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Osteocytes are capable of remodeling their perilacunar bone matrix, which causes considerable variations in the shape and size of their lacunae. If these variations in lacunar morphology cause changes in the mechanical environment of the osteocytes, in particular local strains, they would subsequently affect bone mechanotransduction, since osteocytes are likely able to directly sense these strains. The purpose of this study is to quantify the effect of alterations in osteocyte lacunar morphology on peri-lacunar bone tissue strains. To this end, we related the actual lacunar shape in fibulae of six young-adult (5-month) and six old (23-month) mice, quantified by high-resolution micro-computed tomography, to microscopic strains, analyzed by micro-finite element modeling. We showed that peak effective strain increased by 12.6% in osteocyte cell bodies (OCYs), 9.6% in pericellular matrix (PCM), and 5.3% in extra cellular matrix (ECM) as the lacunae volume increased from 100-200 μm 3 to 500-600 μm 3 . Lacunae with a larger deviation (>8°) in orientation from the longitudinal axis of the bone are exposed to 8% higher strains in OCYs, 6.5% in PCM, 4.2% in ECM than lacunae with a deviation in orientation below 8°. Moreover, increased lacuna sphericity from 0 to 0.5 to 0.7-1 led to 25%, 23%, and 13% decrease in maximum effective strains in OCYs, PCM, and ECM, respectively. We further showed that due to the presence of smaller and more round lacunae in old mice, local bone tissue strains are on average 5% lower in the vicinity of lacunae and their osteocytes of old mice compared to young. Understanding how changes in lacunar morphology affect the micromechanical environment of osteocytes presents a first step in unraveling their potential role in impaired bone mechanoresponsiveness with e.g. aging., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Fluid flow dynamics and oxygen-concentration in 3D-printed scaffolds within perfusion bioreactors are sensitive to controllable bioreactor parameters such as inlet flow rate. Here we aimed to determine fluid flow dynamics, oxygen-concentration, and cell proliferation and distribution in 3D-printed scaffolds as a result of different inlet flow rates of perfusion bioreactors using experiments and finite element modeling. Pre-osteoblasts were treated with 1 h pulsating fluid flow with low (0.8 Pa; PFF low ) or high peak shear stress (6.5 Pa; PFF high ), and nitric oxide (NO) production was measured to validate shear stress sensitivity. Computational analysis was performed to determine fluid flow between 3D-scaffold-strands at three inlet flow rates (0.02, 0.1, 0.5 ml/min) during 5 days. MC3T3-E1 pre-osteoblast proliferation, matrix production, and oxygen-consumption in response to fluid flow in 3D-printed scaffolds inside a perfusion bioreactor were experimentally assessed. PFF high more strongly stimulated NO production by pre-osteoblasts than PFF low . 3D-simulation demonstrated that dependent on inlet flow rate, fluid velocity reached a maximum (50-1200 μm/s) between scaffold-strands, and fluid shear stress (0.5-4 mPa) and wall shear stress (0.5-20 mPa) on scaffold-strands surfaces. At all inlet flow rates, gauge fluid pressure and oxygen-concentration were similar. The simulated cell proliferation and distribution, and oxygen-concentration data were in good agreement with the experimental results. In conclusion, varying a perfusion bioreactor's inlet flow rate locally affects fluid velocity, fluid shear stress, and wall shear stress inside 3D-printed scaffolds, but not gauge fluid pressure, and oxygen-concentration, which seems crucial for optimized bone tissue engineering strategies using bioreactors, scaffolds, and cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In bone tissue engineering, prediction of forces induced to the native bone during normal functioning is important in the design, fabrication, and integration of a scaffold with the host. The aim of this study was to customize the mechanical properties of a layer-by-layer 3D-printed poly(ϵ-caprolactone) (PCL) scaffold estimated by finite element (FE) modeling in order to match the requirements of the defect, to prevent mechanical failure, and ensure optimal integration with the surrounding tissue. Forces and torques induced on the mandibular symphysis during jaw opening and closing were predicted by FE modeling. Based on the predicted forces, homogeneous-structured PCL scaffolds with 3 different void sizes (0.3, 0.6, and 0.9 mm) were designed and 3D-printed using an extrusion based 3D-bioprinter. In addition, 2 gradient-structured scaffolds were designed and 3D-printed. The first gradient scaffold contained 2 regions (0.3 mm and 0.6 mm void size in the upper and lower half, respectively), whereas the second gradient scaffold contained 3 regions (void sizes of 0.3, 0.6, and 0.9 mm in the upper, middle and lower third, respectively). Scaffolds were tested for their compressive and tensile strength in the upper and lower halves. The actual void size of the homogeneous scaffolds with designed void size of 0.3, 0.6, and 0.9 mm was 0.20, 0.59, and 0.95 mm, respectively. FE modeling showed that during opening and closing of the jaw, the highest force induced on the symphysis was a compressive force in the transverse direction. The compressive force was induced throughout the symphyseal line and reduced from top (362.5 N, compressive force) to bottom (107.5 N, tensile force) of the symphysis. Compressive and tensile strength of homogeneous scaffolds decreased by 1.4-fold to 3-fold with increasing scaffold void size. Both gradient scaffolds had higher compressive strength in the upper half (2 region-gradient scaffold: 4.9 MPa; 3 region-gradient scaffold: 4.1 MPa) compared with the lower half (2 region-gradient scaffold: 2.5 MPa; 3 region-gradient scaffold: 2.7 MPa) of the scaffold. 3D-printed PCL scaffolds had higher compressive strength in the scaffold layer-by-layer building direction compared with the side direction, and a very low tensile strength in the scaffold layer-by-layer building direction. Fluid shear stress and fluid pressure distribution in the gradient scaffolds were more homogeneous than in the 0.3 mm void size scaffold and similar to the 0.6 mm and 0.9 mm void size scaffolds. In conclusion, these data show that the mechanical properties of 3D-printed PCL scaffolds can be tailored based on the predicted forces on the mandibular symphysis. These 3D-printed PCL scaffolds had different mechanical properties in scaffold building direction compared with the side direction, which should be taken into account when placing the scaffold in the defect site. Our findings might have implications for improved performance and integration of scaffolds with native tissue., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to describe the surgical technique of immediate dental implant placement in calvarial grafts for augmentation of the severely resorbed maxilla and to assess the treatment results., Methods: In 13 patients the maxilla was augmented with calvarial bone followed by simultaneous dental implant placement (total: 68 implants). In the frontal "knife edge" region, implants were inserted in the buccal plated area. In the maxillary sinus area, implants were inserted into alveolar bone that was plated buccally or palatally through the sinus window. After 4 months, the implants were retrieved and subsequently loaded. Per-operative and post-operative variables were scored. One bone biopsy sample was taken for histological analysis., Results: The surgical procedure and wound healing was uneventful. During abutment connection after 4 months, all implants were fully osseointegrated with no signs of graft resorption. Radiographically, the mean (±SD) peri-implant bone loss after 1 year of functional loading was 0.23 ± 0.44 mm. No implants were lost. Histological examination revealed vital calvarial and maxillary bone with active remodeling., Conclusion: Immediate dental implant placement in calvarial bone grafts to rehabilitate severely resorbed maxilla is technically feasible and seems to have a high success rate., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Biocompatibility of biomedical devices can be improved by endothelialization of blood-contacting parts mimicking the vascular endothelium's function. Improved endothelialization might be obtained by using biomimetic coatings that allow local sustained release of biologically active molecules, e.g. anti-thrombotic and growth-inducing agents, from nanoliposomes. We aimed to test whether incorporation of growth-inducing nanoliposomal growth hormone (nGH) and anti-thrombotic nanoliposomal sodium nitrite (nNitrite) into collagen coating of silicone tubes enhances endothelialization by stimulating endothelial cell proliferation and inhibiting platelet adhesion. Collagen coating stably immobilized on acrylic acid-grafted silicone tubes decreased the water contact angle from 102° to 56°. Incorporation of 50 or 500nmol/ml nNitrite and 100 or 1000ng/ml nGH into collagen coating decreased the water contact angle further to 48°. After 120h incubation, 58% nitrite and 22% GH of the initial amount of sodium nitrite and GH in nanoliposomes were gradually released from the nNitrite-nGH-collagen coating. Endothelial cell number was increased after surface coating of silicone tubes with collagen by 1.6-fold, and with nNitrite-nGH-collagen conjugate by 1.8-3.9-fold after 2days. After 6days, endothelial cell confluency in the absence of surface coating was 22%, with collagen coating 74%, and with nNitrite-nGH-collagen conjugate coating 83-119%. In the absence of endothelial cells, platelet adhesion was stimulated after collagen coating by 1.3-fold, but inhibited after nNitrite-nGH-collagen conjugate coating by 1.6-3.7-fold. The release of anti-thrombotic prostaglandin I 2 from endothelial cells was stimulated after nNitrite-nGH-collagen conjugate coating by 1.7-2.2-fold compared with collagen coating. Our data shows improved endothelialization and blood compatibility using nNitrite-nGH-collagen conjugate coating on silicone tubes suggesting that these coatings are highly suitable for use in blood-contacting parts of biomedical devices., (Copyright © 2017. Published by Elsevier B.V.)

Anabolic resistance reflects the inability of skeletal muscle to maintain protein mass by appropriate stimulation of protein synthesis. We hypothesized that endoplasmic reticulum (ER) stress contributes to anabolic resistance in skeletal muscle with aging. Muscles were isolated from adult (8 mo) and old (26 mo) mice and weighed. ER stress markers in each muscle were quantified, and the anabolic response to leucine was assessed by measuring the phosphorylation state of S6K1 in soleus and EDL using an ex vivo muscle model. Aging reduced the muscle-to-body weight ratio in soleus, gastrocnemius, and plantaris, but not in EDL and tibialis anterior. Compared to adult mice, the expression of ER stress markers BiP and IRE1α was higher in EDL, and phospho-eIF2α was higher in soleus and EDL of old mice. S6K1 response to leucine was impaired in soleus, but not in EDL, suggesting that anabolic resistance contributes to soleus weight loss in old mice. Pre-incubation with ER stress inducer tunicamycin before leucine stimulation increased S6K1 phosphorylation beyond the level reached by leucine alone. Since tunicamycin did not impair leucine-induced S6K1 response, and based on the different ER stress marker regulation patterns, ER stress is probably not involved in anabolic resistance in skeletal muscle with aging., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: The gain of mineralized bone was compared between deproteinized bovine bone allograft (DBA) and biphasic calcium phosphate (BCP) for dental implant placement., Study Design: Five patients with atrophic maxillae underwent bilateral sinus elevation with DBA (Bio-Oss) and BCP (Straumann BoneCeramic). After 3 to 8 months, 32 Camlog implants were placed, and biopsies were retrieved. Bone and graft volume, degree of bone mineralization, and graft degradation gradient were determined using micro-computed tomography, and bone formation and resorption parameters were measured using histomorphometry. Implant functioning and peri-implant mucosa were evaluated up to 4 years., Results: Patients were prosthetically successfully restored. All but one of the implants survived, and peri-implant mucosa showed healthy appearance and stability. Bone volume, graft volume, degree of bone mineralization, and osteoclast and osteocyte numbers were similar, but BCP-grafted biopsies had relatively more osteoid than DBA-grafted biopsies., Conclusions: The BCP and DBA materials showed similar osteoconductive patterns and mineralized bone, although signs of more active bone formation and remodeling were observed in BCP- than in DBA-grafted biopsies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Inflammatory diseases such as rheumatoid arthritis are often accompanied by higher plasma and synovial fluid levels of interleukin-1β (IL-1β), and by increased bone resorption. Since osteocytes are known to regulate bone resorption in response to changes in mechanical stimuli, we investigated whether IL-1β affects osteocyte-modulated osteoclastogenesis in the presence or absence of mechanical loading of osteocytes. MLO-Y4 osteocytes were pre-incubated with IL-1β (0.1-1 ng/ml) for 24h. Cells were either or not subjected to mechanical loading by 1h pulsating fluid flow (PFF; 0.7 ± 0.3 Pa, 5 Hz) in the presence of IL-1β (0.1-1 ng/ml). Conditioned medium was collected after 1h PFF or static cultures. Subsequently mouse bone marrow cells were seeded on top of the IL-1β-treated osteocytes to determine osteoclastogenesis. Conditioned medium from mechanically loaded or static IL-1β-treated osteocytes was added to co-cultures of untreated osteocytes and mouse bone marrow cells. Gene expression of cysteine-rich protein 61 (CYR61/CCN1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) by osteocytes was determined immediately after PFF. Incubation of osteocytes with IL-1β, as well as conditioned medium from static IL-1β-treated osteocytes increased the formation of osteoclasts. However, conditioned medium from mechanically loaded IL-1β-treated osteocytes prevented osteoclast formation. Incubation with IL-1β upregulated RANKL and downregulated OPG gene expression by static osteocytes. PFF upregulated CYR61, RANKL, and OPG gene expression by osteocytes. Our results suggest that IL-1β increases osteocyte-modulated osteoclastogenesis, and that mechanical loading of osteocytes may abolish IL-1β-induced osteoclastogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing., (Copyright © 2011 Elsevier Inc. All rights reserved.)

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) 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)(2)D(3) affects NO production by osteoblasts in the presence or absence of mechanical stimulation. We hypothesised that 1,25(OH)(2)D(3) stimulates NO production via nuclear actions of the vitamin D receptor (VDR), which requires hours of incubation with 1,25(OH)(2)D(3) 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)(2)D(3) (10(-13)-10(-9)M), followed by 30 min pulsating fluid flow (PFF; 0.7±0.3 Pa, 5 Hz) or static culture with or without 1,25(OH)(2)D(3). NO production and NO synthase (NOS) expression were quantified. 10(-11)M 1,25(OH)(2)D(3) for 24h, but not 30 min, stimulated NO production by MC3T3-E1 osteoblasts (eightfold). 1,25(OH)(2)D(3) for 24h increased inducible-NOS gene-expression (twofold), suggesting that 1,25(OH)(2)D(3) 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)(2)D(3) for 24h, or during PFF only. Our results suggest that 1,25(OH)(2)D(3) 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)(2)D(3) may affect mechanical loading-induced NO production independent of genomic VDR action, since 1,25(OH)(2)D(3) diminished PFF-induced NO production in VDR(-/-) bone cells. In conclusion, 1,25(OH)(2)D(3) and mechanical loading interact at the level of mechanotransduction, whereby 1,25(OH)(2)D(3) seems to act independently of VDR genomic mechanism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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 ± 0.3 Pa, 5 Hz) 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.

Background Aims: Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC lineage commitment and the mechanisms activated upon Wnt5a binding are unknown. We examined whether Wnt5a induces osteogenic and/or adipogenic differentiation of hASC., Methods: hASC were incubated for 7 days with or without Wnt5a, rho-associated kinase (ROCK)-activity inhibitor Y27632 or Wnt3a. Cells were lysed for total RNA isolation, DNA content and alkaline phosphatase (ALP) activity. Mineralized nodule formation and gene expression of osteogenic markers osteocalcin and runt-related protein-2 (RUNX2), and adipogenic markers peroxisome proliferator activator receptor-γ (PPARγ) and transcription factor apetala-2 (aP2), were analyzed. hASC were incubated with Wnt5a or Wnt3a to determine activation of canonical and/or non-canonical Wnt signaling pathways, and protein kinase C activity (PKC), total ß-catenin content and gene expression of connexin 43 and cyclin D1 were quantified., Results: Wnt5a increased ALP activity and RUNX2 and osteocalcin gene expression, and down-regulated adipogenic markers through ROCK activity. Wnt5a also induced mineralized nodule formation. Wnt3a only enhanced RUNX2 and osteocalcin gene expression, and did not induce osteogenic differentiation. Wnt5a activated the non-canonical Wnt signaling pathway by increasing PKC activity, while Wnt3a mildly activated the Wnt canonical pathway by increasing total ß-catenin content and connexin 43 and cyclin D1 gene expression., Conclusions: Our data illustrate the importance of Wnt5a as a stimulator of hASC osteogenic differentiation, and show that changes in actin cytoskeleton controlled by ROCK are determinants for Wnt5a-induced osteogenic differentiation of hASC.

Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/beta-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not well understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced beta-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7+/-0.3 Pa, 5 Hz) for 30 min induced beta-catenin stabilization and activation of the Wnt/beta-catenin signaling pathway. The PFF-induced stabilization of beta-catenin and activation of the beta-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 microM), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 microM). Addition of nitric oxide synthase inhibitor L-NAME (1.0mM) also abolished PFF-induced stabilization of beta-catenin. This suggests that mechanical loading activates the beta-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of beta-catenin in mechanical adaptation of bone., (Copyright 2009 Elsevier Inc. All rights reserved.)

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.

Fluid flowing through the bone porosity might be a primary stimulus for functional adaptation of bone. Osteoblasts, and osteocytes in particular, respond to fluid flow in vitro with enhanced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release; both of these signaling molecules mediate mechanically-induced bone formation. Because the cell cytoskeleton is involved in signal transduction, we hypothesized that the pulsatile fluid flow-induced release of NO and PGE(2) in both osteoblastic and osteocytic cells involves the actin and microtubule cytoskeleton. In testing this hypothesis we found that fluid flow-induced NO response in osteoblasts was accompanied by parallel alignment of stress fibers, whereas PGE(2) response was related to fluid flow stimulation of focal adhesions formed after cytoskeletal disruption. Fluid flow-induced PGE(2) response in osteocytes was inhibited by cytoskeletal disruption, whereas in osteoblasts it was enhanced. These opposite PGE(2) responses are likely related to differences in cytoskeletal composition (osteocyte structure was more dependent on actin), but may occur via cytoskeletal modulation of shear/stretch-sensitive ion channels that are known to be dominant in osteocyte (and not osteoblast) response to mechanical loading.

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

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.