Microfluidics-Based Technologies for the Assessment of Castration-Resistant Prostate Cancer.

Castration-resistant prostate cancer remains a significant clinical challenge, wherein patients display no response to existing hormone therapies. The standard of care often includes aggressive treatment options using chemotherapy, radiation therapy and various drugs to curb the growth of additional metastases. As such, there is a dire need for the development of innovative technologies for both its diagnosis and its management. Traditionally, scientific exploration of prostate cancer and its treatment options has been heavily reliant on animal models and two-dimensional (2D) in vitro technologies. However, both laboratory tools often fail to recapitulate the dynamic tumor microenvironment, which can lead to discrepancies in drug efficacy and side effects in a clinical setting. In light of the limitations of traditional animal models and 2D in vitro technologies, the emergence of microfluidics as a tool for prostate cancer research shows tremendous promise. Namely, microfluidics-based technologies have emerged as powerful tools for assessing prostate cancer cells, isolating circulating tumor cells, and examining their behaviour using tumor-on-a-chip models. As such, this review aims to highlight recent advancements in microfluidics-based technologies for the assessment of castration-resistant prostate cancer and its potential to advance current understanding and to improve therapeutic outcomes.

Investigating the effects of Ceylon cinnamon water extract on HepG2 cells for Type 2 diabetes therapy.

Cinnamon and its extracts have been used as herbal remedies for many ailments, including for reducing insulin resistance and diabetes complications. Type 2 diabetes mellitus (T2DM) is a rapidly growing health concern around the world. Although many drugs are available for T2DM treatment, side effects and costs can be considerable, and there is increasing interest in natural products for managing chronic health conditions. Cinnamon may decrease the expression of genes associated with T2DM risk. The purpose of this study was to evaluate the effects of cinnamon water extract (CWE) compared with metformin on T2DM-related gene expression. HepG2 human hepatoma cells, widely used in drug metabolism and hepatotoxicity studies, were treated with different concentrations of metformin or CWE for 24 or 48 h. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and glucose uptake was compared in untreated and CWE- or metformin-treated cells under high-glucose conditions. Finally, total RNA was extracted and analyzed by RNA sequencing (RNA-seq), and bioinformatics analyses were performed to compare the transcriptional effects of CWE and metformin. We found cell viability was better in cells treated with CWE than in metformin-treated cells, demonstrating that CWE was not toxic at tested doses. CWE significantly increased glucose uptake in HepG2 cells, to the same degree as metformin (1.4-fold). RNA-seq data revealed CWE and metformin both induced significantly increased (1.3- to 1.4-fold) glucose uptake gene expression compared with untreated controls. Transcriptional differences between CWE and metformin were not significant. The effects of 0.125 mg mL-1 CWE on gene expression were comparable to 1.5 mg mL-1 (9.5 mM) metformin. In addition, gene expression at 0.125 mg mL-1 CWE was comparable to 1.5 mg mL-1 (9.5 mM) metformin. Our results reveal that CWE's effects on cell viability, glucose uptake, and gene expression in HepG2 cells are comparable to those of metformin, suggesting CWE may be an effective dietary supplement for mitigating T2DM-related metabolic dysfunction.

Investigation of calcium variations in single cells and the impact of Yoda1 on osteocytes by ICP-OES.

BACKGROUND: Detection of elements in individual cells by inductively coupled plasma (ICP) spectrometry has recently attracted significant interest in biological research, due to the unique ability of ICP spectrometry for trace element analysis. However, performing single-cell analysis using ICP optical emission spectrometry (ICP-OES) remains a challenge due to the small size and discrete nature of cells. This is while ICP-OES can serve as a cost-effective and label-free method for this purpose. Therefore, it is necessary to improve the current ICP-OES technique to facilitate the detection of elements in single cells, thereby unlocking novel applications. RESULTS: A new conical ICP torch, which has been illustrated to offer better analytical performance than the conventional ones, was applied to achieve the detection of calcium in single micro-sized cells. A new heated chamber was designed and coupled with a high-efficiency nebulizer as the sample introduction system. For the detection of single SiO2 particles, the number of particle events obtained by the new sample introduction system was found to be up to 9 times higher than that of the conventional system without sacrificing the signal intensity. Subsequently, calcium in human breast cancer cells (MDA-MB-231), mice breast cancer cells (Py8119), and mice osteocytes (MLO-Y4) was successfully detected using the new ICP-OES system. The cell detection efficiency turned out to be around 2%-3% which is much higher than that the reported values in previous single-cell ICP-OES research. Finally, as a new application, the effect of Yoda1, a recently identified activator of Piezo1 calcium channel, on osteocytes was investigated. The calcium content in Yoda1-treated MLO-Y4 cells was seen increase by 36% compared to the control sample. SIGNIFICANCE: This research reveals the capability of ICP-OES in single-cell analysis for micro-sized cells which was made possible by the new conical ICP torch and the new sample introduction system. The ability to detect calcium in single mammalian cells enables the first ever application of this technique to assess the impact of the Yoda1 activator on the calcium level in osteocytes.

Reduction of breast cancer extravasation via vibration activated osteocyte regulation.

Physical exercise benefits breast cancer patients by reducing cancer progression and promoting bone health. However, intense exercise is physically challenging for bedridden, disabled, or aged patients. As an exercise surrogate, low-magnitude (<1 g) high-frequency (>30 Hz) (LMHF) vibration has gained growing interest in recent years, but its effects on bone metastasis remain unknown. We used a microfluidic co-culture platform that mimics bone-cancer environments to study the impact of vibration on breast cancer extravasation. LMHF vibration activated osteocytes, the primary mechanosensing cells in bones, which reduced cancer extravasation by 43%. We further studied the vibration mechanism by demonstrating the important role of the Piezo1 ion channel in osteocyte mechanotransduction. Chemical activation of Piezo1 enhanced osteocyte inhibition of cancer extravasation under vibration at the early time point. These data indicated that LMHF vibration could inhibit cancer extravasation, suggesting that vibration may suppress bone metastasis in breast cancer patients.

Microfluidic Co-culture Platforms for Studying Osteocyte Regulation of Other Cell Types under Dynamic Mechanical Stimulation.

PURPOSE OF REVIEW: Osteocytes are the most abundant cell type in bone. These unique cells act primarily as mechanosensors and play crucial roles in the functional adaptation of bone tissue. This review aims to summarize the recent microfluidic studies on mechanically stimulated osteocytes in regulating other cell types. RECENT FINDINGS: Microfluidics is a powerful technology that has been widely employed in recent years. With the advantages of microfluidic platforms, researchers can mimic multicellular environments and integrate dynamic systems to study osteocyte regulation under mechanical stimulation. Microfluidic platforms have been developed to investigate mechanically stimulated osteocytes in the direct regulation of multiple cell types, including osteoclasts, osteoblasts, and cancer cells, and in the indirect regulation of cancer cells via endothelial cells. Overall, these microfluidic studies foster the development of treatment approaches targeting osteocytes under mechanical stimulation.

Yoda1 Enhanced Low-Magnitude High-Frequency Vibration on Osteocytes in Regulation of MDA-MB-231 Breast Cancer Cell Migration.

Low-magnitude (≤1 g) high-frequency (≥30 Hz) (LMHF) vibration has been shown to enhance bone mineral density. However, its regulation in breast cancer bone metastasis remains controversial for breast cancer patients and elder populations. Yoda1, an activator of the mechanosensitive Piezo1 channel, could potentially intensify the effect of LMHF vibration by enhancing the mechanoresponse of osteocytes, the major mechanosensory bone cells with high expression of Piezo1. In this study, we treated osteocytes with mono- (Yoda1 only or vibration only) or combined treatment (Yoda1 and LMHF vibration) and examined the further regulation of osteoclasts and breast cancer cells through the conditioned medium. Moreover, we studied the effects of combined treatment on breast cancer cells in regulation of osteocytes. Combined treatment on osteocytes showed beneficial effects, including increasing the nuclear translocation of Yes-associated protein (YAP) in osteocytes (488.0%, p < 0.0001), suppressing osteoclastogenesis (34.3%, p = 0.004), and further reducing migration of MDA-MB-231 (15.1%, p = 0.02) but not Py8119 breast cancer cells (4.2%, p = 0.66). Finally, MDA-MB-231 breast cancer cells subjected to the combined treatment decreased the percentage of apoptotic osteocytes (34.5%, p = 0.04) but did not affect the intracellular calcium influx. This study showed the potential of stimulating Piezo1 in enhancing the mechanoresponse of osteocytes to LMHF vibration and further suppressing breast cancer migration via osteoclasts.

A cannabidiol-containing alginate based hydrogel as novel multifunctional wound dressing for promoting wound healing.

In addition to preventing infection and promoting angiogenesis, novel hydrogel dressings are highly expected to possess the potential to scavenge reactive oxygen species (ROS) and reduce inflammatory responses during the wound healing process. In this study, we designed and fabricated a hydrogel dressing (CBD/Alg@Zn) containing cannabidiol (CBD) based on the ion crosslinked interaction between Zn2+ ions and the alginate polymer (Alg). The as-fabricated hydrogel exhibited a suitable swelling ratio, sufficient thermal stability, and stable rheological property. In vitro biological activity experiments indicated that the hydrogel has good biocompatibility, antibacterial activity, and angiogenesis properties. Moreover, it could significantly scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals and reduce the inflammatory response. In vivo studies revealed that the CBD/Alg@Zn hydrogel significantly facilitated the wound healing process by controlling the inflammatory infiltration, promoting collagen deposition and the granulation tissue, and benefiting the formation of blood vessels. We, therefore, suggested that CBD/Alg@Zn hydrogel should be a potential candidate material for wound dressing and skin tissue engineering.

Moderate tibial loading and treadmill running, but not overloading, protect adult murine bone from destruction by metastasized breast cancer.

Osteolytic bone lesions, which develop in many metastatic breast cancer patients, impair bone integrity and lead to adverse skeletal related events that are difficult to treat and sometimes fatal. Moderate mechanical loading has been shown to suppress osteolysis in young mice with breast cancer. In this study, we aimed to investigate the dose-dependent effects of mechanical loading on protecting the integrity of adult skeletons with breast cancer. Localized tibial loading and aerobic treadmill running with three levels of varying intensity were tested in a syngeneic mammary tumor bone metastasis model. Adult C57BL/6J female mice (14-week-old, N = 88 mice) received intra-tibial injections of Py8119 triple-negative murine breast cancer cells or PBS and underwent 4 to 5 weeks of exercise or acted as sedentary/non-loaded controls. The bone structure was monitored longitudinally with weekly in vivo micro-computed tomography imaging, while the cellular responses in bone and marrow were examined using immunohistochemistry. Moderate treadmill running (16 m/min, 50 min/day, 5 days/week, and 5 weeks) and tibial loading (4.5 N, 630 µÎµ, 4 Hz, 300 cycles/day, 5 days/week, and 4 weeks) suppressed tumor-induced bone destruction, as evaluated by full-thickness perforation of tibial cortex and the volume of osteolytic lesions in the cortex. In contrast, tibial loading at higher magnitude (8 N, 1100 µÎµ) induced woven bone and accelerated bone destruction, compared with the non-loaded controls. The three exercise regimens differentially affected osteocyte apoptosis, osteocyte hypoxia, osteoclast activity, bone marrow vasculature, and tumor proliferation. In conclusion, the relationship between exercise intensity and the risk of breast cancer-induced osteolysis was found to follow a J-shaped curve in a preclinical model, suggesting the need to optimize exercise parameters in order to harness the skeletal benefits of exercise in metastatic breast cancers.

The migration of metastatic breast cancer cells is regulated by matrix stiffness via YAP signalling.

Matrix stiffness is a driver of breast cancer progression and mechanosensitive transcriptional activator YAP plays an important role in this process. However, the interplay between breast cancer and matrix stiffness, and the significance of this interplay remained largely unknown. Here, we showed an increase in YAP nuclear localization and a higher proliferation rate in both highly metastatic MDA-MB-231 cells and the non-metastatic counterpart MCF-7 cells when they were exposed to the stiff matrix. However, in response to the stiff matrix highly metastatic MDA-MB-231 cells instead of MCF-7 cells exhibited upregulated mobility, which was shown to be YAP-dependent. Consistently, MDA-MB-231 cells exhibited different focal adhesion dynamics from MCF-7 cells in response to matrix stiffness. These results suggested a YAP-dependent mechanism through which matrix stiffness regulates the migratory potential of metastatic breast cancer cells.

Local stimulation of osteocytes using a magnetically actuated oscillating beam.

Mechanical loading on bone tissue is an important physiological stimulus that plays a key role in bone growth, fracture repair, and treatment of bone diseases. Osteocytes (bone cells embedded in bone matrix) are well accepted as the sensor cells to mechanical loading and play a critical role in regulating the bone structure in response to mechanical loading. To understand the response of osteocytes to differential mechanical stimulation in physiologically relevant arrangements, there is a need for a platform which can locally stimulate bone cells with different levels of fluid shear stress. In this study, we developed a device aiming to achieve non-contact local mechanical stimulation of osteocytes with a magnetically actuated beam that generates the fluid shear stresses encountered in vivo. The stimulating beam was made from a composite of magnetic powder and polymer, where a magnetic field was used to precisely oscillate the beam in the horizontal plane. The beam is placed above a cell-seeded surface with an estimated gap height of 5 µm. Finite element simulations were performed to quantify the shear stress values and to generate a shear stress map in the region of interest. Osteocytes were seeded on the device and were stimulated while their intracellular calcium responses were quantified and correlated with their position and local shear stress value. We observed that cells closer to the oscillating beam respond earlier compared to cells further away from the local shear stress gradient generated by the oscillating beam. We have demonstrated the capability of our device to mimic the propagation of calcium signalling to osteocytes outside of the stimulatory region. This device will allow for future studies of osteocyte network signalling with a physiologically accurate localized shear stress gradient.

Magnetic Measurement and Stimulation of Cellular and Intracellular Structures.

From single-pole magnetic tweezers to robotic magnetic-field generation systems, the development of magnetic micromanipulation systems, using electromagnets or permanent magnets, has enabled a multitude of applications for cellular and intracellular measurement and stimulation. Controlled by different configurations of magnetic-field generation systems, magnetic particles have been actuated by an external magnetic field to exert forces/torques and perform mechanical measurements on the cell membrane, cytoplasm, cytoskeleton, nucleus, intracellular motors, etc. The particles have also been controlled to generate aggregations to trigger cell signaling pathways and produce heat to cause cancer cell apoptosis for hyperthermia treatment. Magnetic micromanipulation has become an important tool in the repertoire of toolsets for cell measurement and stimulation and will continue to be used widely for further explorations of cellular/intracellular structures and their functions. Existing review papers in the literature focus on fabrication and position control of magnetic particles/structures (often termed micronanorobots) and the synthesis and functionalization of magnetic particles. Differently, this paper reviews the principles and systems of magnetic micromanipulation specifically for cellular and intracellular measurement and stimulation. Discoveries enabled by magnetic measurement and stimulation of cellular and intracellular structures are also summarized. This paper ends with discussions on future opportunities and challenges of magnetic micromanipulation in the exploration of cellular biophysics, mechanotransduction, and disease therapeutics.

Novel in vitro microfluidic platform for osteocyte mechanotransduction studies.

Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-µm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.

Mechanical segregation and capturing of clonal circulating plasma cells in multiple myeloma using micropillar-integrated microfluidic device.

Multiple myeloma (MM), the disorder of plasma cells, is the second most common type of hematological cancer and is responsible for approximately 20% of deaths from hematological malignancies. The current gold standard for MM diagnosis includes invasive bone marrow aspiration. However, it lacks the sensitivity to detect minimal residual disease, and the nonuniform distribution of clonal plasma cells (CPCs) within bone marrow also often results in inaccurate reporting. Serum and urine assessment of monoclonal proteins, such as Kappa light chains, is another commonly used approach for MM diagnosis. Although it is noninvasive, the level of paraprotein elevation is still too low for detecting minimal residual disease and nonsecretive MM. Circulating CPCs (cCPCs) have been reported to be present in the peripheral blood of MM patients, and high levels of cCPCs were shown to correlate with poor survival. This suggests a potential noninvasive approach for MM disease progress monitoring and prognosis. In this study, we developed a mechanical property-based microfluidic platform to capture cCPCs. Using human myeloma cancer cell lines spiked in healthy donor blood, the microfluidic platform demonstrates high enrichment ratio (>500) and sufficient capture efficiency (40%-55%). Patient samples were also assessed to investigate the diagnostic potential of cCPCs for MM by correlating with the levels of Kappa light chains in patients.

Microfluidic platform for studying osteocyte mechanoregulation of breast cancer bone metastasis.

Bone metastasis is a common, yet serious, complication of breast cancer. Breast cancer cells that extravasate from blood vessels to the bone devastate bone quality by interacting with bone cells and disrupting the bone remodeling balance. Although exercise is often suggested as a cancer intervention strategy and mechanical loading during exercise is known to regulate bone remodeling, its role in preventing bone metastasis remains unknown. We developed a novel in vitro microfluidic tissue model to investigate the role of osteocytes in the mechanical regulation of breast cancer bone metastasis. Metastatic MDA-MB-231 breast cancer cells were cultured inside a 3D microfluidic lumen lined with human umbilical vein endothelial cells (HUVECs), which is adjacent to a channel seeded with osteocyte-like MLO-Y4 cells. Physiologically relevant oscillatory fluid flow (OFF) (1 Pa, 1 Hz) was applied to mechanically stimulate the osteocytes. Hydrogel-filled side channels in-between the two channels allowed real-time, bi-directional cellular signaling and cancer cell extravasation over 3 days. The applied OFF was capable of inducing intracellular calcium responses in osteocytes (82.3% cells responding with a 3.71 fold increase average magnitude). Both extravasation distance and percentage of extravasated side-channels were significantly reduced with mechanically stimulated osteocytes (32.4% and 53.5% of control, respectively) compared to static osteocytes (102.1% and 107.3% of control, respectively). This is the first microfluidic device that has successfully integrated stimulatory bone fluid flow, and demonstrated that mechanically stimulated osteocytes reduced breast cancer extravasation. Future work with this platform will determine the specific mechanisms involved in osteocyte mechanoregulation of breast cancer bone metastasis, as well as other types of cancer metastasis and diseases.

OCY454 Osteocytes as an in Vitro Cell Model for Bone Remodeling Under Mechanical Loading.

Osteocytes' mechano-regulation of bone formation and resorption is key to maintaining appropriate bone health. Although extensive in vitro studies have explored osteocyte mechanobiology using the well-established MLO-Y4 cell model, the low amount of sclerostin secreted by this cell line renders it inadequate for studying cross-talk between osteocytes and osteoblasts under mechanical loading. Here, we investigated the potential of the sclerostin-expressing OCY454 osteocyte cell model in fulfilling this role. Fully differentiated OCY454 cells were tested for mechano-sensitivity by measuring changes in protein secretion, total adenosine triphosphate (ATP) content, and intracellular calcium in response to oscillatory fluid flow. Increases in sclerostin expression and total ATP content were observed. However, very low levels of receptor activator of the nuclear factor κ-B ligand were detected, and there was a great inconsistency in calcium response. Conditioned medium (CM) from OCY454 cells was then used to culture osteoblast and osteoclast precursors. Osteoblast activity was quantified with alkaline phosphatase (ALP) and Alizarin Red S stain, while osteoclast differentiation was quantified with tartrate-resistant acid phosphatase (TRAP) staining. We demonstrated that mechanically stimulated OCY454 cells released soluble factors that increased osteoblasts' ALP activity (p < 0.05) and calcium deposition (p < 0.05). There was also a significant decrease of large-sized TRAP-positive osteoclasts when osteoclast precursors were treated with CM from flow-stimulated OCY454 cells (p < 0.05). Results from this study suggest that OCY454 cells respond to mechanical loading with the release of key factors such as sclerostin to regulate downstream bone cells, thus demonstrating its potential as a novel cell model for in vitro osteocyte mechanobiology studies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1681-1689, 2019.

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells.

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.

Mechanical regulation of breast cancer migration and apoptosis via direct and indirect osteocyte signaling.

Bone metastases, the migration of cancers to bone, occur in 65-80% of patients with advanced breast cancer. Metastasized cancer cells interact with cells such as the bone-resorbing osteoclasts to alter bone remodeling. Exercise, often suggested as an intervention for cancer patients, regulates bone remodeling via osteocytes. Osteocytes also signal to endothelial cells, which may affect cancer cell extravasation. Therefore, we hypothesize that mechanically stimulated osteocytes can regulate processes in breast cancer bone metastasis. To test this, we exposed osteocytes to oscillatory fluid flow in vitro using parallel-plate flow chambers. We observed that conditioned medium from flow-stimulated osteocytes increased migration (by 45%) and reduced apoptosis (by 12%) of breast cancer cells. Conditioned medium from osteoclasts conditioned in flowed osteocytes' conditioned medium reduced migration (by 47%) and increased apoptosis (by 55%) of cancer cells. Cancer cell trans-endothelial migration was reduced by 34% toward flowed osteocytes' conditioned medium. This difference was abolished with ICAM-1 or IL-6 neutralizing antibodies. Conditioned medium from endothelial cells conditioned in flowed osteocytes' conditioned medium increased cancer cell apoptosis by 29%. To summarize, this study demonstrated mechanically stimulated osteocytes' potential to affect breast cancer cells not only through direct signaling, but also through osteoclasts and endothelial cells. The anti-metastatic potential of the indirect signalings is particularly exciting since osteocytes are further away from metastasizing cancer cells than osteoclasts and endothelial cells. Future studies into the effect of bone mechanical loading on metastases and its mechanism will assist in designing cancer intervention programs that lowers the risk for bone metastases.

Microfluidics approach to investigate the role of dynamic similitude in osteocyte mechanobiology.

Fluid flow is an important regulator of cell function and metabolism in many tissues. Fluid shear stresses have been used to level the mechanical stimuli applied in vitro with what occurs in vivo. However, these experiments often lack dynamic similarity, which is necessary to ensure the validity of the model. For interstitial fluid flow, the major requirement for dynamic similarity is the Reynolds number (Re), the ratio of inertial to viscous forces, is the same between the system and model. To study the necessity of dynamic similarity for cell mechanotransduction studies, we investigated the response of osteocyte-like MLO-Y4 cells to different Re flows at the same level of fluid shear stress. Osteocytes were chosen for this study as flows applied in vitro and in vivo have Re that are orders of magnitude different. We hypothesize that osteocytes' response to fluid flow is Re dependent. We observed that cells exposed to lower and higher Re flows developed rounded and triangular morphologies, respectively. Lower Re flows also reduced apoptosis rates compared to higher Re flows. Furthermore, MLO-Y4 cells exposed to higher Re flows had stronger calcium responses compared to lower Re flows. However, by also controlling for flow rate, the lower Re flows induced a stronger calcium response; while degradation of components of the osteocyte glycocalyx reversed this effect. This work suggests that osteocytes are highly sensitive to differences in Re, independent of just shear stresses, supporting the need for improved in vitro flow platforms that better recapitulate the physiological environment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:663-671, 2018.

Mechanical loading up-regulates early remodeling signals from osteocytes subjected to physical damage.

In the mineralized bone matrix, mechanical loading causes micrometer-sized cracks. These cracks trigger targeted remodeling along the micro-crack. Physical damage to osteocytes was shown to be involved in the initiation of this remodeling process. However, the role of subsequent mechanical loading osteocyte response to physical damage is unclear. In this study, we have designed and developed an in vitro cell model to study the impact of mechanical loading on osteocytes with physical damage. Specifically, a system was developed to create sub-cellular physical damage on MLO-Y4 osteocytes in vitro. This model re-created the spatial distribution of non-viable cells and VEGF expression around microdamage as reported in vivo. Using this system, the short term (24h) effects of fluid shear stress in regulation of osteocyte response to physical damage were investigated. We have observed that the mechanical stimuli had an additive effect in terms of COX-2, VEGF mRNA expressions, as well as PGE2, VEGF concentrations in the media. Interestingly, other inflammatory signals such as IL-6 and TNF-α did not change with these stimuli, at this time point. Moreover, fluid shear also had a modulating effect in regulation of osteoclast differentiation by osteocyte with physical damage. These results show that (1) subcellular physical damage upregulates remodeling signals in osteocytes at early time point, (2) mechanical loading substantially upregulates these signals for remodeling in osteocytes with physical damage.

Bone's responses to mechanical loading are impaired in type 1 diabetes.

Diabetes adversely impacts many organ systems including the skeleton. Clinical trials have revealed a startling elevation in fracture risk in diabetic patients. Bone fractures can be life threatening: nearly 1 in 6 hip fracture patients die within one year. Because physical exercise is proven to improve bone properties and reduce fracture risk in non-diabetic subjects, we tested its efficacy in type 1 diabetes. We hypothesized that diabetic bone's response to anabolic mechanical loading would be attenuated, partially due to impaired mechanosensing of osteocytes under hyperglycemia. Heterozygous C57BL/6-Ins2(Akita)/J (Akita) male and female diabetic mice and their age- and gender-matched wild-type (WT) C57BL/6J controls (7-month-old, N=5-7 mice/group) were subjected to unilateral axial ulnar loading with a peak strain of 3500 µÎµ at 2 Hz and 3 min/day for 5 days. The Akita female mice, which exhibited a relatively normal body weight and a mild 40% elevation of blood glucose level, responded with increased bone formation (+6.5% in Ct.B.Ar, and 4 to 36-fold increase in Ec.BFR/BS and Ps.BFR/BS), and the loading effects, in terms of changes of static and dynamic indices, did not differ between Akita and WT females (p ≥ 0.1). However, loading-induced anabolic effects were greatly diminished in Akita males, which exhibited reduced body weight, severe hyperglycemia (+230%), diminished bone formation (ΔCt.B.Ar: 0.003 vs. 0.030 mm(2), p=0.005), and suppressed periosteal bone appositions (ΔPs.BFR/BS, p=0.02). Hyperglycemia (25 mM glucose) was further found to impair the flow-induced intracellular calcium signaling in MLO-Y4 osteocytes, and significantly inhibited the flow-induced downstream responses including reduction in apoptosis and sRANKL secretion and PGE2 release. These results, along with previous findings showing adverse effects of hyperglycemia on osteoblasts and mesenchymal stem cells, suggest that failure to maintain normal glucose levels may impair bone's responses to mechanical loading in diabetics.