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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.

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. ispartof: EXPERIMENTAL CELL RESEARCH vol:417 issue:1 ispartof: location:United States status: published

During myofiber regeneration, myoblasts are continuously subjected to shear stress. It is currently not known whether shear stress affects the regenerative capacity of myoblasts when extracellular matrix (ECM) stiffness increases (e.g. upon aging). Therefore, we aimed to assess (1) whether matrix stiffness and pulsating fluid shear stress affect myoblast proliferation and/or expression of differentiation-associated genes in myoblasts, and (2) whether matrix stiffness modulates the mechanoresponse of myoblasts to pulsating fluid shear stress. Myoblasts were seeded on matrigel-coated polyacrylamide gel matrices of varying stiffness, mimicking young (“soft”, 0.5 kPa) and old ECM (“stiff”, 20 kPa), as well as on matrigel-coated glass matrices with very high stiffness (40 ϺPa), and subjected to 1 h pulsating fluid shear stress (3 Pa/s or 4 Pa/s, 1 Hz). We found enhanced proliferation of myoblasts on stiff matrices, but reduced differentiation compared to myoblasts on soft matrices. Pulsating fluid shear stress significantly upregulated gene expression of proliferation-associated genes C-fos and Il-6, as well as expression of cytoskeletal α-actin in myoblasts seeded on glass. In contrast, pulsating fluid shear stress significantly downregulated gene expression of α-actin and Myogenin in myoblasts seeded on soft matrices. In conclusion, these results suggest that age and disease-associated increased ECM stiffness may contribute to declined regenerative capacity of myoblasts, by reducing their capacity to differentiate into new muscular tissue, at least in the absence of mechanical stimulation.

Objective: To investigate the diagnosis and treatment procedure of synovial chondromatosis (SC) of the temporomandibular joint (TMJ). Methods: Clinical features, imaging features, surgical methods, and prognosis of 7 patients with SC of the TMJ were analyzed. We also reviewed and analyzed surgery-relevant literature included in the Pubmed database in the past decade using the search terms “synovial chondromatosis” and “temporomandibular joint”, and found 181 cases. Results: There was no specific difference in the symptoms of SC in the TMJ in different Milgram’s stages in our cases and the cases mentioned in the literature. The main symptoms of SC in the TMJ were pain (100%, 7/7; 64.64%, 117/181), limited mouth opening (57.14%, 4/7; 53.59%, 97/181), swelling (14.29%, 1/7; 28.18%, 51/181), crepitus (28.57%, 2/7; 19.34%, 35/181), and clicking (14.29%, 1/7; 9.94%, 18/181) in our cases and cases from literature separately. The imaging features of SC were occupying lesions (including loose bodies or masses) (71.42%, 5/7; 37.57%, 68/181), bone change in condyle or glenoid fossa (1/7, 14.29%; 34.81%, 63/181), effusion (42.86%, 3/7; 20.99%, 38/181), joint space changes (42.86%, 3/7; 11.05%, 20/181) in our cases and cases from literature separately. The surgical procedures seem to depend mainly on the involved structures and the extension of the lesion rather than the Milgram’s stage. Conclusions: The clinical features of SC in the TMJ are nonspecific and easy to be misdiagnosed. MRI is helpful in the diagnosis of SC in the TMJ. The surgical procedures mainly depend on the involved structures and the extension of the lesion.

© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.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.

Custom‐made polymethyl methacrylate (PMMA) bone cement is used to treat cranial bone defects but whether it is cytotoxic is still unsure. Possible PMMA‐induced adverse effects in vivo affect mesenchymal stem cells and osteoblasts at the implant site. We aimed to investigate whether PMMA affects osteogenic and osteoclast activation potential of human mesenchymal stem cells and/or osteoblasts. Immediately after polymerization, PMMA was added to cultured human adipose stem cells (hASCs) or human osteoblasts (hOBs). Medium lactate dehydrogenase was measured (day 1), metabolic activity, proliferation, osteogenic and osteoclast‐activation marker expression (day 1 and 7), and mineralization (day 14). PMMA did not affect lactate dehydrogenase, KI67 gene expression, or metabolic activity in hASCs and hOBs. PMMA transiently decreased DNA content in hOBs only. PMMA increased COL1 gene expression in hASCs, but decreased RUNX2 in hOBs. PMMA did not affect osteocalcin or alkaline phosphatase (ALP) expression, ALP activity, or mineralization. Only in hOBs, PMMA decreased RANKL/OPG ratio. In conclusion, PMMA is not cytotoxic and does not adversely affect the osteogenic potential of hASCs or hOBs. Moreover, PMMA does not enhance production of osteoclast factors by hASCs and hOBs in vitro. Therefore, PMMA bone cement seems highly suitable to treat patients with cranial bone defects.

Biomaterial integration into bone requires optimal surface conditions to promote osteoprogenitor behavior, which is affected by integrin-binding via arginine-glycine-aspartate (RGD). RGD-functionalized supported lipid bilayers (SLBs) might be interesting as biomaterial coating in bone regeneration, because they allow integration of proteins, for example, growth factors, cytokines, and/or antibacterial agents. Since it is unknown whether and how they affect osteoprogenitor adhesion and differentiation, the aim was to investigate adhesion, focal adhesion formation, morphology, proliferation, and osteogenic potential of pre-osteoblasts cultured on RGD-functionalized SLBs compared to unfunctionalized SLBs and poly-l-lysine (PLL). After 17 hr, pre-osteoblast density on SLBs without or with RGD was similar, but lower than on PLL. Cell surface area, elongation, and number and size of phospho-paxillin clusters were also similar. Cells on SLBs without or with RGD were smaller, more elongated, and had less and smaller phospho-paxillin clusters than on PLL. OPN expression was increased on SLBs with RGD compared to PLL. Moreover, after 1 week, COL1a1 expression was increased on SLBs without or with RGD. In conclusion, pre-osteoblast adhesion and enhanced differentiation were realized for the first time on RGD-functionalized SLBs, pointing to a new horizon in the management of bone regeneration using biomaterials. Together with SLBs nonfouling nature and the possibility of adjusting SLB fluidity and peptide content make SLBs highly promising as substrate to develop innovative biomimetic coatings for biomaterials in bone regeneration.

Standard cell cultures may not predict the proliferation and differentiation potential of human mesenchymal stromal cells (MSCs) after seeding on a scaffold and implanting this construct in a bone defect. We aimed to develop a more biologically relevant in vitro 3D-model for preclinical studies on the bone regeneration potential of MSCs. Human adipose tissue-derived mesenchymal stromal cells (hASCs; five donors) were seeded on biphasic calcium phosphate (BCP) granules and cultured under hypoxia (1% O2) for 14 days with pro-inflammatory TNFα, IL4, IL6, and IL17F (10 mg/mL each) added during the first three days, simulating the early stages of repair (bone construct model). Alternatively, hASCs were cultured on plastic, under 20% O2 and without cytokines for 14 days (standard cell culture). After two days, the bone construct model decreased total DNA (3.9-fold), COL1 (9.8-fold), and RUNX2 expression (19.6-fold) and metabolic activity (4.6-fold), but increased VEGF165 expression (38.6-fold) in hASCs compared to standard cultures. After seven days, the bone construct model decreased RUNX2 expression (64-fold) and metabolic activity (2.3-fold), but increased VEGF165 (54.5-fold) and KI67 expression (5.7-fold) in hASCs compared to standard cultures. The effect of the bone construct model on hASC proliferation and metabolic activity could be largely mimicked by culturing on BCP alone (20% O2, no cytokines). The effect of the bone construct model on VEGF165 expression could be mimicked by culturing hASCs under hypoxia alone (plastic, no cytokines). In conclusion, we developed a new, biologically relevant in vitro 3D-model to study the bone regeneration potential of MSCs. Our model is likely more suitable for the screening of novel factors to enhance bone regeneration than standard cell cultures.

Objective: To study the correlation between condylar morphology and clinical manifestations in patients with degenerative joint disease (DJD) of the temporomandibular joint (TMJ). Methods: A total of 175 joints of 131 patients with DJD were included. Data on patients’ basic information and symptoms were collected and analyzed. Condylar morphology was evaluated using cone beam computed tomography (CBCT). The correlation between the condylar morphology and clinical manifestations was analyzed. Results: The prevalence of joint noises, clicks, and crepitus was 93/175 (53%), 73/175 (42%), and 20/175 (11%), respectively. Condylar anteroposterior diameter and condylar height were correlated with pain. There was a correlation between the shape of the condyle in the sagittal plane and joint noise. Conclusion: Condylar morphology and clinical features of DJD were correlated to some extent.

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-alpha 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. Funding Agencies|MechanoCHIP

During myofiber regeneration, myoblasts are continuously subjected to shear stress. It is currently not known whether shear stress affects the regenerative capacity of myoblasts when extracellular matrix (ECM) stiffness increases (e.g. upon aging). Therefore, we aimed to assess (1) whether matrix stiffness and pulsating fluid shear stress affect myoblast proliferation and/or expression of differentiation-associated genes in myoblasts, and (2) whether matrix stiffness modulates the mechanoresponse of myoblasts to pulsating fluid shear stress. Myoblasts were seeded on matrigel-coated polyacrylamide gel matrices of varying stiffness, mimicking young ("soft", 0.5 kPa) and old ECM ("stiff", 20 kPa), as well as on matrigel-coated glass matrices with very high stiffness (40 ϺPa), and subjected to 1 h pulsating fluid shear stress (3 Pa/s or 4 Pa/s, 1 Hz). We found enhanced proliferation of myoblasts on stiff matrices, but reduced differentiation compared to myoblasts on soft matrices. Pulsating fluid shear stress significantly upregulated gene expression of proliferation-associated genes C-fos and Il-6, as well as expression of cytoskeletal α-actin in myoblasts seeded on glass. In contrast, pulsating fluid shear stress significantly downregulated gene expression of α-actin and Myogenin in myoblasts seeded on soft matrices. In conclusion, these results suggest that age and disease-associated increased ECM stiffness may contribute to declined regenerative capacity of myoblasts, by reducing their capacity to differentiate into new muscular tissue, at least in the absence of mechanical stimulation.

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.

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.Summary 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.

Bone mass increases after error‐loading, even in the absence of osteocytes. Loaded osteoblasts may produce a combination of growth factors affecting adjacent osteoblast differentiation. We hypothesized that osteoblasts respond to a single load in the short‐term (minutes) by changing F‐actin stress fiber distribution, in the intermediate‐term (hours) by signaling molecule production, and in the long‐term (days) by differentiation. Furthermore, growth factors produced during and after mechanical loading by pulsating fluid flow (PFF) will affect osteogenic differentiation. MC3T3‐E1 pre‐osteoblasts were either/not stimulated by 60 min PFF (amplitude, 1.0 Pa; frequency, 1 Hz; peak shear stress rate, 6.5 Pa/s) followed by 0–6 h, or 21/28 days of post‐incubation without PFF. Computational analysis revealed that PFF immediately changed distribution and magnitude of fluid dynamics over an adherent pre‐osteoblast inside a parallel‐plate flow chamber (immediate impact). Within 60 min, PFF increased nitric oxide production (5.3‐fold), altered actin distribution, but did not affect cell pseudopodia length and cell orientation (initial downstream impact). PFF transiently stimulated Fgf2, Runx2, Ocn, Dmp1, and Col1⍺1 gene expression between 0 and 6 h after PFF cessation. PFF did not affect alkaline phosphatase nor collagen production after 21 days, but altered mineralization after 28 days. In conclusion, a single bout of PFF with indirect associated release of biochemical factors, stimulates osteoblast differentiation in the long‐term, which may explain enhanced bone formation resulting from mechanical stimuli., Pulsating fluid flow has distinct temporal impact on pre‐osteoblast behavior and osteogenic differentiation. Initially, PFF increased nitric oxide production, followed in the short‐term by F‐actin stress fiber changes. In the long‐term, PFF did not enhance collagen production, but increased mineralization. This indicates that a single bout of mechanical loading, triggering release of soluble factors, stimulates mineralization in the long‐term.

Aging-associated muscle wasting and impaired regeneration are caused by deficiencies in muscle stem cell (MuSC) number and function. We postulated that aged MuSCs are intrinsically impaired in their responsiveness to omnipresent mechanical cues through alterations in MuSC morphology, mechanical properties, and number of integrins, culminating in impaired proliferative capacity. Here we show that aged MuSCs exhibited significantly lower growth rate and reduced integrin-α7 expression as well as lower number of phospho-paxillin clusters than young MuSCs. Moreover, aged MuSCs were less firmly attached to matrigel-coated glass substrates compared to young MuSCs, as 43% of the cells detached in response to pulsating fluid shear stress (1 Pa). YAP nuclear localization was 59% higher than in young MuSCs, yet YAP target genes Cyr61 and Ctgf were substantially downregulated. When subjected to pulsating fluid shear stress, aged MuSCs exhibited reduced upregulation of proliferation-related genes. Together these results indicate that aged MuSCs exhibit impaired mechanosensitivity and growth potential, accompanied by altered morphology and mechanical properties as well as reduced integrin-α7 expression. Aging-associated impaired muscle regenerative capacity and muscle wasting is likely due to aging-induced intrinsic MuSC alterations and dysfunctional mechanosensitivity. ispartof: AGING-US vol:14 issue:1 pages:28-53 ispartof: location:United States status: published

Mechanical loading preserves bone mass and function&mdash, 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 &micro, m3. After PFF, &alpha, 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-&alpha, 5 (2.8-fold), but did not increase mRNA expression of paxillin-a (PXNA), paxillin-b (PXNB), integrin-&alpha, 5 (ITGA51), or &alpha, 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.

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-&alpha, ) 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-&Beta, 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.

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.

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 µε, 5 Hz) by quantifying loading-related changes in Sost mRNA expression (qPCR) and sclerostin and β-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 β-catenin expression in osteocytes in lactating mice by 3.5-fold (± 0.2, n = 6) which is significantly (p

Osteocyte lacunae are small cavities within the bone matrix. Their dimensions and spatial arrangement affect bone mechanical properties. Furthermore, their size and shape affect the strain in bone tissue close to the lacunae; hence, they are supposed to affect the mechanosensory function of the osteocytes residing in the lacunae. It was the purpose of this study to quantify the morphological features of osteocyte lacunae, whether these are affected by aging and whether these vary among different anatomical location. In addition, we aimed at quantifying the vascular canals as these affect bone's microporosity too. We quantified the microporosity in the fibular midshaft of young-adult and old female C57BL/6 mice. Using micro-computed tomography (μCT), we found that advanced age was associated with a significantly decreased vascular canal number and volume density. In aged mice, the mean volume of the lacuna was significantly smaller than in young animals and they were more round. Lacuna number density close to the neutral axis of the fibula was higher in older mice than in young ones. The characterization of bone microporosity presents a first step in further unraveling their potential role in age-related reductions in bone strength.

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-β, MAPK, and Wnt signaling as core signaling pathways up-regulated by shear. Inhibitors of TGF-β 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-β 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-β, 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.

We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability-induced osteoclast differentiatio ...

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.

BackgroundMuscle 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 consisting of the host myofiber and neighbouring cells. A multitude of biochemical stimuli are known to regulate fate and function of MuSCs. However, in addition to biochemical factors, it is conceivable that MuSCs residing between basal lamina and sarcolemma of myofibers are subjected to mechanical forces during muscle stretch-shortening cycles due to myofascial connections between MuSCs and myofibers. MuSCs have been shown to respond to mechanical forcesin vitrobut 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.MethodsMuSCs deformation in their native niche resulting from mechanical loading ofex vivomyofiber bundles were visualized utilizingmT/mGdouble-fluorescent Cre-reporter mouse and multiphoton microscopy. MuSCs were subjected to 1 hour pulsating fluid shear stress with a peak shear stress rate of 8.8 Pa/s. After treatment, nitric oxide and mRNA expression levels of genes involved in regulation of MuSC proliferation and differentiation were determined.ResultsEx vivostretching 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 pulsating fluid shear stressin vitrowith increased nitric oxide production and an upward trend iniNOSmRNA levels, whilenNOSexpression was unaltered. Pulsating fluid shear stress enhanced gene expression ofc-Fos, Cdk4, andIL-6, while expression ofWnt1, MyoD, Myog, Wnt5a, COX2, Rspo1, Vangl2, Wnt10b, andMGFremained unchanged.ConclusionsWe conclude that MuSCs in their native niche are subjected to force-induced deformations due to myofiber stretch-shortening. Moreover, MuSCs are mechanoresponsive as evident by pulsating fluid shear stress-mediated expression of factors by MuSCs known to promote proliferation.

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.

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‐1β (IL‐1β), 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.

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-1&beta, a cytokine highly expressed in arthritic joints, on TMJ fibrocartilage-derived cells, and we investigated the modulatory effect of mechanical loading on IL-1&beta, induced expression of catabolic enzymes. TMJ cartilage degradation was analyzed in 8&ndash, 11-week-old mice deficient for IL-1 receptor antagonist (IL-1RA&minus, /&minus, ) and wild-type controls. Cells were isolated from the juvenile porcine condyle, fossa, and disc, grown in agarose gels, and subjected to IL-1&beta, (0.1&ndash, 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-1&beta, for 12 h with IL-1&beta, 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&minus, mice. In porcine cells, IL-1&beta, 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-1&beta, induced MMP-13 gene expression by condylar fibrochondrocytes. IL-1&beta, induces TMJ condylar cartilage damage, possibly by enhancing MMP-13 production. Mechanical loading reduces IL-1&beta, induced MMP-13 gene expression, suggesting that mechanical stimuli may prevent cartilage damage of the TMJ in arthritic patients.

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.

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. Summary: 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.

Introduction: Fibrin-matrices of different stiffness can be used for tissue engineering. The differentiation and extracellular matrix (ECM) remodeling properties of mesenchymal stem cells can be influenced by matrix stiffness. We hypothesized that stiffer fibrin matrices slow matrix degradation and favor the osteogenic differentiation of human adipose-derived stem cells (hASCs). Materials and Methods: hASCs were incorporated at different densities into soft and stiff fibrin matrices composed of 2 mg/ml fibrinogen and 0.1 or 1.0 IU/ml thrombin. The Young's moduli of the matrices were determined by nano-indentation. Fibrin degradation was determined during a 14 day culture period by ELISA. qPCR and histology were used to assess ECM remodeling and osteogenic differentiation. Results: Fibrin matrices polymerized with 1.0 IU/ml thrombin were 69% stiffer than those polymerized with 0.1 IU/ml. Stiffer matrices degraded more than soft matrices. Higher cell seeding densities increased matrix degradation. Cells in stiffer matrices produced more Alkaline Phosphatase and ECM than cells in softer matrices. RUNX-2 expression was almost ten times higher in stiff matrices than in soft matrices. Discussion: Only stiff fibrin matrices induced osteogenic differentiation of hASCs. Unexpectedly, this was accompanied by enhanced cell-mediated matrix remodeling. These results suggest that a mechanical threshold for differentiation and ECM-remodeling was reached for cells embedded in the stiff matrices.

The skeleton fulfils its mechanical functions through structural organization and material properties of individual bones. It is stated that both cortical and trabecular morphology and mass can be (re)modelled in response to changes in mechanical strains engendered by load-bearing. To address this, animal models that enable the application of specific loads to individual bones have been developed. These are useful in defining how loading modulates (re)modeling and allow examination of the mechanisms that coordinate these events. This chapter describes how to apply mechanical loading to murine bones through points of articulation, which allows changes in endosteal, periosteal as well as trabecular bone to be revealed at multiple hierarchies, by a host of methodologies, including double fluorochrome labeling and computed tomography.

The skeleton fulfils its mechanical functions through structural organization and material properties of individual bones. It is stated that both cortical and trabecular morphology and mass can be (re)modelled in response to changes in mechanical strains engendered by load-bearing. To address this, animal models that enable the application of specific loads to individual bones have been developed. These are useful in defining how loading modulates (re)modeling and allow examination of the mechanisms that coordinate these events. This chapter describes how to apply mechanical loading to murine bones through points of articulation, which allows changes in endosteal, periosteal as well as trabecular bone to be revealed at multiple hierarchies, by a host of methodologies, including double fluorochrome labeling and computed tomography.

Critical-size bone defects are a common clinical problem. The golden standard to treat these defects is autologous bone grafting. Besides the limitations of availability and co-morbidity, autografts have to be manually adapted to fit in the defect, which might result in a sub-optimal fit and impaired healing. Scaffolds with precise dimensions can be created using 3-dimensional (3D) printing, enabling the production of patient-specific, ‘tailor-made’ bone substitutes with an exact fit. Calcium phosphate (CaP) is a popular material for bone tissue engineering due to its biocompatibility, osteoconductivity, and biodegradable properties. To enhance bone formation, a bioactive 3D-printed CaP scaffold can be created by combining the printed CaP scaffold with biological components such as growth factors and cytokines, e.g., vascular endothelial growth factor (VEGF), bone morphogenetic protein-2 (BMP-2), and interleukin-6 (IL-6). However, the 3D-printing of CaP with a biological component is challenging since production techniques often use high temperatures or aggressive chemicals, which hinders/inactivates the bioactivity of the incorporated biological components. Therefore, in our laboratory, we routinely perform extrusion-based 3D-printing with a biological binder at room temperature to create porous scaffolds for bone healing. In this method paper, we describe in detail a 3D-printing procedure for CaP paste with K-carrageenan as a biological binder.

Bone remodeling can be disturbed in active rheumatoid arthritis (RA), possibly as a result of elevated levels of circulating inflammatory cytokines. Osteocyte-specific proteins and cytokines play a vital role in bone remodeling by orchestrating bone formation and/or bone resorption. Therefore, we aimed to investigate the effect of RA-serum or inflammatory cytokines on expression of human osteocyte-specific proteins and cytokines. Human trabecular bone chips were cultured with RA-serum or inflammatory cytokines for 7-days. Live-dead staining was performed to assess cell viability. Gene expression of osteocyte-specific proteins and cytokines was analyzed by qPCR. Immuno-staining was performed for osteocyte-specific markers. Approximately 60 % of the osteocytes on the bone chips were alive at day-7. Cells in or on the bone chips did express the gene for osteocyte markers SOST, FGF23, DMP1, and MEPE, and the cytokines IL-1β, IL-6, and TNFα at day 0 and 7. Active RA-serum treatment enhanced IL-1β, TNFα, SOST, and DKK1 gene expression. IL-1β treatment enhanced IL-1β, TNFα, IL-6, IL-8, FGF23, and SOST gene expression. TNFα treatment enhanced IL-1β, TNFα, IL-6, IL-8, and FGF23 gene expression. IL-8 treatment enhanced TNFα, IL-8, and FGF23 gene expression. A combination of IL-1β, IL-6, and TNFα treatment synergistically upregulated IL-1β, IL-6, and IL-8 gene expression, as well as enhanced TNFα, OPG, SOST, and FGF23, and inhibited DKK1 gene expression. In conclusion, gene expression of human osteocyte-specific proteins and cytokines was affected by RA-serum, and exogenous recombinant cytokines treatment suggesting that osteocytes could provide a new target to prevent systemic inflammation-induced bone loss in RA.

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. ispartof: Biochemical and Biophysical Research Communications vol:468 issue:4 pages:702-707 ispartof: location:United States status: published

Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1–100 ng/ml) for 0.5–6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts.

Tuzuner, Tamer/0000-0001-5817-5928 WOS:000448648700005 Objective: To compare the importance of storage time and the tooth type for isolation of dental pulp cells (DPCs) from extracted human teeth. Methods: 35 human teeth were used in this study. The teeth were stored in phosphate buffered saline (PBS) after extraction and divided into two groups randomly according to the time elapsed between extraction and isolation. In group one, the isolation was performed within 2 hours and in the other group it was performed 24 hours after extraction. Results: No significant differences between isolation time and total cell counts (p=0.483) and between isolation time and viable cells (p=0.341). No significant differences between the first molar and the premolar related cell counts and viable cells, but both teeth groups showed significant higher viability and had higher total cell amounts than third molars after isolation. Statistically significant correlations were found between age of donors and viable cells and viability after 24 hours isolation time. Conclusion: The immediate isolation of DPCs is not necessary after the tooth extraction. The tooth can be stored in PBS at room temperature up to twenty four hours after the extraction without a significant reduction in cell viability and counts. The cells obtained from younger donors might have more chance for more viability even if storage time was extended. Premolars and first molars were better donors than the third molars for DPCs isolations and the high number of success revascularization rate in premolars with necrotic immature premolars might be because of their high cell viability potentials.

During aging, skeletal muscle tissue progressively declines in mass, strength, and regenerative capacity. Decreased muscle stem cell (MuSC) number and impaired function might underlie the aging‐related muscle wasting and impaired regenerative capacity. As yet, the search for factors that regulate MuSC fate and function has revealed several biochemical factors within the MuSC niche that may be responsible for the decline in MuSC regenerative capacity. This decline cannot be explained by environmental factors solely, as the MuSC potential to regenerate muscle tissue is not reversed by changing the biochemical MuSC niche composition. Here we discuss the likeliness that during physical exercise, MuSCs within their niche are subjected to mechanical loads, in particular pressure and shear stress, as well as associated deformations. We postulate that these physical cues are involved in the activation and differentiation of MuSCs as these cells contain several transmembrane sensor proteins that have been shown to be mechanosensitive in other cell types, that is, endothelial cells and osteoprogenitors. We will specifically address age‐related changes in mechanosensing in MuSCs and their niche. Insight in the physical cues applied to the MuSCs in vivo, and how these cues affect MuSC fate and function, helps to develop new therapeutic interventions to counterbalance age‐related muscle loss. This requires an approach combining two‐ and three‐dimensional live cell imaging of MuSCs within contracting muscle tissue, mathematical finite element modeling, and cell biology. © 2017 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:632–641, 2018.

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.

Aging reduces bone mass as well as the anabolic response of bone to mechanical stimuli, resulting in osteopenia. Endoplasmic reticulum (ER) stress impairs the response of myogenic cells to anabolic stimuli, and is involved in sarcopenia, but whether ER stress also contributes to osteopenia is unknown. Therefore, we tested whether ER stress exists in bones of aged mice, and whether this impairs the osteocyte response to mechanical stimulation. Primary osteocytes were obtained from long bones of adult (8 months) and old (24-26 months) mice, treated with or without the pharmacological ER stress inducer tunicamycin, and either or not subjected to mechanical loading by pulsating fluid flow (PFF). The osteocyte response to PFF was assessed by measuring cyclooxygenase-2 (Cox-2) mRNA levels and nitric oxide (NO) production. mRNA levels of ER stress markers were higher in old versus adult osteocytes (+40% for activating transcription factor-4, +120% for C/EBP homologous protein, and +120% for spliced X-box binding protein-1, p < 0.05). The Cox-2 response to PFF was fourfold decreased in cells from old bones (p < 0.001), while tunicamycin decreased PFF-induced Cox-2 expression by threefold in cells from adult bones (p < 0.01). PFF increased NO production by 50% at 60 min in osteocytes from old versus adult bones (p < 0.01). In conclusion, our data indicate that the expression of several ER stress markers was higher in osteocytes from bones of old compared to adult mice. Since ER stress altered the response of osteocytes to mechanical loading, it could be a novel factor contributing to osteopenia. ispartof: Calcified Tissue International vol:96 issue:2 pages:123-128 ispartof: location:United States status: published

Multiple factors contribute to bone loss in inflammatory diseases such as rheumatoid arthritis (RA), but circulating inflammatory factors and immobilization play a crucial role. Mechanical loading prevents bone loss in the general population, but the effects of mechanical loading in patients with RA are less clear. Therefore, we aimed to investigate whether mechanical stimuli reverse the stimulatory effect of RA serum on osteocyte-to-osteoclast communication. Human primary osteocytes were pretreated with 10 % RA serum or healthy control serum for 7 days, followed by 1 h ± mechanical loading by pulsating fluid flow (PFF). Nitric oxide (NO) and prostaglandin E2 were measured in the medium. Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), interleukin-6 (IL-6), cyclooxygenase-2 (COX2), matrix-extracellular phosphoglycoprotein (MEPE), cysteine-rich protein 61 (CYR61), and SOST gene expression was quantified by qPCR. Osteoclast precursors were cultured with PFF-conditioned medium (PFF-CM) or static-conditioned medium (stat-CM), and osteoclast formation was assessed. RA serum alone did not affect IL-6, CYR61, COX2, MEPE, or SOST gene expression in osteocytes. However, RA serum enhanced the RANKL/OPG expression ratio by 3.4-fold, while PFF nullified this effect. PFF enhanced NO production to the same extent in control serum (2.6-3.5-fold) and RA serum-pretreated (2.7-3.6-fold) osteocytes. Stat-CM from RA serum-pretreated osteocytes enhanced osteoclastogenesis compared with stat-CM from control serum-pretreated osteocytes, while PFF nullified this effect. In conclusion, RA serum, containing inflammatory factors, did not alter the intrinsic capacity of osteocytes to sense mechanical stimuli, but upregulated osteocyte-to-osteoclast communication. Mechanical loading nullified this upregulation, suggesting that mechanical stimuli could contribute to the prevention of osteoporosis in inflammatory disease. ispartof: Calcified Tissue International vol:97 issue:2 pages:169-178 ispartof: location:United States status: published

Insulin-like growth factor 1 (IGF-1) and interleukin 6 (IL-6) play an important role in the adaptation of both muscle and bone to mechanical stimuli. Here, we provide an overview of the functions of IL-6 and IGF-1 in bone and muscle metabolism, and the intracellular signaling pathways that are well known to mediate these functions. In particular, we discuss the Akt/mammalian target of rapamycin (mTOR) pathway which in skeletal muscle is known for its key role in regulating the rate of mRNA translation (protein synthesis). Since the role of the mTOR pathway in bone is explored to a much lesser extent, we discuss what is known about this pathway in bone and the potential role of this pathway in bone remodeling. We will also discuss the possible ways of influencing IGF-1 or IL-6 signaling by osteocytes and the clinical implications of pharmacological or nutritional modulation of the Akt/mTOR pathway.

During aging, skeletal muscle tissue progressively declines in mass, strength, and regenerative capacity. Decreased muscle stem cell (MuSC) number and impaired function might underlie the aging-related muscle wasting and impaired regenerative capacity. As yet, the search for factors that regulate MuSC fate and function has revealed several biochemical factors within the MuSC niche that may be responsible for the decline in MuSC regenerative capacity. This decline cannot be explained by environmental factors solely, as the MuSC potential to regenerate muscle tissue is not reversed by changing the biochemical MuSC niche composition. Here we discuss the likeliness that during physical exercise, MuSCs within their niche are subjected to mechanical loads, in particular pressure and shear stress, as well as associated deformations. We postulate that these physical cues are involved in the activation and differentiation of MuSCs as these cells contain several transmembrane sensor proteins that have been shown to be mechanosensitive in other cell types, i.e. endothelial cells and osteoprogenitors. We will specifically address age-related changes in mechanosensing in MuSCs and their niche. Insight in the physical cues applied to the MuSCs in vivo, and how these cues affect MuSC fate and function, helps to develop new therapeutic interventions to counterbalance age-related muscle loss. This requires an approach combining two- and three-dimensional live cell imaging of MuSCs within contracting muscle tissue, mathematical finite element modeling, and cell biology. This article is protected by copyright. All rights reserved

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. ispartof: Current Osteoporosis Reports vol:15 issue:5 pages:401-411 ispartof: location:United States status: published

Bone's microporosity plays important roles in bone biology and bone mechanical quality. In this study, we explored the accuracy and reproducibility of nondestructive desktop μCT for 3D visualization and subsequent morphometric analysis of mouse cortical bone microporosity including the vascular canal network and osteocyte lacunae. The accuracy of measurements was evaluated in five murine fibula using confocal laser scanning microscopy (CLSM) in conjunction with Fluorescein isothiocyanate (FITC) staining as the reference method. The reproducibility of μCT-derived cortical bone microstructural indices was examined in 10 fibulae of C57Bl/6J male mice at a nominal resolution of 700 nanometer. Three repeated measurements were made on different days. An excellent correlation between μCT and CLSM was observed for both mean lacuna volume (r = 0.98, p = 0.002) and for mean lacuna orientation (r = 0.93, p = 0.02). Whereas the two techniques showed no significant differences for these parameters, the mean lacuna sphericity acquired from μCT was significantly higher than CLSM (p = 0.01). Reproducibility was high, with precision errors (PE) of 1.57-4.69% for lacuna parameters, and of 1.01-9.45% for vascular canal parameters. Intraclass correlation coefficient (ICC) showed a high reliability of the measurements, ranging from 0.998-1.000 for cortical parameters, 0.973-0.999 for vascular canal parameters and 0.755-0.991 for lacuna parameters. In conclusion, desktop μCT is a valuable tool to quantify the 3D characteristics of bone vascular canals as well as lacunae which can be applied to intact murine bones with high accuracy and reproducibility. Thus, μCT might be an important tool to improve our understanding of the physiological and biomechanical significance of these cannular and lacunar structure in cortical bone. ispartof: PLoS One vol:12 issue:8 ispartof: location:United States status: published

We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability-induced osteoclast differentiation and peri-implant bone loss. MLO-Y4-osteocytes were mechanically stimulated for 1 h by fluid shear stress using regimes simulating: (i) supraphysiological loading in the peri-prosthetic interface (2.9 ± 2.9 Pa, 1 Hz, square wave); (ii) physiologic loading in the cortical bone (0.7 ± 0.7 Pa, 5 Hz, sinusoidal wave); and (iii) stress shielding. Cellular morphological parameters, membrane-bound RANKL expression, gene expression influencing osteoclast differentiation, nitric oxide release and caspase 3/7-activity were determined. Either Mouse bone marrow cells were cultured on top of loaded osteocytes or osteocyte-conditioned medium was added to bone marrow cells. Osteoclast differentiation was assessed after 6 days. We found that osteocytes subjected to supraphysiological loading showed similar morphology and caspase 3/7-activity compared to simulated physiological loading or stress shielding. Supraphysiological stimulation of osteocytes enhanced osteoclast differentiation by 1.9-fold compared to physiological loading when cell-to-cell contact was permitted. In addition, it enhanced the number of osteoclasts using conditioned medium by 1.7-fold, membrane-bound RANKL by 3.3-fold, and nitric oxide production by 3.2-fold. The stimulatory effect of supraphysiological loading on membrane-bound RANKL and nitric oxide production was higher than that achieved by stress shielding. In conclusion, the in vitro model developed recapitulated the catabolic biological situation in the peri-prosthetic interface during instability that is associated with osteoclast differentiation and enhanced RANKL expression. The model thus provides a platform for pre-clinical testing of pharmacological interventions with potential to stop instability-induced bone implant loosening. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1425-1434, 2018.

Osteoclasts strongly contribute to proper bone quality and function. Their actions are largely coordinated by osteocytes, i.e., osteocytes devoid of mechanical stimuli produce a multitude of membrane bound and soluble factors that stimulate the formation of osteoclasts. Among these are inflammatory factors, the most well known being receptor activator of nuclear factor κB ligand, matricellular proteins, and growth factors/morphogens such as WNTs. The intimate relationship between osteocytes and osteoclasts suggests that a better knowledge of the factors produced by osteocytes could lead to a better understanding of osteoclast formation and activity.

Skeletal muscle fibers have the ability to increase their size in response to a mechanical overload. Finite element modeling data suggest that mechanically loaded muscles in vivo may experience not only tensile strain but also shear stress. However, whether shear stress affects biological pathways involved in muscle fiber size adaptation in response to mechanical loading is unknown. Therefore, our aim was twofold: (1) to determine whether shear stress affects growth factor expression and nitric oxide (NO) production by myotubes, and (2) to explore the mechanism by which shear stress may affect myotubes in vitro. C2C12 myotubes were subjected to a laminar pulsating fluid flow (PFF; mean shear stress 0.4, 0.7 or 1.4 Pa, 1 Hz) or subjected to uni-axial cyclic strain (CS; 15 % strain, 1 Hz) for 1 h. NO production during 1-h PFF or CS treatment was quantified using Griess reagent. The glycocalyx was degraded using hyaluronidase, and stretch-activated ion channels (SACs) were blocked using GdCl3. Gene expression was analyzed immediately after 1-h PFF (1.4 Pa, 1 Hz) and at 6 h post-PFF treatment. PFF increased IGF-I Ea, MGF, VEGF, IL-6, and COX-2 mRNA, but decreased myostatin mRNA expression. Shear stress enhanced NO production in a dose-dependent manner, while CS induced no quantifiable increase in NO production. Glycocalyx degradation and blocking of SACs ablated the shear stress-stimulated NO production. In conclusion, shear stress activates signaling pathways involved in muscle fiber size adaptation in myotubes, likely via membrane-bound mechanoreceptors. These results suggest that shear stress exerted on myofiber extracellular matrix plays an important role in mechanotransduction in muscle.

Introduction: The periodontal ligament (PDL) connects the tooth to the alveolar bone. For PDL regeneration after tissue damage, we propose human adipose-derived stem cells (hASCs) embedded in fibrin. We showed previously that hASCs in fibrin extensively produce collagen, but in a non-functional, random orientation. Also, hASCs produce undesirable factors associated with matrix calcification. We hypothesize that with dynamic strain the matrix will align, and the calcification potential of hASCs reduces. Materials and Methods: Starting at day 0 (immediate) or day 4 (delayed), hASCs-seeded fibrin matrices were statically or dynamically strained for 3 days. Fiber and cell alignment, ECM production, and calcification-associated gene-expression were assessed. Results: Cell and fibrin fiber orientation was random in non-strained fibrin matrices. Regardless of straining type and onset, cells and fibers aligned in the strain direction. Immediate static strain decreased ALP, RUNX-2, type III collagen and elastin expression. Immediate dynamic strain decreased RUNX-2 and osteonectin expression. Delayed strain hardly affected gene expression. Discussion: Provided that dynamic loading commences immediately, expression of calcification-associated genes by hASCs reduces, while matrix and cells align without compromising their regenerative capacity. These results combined confirm the potential for hASC-seeded fibrin matrices for PDL regeneration

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 −/− cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling. Electronic supplementary material The online version of this article (doi:10.1007/s00018-017-2460-x) contains supplementary material, which is available to authorized users.