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In this in vitro study the effect of XZ.700, a new endolysin, on methicillin resistant Staphylococcus aureus (MRSA) biofilms grown on titanium was evaluated. Biofilms of S. aureus USA300 were grown statically and under flow, and treatment with XZ.700 was compared with povidone-iodine (PVP-I) and gentamicin. To evaluate the cytotoxic effects of XZ.700 and derived biofilm lysates, human osteocyte-like cells were exposed to biofilm supernatants, and metabolism and proliferation were quantified. XZ.700 showed a significant, concentration dependent reduction in biofilm viability, compared with carrier controls. Metabolism and proliferation of human osteocyte-like cells were not affected by XZ.700 or lysates, unlike PVP-I and gentamicin lysates which significantly inhibited proliferation. Using time-lapse microscopy, rapid biofilm killing and removal was observed for XZ.700. In comparison, PVP-I and gentamicin showed slower biofilm killing, with no apparent biofilm removal. In conclusion, XZ.700 reduced MRSA biofilms, especially under flow condition, without toxicity for surrounding bone cells. [ABSTRACT FROM AUTHOR]

Osteocytes are the most abundant (~95%) cells in bone with the longest half-life (~25 years) in humans. In the past osteocytes have been regarded as vestigial cells in bone, since they are buried inside the tough bone matrix. However, during the last 30 years it has become clear that osteocytes are as important as bone forming osteoblasts and bone resorbing osteoclasts in maintaining bone homeostasis. The osteocyte cell body and dendritic processes reside in bone in a complex lacuno-canalicular system, which allows the direct networking of osteocytes to their neighboring osteocytes, osteoblasts, osteoclasts, bone marrow, blood vessels, and nerves. Mechanosensing of osteocytes translates the applied mechanical force on bone to cellular signaling and regulation of bone adaptation. The osteocyte lacuno-canalicular system is highly efficient in transferring external mechanical force on bone to the osteocyte cell body and dendritic processes via displacement of fluid in the lacuno-canalicular space. Osteocyte mechanotransduction regulates the formation and function of the osteoblasts and osteoclasts to maintain bone homeostasis. Osteocytes produce a variety of proteins and signaling molecules such as sclerostin, cathepsin K, Wnts, DKK1, DMP1, IGF1, and RANKL/OPG to regulate osteoblast and osteoclast activity. Various genetic abnormality-associated rare bone diseases are related to disrupted osteocyte functions, including sclerosteosis, van Buchem disease, hypophosphatemic rickets, and WNT1 and plastin3 mutation-related disorders. Meticulous studies during the last 15 years on disrupted osteocyte function in rare bone diseases guided for the development of various novel therapeutic agents to treat bone diseases. Studies on genetic, molecular, and cellular mechanisms of sclerosteosis and van Buchem disease revealed a role for sclerostin in bone homeostasis, which led to the development of the sclerostin antibody to treat osteoporosis and other bone degenerative diseases. The mechanism of many other rare bone diseases and the role of the osteocyte in the development of such conditions still needs to be investigated. In this review, we mainly discuss the knowledge obtained during the last 30 years on the role of the osteocyte in rare bone diseases. We speculate about future research directions to develop novel therapeutic drugs targeting osteocyte functions to treat both common and rare bone diseases. [ABSTRACT FROM AUTHOR]

Adaptation of bone to mechanical stresses normally produces a bone architecture that combines a proper resistance against failure with a minimal use of material. This adaptive process is governed by mechanosensitive osteocytes that transduce the mechanical signals into chemical responses, i.e. the osteocytes release signaling molecules, which orchestrate the recruitment and activity of bone forming osteoblasts and/or bone resorbing osteoclasts. Computer models have shown that the maintenance of a mechanically-efficient bone architecture depends on the intensity and spatial distribution of the mechanical stimulus as well as on the osteocyte response. Osteoporosis is a condition characterized by a reduced bone mass and a compromized resistance of bone against mechanical loads, which has led us to hypothesize that mechanotransduction by osteocytes is altered in osteoporosis. One of the major causal factors for osteoporosis is the loss of estrogen, the major hormonal regulator of bone metabolism. Loss of estrogen may increase osteocyte-mediated activation of bone remodeling, resulting in impaired bone mass and architecture. In this review we highlight current insights on how osteocytes perceive mechanical stimuli placed on whole bones. Particular emphasis is placed on the role of estrogen in signaling pathway activation by mechanical stimuli, and on computer simulation in combination with cell biology to unravel biological processes contributing to bone strength. [ABSTRACT FROM AUTHOR]

Titanium–nitride coating is used to improve cobalt–chromium–molybdenum implant survival in total knee arthroplasty, but its effect on osteoconduction is unknown. Chromium and cobalt ions negatively affect the growth and metabolism of cultured osteoblasts while enhancing osteoclastogenic cytokine production. Therefore, it was hypothesized that a titanium–nitride surface would enhance osteoblast proliferation and/or differentiation and reduce osteoclastogenic cytokine production compared with a cobalt–chromium–molybdenum surface. MC3T3-E1 osteoblasts showed increased proliferation and decreased differentiation on titanium–nitride, while cytokine interleukin-6 production was higher on porous cobalt–chromium–molybdenum (p < 0.05), though interleukin-1β was occasionally detected on both surfaces. These findings suggest improved osteoconduction on titanium–nitride compared with cobalt–chromium–molybdenum surface. [ABSTRACT FROM AUTHOR]

Background aims. For 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. Methods. Human 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. Results. We 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. Conclusions. These 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. [ABSTRACT FROM AUTHOR]

Healthy bones combine a proper resistance against fracture with a minimum use of material. This property is likely brought about by osteocytes in response to mechanical cues, but it is still unknown how whole bone loads are translated into a signal that can be sensed by the osteocytes. The goal of this chapter is to critically analyze our current knowledge on how bone transduction takes place from the level of mechanical loads placed upon the bone as an organ to activation of a cell. [ABSTRACT FROM AUTHOR]

In regions of high bone loading, the mechanoresponsive osteocytes inhibit osteoclastic bone resorption by producing signaling molecules. One possible candidate is matrix extracellular phosphoglycoprotein (MEPE) because acidic serine- and aspartate-rich MEPE-associated motif peptides upregulate osteoprotegerin (OPG) gene expression, a negative regulator of osteoclastogenesis. These peptides are cleaved from MEPE when relatively more MEPE than PHEX (phosphate-regulating gene with homology to endopeptidases on the X chromosome) is present. We investigated whether mechanical loading of osteocytes affects osteocyte-stimulated osteoclastogenesis by involvement of MEPE. MLO-Y4 osteocytes were mechanically loaded by 1-h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) or kept under static control conditions. Recombinant MEPE (0.05, 0.5, or 5 μg/ml) was added to some static cultures. Mouse bone marrow cells were seeded on top of the osteocytes to determine osteoclastogenesis. Gene expression of MEPE, PHEX, receptor activator of nuclear factor kappa-B ligand (RANKL), and OPG by osteocytes was determined after PFF. Osteocytes supported osteoclast formation under static control conditions. Both PFF and recombinant MEPE inhibited osteocyte-stimulated osteoclastogenesis. PFF upregulated MEPE gene expression by 2.5-fold, but not PHEX expression. PFF decreased the RANKL/OPG ratio at 1-h PFF treatment. Our data suggest that mechanical loading induces changes in gene expression by osteocytes, which likely contributes to the inhibition of osteoclastogenesis after mechanical loading of bone. Because mechanical loading upregulated gene expression of MEPE but not PHEX, possibly resulting in the upregulation of OPG gene expression, we speculate that MEPE is a soluble factor involved in the inhibition of osteoclastogenesis by osteocytes. [ABSTRACT FROM AUTHOR]

Kraft DCE, Bindslev DA, Melsen B, Abdallah BM, Kassem M, Klein-Nulend J. Mechanosensitivity of dental pulp stem cells is related to their osteogenic maturity. Eur J Oral Sci 2010; 118: 29–38. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci For engineering bone tissue, mechanosensitive cells are needed for bone (re)modelling. Local bone mass and architecture are affected by mechanical loading, which provokes a cellular response via loading-induced interstitial fluid flow. We studied whether human dental pulp-derived mesenchymal stem cells (PDSCs) portraying mature (PDSC-mature) or immature (PDSC-immature) bone cell characteristics are responsive to pulsating fluid flow (PFF) in vitro. We also assessed bone formation by PDSCs on hydroxyapatite-tricalcium phosphate granules after subcutaneous implantation in mice. Cultured PDSC-mature exhibited higher osteocalcin and alkaline phosphatase gene expression and activity than PDSC-immature. Pulsating fluid flow (PFF) stimulated nitric oxide production within 5 min by PDSC-mature but not by PDSC-immature. In PDSC-mature, PFF induced prostaglandin E2 production, and cyclooxygenase 2 gene expression was higher than in PDSC-immature. Implantation of PDSC-mature resulted in more osteoid deposition and lamellar bone formation than PDSC-immature. We conclude that PDSCs with a mature osteogenic phenotype are more responsive to pulsating fluid shear stress than osteogenically immature PDSCs and produce more bone in vivo. These data suggest that PDSCs with a mature osteogenic phenotype might be preferable for bone tissue engineering to restore, for example, maxillofacial defects, because they might be able to perform mature bone cell-specific functions during bone adaptation to mechanical loading in vivo. [ABSTRACT FROM AUTHOR]

Stochastic resonance is exhibited by many biological systems, where the response to a small stimulus is enhanced with the aid of noise. This intriguing possibility provides a novel paradigm for understanding previously reported osteogenic benefits of low amplitude dynamic loading. However, it is unknown whether bone cell mechanosensitivity is enhanced by noise as an alternative mechanism for an amplified response to small stresses. We studied whether noise of varying intensities enhanced the mechanosensitivity of MC3T3-E1 cells. Nitric oxide (NO) production was measured as the parameter for bone cell activation. Dynamic fluid shear stress stimulated bone cells provided an initial-stress kick was implemented. Without the initial stress-kick bone cells did not release a significant amount of NO demonstrating an essential non-linearity to bone cell responses to stress and the possibility of stochastic resonance in bone cell mechanosensitivity. The rapid NO response of MC3T3-E1 cells to a small periodic fluid shear stress was increased with the addition of noise compared to the response to stress with only noise. This confirms the possibility of stochastic resonance enhancement of NO production by bone cells. Since NO regulate bone formation as well as resorption, our results suggest that noise enhances the activity of bone cells in driving the mechanical adaptation of bone. [ABSTRACT FROM AUTHOR]

For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) display a bone cell-like response to mechanical loading. We have shown earlier that this response includes increased nitric oxide (NO) production and cyclooxygenase-2 (COX-2) gene expression, both of which are intimately involved in mechanical adaptation of bone. COX-2 gene expression is likely regulated by polyamines, which are organic cations implicated in cell proliferation and differentiation. This has led to the hypothesis that polyamines may play a role in the response of adipose tissue-derived MSCs (AT-MSCs) to mechanical loading. The aim of this study was to investigate whether genes involved in polyamine metabolism are regulated by mechanical loading and to study whether polyamines modulate mechanical loading-induced NO production and COX-2 gene expression in human AT-MSCs. Human AT-MSCs displayed a bone cell-like response to mechanical loading applied by pulsating fluid flow (PFF), as demonstrated by increased NO production and increased gene expression of COX-2. Furthermore, PFF increased gene expression of spermidine/spermine N (1)-acetyltransferase, which is involved in polyamine catabolism, suggesting that mechanical loading modulates polyamine levels. Finally, the polyamine spermine was shown to inhibit both PFF-induced NO production and COX-2 gene expression, suggesting that polyamines modulate the response of human AT-MSCs to mechanical loading. In conclusion, this is the first study implicating polyamines in the response of human AT-MSCs to mechanical loading, creating opportunities for the use of polyamines in tissue engineering approaches targeting skeletal defects. [ABSTRACT FROM AUTHOR]

Loading frequency is an important parameter for the stimulation of bone formation in vivo. It is still unclear how the information of external loading characteristics is conveyed to osteoblasts and osteoclasts. Osteocytes are thought to detect mechanical loads by sensing fluid flow through the lacuno-canalicular network within bone and to translate this information into chemical signals. The signaling molecules nitric oxide (NO) and prostaglandin E2 (PGE2) are known to play important roles in the adaptive response of bone to mechanical loads. We have investigated the effects of fluid flow frequency on the production of PGE2 and NO in bone cells in vitro. Pulsatile fluid flow with different frequencies stimulated the release of NO by MC3T3-E1 osteoblasts in a dose-dependent manner. In contrast, PGE2 production was enhanced consistently by all fluid flow regimes, independent of flow frequency. This implies that the NO response may play a role in mediating the differential effects of the various loading patterns on bone. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1170-1177, 2006 [ABSTRACT FROM AUTHOR]

Mechanosensing by cells directs changes in bone mass and structure in response to the challenges of mechanical loading. Low-amplitude, high-frequency loading stimulates bone growth by enhancing bone formation and inhibiting disuse osteoporosis. However, how bone cells sense vibration stress is unknown. Hence, we investigated bone cell responses to vibration stress at a wide frequency range (5-100 Hz). We used NO and prostaglandin E2 (PGE2) release, and COX-2 mRNA expression as parameters for bone cell response since these molecules regulate bone adaptation to mechanical loading. NO release positively correlated whereas PGE2 release negatively correlated to the maximum acceleration rate of the vibration stress. COX-2 mRNA expression increased in a frequency-dependent manner, which relates to increased NO release at high frequencies, confirming our previous results. The negatively correlated release of NO and PGE2 suggests that these signaling molecules play different roles in bone adaptation to high-frequency loading. The maximum acceleration rate is proportional to to3 (frequency=ω/2π), which is commensurate with the Stokes-Einstein relation for modeling cell nucleus motion within the cytoplasm due to vibration stress. Correlations of NO and PGE2 with the maximum acceleration rate then relate to nucleus oscillations, providing a physical basis for cellular mechanosensing of high-frequency loading. [ABSTRACT FROM AUTHOR]

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 6× more often than periosteal fibroblasts and 3× 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. [Copyright &y& Elsevier]

Tests the hypothesis that estrogen enhances mechanical stress-induced prostaglandin production by bone cells from elderly women. Effects of pulsating fluid flow on prostaglandin production by bone cells from individual women; Shifting of the 'set point' for bone mass adaptation to mechanical loads by estrogen deficiency.

Highlights a study which analyzed the response of the bone cell cultures from human bone biopsies to stress, in contrast to the animal bone cells. Questioning whether the osteoporotic patients' bone cells responses were similar to the nonosteoporotic donors; Outgrowth of the bone cell cultures; Methodology used to conduct the study; Results of the study.

Presents a comparison of strain and fluid shear stress in stimulating bone cell responses. Development of a computational model of an adherent cell; Displacements of the cell model caused by fluid shear stress; Deformation of the membrane caused by fluid shear stress.

The article discusses various reports published within the issue, including one on mechanisms of osteocyte mechanotransduction, one on the application of bioimaging to osteocyte biology and another on stress response by bone cells and implications on microgravity environment.

Abstract: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) strongly mediates bone mass. Mechanical stimulation also affects bone mass, partly via enhancing nitric oxide (NO) production by osteoblasts. We aimed to determine whether 1,25(OH)2D3 affects NO production by osteoblasts in the presence or absence of mechanical stimulation. We hypothesised that 1,25(OH)2D3 stimulates NO production via nuclear actions of the vitamin D receptor (VDR), which requires hours of incubation with 1,25(OH)2D3 to occur. MC3T3-E1 osteoblasts and long-bone osteoblasts of adult wildtype and VDR−/− mice were pre-incubated for 24h with or without 1,25(OH)2D3 (10−13–10−9 M), followed by 30min pulsating fluid flow (PFF; 0.7±0.3Pa, 5Hz) or static culture with or without 1,25(OH)2D3. NO production and NO synthase (NOS) expression were quantified. 10−11 M 1,25(OH)2D3 for 24h, but not 30min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH)2D3 for 24h increased inducible-NOS gene-expression (twofold), suggesting that 1,25(OH)2D3 stimulated NO production via activation of NOS gene transcription. PFF rapidly increased NO production by MC3T3-E1 osteoblasts, wildtype osteoblasts, and VDR−/− osteoblasts. This PFF effect was abolished after incubation with 1,25(OH)2D3 for 24h, or during PFF only. Our results suggest that 1,25(OH)2D3 stimulates inducible-NOS expression and NO production by osteoblasts in the absence of mechanical stimulation, likely via genomic VDR action. In contrast, 1,25(OH)2D3 may affect mechanical loading-induced NO production independent of genomic VDR action, since 1,25(OH)2D3 diminished PFF-induced NO production in VDR−/− bone cells. In conclusion, 1,25(OH)2D3 and mechanical loading interact at the level of mechanotransduction, whereby 1,25(OH)2D3 seems to act independently of VDR genomic mechanism. [Copyright &y& Elsevier]

Abstract: Introduction: Osteogenic cell proliferation and differentiation play an important role in adequate fracture healing, and is target for osteoinductive therapies in delayed fracture healing. The aim of this study was to investigate whether low-intensity pulsed ultrasound enhances fracture healing at the tissue level in patients with a delayed union of the osteotomized fibula through an effect on the presence of RUNX2 immunopositive osteogenic cells. The effect was studied in both atrophic and hypertrophic delayed unions. Materials and methods: Biopsies were obtained from 6 female and 1 male patient (age 43–63) with a delayed union of the osteotomized fibula after a high tibial osteotomy treated for 2–4 months with or without low-intensity pulsed ultrasound in a randomized prospective double-blind placebo-controlled trial. Immunolocalization of RUNX2 protein was performed to identify osteogenic cells. Histomorphometrical analysis was performed to determine the number of cells expressing RUNX2 located within and around the newly formed woven bone at the fracture end (area of new bone formation), and up to 3 mm distant from the fracture end. Results: Cells expressing RUNX2 were present in all histological sections of control and low-intensity pulsed ultrasound-treated bone evaluated. Within the area of new bone formation, RUNX2 immunopositive cells were found in the undifferentiated soft connective tissue, at the bone surface (presumably osteoblasts), and within the newly formed woven bone. Low-intensity pulsed ultrasound treatment of fibula delayed unions significantly reduced the number of RUNX2 immunopositive cells within the soft connective tissue at the fracture ends, whereas the number of RUNX2 immunopositive cells at the bone surface was not affected. The number of RUNX2 immunopositive cells was similar for the atrophic and hypertrophic delayed unions. Conclusions: Immunolocalization of RUNX2 positive cells in delayed unions of the fibula reveals that delayed clinical fracture healing does not result in impairment of osteogenic cell proliferation and/or differentiation at the tissue level, even if delayed unions are clinically regarded as atrophic. Reduced number of osteogenic RUNX2 immunopositive cells within the soft connective tissue, and unchanged number of RUNX2 immunopositive cells at the bone surface, implicate that low-intensity pulsed ultrasound does not increase osteogenic cell presence, but likely affects osteogenic cell differentiation. [Copyright &y& Elsevier]

Abstract: Matrix strains due to external loading are different in bones of different pathologies with different bone mineral density (BMD), and are likely sensed by the osteocytes, the putative bone mechanosensors. The mechanosensitivity of osteocytes appears to be strongly influenced by their morphology. In this study, we explored the possibility that osteocyte morphology might play a role in various bone pathologies with different BMD. Confocal laser scanning microscopy and nano-CT were used to quantitatively determine 3D morphology and alignment of osteocytes and osteocyte lacunae in human proximal tibial bone with relatively low (osteopenic), medium (osteoarthritic), and high (osteopetrotic) BMD. Osteopenic osteocytes were relatively large and round (lengths 8.9:15.6:13.4 μm), osteopetrotic osteocytes were small and discoid shaped (lengths 5.5:11.1:10.8 μm), and osteoarthritic osteocytes were large and elongated (lengths 8.4:17.3:12.2 μm). Osteopenic osteocyte lacunae showed 3.5 fold larger volume and 2.2 fold larger surface area than osteoarthritic lacunae, whereas osteopetrotic lacunae were 1.9 fold larger and showed 1.5 fold larger surface area than osteoarthritic lacunae. Osteopetrotic osteocyte lacunae had lower alignment than osteopenic and osteoarthritic lacunae as indicated by their lower degree of anisotropy. The differences in 3D morphology of osteocytes and their lacunae in long bones of different pathologies with different BMD might reflect an adaptation to matrix strain due to different external loading conditions. Moreover, since direct mechanosensing of matrix strain likely occurs by the cell bodies, the differences in osteocyte morphology and their lacunae might indicate differences in osteocyte mechanosensitivity. The exact relationship between osteocyte morphology and bone architecture, however, is complex and deserves further study. [Copyright &y& Elsevier]

Abstract: Introduction: External mechanical forces on cells are known to influence cytoskeletal structure and thus cell shape. Mechanical loading in long bones is unidirectional along their long axes, whereas the calvariae are loaded at much lower amplitudes in different directions. We hypothesised that if osteocytes, the putative bone mechanosensors, can indeed sense matrix strains directly via their cytoskeleton, the 3D shape and the long axes of osteocytes in fibulae and calvariae will bear alignment to the different mechanical loading patterns in the two types of bone. Materials and methods: We used confocal laser scanning microscopy and nano-computed tomography to quantitatively determine the 3D morphology and alignment of long axes of osteocytes and osteocyte lacunae in situ. Results: Fibular osteocytes showed a relatively elongated morphology (ratio lengths 5.9:1.5:1), whereas calvarial osteocytes were relatively spherical (ratio lengths 2.1:1.3:1). Osteocyte lacunae in fibulae had higher unidirectional alignment than the osteocyte lacunae in calvariae as demonstrated by their degree of anisotropy (3.33 and 2.10, respectively). The long axes of osteocyte lacunae in fibulae were aligned parallel to the principle mechanical loading direction, whereas those of calvarial osteocyte lacunae were not aligned in any particular direction. Conclusions: The anisotropy of osteocytes and their alignment to the local mechanical loading condition suggest that these cells are able to directly sense matrix strains due to external loading of bone. This reinforces the widely accepted role of osteocytes as mechanosensors, and suggests an additional mode of mechanosensing besides interstitial fluid flow. The relatively spherical morphology of calvarial osteocytes suggests that these cells are more mechanosensitive than fibular osteocytes, which provides a possible explanation of efficient physiological load bearing for the maintenance of calvarial bone despite its condition of relative mechanical disuse. [Copyright &y& Elsevier]

Abstract: Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70±0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-κB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N G -nitro-l-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption. [Copyright &y& Elsevier]

Abstract: Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation. [Copyright &y& Elsevier]

Shear stress due to mechanical loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. Moreover, the rate (determined by frequency and magnitude) of mechanical loading determines the amount of bone formation. Whether the bone cells’ response to fluid shear stress is rate dependent is unknown. Here we investigated whether bone cell activation by fluid shear stress is rate dependent. MC3T3-E1 osteoblastic cells were subjected for 15 min to fluid shear stress of varying frequencies and amplitudes, resulting in peak fluid shear stress rates ranging from 0 to 39.6 Pa-Hz. Nitric oxide production, a parameter for bone cell activation, was found to be linearly dependent on the fluid shear stress rate; the slope was steepest at 5 min (0.11 Pa-Hz−1) and decreased to 0.03 Pa-Hz−1 at 15 min. We conclude that the fluid shear stress rate is an important parameter for bone cell activation. [Copyright &y& Elsevier]