Paxillin localisation in osteocytes--is it determined by the direction of loading?

External mechanical loading of cells aligns cytoskeletal stress fibres in the direction of principle strains and localises paxillin to the mechanosensing region. If the osteocyte cell body can indeed directly sense matrix strains, then cytoskeletal alignment and distribution of paxillin in osteocytes in situ will bear alignment to the different mechanical loading patterns in fibulae and calvariae. We used confocal microscopy to visualise the immunofluorescence-labelled actin cytoskeleton in viable osteocytes and paxillin distribution in fixated osteocytes in situ. In fibular osteocyte cell bodies, actin cytoskeleton and nuclei were elongated and aligned parallel to the principal (longitudinal) mechanical loading direction. Paxillin was localised to the 'poles' of elongated osteocyte cell bodies. In calvarial osteocyte cell bodies, actin cytoskeleton and nuclei were relatively more round. Paxillin was distributed evenly in the osteocyte cell bodies. Thus in osteocyte cell bodies in situ, the external mechanical loading pattern likely determines the orientation of the actin cytoskeleton, and focal adhesions mediate direct mechanosensation of matrix strains.

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide.

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7+/-0.3Pa, 5Hz, 1h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.

Fluid shear stress inhibits TNFalpha-induced osteocyte apoptosis.

Bone tissue can adapt to orthodontic load. Mechanosensing in bone is primarily a task for the osteocytes, which translate the canalicular flow resulting from bone loading into osteoclast and osteoblast recruiting signals. Apoptotic osteocytes attract osteoclasts, and inhibition of osteocyte apoptosis can therefore affect bone remodeling. Since TNF-alpha is a pro-inflammatory cytokine with apoptotic potency, and elevated levels are found in the gingival sulcus during orthodontic tooth movement, we investigated if mechanical loading by pulsating fluid flow affects TNF-alpha-induced apoptosis in chicken osteocytes, osteoblasts, and periosteal fibroblasts. During fluid stasis, TNF-alpha increased apoptosis by more than two-fold in both osteocytes and osteoblasts, but not in periosteal fibroblasts. One-hour pulsating fluid flow (0.70 +/- 0.30 Pa, 5 Hz) inhibited (-25%) TNF-alpha-induced apoptosis in osteocytes, but not in osteoblasts or periosteal fibroblasts, suggesting a key regulatory role for osteocyte apoptosis in bone remodeling after the application of an orthodontic load.

Pulsating fluid flow stimulates prostaglandin release and inducible prostaglandin G/H synthase mRNA expression in primary mouse bone cells.

Bone tissue responds to mechanical stress with adaptive changes in mass and structure. Mechanical stress produces flow of fluid in the osteocyte lacunar-canalicular network, which is likely the physiological signal for bone cell adaptive responses. We examined the effects of 1 h pulsating fluid flow (PFF; 0.7 +/- 0.02 Pa, 5 Hz) on prostaglandin (PG) E2, PGI2, and PGF2alpha production and on the expression of the constitutive and inducible prostaglandin G/H synthases, PGHS-1, and PGHS-2, the major enzymes in the conversion of arachidonic acid to prostaglandins, using mouse calvarial bone cell cultures. PFF treatment stimulated the release of all three prostaglandins under 2% serum conditions, but with a different time course and to a different extent. PGF2alpha was rapidly increased 5-10 minutes after the onset of PFF. PGE2 release increased somewhat more slowly (significant after 10 minutes), but continued throughout 60 minutes of treatment. The response of PGI2 was the slowest, and only significant after 30 and 60 minutes of treatment. In addition, PFF induced the expression of PGHS-2 but not PGHS-1. One hour of PFF treatment increased PGHS-2 mRNA expression about 2-fold relative to the induction by 2% fresh serum given at the start of PFF. When the addition of fresh serum was reduced to 0.1%, the induction of PGHS-2 was 8- to 9-fold in PFF-treated cells relative to controls. This up-regulation continued for at least 1 h after PFF removal. PFF also markedly increased PGHS activity, measured as the conversion of arachidonic acid into PGE2. One hour after PFF removal, the production of all three prostaglandins was still enhanced. These results suggest that prostaglandins are important early mediators of the response of bone cells to mechanical stress. Prostaglandin up-regulation is associated with an induction of PGHS-2 enzyme mRNA, which may subsequently provide a means for amplifying the cellular response to mechanical stress.

Opposite effects of osteogenic protein and transforming growth factor beta on chondrogenesis in cultured long bone rudiments.

Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-beta superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-beta 1 and porcine TGF-beta 1 and beta 2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35SO4 into glycosaminoglycans. Mineralization was monitored by 45Ca incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondrocytes, resulting in increased appositional growth. In contrast to OP-1, TGF-beta 1 and beta 2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-beta inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-beta exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-beta have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development.

Decreased mineralization and increased calcium release in isolated fetal mouse long bones under near weightlessness.

Mechanical loading plays an important role in the development and maintenance of skeletal tissues. Subnormal mechanical stress as a result of bed rest, immobilization, but also in spaceflight, results in a decreased bone mass and disuse osteoporosis, whereas supranormal loads upon extremities result in an increased bone mass. In this first in vitro experiment with complete fetal mouse cartilaginous long bones, cultured under microgravity conditions, we studied growth, glucose utilization, collagen synthesis, and mineral metabolism, during a 4-day culture period in space. There was no change in percent length increase and collagen synthesis under microgravity compared with in-flight 1x gravity. Glucose utilization and mineralization were decreased under microgravity. In addition, mineral resorption, as measured by 45Ca release, was increased. These data suggest that weightlessness has modulating effects on skeletal tissue cells. Loss of bone during spaceflight could be the result of both impaired mineralization as well as increased resorption.

Stimulation of cartilage differentiation by osteogenic protein-1 in cultures of human perichondrium.

Exposure of progenitor cells with chondrogenic potential to recombinant human osteogenic protein-1 [rhOP-1, or bone morphogenetic protein-7 (BMP-7] may be of therapeutic interest in the regeneration of articular cartilage. Therefore, in this study, we examined the influence of rhOP-1 on cartilage formation by human perichondrium tissue containing progenitor cells with chondrogenic potential in vitro. Fragments of outer ear perichondrium tissue were embedded in clotting autologous blood to which rhOP-1 had been added or not (controls), and the resulting explant was cultured for 3 weeks without further addition of rhOP-1. Cartilage formation was monitored biochemically by measuring [³5;S]sulfate incorporation into proteoglycans and histologically by monitoring the presence of metachromatic matrix with cells in nests. The presence of rhOP-1 in the explant at the beginning of culture stimulated [³5;S]sulfate incorporation into proteoglycans in a dose-dependent manner after 3 weeks of culture. Maximal stimulation was reached at 40 microgram/ml. Histology revealed that explants treated with 20-200 microgram/ml rhOP-1, but not untreated control explants, contained areas of metachromatic-staining matrix with chondrocytes in cell nests. These results suggest that rhOP-1 stimulates differentiation of cartilage from perichondrium tissue. The direct actions of rhOP-1 on perichondrium cells to stimulate chondrocytic differentiation and production of cartilage matrix in vitro provide a cellular mechanism for the induction of cartilage formation by rhOP-1 in vivo. Thus, rhOP-1 may promote early steps in the cascade of events leading to cartilage formation. Therefore, rhOP-1 could be an interesting factor for regeneration of cartilage in articular cartilage defects.

Low-intensity ultrasound stimulates endochondral ossification in vitro.

Animal and clinical studies have shown an acceleration of bone healing by the application of low-intensity ultrasound. The objective of this study was to examine in vitro the influence of low-intensity ultrasound on endochondral ossification of 17-day-old fetal mouse metatarsal rudiments. Forty-six triplets of paired metatarsal rudiments were resected 'en block' and cultured for 7 days with and without low-intensity ultrasound stimulation (30 mw/cm2). At days 1, 3, 5, and 7, the total length of the metatarsal rudiments, as well as the length of the calcified diaphysis were measured. Histology of the tissue was performed to examine its vitality. The increase in length of the calcified diaphysis during 7 days of culture was significantly higher in the ultrasound-treated rudiments compared to the untreated controls (P = 0.006). The growth of the control diaphysis was 180 +/- 30 microm (mean +/- SEM), while the growth of the ultrasound-treated diaphysis was 530 +/- 120 microm. The total length of the metatarsal rudiments was not affected by ultrasound treatment. Histology revealed a healthy condition of both ultrasound-treated and control rudiments. In conclusion, low-intensity ultrasound treatment stimulated endochondral ossification of fetal mouse metatarsal rudiments. This might be due to stimulation of activity and/or differentiation of osteoblasts and hypertrophic chondrocytes. Our results support the hypothesis that low-intensity ultrasound activates ossification via a direct effect on osteoblasts and ossifying cartilage.

Stimulation of bone cell differentiation by low-intensity ultrasound--a histomorphometric in vitro study.

Several investigations have established a stimulatory effect of low-intensity ultrasound treatment on osteogenesis and fracture healing. The objective of this study was to examine whether the stimulatory effect of low-intensity ultrasound results in increased bone cell activity and/or proliferation. Twenty-four paired triplets of metatarsal bone rudiments of twelve 17-days-old fetal mice were dissected and divided into two groups. One group of bone rudiments was treated with pulsating low-intensity ultrasound (30 mW/cm(2); 1.5 MHz) for 20 min/day for a period of 3 or 6 days. The other group served as controls. After culture, the metatarsal bone rudiments were prepared for computer aided light microscopy. The following histomorphometric parameters were determined: length, width and volume of the calcified cartilage and of the bone collar, and cell number. GLM analysis demonstrated that bone collar volume and calcified cartilage percentage were significantly higher in the ultrasound-stimulated rudiments compared to untreated controls. Further, the calcified cartilage volume bordering the hypertrophic zone was significantly higher than in the center of the bone rudiment. Ultrasound treatment did not change the number of the cells. These results suggest that the stimulatory effect of low-intensity ultrasound on endochondral ossification is likely due to stimulation of bone cell differentiation and calcified matrix production, but not to changed cell proliferation.

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.

Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: a model for cell-mechanics studies.

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.

Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro.

We investigated whether low-intensity pulsed ultrasound (LIPUS) stimulates chondrocyte proliferation and matrix production in explants of human articular cartilage obtained from donors suffering from unicompartimental osteoarthritis of the knee, as well as in isolated human chondrocytes in vitro. Chondrocytes and explants were exposed to LIPUS (30 mW/cm(2); 20 min/day, 6 days). Stimulation of [35S]-sulphate incorporation into proteoglycans by LIPUS was 1.3-fold higher in degenerative than in collateral monolayers as assessed biochemically and 1.9-fold higher in explants as assessed by autoradiography. LIPUS decreased the number of cell nests containing 1-3 chondrocytes by 1.5 fold in collateral and by 1.6 fold in degenerative explants. LIPUS increased the number of nests containing 4-6 chondrocytes by 4.8 fold in collateral and by 3.9 fold in degenerative explants. This suggests that LIPUS stimulates chondrocyte proliferation and matrix production in chondrocytes of human articular cartilage in vitro. LIPUS might provide a feasible tool for cartilage tissue repair in osteoarthritic patients, since it stimulates chondrocyte proliferation and matrix production.

Prostaglandin mediated modulation of transforming growth factor-beta metabolism in primary mouse osteoblastic cells in vitro.

Prostaglandins and transforming growth factor-beta (TGF-beta) are both important local regulators of bone metabolism, but their actions on bone are complex. Prostaglandins mediate bone loss due to immobilization, but prostaglandin E2 (PGE2) treatment stimulates bone formation in vivo. TGF-beta may have both anabolic and catabolic effects on bone in vitro. In this study, we tested the effects of PGE2 on TGF-beta release and on TGF-beta messenger RNA (mRNA) levels in neonatal mouse calvarial cell cultures. We also examined the relationship between endogenous prostaglandin production as a result of mechanical stress and the release of TGF-beta. Addition of PGE2 (10(-8)-10(-6)M) to the culture medium stimulated the release of TGF-beta peptide (active plus latent) after 24 and 48 h in a dose-related manner. This upregulation was paralleled by an increased expression of TGF-beta mRNA levels. Mechanical stimulation by 1 h treatment with pulsating fluid flow (producing a shear stress of 0.5 +/- 0.02 Pa at 5 Hz) resulted 1 h posttreatment in increased production of PGE2, prostaglandin l2 (PGI2), and prostaglandin F2a. In addition, the release of TGF-beta activity but not TGF-beta peptide was decreased 24 h after PFF treatment. Addition of indomethacin, which blocks endogenous prostaglandin production, neutralized the effect of PFF treatment on TGF-beta activity, indicating that the effect of stress was mediated by endogenous prostaglandins. These results suggest that PGE2 and other prostaglandins (probably PGI2 and/or PGF2a) have opposite effects on TGF-beta metabolism in bone cells, as PGE2 upregulates TGF-beta expression and synthesis while other prostaglandins downregulate TGF-beta activation.

Different responsiveness of cells from adult and neonatal mouse bone to mechanical and biochemical challenge.

Neonatal rodent calvarial bone cell cultures are often used to study bone cell responsiveness to biochemical and mechanical signals. However, mechanical strains in the skull are low compared to the axial and appendicular skeleton, while neonatal, rapidly growing bone has a more immature cell composition than adult bone. In the present study, we tested the hypothesis that bone cell cultures from neonatal and adult mouse calvariae, as well as adult mouse long bones, respond similarly to treatment with mechanical stress or 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3). Treatment with pulsating fluid shear stress (0.6 +/- 0.3 Pa, 5 Hz) caused a rapid (within 5 min) 2-4-fold increase in NO production in all cases, without significant differences between the three cell preparations. However, basal NO release was significantly higher in neonatal calvarial cells than adult calvarial and long bone cells. The response to 1,25(OH)2 D3), measured as increased alkaline phosphatase activity, was about three times higher in the neonatal cells than the adult cell cultures. We conclude that all three types of primary bone cell cultures responded similarly to fluid shear stress, by rapid production of NO. However, the neonatal cell cultures were different in basal metabolism and vitamin D3 responsiveness, suggesting that cell cultures from adult bone are best used for in vitro studies on bone cell biology.

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.

Mechanical loading stimulates the release of transforming growth factor-beta activity by cultured mouse calvariae and periosteal cells.

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) inhibits bone resorption and stimulates bone formation in cultured fetal mouse calvariae (Klein-Nulend et al., 1986, Arthritis Rheum., 29: 1002-1009). The production of soluble bone factors by such calvariae is also modified (Klein-Nulend et al., 1993, Cell Tissue Res., 271:513-517). Transforming growth factor-beta (TGF-beta) is an important local regulator of bone metabolism and is produced by osteoblasts. In this study, the release of TGF-beta activity as a result of mechanical stress was examined in organ cultures of neonatal mouse calvariae, in primary cultures of calvariae-derived osteoprogenitor (OPR) cells, and in more differentiated osteoblastic (OB) cells. Whole calvariae and calvariae-derived cells were cultured in the presence or absence of IHC for 1-7 days and medium concentrations of active as well as total TGF-beta were measured using a bioassay. IHC (maximum 13 kPa, maximal pressure rate 32.5 kPa/sec) was generated by intermittently (0.3 Hz) compressing the gas phase above the cultures. We found that mechanical loading by IHC stimulated the release of TGF-beta activity from cultured calvariae by twofold after 1 day. IHC also stimulated the release of TGF-beta activity from calvariae-derived cells after 1 and 3 days. The absolute amounts of TGF-beta activity released were lower in OPR cells than in OB cells, but the stimulatory effect of IHC was greater in OPR cells. Total TGF-beta (active and bound) released into the medium was not affected by IHC. IHC did not change the dry weight of the organ cultures, nor the DNA or protein content of the cell cultures. These data show that mechanical perturbation of bone cells, particularly OPR cells, enhances the activation of released TGF-beta. We conclude that modulation of TGF-beta metabolism may be part of the response of bone tissue to mechanical stress.

Effect of mechanical stimulation on the production of soluble bone factors in cultured fetal mouse calvariae.

Mechanical stimulation by intermittent compressive force (ICF) stimulates bone formation and inhibits bone resorption in cultured fetal mouse bone. Fetal bone tissue can produce autocrine factors that stimulate bone cell replication and matrix formation, and paracrine factors that increase the formation of osteoclast precursor-like cells from bone marrow. In the present study, we have tested whether ICF affects the production of such local factors in fetal mouse calvariae. Calvariae were cultured for 4 days in the presence and absence of ICF (130 mbar, 0.3 Hz). Conditioned medium was collected daily and pooled. We found that conditioned medium from ICF-exposed cultures stimulated [3H]-TdR incorporation into DNA, and [3H]-proline incorporation into collagenase digestible protein but not into non-collagen protein in fresh calvarial cultures. Treatment with conditioned medium from ICF-exposed cultures had earlier effects on [3H]-TdR and [3H]-proline incorporation than direct treatment with ICF. Conditioned medium from ICF-exposed cultures decreased the number of osteoclast precursor-like cells in bone marrow cultures stained for tartrate-resistant acid phosphatase. We conclude that ICF stimulates the release (activity) of an autocrine growth-factor from bone. In addition, ICF can stimulate the release (activity) of a paracrine factor, inhibiting the growth and/or differentiation of osteoclast precursor-like cells. These data suggest that mechanical forces may modulate skeletal (re)modeling by affecting the production of local growth factors.

Osteopontin-deficient bone cells are defective in their ability to produce NO in response to pulsatile fluid flow.

Osteopontin (OPN) is a noncollagenous component of bone matrix. It mediates cell attachment and activates signal transduction pathways. In this work, bone cells, cultured from fragments of long bones derived from wild-type and OPN-/- ("knock-out") mice, were exposed to pulsatile fluid flow (PFF) over a 60-min period. The medium was assayed periodically for nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release. OPN+/+ cells exhibited a peak of NO production 5-10 min after the onset of PFF, decreasing to a stable plateau at 15 min; much less NO was produced by the OPN-/- cells. PFF resulted in reduced PGE(2) release by both cell types, although the reduction was less for the OPN-/- cells in the 15-30 min window. Both cell types exhibited a similar enhancement of cyclooxygenase2 mRNA levels 60 min after initiation of PFF. These results suggest that bone cells require OPN to respond fully to PFF as assessed by increased NO and reduced PGE(2) synthesis.

Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts--correlation with prostaglandin upregulation.

Osteocytes are extremely sensitive to fluid shear stress, a phenomenon that may be related to mechanical adaptation of bone (FASEB J 9:441,1995). Here we examined the effect of pulsating fluid flow (PFF, 0.5 +/- 0.02 Pa, 5 Hz, 0.4 Pa/sec) on the release of NO, in relation with upregulation of prostaglandin E2 (PGE2). Chicken calvarial osteocytes, but not periosteal fibroblasts, as well as mouse calvarial cells responded to PFF with a rapid and transient 2 to 3-fold stimulation of NO release. The effect was maximal after 5 min and leveled off thereafter. PFF also stimulated PGE2 release. This effect was significant after 10 min and continued throughout 60 min PFF treatment. Inhibition of NO release by NG-monomethyl-L-arginine prevented the effect of PFF on NO as well as PGE2 release. These results suggest that NO is a mediator of mechanical effects in bone, leading to enhanced PGE2 release. They further strengthen the hypothesis that fluid flow through the osteocyte canalicular network provides the physical stimulus for mechanosensation in bone.

Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow.

Mechanical stress produces flow of fluid in the osteocytic lacunar-canalicular network, which is likely the physiological signal for the adaptive response of bone. We compared the induction of prostaglandin G/H synthase-2 (PGHS-2) by pulsating fluid flow (PFF) and serum in osteocytes, osteoblasts, and periosteal fibroblasts, isolated from 18-day-old fetal chicken calvariae. A serum-deprived mixed population of primarily osteocytes and osteoblasts responded to serum with a two- to threefold induction of PGHS-2 mRNA. Serum stimulated PGHS-2-derived PGE(2) release from osteoblasts and osteocytes but not from periosteal fibroblasts as NS-398, a PGHS-2 blocker, inhibited PGE(2) release from osteocytes and osteoblasts with 65%, but not that from periosteal fibroblasts. On the other hand PFF (0.7 Pa, 5 Hz) stimulated (3 fold) PGHS-2 mRNA only in OCY. The related PGE(2) response could be completely inhibited by NS-398. We conclude that osteocytes have a higher intrinsic sensitivity for loading-derived fluid flow than osteoblasts or periosteal fibroblasts.