopontin mRNA expression and synthesis in bone cell cultures

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell-matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast-like cell line MC3T3-E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3-E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor-like cells. Also, in both proliferating and differentiating MC3T3-E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down-regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley-Liss, Inc.     

The effect of environmental factors on floating fresh marsh end-of-season biomass

We used both stepwise and quantile regression to determine the sources of environmental variation that explained the observed inter-annual variation in end-of-season freshwater floating marsh aboveground biomass over an 18-year period. The vegetation at our study site had high species diversity with an average of 20 species recorded from 10 0.25 m⁻² plots. However, Panicum hemitomon was clearly the dominant contributing 74% of the total biomass. Only three other species (Solidago sempervirens, Vigna luteola, and Thelypteris palustris) were so common that they were sampled in all years. We expected that the most important factors controlling interannual variation in aboveground biomass are temperature and nitrogen availability. We also expected that nitrogen availability to the plants is affected by water movement through and under the mat driven by precipitation (lower N), evaporation (transportation of higher N waters to roots), and local runoff (higher N). Stepwise regression analysis indicated that P. hemitomon average biomass was negatively related to average water level and positively related to maximum water level and had a curvilinear response with TKN. Using quantile regression the best fit for P. hemitomon maximum-biomass with two parameters was obtained using hot days (positive relationship) and maximum water level (negative relationship). Both analytical methods showed maximum water level (negative relationship) and cold front passage (positive relationship) to be the environmental parameters that best explained interannual variation in S. sempervirens biomass. V. luteola biomass was positively related to temperature. Stepwise regression added chloride concentration as an additional positive parameter explaining V. luteola biomass, while quantile regression identified nitrogen as an important positive parameter. Both analytical methods identified pH, TKN, and water level as environmental parameters that were negatively correlated with T. palustris biomass. The overall negative effect of water level on all species was unexpected in this floating mat system. We initially assumed that higher water levels were due to higher runoff which should have a positive effect on biomass. However, higher water levels may also be related to a higher retention time in this fresh-water tidal system, which decreases water exchange and nutrient replenishment.     

Interactive effects of PTH and mechanical stress on nitric oxide and PGE2 production by primary mouse osteoblastic cells

Parathyroid hormone (PTH) and mechanical stress both stimulate bone formation but have opposite effects on bone resorption. PTH increased loading-induced bone formation in a rat model, suggesting that there is an interaction of these stimuli, possibly at the cellular level. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of 10-9 M human PTH-(1-34) on fluid flow-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production by primary mouse osteoblastic cells in vitro. Mechanical stress applied by means of a pulsating fluid flow (PFF; 0.6 +/- 0.3 Pa at 5 Hz) stimulated both NO and PGE2 production twofold. In the absence of stress, PTH also caused a twofold increase in PGE2 production, but NO release was not affected and remained low. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, whereas PGE2 production was again stimulated only twofold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2 by stress, leaving only the NO-independent PGE2 upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction.