Shear loaded osteocyte-like-cells affect epithelial and mesenchymal gene expression in DU145 prostate cancer cells, while decreasing their invasion in vitro.

Once prostate cancer (PC) metastasizes towards bone the 5-year survival rates drop with 70%, but it is largely unknown why. Bone is continuously mechanically loaded, which likely modulates the paracrine signaling from osteocytes towards PC cells to affect tumor behavior. We hypothesize that shear loaded osteocytes affect PC cell proliferation, invasion and epithelial and mesenchymal-related gene and protein expression. We cultured human DU145 cells, a commonly used cell line for prostate cancer metastases, in the conditioned medium (CM) from shear loaded or unloaded human osteocyte-like-cells (OCYLCs) for 1 and 3 days and assessed their number by staining nuclei with DAPI, their invasion by performing an invasion assay, and epithelial-to-mesenchymal (EMT)-related gene and protein expression by qPCR and immunocytochemistry. CM of shear loaded OCYLCs did not affect DU145 cell number compared to CM of static cultured OCYLCs, but decreased their invasion 1.34-fold. CM of shear loaded OCYLCs enhanced expression of epithelial genes: SYND1 and CDH1 after day 1, while it also enhanced CDH1 after day 3. CM of shear loaded osteocytes enhanced mesenchymal genes: VMN, Snail and MIP2 after day 1, while it decreased expression of mesenchymal CYR61 after day 3. We conclude that CM of shear loaded OCYLCs does not affect DU145 cell proliferation, but decreases their invasion, and differentially affects their EMT-related gene expression. Identifying paracrine signals from shear loaded osteocytes that decrease PC cell invasion may provide novel leads in developing treatments for bone metastases from PC.

Fluid shear stress-induced mechanotransduction in myoblasts: Does it depend on the glycocalyx?

Muscle stem cells (MuSCs) are involved in muscle maintenance and regeneration. Mechanically loaded MuSCs within their native niche undergo tensile and shear deformations, but how MuSCs sense mechanical stimuli and translate these into biochemical signals regulating function and fate is still poorly understood. We aimed to investigate whether the glycocalyx is involved in the MuSC mechanoresponse, and whether MuSC morphology affects mechanical loading-induced pressure, shear stress, and fluid velocity distribution. FSS-induced deformation of active proliferating MuSCs (myoblasts) with intact or degraded glycocalyx was assessed by live-cell imaging. Glycocalyx-degradation did not significantly affect nitric oxide production, but reduced FSS-induced myoblast deformation and modulated gene expression. Finite-element analysis revealed that the distribution of FSS-induced pressure, shear stress, and fluid velocity on myoblasts was non-uniform, and the magnitude depended on myoblast morphology and apex-height. In conclusion, our results suggest that the glycocalyx does not play a role in NO production in myoblasts but might impact mechanotransduction and gene expression, which needs further investigation. Future studies will unravel the underlying mechanism by which the glycocalyx affects FSS-induced myoblast deformation, which might be related to increased drag forces. Moreover, MuSCs with varying apex-height experience different levels of FSS-induced pressure, shear stress, and fluid velocity, suggesting differential responsiveness to fluid shear forces.

Compressed Prostate Cancer Cells Decrease Osteoclast Activity While Enhancing Osteoblast Activity In Vitro.

Once prostate cancer cells metastasize to bone, they perceive approximately 2 kPa compression. We hypothesize that 2 kPa compression stimulates the epithelial-to-mesenchymal transition (EMT) of prostate cancer cells and alters their production of paracrine signals to affect osteoclast and osteoblast behavior. Human DU145 prostate cancer cells were subjected to 2 kPa compression for 2 days. Compression decreased expression of 2 epithelial genes, 5 out of 13 mesenchymal genes, and increased 2 mesenchymal genes by DU145 cells, as quantified by qPCR. Conditioned medium (CM) of DU145 cells was added to human monocytes that were stimulated to differentiate into osteoclasts for 21 days. CM from compressed DU145 cells decreased osteoclast resorptive activity by 38% but did not affect osteoclast size and number compared to CM from non-compressed cells. CM was also added to human adipose stromal cells, grown in osteogenic medium. CM of compressed DU145 cells increased bone nodule production (Alizarin Red) by osteoblasts from four out of six donors. Compression did not affect IL6 or TNF-α production by PC DU145 cells. Our data suggest that compression affects EMT-related gene expression in DU145 cells, and alters their production of paracrine signals to decrease osteoclast resorptive activity while increasing mineralization by osteoblasts is donor dependent. This observation gives further insight in the altered behavior of PC cells upon mechanical stimuli, which could provide novel leads for therapies, preventing bone metastases.

Myofiber stretch induces tensile and shear deformation of muscle stem cells in their native niche.

Muscle stem cells (MuSCs) are requisite for skeletal muscle regeneration and homeostasis. Proper functioning of MuSCs, including activation, proliferation, and fate decision, is determined by an orchestrated series of events and communication between MuSCs and their niche. A multitude of biochemical stimuli are known to regulate MuSC fate and function. However, in addition to biochemical factors, it is conceivable that MuSCs are subjected to mechanical forces during muscle stretch-shortening cycles because of myofascial connections between MuSCs and myofibers. MuSCs respond to mechanical forces in vitro, but it remains to be proven whether physical forces are also exerted on MuSCs in their native niche and whether they contribute to the functioning and fate of MuSCs. MuSC deformation in their native niche resulting from mechanical loading of ex vivo myofiber bundles was visualized utilizing mT/mG double-fluorescent Cre-reporter mouse and multiphoton microscopy. MuSCs were subjected to 1 h pulsating fluid shear stress (PFSS) with a peak shear stress rate of 6.5 Pa/s. After PFSS treatment, nitric oxide, messenger RNA (mRNA) expression levels of genes involved in regulation of MuSC proliferation and differentiation, ERK 1/2, p38, and AKT activation were determined. Ex vivo stretching of extensor digitorum longus and soleus myofiber bundles caused compression as well as tensile and shear deformation of MuSCs in their niche. MuSCs responded to PFSS in vitro with increased nitric oxide production and an upward trend in iNOS mRNA levels. PFSS enhanced gene expression of c-Fos, Cdk4, and IL-6, whereas expression of Wnt1, MyoD, Myog, Wnt5a, COX2, Rspo1, Vangl2, Wnt10b, and MGF remained unchanged. ERK 1/2 and p38 MAPK signaling were also upregulated after PFSS treatment. We conclude that MuSCs in their native niche are subjected to force-induced deformations due to myofiber stretch-shortening. Moreover, MuSCs are mechanoresponsive, as evidenced by PFSS-mediated expression of factors by MuSCs known to promote proliferation.

High shear stress amplitude in combination with prolonged stimulus duration determine induction of osteoclast formation by hematopoietic progenitor cells.

To date, it is unclear how fluid dynamics stimulate mechanosensory cells to induce an osteoprotective or osteodestructive response. We investigated how murine hematopoietic progenitor cells respond to 2 minutes of dynamic fluid flow stimulation with a precisely controlled sequence of fluid shear stresses. The response was quantified by measuring extracellular adenosine triphosphate (ATP), immunocytochemistry of Piezo1, and sarcoplasmic/endoplasmic Ca2+ reticulum ATPase 2 (SERCA2), and by the ability of soluble factors produced by mechanically stimulated cells to modulate osteoclast differentiation. We rejected our initial hypothesis that peak wall shear stress rate determines the response of hematopoietic progenitor cells to dynamic fluid shear stress, as it had only a minor correlation with the abovementioned parameters. Low stimulus amplitudes corresponded to activation of Piezo1, SERCA2, low concentrations of extracellular ATP, and inhibition of osteoclastogenesis and resorption area, while high amplitudes generally corresponded to osteodestructive responses. At a given amplitude (3 Pa) and waveform (square), the duration of individual stimuli (duty cycle) showed a strong correlation with the release of ATP and osteoclast number and resorption area. Collectively, our data suggest that hematopoietic progenitor cells respond in a viscoelastic manner to loading, since a combination of high shear stress amplitude and prolonged duty cycle is needed to trigger an osteodestructive response. PLAIN LANGUAGE SUMMARY: In case of painful joints or missing teeth, the current intervention is to replace them with an implant to keep a high-quality lifestyle. When exercising or chewing, the cells in the bone around the implant experience mechanical loading. This loading generally supports bone formation to strengthen the bone and prevent breaking, but can also stimulate bone loss when the mechanical loading becomes too high around orthopedic and dental implants. We still do not fully understand how cells in the bone can distinguish between mechanical loading that strengthens or weakens the bone. We cultured cells derived from the bone marrow in the laboratory to test whether the bone loss response depends on (i) how fast a mechanical load is applied (rate), (ii) how intense the mechanical load is (amplitude), or (iii) how long each individual loading stimulus is applied (duration). We mimicked mechanical loading as it occurs in the body, by applying very precisely controlled flow of fluid over the cells. We found that a mechanosensitive receptor Piezo1 was activated by a low amplitude stimulus, which usually strengthens the bone. The potential inhibitor of Piezo1, namely SERCA2, was only activated by a low amplitude stimulus. This happened regardless of the rate of application. At a constant high amplitude, a longer duration of the stimulus enhanced the bone-weakening response. Based on these results we deduce that a high loading amplitude tends to be bone weakening, and the longer this high amplitude persists, the worse it is for the bone.

The novel endolysin XZ.700 effectively treats MRSA biofilms in two biofilm models without showing toxicity on human bone cells in vitro.

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.

Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells.

Osteoblasts derived from mouse skulls have increased osteoclastogenic potential compared to long bone osteoblasts when stimulated with 1,25(OH)2 vitamin D3 (vitD3). This indicates that bone cells from specific sites can react differently to biochemical signals, e.g., during inflammation or as emitted by bioactive bone tissue-engineering constructs. Given the high turn-over of alveolar bone, we hypothesized that human alveolar bone-derived osteoblasts have an increased osteogenic and osteoclastogenic potential compared to the osteoblasts derived from long bone. The osteogenic and osteoclastogenic capacity of alveolar bone cells and long bone cells were assessed in the presence and absence of osteotropic agent vitD3. Both cell types were studied in osteogenesis experiments, using an osteogenic medium, and in osteoclastogenesis experiments by co-culturing osteoblasts with peripheral blood mononuclear cells (PBMCs). Both osteogenic and osteoclastic markers were measured. At day 0, long bones seem to have a more late-osteoblastic/preosteocyte-like phenotype compared to the alveolar bone cells as shown by slower proliferation, the higher expression of the matrix molecule Osteopontin (OPN) and the osteocyte-enriched cytoskeletal component Actin alpha 1 (ACTA1). This phenotype was maintained during the osteogenesis assays, where long bone-derived cells still expressed more OPN and ACTA1. Under co-culture conditions with PBMCs, long bone cells also had a higher Tumor necrose factor-alfa (TNF-α) expression and induced the formation of osteoclasts more than alveolar bone cells. Correspondingly, the expression of osteoclast genes dendritic cell specific transmembrane protein (DC-STAMP) and Receptor activator of nuclear factor kappa-Β ligand (RankL) was higher in long bone co-cultures. Together, our results indicate that long bone-derived osteoblasts are more active in bone-remodeling processes, especially in osteoclastogenesis, than alveolar bone-derived cells. This indicates that tissue-engineering solutions need to be specifically designed for the site of application, such as defects in long bones vs. the regeneration of alveolar bone after severe periodontitis.

Shear Stress Modulates Osteoblast Cell and Nucleus Morphology and Volume.

Mechanical loading preserves bone mass and function-yet, little is known about the cell biological basis behind this preservation. For example, cell and nucleus morphology are critically important for cell function, but how these morphological characteristics are affected by the physiological mechanical loading of bone cells is under-investigated. This study aims to determine the effects of fluid shear stress on cell and nucleus morphology and volume of osteoblasts, and how these effects relate to changes in actin cytoskeleton and focal adhesion formation. Mouse calvaria 3T3-E1 (MC3T3-E1) osteoblasts were treated with or without 1 h pulsating fluid flow (PFF). Live-cell imaging was performed every 10 min during PFF and immediately after PFF. Cytoskeletal organization and focal adhesions were visualized, and gene and protein expression quantified. Two-dimensional (2D) and three-dimensional (3D) morphometric analyses were made using MeasureStack and medical imaging interaction toolkit (MITK) software. 2D-images revealed that 1 h PFF changed cell morphology from polygonal to triangular, and nucleus morphology from round to ellipsoid. PFF also reduced cell surface area (0.3-fold), cell volume (0.3-fold), and nucleus volume (0.2-fold). During PFF, the live-cell volume gradually decreased from 6000 to 3000 µm3. After PFF, α-tubulin orientation was more disorganized, but F-actin fluorescence intensity was enhanced, particularly around the nucleus. 3D-images obtained from Z-stacks indicated that PFF increased F-actin fluorescence signal distribution around the nucleus in the XZ and YZ direction (2.3-fold). PFF increased protein expression of phospho-paxillin (2.0-fold) and integrin-α5 (2.8-fold), but did not increase mRNA expression of paxillin-a (PXNA), paxillin-b (PXNB), integrin-α5 (ITGA51), or α-tubulin protein expression. In conclusion, PFF induced substantial changes in osteoblast cytoskeleton, as well as cell and nucleus morphology and volume, which was accompanied by elevated gene and protein expression of adhesion and structural proteins. More insights into the mechanisms whereby mechanical cues drive morphological changes in bone cells, and thereby, possibly in bone cell behavior, will aid the guidance of clinical treatment, particularly in the field of orthodontics, (oral) implantology, and orthopedics.

Burst, Short, and Sustained Vitamin D3 Applications Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells.

Incorporation of 1,25(OH)2 vitamin D3 (vitD3) into tissue-engineered scaffolds could aid the healing of critical-sized bone defects. We hypothesize that shorter applications of vitD3 lead to more osteogenic differentiation of mesenchymal stem cells (MSCs) than a sustained application. To test this, release from a scaffold was mimicked by exposing MSCs to exactly controlled vitD3 regimens. Human adipose stem cells (hASCs) were seeded onto calcium phosphate particles, cultured for 20 days, and treated with 124 ng vitD3, either provided during 30 min before seeding ([200 nM]), during the first two days ([100 nM]), or during 20 days ([10 nM]). Alternatively, hASCs were treated for two days with 6.2 ng vitD3 ([10 nM]). hASCs attached to the calcium phosphate particles and were viable (~75%). Cell number was not affected by the various vitD3 applications. VitD3 (124 ng) applied over 20 days increased cellular alkaline phosphatase activity at Days 7 and 20, reduced expression of the early osteogenic marker RUNX2 at Day 20, and strongly upregulated expression of the vitD3 inactivating enzyme CYP24. VitD3 (124 ng) also reduced RUNX2 and increased CYP24 applied at [100 nM] for two days, but not at [200 nM] for 30 min. These results show that 20-day application of vitD3 has more effect on hASCs than the same total amount applied in a shorter time span.

Mechanical loading releases osteoclastogenesis-modulating factors through stimulation of the P2X7 receptor in hematopoietic progenitor cells.

Mechanical instability of bone implants stimulate osteoclast differentiation and peri-implant bone loss, leading to prosthetic loosening. It is unclear which cells at the periprosthetic interface transduce mechanical signals into a biochemical response, and subsequently facilitate bone loss. We hypothesized that mechanical overloading of hematopoietic bone marrow progenitor cells, which are located near to the inserted bone implants, stimulates the release of osteoclast-inducing soluble factors. Using a novel in vitro model to apply mechanical overloading, we found that hematopoietic progenitor cells released adenosine triphosphate (ATP) after only 2 min of mechanical loading. The released ATP interacts with its specific receptor P2X7 to stimulate the release of unknown soluble factors that inhibit (physiological loading) or promote (supraphysiological loading) the differentiation of multinucleated osteoclasts derived from bone marrow cultures. Inhibition of ATP-receptor P2X7 by Brilliant Blue G completely abolished the overloading-induced stimulation of osteoclast formation. Likewise, stimulation of P2X7 receptor on hematopoietic cells by BzATP enhanced the release of osteoclastogenesis-stimulating signaling molecules to a similar extent as supraphysiological loading. Supraphysiological loading affected neither gene expression of inflammatory markers involved in aseptic implant loosening (e.g., interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α, and PTGES2) nor expression of the osteoclast modulators receptor activator of nuclear factor κ-Β ligand and osteoprotegerin. Our findings suggest that murine hematopoietic progenitor cells are a potential key player in local mechanical loading-induced bone implant loosening via the ATP/P2X7-axis. Our approach identifies potential therapeutic targets to prevent prosthetic loosening.

IL-6 counteracts the inhibitory effect of IL-4 on osteogenic differentiation of human adipose stem cells.

Fracture repair is characterized by cytokine production and hypoxia. To better predict cytokine modulation of mesenchymal stem cell (MSC)-aided bone healing, we investigated whether interleukin 4 (IL-4), IL-6, and their combination, affect osteogenic differentiation, vascular endothelial growth factor (VEGF) production, and/or mammalian target of rapamycin complex 1 (mTORC1) activation by MSCs under normoxia or hypoxia. Human adipose stem cells (hASCs) were cultured with IL-4, IL-6, or their combination for 3 days under normoxia (20% O 2 ) or hypoxia (1% O 2 ), followed by 11 days without cytokines under normoxia or hypoxia. Hypoxia did not alter IL-4 or IL-6-modulated gene or protein expression by hASCs. IL-4 alone decreased runt-related transcription factor 2 (RUNX2) and collagen type 1 (COL1) gene expression, alkaline phosphatase (ALP) activity, and VEGF protein production by hASCs under normoxia and hypoxia, and decreased mineralization of hASCs under hypoxia. In contrast, IL-6 increased mineralization of hASCs under normoxia, and enhanced RUNX2 gene expression under normoxia and hypoxia. Neither IL-4 nor IL-6 affected phosphorylation of the mTORC1 effector protein P70S6K. IL-4 combined with IL-6 diminished the inhibitory effect of IL-4 on ALP activity, bone nodule formation, and VEGF production, and decreased RUNX2 and COL1 expression, similar to IL-4 alone, under normoxia and hypoxia. In conclusion, IL-4 alone, but not in combination with IL-6, inhibits osteogenic differentiation and angiogenic stimulation potential of hASCs under normoxia and hypoxia, likely through pathways other than mTORC1. These results indicate that cytokines may differentially affect bone healing and regeneration when applied in isolation or in combination.

Studies on Osteocytes in Their 3D Native Matrix Versus 2D In Vitro Models.

PURPOSE OF REVIEW: Osteocytes are responsible for mechanosensing and mechanotransduction in bone and play a crucial role in bone homeostasis. They are embedded in a calcified collagenous matrix and connected with each other through the lacuno-canalicular network. Due to this specific native environment, it is a challenge to isolate primary osteocytes without losing their specific characteristics in vitro. This review summarizes the commonly used and recently established models to study the function of osteocytes in vitro. RECENT FINDINGS: Osteocytes are mostly studied in monolayer culture, but recently, 3D models of osteocyte-like cells and primary osteocytes in vitro have been established as well. These models mimic the native environment of osteocytes and show superior osteocyte morphology and behavior, enabling the development of human disease models. Osteocyte-like cell lines as well as primary osteocytes isolated from bone are widely used to study the role of osteocytes in bone homeostasis. Both cells lines and primary cells are cultured in 2D-monolayer and 3D-models. The use of these models and their advantages and shortcomings are discussed in this review.

Physicochemical Niche Conditions and Mechanosensing by Osteocytes and Myocytes.

PURPOSE OF REVIEW: Bone and muscle mass increase in response to mechanical loading and biochemical cues. Bone-forming osteoblasts differentiate into early osteocytes which ultimately mature into late osteocytes encapsulated in stiff calcified matrix. Increased muscle mass originates from muscle stem cells (MuSCs) enclosed between their plasma membrane and basal lamina. Stem cell fate and function are strongly determined by physical and chemical properties of their microenvironment, i.e., the cell niche. RECENT FINDINGS: The cellular niche is a three-dimensional structure consisting of extracellular matrix components, signaling molecules, and/or other cells. Via mechanical interaction with their niche, osteocytes and MuSCs are subjected to mechanical loads causing deformations of membrane, cytoskeleton, and/or nucleus, which elicit biochemical responses and secretion of signaling molecules into the niche. The latter may modulate metabolism, morphology, and mechanosensitivity of the secreting cells, or signal to neighboring cells and cells at a distance. Little is known about how mechanical loading of bone and muscle tissue affects osteocytes and MuSCs within their niches. This review provides an overview of physicochemical niche conditions of (early) osteocytes and MuSCs and how these are sensed and determine cell fate and function. Moreover, we discuss how state-of-the-art imaging techniques may enhance our understanding of these conditions and mechanisms.

IL-1ß Damages Fibrocartilage and Upregulates MMP-13 Expression in Fibrochondrocytes in the Condyle of the Temporomandibular Joint.

The temporomandibular joint (TMJ), which differs anatomically and biochemically from hyaline cartilage-covered joints, is an under-recognized joint in arthritic disease, even though TMJ damage can have deleterious effects on physical appearance, pain and function. Here, we analyzed the effect of IL-1ß, a cytokine highly expressed in arthritic joints, on TMJ fibrocartilage-derived cells, and we investigated the modulatory effect of mechanical loading on IL-1ß-induced expression of catabolic enzymes. TMJ cartilage degradation was analyzed in 8-11-week-old mice deficient for IL-1 receptor antagonist (IL-1RA-/-) and wild-type controls. Cells were isolated from the juvenile porcine condyle, fossa, and disc, grown in agarose gels, and subjected to IL-1ß (0.1-10 ng/mL) for 6 or 24 h. Expression of catabolic enzymes (ADAMTS and MMPs) was quantified by RT-qPCR and immunohistochemistry. Porcine condylar cells were stimulated with IL-1ß for 12 h with IL-1ß, followed by 8 h of 6% dynamic mechanical (tensile) strain, and gene expression of MMPs was quantified. Early signs of condylar cartilage damage were apparent in IL-1RA-/- mice. In porcine cells, IL-1ß strongly increased expression of the aggrecanases ADAMTS4 and ADAMTS5 by fibrochondrocytes from the fossa (13-fold and 7-fold) and enhanced the number of MMP-13 protein-expressing condylar cells (8-fold). Mechanical loading significantly lowered (3-fold) IL-1ß-induced MMP-13 gene expression by condylar fibrochondrocytes. IL-1ß induces TMJ condylar cartilage damage, possibly by enhancing MMP-13 production. Mechanical loading reduces IL-1ß-induced MMP-13 gene expression, suggesting that mechanical stimuli may prevent cartilage damage of the TMJ in arthritic patients.

Comprehensive transcriptome analysis of fluid shear stress altered gene expression in renal epithelial cells.

Renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. Shear stress is altered in renal diseases caused by tubular dilation, obstruction, and hyperfiltration, which occur to compensate for lost nephrons. Fundamental in regulation of shear stress are primary cilia and other mechano-sensors, and defects in cilia formation and function have profound effects on development and physiology of kidneys and other organs. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in renal epithelial cells. Functional enrichment-analysis revealed TGF-ß, MAPK, and Wnt signaling as core signaling pathways up-regulated by shear. Inhibitors of TGF-ß and MAPK/ERK signaling modulate a wide range of mechanosensitive genes, identifying these pathways as master regulators of shear-induced gene expression. However, the main down-regulated pathway, that is, JAK/STAT, is independent of TGF-ß and MAPK/ERK. Other up-regulated cytokine pathways include FGF, HB-EGF, PDGF, and CXC. Cellular responses to shear are modified at several levels, indicated by altered expression of genes involved in cell-matrix, cytoskeleton, and glycocalyx remodeling, as well as glycolysis and cholesterol metabolism. Cilia ablation abolished shear induced expression of a subset of genes, but genes involved in TGF-ß, MAPK, and Wnt signaling were hardly affected, suggesting that other mechano-sensors play a prominent role in the shear stress response of renal epithelial cells. Modulations in signaling due to variations in fluid shear stress are relevant for renal physiology and pathology, as suggested by elevated gene expression at pathological levels of shear stress compared to physiological shear.

Mechanical Loading Differentially Affects Osteocytes in Fibulae from Lactating Mice Compared to Osteocytes in Virgin Mice: Possible Role for Lacuna Size.

Hormonal changes during lactation are associated with profound changes in bone cell biology, such as osteocytic osteolysis, resulting in larger lacunae. Larger lacuna shape theoretically enhances the transmission of mechanical signals to osteocytes. We aimed to provide experimental evidence supporting this theory by comparing the mechanoresponse of osteocytes in the bone of lactating mice, which have enlarged lacunae due to osteocytic osteolysis, with the response of osteocytes in bone from age-matched virgin mice. The osteocyte mechanoresponse was measured in excised fibulae that were cultured in hormone-free medium for 24 h and cyclically loaded for 10 min (sinusoidal compressive load, 3000 µÎµ, 5 Hz) by quantifying loading-related changes in Sost mRNA expression (qPCR) and sclerostin and ß-catenin protein expression (immunohistochemistry). Loading decreased Sost expression by ~ threefold in fibulae of lactating mice. The loading-induced decrease in sclerostin protein expression by osteocytes was larger in lactating mice (55% decrease ± 14 (± SD), n = 8) than virgin mice (33% decrease ± 15, n = 7). Mechanical loading upregulated ß-catenin expression in osteocytes in lactating mice by 3.5-fold (± 0.2, n = 6) which is significantly (p < 0.01) higher than the 1.6-fold increase in ß-catenin expression by osteocytes in fibulae from virgin mice (± 0.12, n = 4). These results suggest that osteocytes in fibulae from lactating mice with large lacunae may respond stronger to mechanical loading than those from virgin mice. This could indicate that osteocytes residing in larger lacuna show a stronger response to mechanical loading.

Effect of mechanical loading on the metabolic activity of cells in the temporomandibular joint: a systematic review.

OBJECTIVES: The purpose of this systematic review was to elucidate how different modalities and intensities of mechanical loading affect the metabolic activity of cells within the fibro-cartilage of the temporomandibular joint (TMJ). MATERIALS AND METHODS: A systematic review was conducted according to PRISMA guidelines using PubMed, Embase, and Web of Science databases. The articles were selected following a priori formulated inclusion criteria (viz., in vivo and in vitro studies, mechanical loading experiments on TMJ, and the response of the TMJ). A total of 254 records were identified. After removal of duplicates, 234 records were screened by assessing eligibility criteria for inclusion. Forty-nine articles were selected for full-text assessment. Of those, 23 were excluded because they presented high risk of bias or were reviews. Twenty-six experimental studies were included in this systematic review: 15 in vivo studies and 11 in vitro ones. CONCLUSION: The studies showed that dynamic mechanical loading is an important stimulus for mandibular growth and for the homeostasis of TMJ cartilage. When this loading is applied at a low intensity, it prevents breakdown of inflamed cartilage. Yet, frequent overloading at excessive levels induces accelerated cell death and an increased cartilage degradation. CLINICAL SIGNIFICANCE: Knowledge about the way temporomandibular joint (TMJ) fibrocartilage responds to different types and intensities of mechanical loading is important to improve existing treatment protocols of degenerative joint disease of the TMJ, and also to better understand the regenerative pathway of this particular type of cartilage.

Cyclic Tensile Strain Reduces TNF-α Induced Expression of MMP-13 by Condylar Temporomandibular Joint Cells.

To investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ-derived cells to tumor necrosis factor-α (TNF-α), and whether mechanical loading affects this response. The effect of TNF-α (0.1-10 ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT-qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12 h with TNF-α (10 ng/ml), followed by 8 h of 6% cyclic tensile strain, and gene expression of MMPs was quantified. TNF-α (10 ng/ml) reduced the expression of the matrix proteins collagen types I and II after 6 h of incubation. Aggrecan gene expression was increased in the presence of 0.1 ng/ml TNF-α. The fossa and disc cells responded to TNF-α with an increased expression of the aggrecanase ADAMTS4. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect. Cells isolated from the different cartilaginous structures reacted differently to TNF-α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF-α induced MMP-13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF-α. J. Cell. Physiol. 232: 1287-1294, 2017. © 2016 Wiley Periodicals, Inc.

Aging, Osteocytes, and Mechanotransduction.

PURPOSE OF REVIEW: The bone is able to adapt its structure to mechanical signals via the bone remodeling process governed by mechanosensitive osteocytes. With aging, an imbalance in bone remodeling results in osteoporosis. In this review, we hypothesized that changes in lacunar morphology underlie the decreased bone mechanoresponsiveness to mechanical loading with aging. RECENT FINDINGS: Several studies have reported considerable variations in the shape of osteocytes and their lacunae with aging. Since osteocytes can sense matrix strain directly via their cell bodies, the variations in osteocyte morphology may cause changes in osteocyte mechanosensitivity. As a consequence, the load-adaptive response of osteocytes may change with aging, even when mechanical loading would remain unchanged. Though extensive quantitative data is lacking, evidence exists that the osteocyte lacunae are becoming smaller and more spherical with aging. Future dedicated studies might reveal whether these changes would affect osteocyte mechanosensation and the subsequent biological response, and whether this is (one of) the pathways involved in age-related bone loss.

Diet and Exercise: a Match Made in Bone.

PURPOSE OF REVIEW: Multiple dietary components have the potential to positively affect bone mineral density in early life and reduce loss of bone mass with aging. In addition, regular weight-bearing physical activity has a strong positive effect on bone through activation of osteocyte signaling. We will explore possible synergistic effects of dietary components and mechanical stimuli for bone health by identifying dietary components that have the potential to alter the response of osteocytes to mechanical loading. RECENT FINDINGS: Several (sub)cellular aspects of osteocytes determine their signaling towards osteoblasts and osteoclasts in response to mechanical stimuli, such as the osteocyte cytoskeleton, estrogen receptor α, the vitamin D receptor, and the architecture of the lacunocanalicular system. Potential modulators of these features include 1,25-dihydroxy vitamin D3, several forms of vitamin K, and the phytoestrogen genistein. Multiple dietary components potentially affect osteocyte function and therefore may have a synergistic effect on bone health when combined with a regime of physical activity.