Mechanical loading intensities affect the release of extracellular vesicles from mouse bone marrow-derived hematopoietic progenitor cells and change their osteoclast-modulating effect.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Β ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g) obtained after low-intensity loading and induced by exosomes (100 000× g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect.

The intricate anatomy of the periodontal ligament and its development: Lessons for periodontal regeneration.

The periodontal ligament (PDL) connects the tooth root and alveolar bone. It is an aligned fibrous network that is interposed between, and anchored to, both mineralized surfaces. Periodontal disease is common and reduces the ability of the PDL to act as a shock absorber, a barrier for pathogens and a sensor of mastication. Although disease progression can be stopped, current therapies do not primarily focus on tissue regeneration. Functional regeneration of PDL may be achieved using innovative techniques, such as tissue engineering. However, the complex fibrillar architecture of the PDL, essential to withstand high forces, makes PDL tissue engineering very challenging. This challenge may be met by studying PDL anatomy and development. Understanding PDL anatomy, development and maintenance provides clues regarding the specific events that need to be mimicked for the formation of this intricate tissue. Owing to the specific composition of the PDL, which develops by self-organization, a different approach than the typical combination of biomaterials, growth factors and regenerative cells is necessary for functional PDL engineering. Most specifically, the architecture of the new PDL to be formed does not need to be dictated by textured biomaterials but can emerge from the local mechanical loading conditions. Elastic hydrogels are optimal to fill the space properly between tooth and bone, may house cells and growth factors to enhance regeneration and allow self-optimization by the alignment to local stresses. We suggest that cells and materials should be placed in a proper mechanical environment to initiate a process of self-organization resulting in a functional architecture of the PDL.

Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts.

The metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) is synthesized from its precursor 25-hydroxyvitamin D (25(OH)D) by human osteoblasts leading to stimulation of osteoblast differentiation in an autocrine or paracrine way. Osteoblast differentiation is also stimulated by mechanical loading through activation of various responses in bone cells such as nitric oxide signaling. Whether mechanical loading affects osteoblast differentiation through an enhanced synthesis of 1,25(OH)2D by human osteoblasts is still unknown. We hypothesized that mechanical loading stimulates the synthesis of 1,25(OH)2D from 25(OH)D in primary human osteoblasts. Since the responsiveness of bone to mechanical stimuli can be altered by various endocrine factors, we also investigated whether 1,25(OH)2D or 25(OH)D affect the response of primary human osteoblasts to mechanical loading. Primary human osteoblasts were pre-incubated in medium with/without 25(OH)D3 (400 nM) or 1,25(OH)2D3 (100 nM) for 24h and subjected to mechanical loading by pulsatile fluid flow (PFF). The response of osteoblasts to PFF was quantified by measuring nitric oxide, and by PCR analysis. The effect of PFF on the synthesis of 1,25(OH)2D3 was determined by subjecting osteoblasts to PFF followed by 24h post-incubation in medium with/without 25(OH)D3 (400 nM). We showed that 1,25(OH)2D3 reduced the PFF-induced NO response in primary human osteoblasts. 25(OH)D3 did not significantly alter the NO response of primary human osteoblasts to PFF, but 25(OH)D3 increased osteocalcin and RANKL mRNA levels, similar to 1,25(OH)2D3. PFF did not increase 1,25(OH)2D3 amounts in our model, even though PFF did increase CYP27B1 mRNA levels and reduced VDR mRNA levels. CYP24 mRNA levels were not affected by PFF, but were strongly increased by both 25(OH)D3 and 1,25(OH)2D3. In conclusion, 1,25(OH)2D3 may affect the response of primary human osteoblasts to mechanical stimuli, at least with respect to NO production. Mechanical stimuli may affect local vitamin D metabolism in primary human osteoblasts. Our results suggest that 1,25(OH)2D3 and mechanical loading, both stimuli of the differentiation of osteoblasts, interact at the cellular level.

Inflammatory factors in the circulation of patients with active rheumatoid arthritis stimulate osteoclastogenesis via endogenous cytokine production by osteoblasts.

SUMMARY: The combination of cytokines present in the circulation of patients with active rheumatoid arthritis might contribute to the generalized bone loss that commonly occurs in these patients, by directly inhibiting osteoblast proliferation and differentiation, but especially by enhancing endogenous cytokine (i.e., receptor activator of nuclear factor-kappa B ligand (RANKL) and interleukin-6 (IL)-6) production by osteoblasts, thereby stimulating osteoclastogenesis. INTRODUCTION: Generalized bone loss, as occurs in patients with rheumatoid arthritis (RA), is related to elevated levels of circulating cytokines. Individual cytokines have deleterious effects on proliferation and differentiation of osteoblast cell lines, but little is known about the effect of the interaction between inflammatory factors in the circulation of patients with active RA on human osteoblast function, including their communication towards other bone cells. We investigated whether serum from patients with active RA enhances cytokine production by osteoblasts, thereby effectively altering osteoblast-stimulated osteoclastogenesis. METHODS: Serum was obtained from 20 patients with active RA (active RA sera) and from the same patients in clinical remission (remission RA sera). To determine osteoclastogenesis, RA serum-pretreated primary human osteoblast cultures were established in direct contact with human osteoclast precursors in the presence or absence of osteoprotegerin (OPG) or IL-6 inhibitor. RESULTS: Compared to remission RA sera, active RA sera inhibited osteoblast proliferation and differentiation in vitro as demonstrated by a reduced DNA content and gene expression of KI-67, collagen type 1, osteopontin, and osteocalcin. Active RA sera inhibited OPG expression and enhanced RANKL and IL-6 expression but did not alter IL-8 expression in osteoblasts. IL-1ß, IL-17, and tumor necrosis factor-α (TNF-α) expression were undetectable. In coculture, active RA sera treatment of osteoblasts stimulated while addition of OPG or IL-6 inhibitory antibodies significantly reduced the number of osteoclasts. CONCLUSION: Active RA sera contain circulating factors, likely cytokines and chemokines, that might contribute to bone loss by directly inhibiting osteoblast proliferation and differentiation, but especially, these factors modulate endogenous cytokine production by osteoblasts, thereby affecting osteoclastogenesis.

IL-6 alters osteocyte signaling toward osteoblasts but not osteoclasts.

Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts.

Nitric oxide signaling in mechanical adaptation of bone.

One of the most serious healthcare problems in the world is bone loss and fractures due to a lack of physical activity in elderly people as well as in bedridden patients or otherwise inactive youth. Crucial here are the osteocytes. Buried within our bones, these cells are believed to be the mechanosensors that stimulate bone formation in the presence of mechanical stimuli and bone resorption in the absence of such stimuli. Intercellular signaling is an important physiological phenomenon involved in maintaining homeostasis in all tissues. In bone, intercellular communication via chemical signals like NO plays a critical role in the dynamic process of bone remodeling. If bones are mechanically loaded, fluid flows through minute channels in the bone matrix, resulting in shear stress on the cell membrane that activates the osteocyte. Activated osteocytes produce signaling molecules like NO, which modulate the activity of the bone-forming osteoblasts and the bone-resorbing osteoclasts, thereby orchestrating bone adaptation to mechanical loading. In this review, we highlight current insights in the role of NO in the mechanical adaptation of bone mass and structure, with emphasis on its role in local bone gain and loss as well as in remodeling supervised by osteocytes. Since mechanical stimuli and NO production enhance bone strength and fracture resistance, these new insights may facilitate the development of novel osteoporosis treatments.

Growth factor gene expression profiles of bone morphogenetic protein-2-treated human adipose stem cells seeded on calcium phosphate scaffolds in vitro.

The secretome of stem cells strongly determines the outcome of tissue engineering strategies. We investigated how the secretome of human adipose stem cells (hASCs) can be affected by substrate, BMP-2 treatment, and degree of differentiation. We hypothesized that as differentiation progresses, hASCs produce increasingly more gene products associated with processes such as angiogenesis and bone remodeling. Human ASCs were treated for 15 min with BMP-2 (10 ng/ml) to enhance osteogenic differentiation, or with vehicle. Subsequently, hASCs were seeded on plastic or on biphasic calcium phosphate (BCP) consisting of 60% hydroxyapatite and 40% ß-tricalcium phosphate. A PCR array for ~150 trophic factors and differentiation-related genes was performed at day 21 of culture. A limited set of factors was quantified by qPCR at days 0, 4, 14 and 21, and/or ELISA at day 21. Compared to plastic, BCP-cultured hASCs showed ≥2-fold higher expression of ~20 factors, e.g. cytokines such as IL-6, growth factors such as FGF7 and adhesion molecules such as VCAM1. Expression of another ~50 genes was decreased ≥2-fold on BCP vs. plastic, even though hASCs differentiate better on BCP than on plastic. BMP-2-treatment increased the expression of ~30 factors by hASCs seeded on BCP, while it decreased the expression of only PGF, PPARG and PTN. Substrate affected hASC secretion of Activin A and seemed to affect P1NP release. No clear association between hASC osteogenic differentiation and growth factor expression pattern was observed. Considering our observed lack of association between the degree of differentiation and the expression of factors associated with angiogenesis and bone remodeling by hASCs, future bone regeneration studies should focus more on systematically orchestrating the secretome of stem cells, rather than on inducing osteogenic differentiation of stem cells only. Short incubation with BMP-2 may be a promising treatment to enhance both osteogenic differentiation and environmental modulation.

Mechanical loading and how it affects bone cells: the role of the osteocyte cytoskeleton in maintaining our skeleton.

Lack of physical activity causes bone loss and fractures not only in elderly people, but also in bedridden patients or otherwise inactive youth. This is fast becoming one of the most serious healthcare problems in the world. Osteocytes, cells buried within our bones, stimulate bone formation in the presence of mechanical stimuli, as well as bone degradation in the absence of such stimuli. As yet, we do not fully comprehend how osteocytes sense mechanical stimuli, and only know a fraction of the whole range of molecules that osteocytes subsequently produce to regulate bone formation and degradation in response to mechanical stimuli. This dramatically hampers the design of bone loss prevention strategies. In this review we will focus on the first step in the cascade of events leading to adaptation of bone mass to mechanical loading, i.e., on how osteocytes are able to perceive mechanical stimuli placed on whole bones. We will place particular emphasis on the role of the osteocyte cytoskeleton in mechanosensing. Given the crucial importance of osteocytes in maintaining a proper resistance against bone fracture, greater knowledge of the molecular mechanisms that govern the adaptive response of osteocytes to mechanical stimuli may lead to the development of new strategies towards fracture prevention and enhanced bone healing.

Tumor necrosis factor alpha and interleukin-1beta modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes.

OBJECTIVE: Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) modulate the osteocyte response to mechanical loading. METHODS: MLO-Y4 osteocytes were incubated with TNFalpha (0.5-30 ng/ml) or IL-1beta (0.1-10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean +/- amplitude 0.7 +/- 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca(2+)](i)) using Fluo-4/AM. Focal adhesions and filamentous actin (F-actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell-generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry. RESULTS: Pulsatile fluid flow increased [Ca(2+)](i) within seconds (in 13% of cells) and NO production within 5 minutes (4.7-fold). TNFalpha and IL-1beta inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow-induced NO production. TNFalpha and IL-1beta affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFalpha and IL-1beta decreased the amount of F-actin. Incubation with IL-1beta for 24 hours stimulated osteocyte apoptosis. CONCLUSION: Our results suggest that TNFalpha and IL-1beta inhibit mechanical loading-induced NO production by osteocytes via abrogation of pulsatile fluid flow-stimulated [Ca(2+)](i), and that IL-1beta stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.

Predictive value of femoral head heterogeneity for fracture risk.

Osteoporosis (OP) is characterized by low bone mass and weak bone structure, which results in increased fracture risk. It has been suggested that osteoporotic bone is strongly adapted to the main loading direction and less adapted to the other directions. In this study, we hypothesized that osteoporotic femoral heads have 1) an increased anisotropy; 2) a more heterogenic distribution of bone volume fraction (BV/TV) throughout the femoral head; and, 3) a more heterogenic distribution of the trabecular thickness (Tb.Th.) throughout the femoral head, as compared to non-osteoporotic bone. To test these hypotheses, we used 7 osteoporotic femoral heads from patients who fractured their femoral neck and 7 non-fractured femoral heads from patients with osteoarthrosis (OA). Bone structural parameters from the entire trabecular region were analyzed using microCT. We found that the degree of anisotropy was higher in the fractured femoral heads, i.e. 1.72, compared to a value of 1.61 in the non-fractured femoral heads. The BV/TV and Tb.Th. and their variations throughout the femoral head, however, were all significantly lower in the fractured group. Hence, the first hypothesis was confirmed, whereas the other two were rejected. Interestingly, the variation of Tb.Th. throughout the femoral head provided a 100% discrimination between the OP and OA groups, i.e. for the same BV/TV, all fractured cases had a less heterogenic distribution. In conclusion, our results suggest that bone loss in OP takes place uniformly throughout the femoral head, leading to an overall decrease in bone mass and trabecular thickness. Furthermore, the variation of Tb.Th. in the femoral head could be an interesting parameter to improve the prediction of fracture risk in the proximal femur.

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.

Different responsiveness to mechanical stress of bone cells from osteoporotic versus osteoarthritic donors.

INTRODUCTION: Osteoporosis (OP) and osteoarthritis (OA) are both common diseases in the elderly, but remarkably seldom coexist. The bone defects that are related to both diseases develop with increasing age, which suggests that they are related to some form of imperfect bone remodeling. Current opinion holds that the bone remodeling process is supervised by bone cells that respond to mechanical stimuli. An imperfect response of bone cells to mechanical stimuli might thus relate to imperfect bone remodeling, which could eventually lead to a lack bone mass and strength, such as in OP patients. MATERIALS: To investigate whether the cellular response to mechanical stress differs between OP and OA patients, we compared the response of bone cells from both groups to fluid shear stress of increasing magnitude. Bone cells from 9 female OP donors (age 60-90 year) and 9 female age-matched OA donors were subjected to pulsating fluid flow (PFF) of low (0.4+/-0.1 Pa at 3 Hz), medium (0.6+/-0.3 Pa at 5 Hz), or high shear stress (1.2+/-0.4 at 9Hz), or were kept under static culture conditions. RESULTS: We found subtle differences in the shear-stress response of the two groups, measured as nitric oxide (NO) and prostaglandin E2 (PGE2) production. The NO-response to shear stress was higher in the OP than the OA cells, while the PGE2-response was higher in the OA cells. CONCLUSIONS: Assuming that NO and PGE2 play a role in cell-cell communication during remodeling, these results suggest that slight differences in mechanotransduction might relate to the opposite bone defects in osteoporosis and osteoarthritis.

Additive effects of estrogen and mechanical stress on nitric oxide and prostaglandin E2 production by bone cells from osteoporotic donors.

Mechanical loading is thought to provoke a cellular response via loading-induced flow of interstitial fluid through the lacuno-canalicular network of osteocytes. This response supposedly leads to an adaptation of local bone mass and architecture. It has been suggested that loss of estrogen during menopause alters the sensitivity of bone tissue to mechanical load, thereby contributing to the rapid loss of bone. The present study aimed to determine whether estrogen modulates the mechanoresponsiveness of bone cells from osteoporotic women. Bone cell cultures from nine osteoporotic women (aged 62-90 years) were pre-cultured for 24 h with 10(-11) mol/l 17beta-estradiol (E2) or vehicle, and subjected to 1 h of pulsating fluid flow (PFF) or static culture. E2 alone enhanced prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production by 2.8-fold and 2.0-fold, respectively, and stimulated endothelial nitric oxide synthase protein expression by 2.5-fold. PFF, in the absence of E2, stimulated PGE(2) production by 3.1-fold and NO production by 3.9-fold. Combined treatment with E2 and PFF increased PGE(2) and NO production in an additive manner. When expressed as PFF-treatment-over-control ratio, the response to fluid shear stress was similar in the absence or presence of E2. These results suggest that E2 does not affect the early response to stress in bone cells. Rather, E2 and shear stress both promote the production of paracrine factors such as NO and PGE(2) in an additive manner.

Mechanotransduction in bone cells proceeds via activation of COX-2, but not COX-1.

Cyclooxygenase (COX) is the key enzyme in the production of prostaglandins, which are essential for the response of bone to mechanical loading. We determined which COX-isoform, COX-1 or COX-2, determines loading-induced prostaglandin production in primary bone cells in vitro. Mouse and human bone cells reacted to 1 h of pulsating fluid flow (PFF, 0.6+/-0.3 Pa at 5 Hz) with an increased prostaglandin E(2) production, which continued 24 h after cessation of PFF. Inhibition of COX-2 activity with NS-398 abolished the stimulating effect of PFF both at 1 h and at 24 h post-incubation, while inhibition of COX-1 by SC-560 affected neither the early nor the late response to flow. PFF rapidly stimulated COX-2 mRNA expression at 1 h but did not affect COX-1 mRNA expression. COX-2 mRNA expression was still significantly enhanced 24 h after cessation of PFF. We conclude that COX-2 is the mechanosensitive form of COX that determines the response of bone tissue to mechanical loading.

[The response of bone cells to shear stress]. / Botcellen reageren op schuifspanning.

Loading-induced flow of fluid is a signal for bone cell adaptive responses, but the nature of the flow-derived stimulus which activates the cell is debated. Candidate stimuli include shear stress, streaming potentials and chemotransport. In this study the nature of the cell stimulus was addressed by varying the shear stress, using nitric oxide (NO) and prostaglandin E2 (PGE2) production as a parameter of bone cell activation. Mouse bone cell cultures were treated for 15 minutes with or without pulsating fluid flow (PFF). In a few experiments, dextran was added to the fluid to increase the shear stress without affecting streaming potentials or chemotransport. NO and PGE2 production were dose-dependently stimulated by PFF. Application of dextran in the flow medium enhanced both NO and PGE2 production by bone cells. It was demonstrated that the production of NO and PGE2 by bone cells is enhanced by fluid flow of increasing shear stress. Therefore, the stimulus leading to NO and PGE2 production is shear stress rather than streaming potentials or chemotransport.

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.

Radiography of the carpal scaphoid. Experimental evaluation of "the carpal box" and first clinical results.

RATIONALE AND OBJECTIVES: The authors describe a new device ("the carpal box") for the radiographic detection of occult scaphoid fracture. METHODS: Fractures in the scaphoid of five cadaver specimens were mechanically produced. Subsequent examinations included conventional scaphoid radiography, multi-angle radiography, and radiography using the new device. The anatomic analysis of the specimens served as the standard for comparison. A pilot study was performed in six consecutive patients with suspected scaphoid fracture. RESULTS: Neither scaphoid radiography nor multi-angle radiography could confirm a fracture in two specimens, whereas all fractures were recognized on the carpal box radiographs. All scaphoid fractures were visualized by carpal box radiography, whereas scaphoid radiography was equivocal in one patient and negative in the other. CONCLUSIONS: Carpal box radiography may have additional value in the diagnosis of occult scaphoid fracture. This may lead to a reduction in costs and inconvenience for patients with clinically suspected scaphoid fracture and negative scaphoid radiography.