TY - JOUR
AU - You,, Lidan
AB - Abstract 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. microfluidic, osteocyte, osteoclast, bone remodeling, fluid shear stress, mechanical loading Insight BOX Our work here provides a novel research tool to enable the study of the bone remodeling process in vitro. This new platform can help provide more insight into how bone cells communicate with each other during mechanical stimulation, and ultimately help develop new clinical approaches to solving bone-related diseases. INTRODUCTION Bone remodeling is an important process that is responsible for bone growth and recovery from injuries to bone tissues. It has been shown that osteocytes play a major mechano-sensing role within the bone matrix, responsible for the coordination of bone formation and bone resorption processes [1]. Many studies have shown the importance of varying levels of signaling factors secreted by osteocytes in response to mechanical loading, such as a receptor activator of nuclear factor-kappaB ligand (RANKL) [2], osteoprotegerin (OPG) [3, 4], sclerostin [5, 6] and prostaglandin E2 (PGE2) [7, 8]. These signaling molecules act as the regulators of downstream osteoclast and osteoblast activities to control the bone remodeling process in accordance with the mechanical forces experienced by the osteocytes. Anomalies in the remodeling process can both lead to and as well as become an accompanying effect to devastating bone diseases such as osteoporosis [9, 10] and osteosarcoma [11, 12]. Hence, a better understanding of the regulatory factors associated with bone remodeling is critical in developing effective clinical solutions to bone diseases, as well as preventative measures against failing of bone health. Current research on osteocyte’s role in bone remodeling has been utilizing both in vivo animal models [13, 14] as well as in vitro flow chamber models [15]. Animal models provide a comprehensive tool for studying overall tissue and whole organ system response to mechanical loading while allowing for accurate compression loading of limbs that mimic exercise conditions. However, it is difficult to study signaling pathways in vivo, as isolating the effect of each factor proves to be difficult and prone to high variance between samples. Furthermore, it is challenging to translate animal models to human tissue behavior, and often, extensive validation experiments are needed. On the other hand, in vitro models provide a more simplistic tool to evaluate single-cell population response to specific levels of mechanical loading in the form of fluid shear stress. Studies using the traditional in vitro flow chamber devices have observed the role of RANKL/OPG in osteocyte regulation of osteoclast [16, 17], as well as osteocyte regulation of breast cancer metastasis [18, 19]. Although lacking the comprehensive tissue level response from in vivo models, in vitro platforms often excel at pinpointing specific functions of signaling factors and how they coordinate with each other to produce the overall response from a population of cells. Troubles arise when using these macroscale flow chambers, which require extensive pumping equipment that are difficult to scale up. As well, device-to-device variations during manual macroflow chamber setup can significantly increase the deviation in experimental results. Most importantly, flow chambers lack the physiologically relevant microenvironments typically experienced by cells in vivo, and with its reliance on conditioned media, it is not possible for real-time signaling between different cell populations. Hence, the development of innovative in vitro platforms is needed to provide more efficient tools for studying osteocyte mechanobiology. The rise of microfluidic devices in modeling biological systems has provided novel experimental tools for studying various cell–cell interactions such as mesenchymal stromal cells-endothelial communication during vasculature formation [20, 21], or endothelial-epithelial interaction in a lung-on-a-chip [22, 23]. In relation to osteocytes, microfluidic devices can be used to simulate different physiological loading conditions (e.g. shear stress, strain, pressure, etc.), accurately controlling their levels and observing specific responses via the secretome of osteocytes. They are also ideal for primary cell studies, allowing large numbers of experimental samples with a limited number of cells. With flexible designs for high-throughput capabilities, microfluidic devices are optimal tools for testing potential signaling factors and provide additional drug screening capabilities before clinical experiments are needed. Our recent work on osteocyte-osteoclast co-culture platform has demonstrated the feasibility of building more complex microfluidic devices for bone cell mechanotransduction studies while maintaining its biological relevance [24]. However, there lack a robust system where multi-physiological flow conditions are applied to osteocytes to study their intercellular communication. Here, we describe a multi-shear co-culture microfluidic device that can be used to study mechanically stimulated osteocyte and their interaction with other bone cells. We validated in our device the changing levels of osteocyte response to different levels of fluid shear stress and confirmed a corresponding change in downstream cell response via osteoclast formation. Moreover, we demonstrated the capability for the microfluidic device to be used for studying drug effect on osteoclast interaction with mechanically stimulated osteocytes. This platform can be developed further into a large array of smaller devices to achieve a gradient of physiological loading conditions for high-throughput experiments on osteocyte mechanotransduction studies, as well as a drug screening tool. METHODS Design and fabrication of co-culture platform The microfluidic device is designed based on standard fluidic principles that utilize changing dimensions to create different flow rates in chambers connected in a parallel circuit (sharing the same inlet). Average wall shear stresses (τA) in each osteocyte chamber are calculated using parallel-plate assumptions based on the following equation: $$\begin{equation} {\tau}_A=\frac{6{U}_A\mu }{h} \end{equation}$$(1) where UA is the average flow velocity, μ is the dynamic viscosity and h is the height of the chamber. Based on the established Hagen–Poiseuille law [25], by changing the length of channels leading up to each chamber, we can proportionally vary the average velocity without affecting the flow profile experienced by the cells (which is dependent on chamber width and height). The experimental design consists of three parallel osteocyte chambers each paired with an adjacent osteoclast chamber (Fig. 1A). Diffusion channels between the two chambers allow for bidirectional transport of signaling molecules between the two cell populations. A sample device is shown in Fig. 1C to demonstrate physical size and layout. Each osteocyte culturing chamber is 4 mm × 16 mm, and each osteoclast culturing chamber is 3 mm × 14 mm. Height for all chambers and channels is 250 μm. This is compared to standard macroflow chamber slides of 75 mm × 51 mm. Circulating volume inside each osteocyte chamber is around 16 μl, while those for macroscale flow chambers can reach 10 ml. Figure 1 Open in new tabDownload slide Basic co-culture microfluidic design. (A) Schematic showing the layout of the device, consisting of three pairs of osteocyte–osteoclast chambers. Each pair of the chamber was connected by 20-μm-wide and 300-μm-long diffusion channels. All three pairs of chambers shared a common inlet, utilizing a single flow input source for the entire device. Scale bar = 4 mm. (B) Flow validation results showing that experimental flow velocities were close to theoretical values designed for the devices. These flow velocities were based on a set pump flow rate and correlated directly with the intended shear stresses. (C) Picture showing device post-fabrication. The green dye highlighted the osteocyte chambers and their inlet/outlet, while the red dye showed the individual osteoclast chambers. Figure 1 Open in new tabDownload slide Basic co-culture microfluidic design. (A) Schematic showing the layout of the device, consisting of three pairs of osteocyte–osteoclast chambers. Each pair of the chamber was connected by 20-μm-wide and 300-μm-long diffusion channels. All three pairs of chambers shared a common inlet, utilizing a single flow input source for the entire device. Scale bar = 4 mm. (B) Flow validation results showing that experimental flow velocities were close to theoretical values designed for the devices. These flow velocities were based on a set pump flow rate and correlated directly with the intended shear stresses. (C) Picture showing device post-fabrication. The green dye highlighted the osteocyte chambers and their inlet/outlet, while the red dye showed the individual osteoclast chambers. Fabrication of the microfluidic devices is based on standard soft lithography techniques [26]. Briefly, a photomask containing the pattern for the device is generated using a laser-etched chromium-lined glass plate. The photomask is then used to transfer the design onto an SU-8 2050-coated silicon wafer using UV exposure. The silicon wafer containing the imprinted design is then used as a negative mold for repeated device fabrication. PDMS at a 10:1 base to curing agent ratio is mixed and poured onto the negative mold before curing at 60°C. The cured PDMS is cut from the negative mold and bonded to glass slides after the plasma treatment of the bonding surfaces to form the final microfluidic device. Fabricated microfluidic device is sterilized using 70% ethanol and UV light before cell seeding. Cell culture and seeding in co-culture platform All cells and devices are incubated at 37°C and 5% CO2 conditions during the incubation period. MLO-Y4 osteocytes are cultured in Alpha Minimum Essential Medium containing 2.5% calf serum, 2.5% fetal bovine serum (FBS) and 1% Penicillin–Streptomycin (PS). RAW264.7 pre-osteoclasts are cultured in Dulbecco’s Modified Eagle Medium containing 10% FBS, 2% Glutamine and 1% PS. MLO-Y4 cells are trypsinized at 80% confluence before seeded into the microfluidic device at 250-k/ml density. Osteocyte chambers in all devices are coated with 0.15-mg/ml type I collagen for 1 h before cell seeding. Air–liquid barriers formed along the posts between the two chambers prevent osteocytes from escaping to the osteoclast chamber. Osteocytes are cultured for 24 h in the device before RAW264.7 cells are added to adjacent chambers. RAW264.7 cells are supplement with 10 ng/ml of recombinant RANKL protein to assist with differentiation. Inlet and outlet of the osteocyte chamber are temporarily sealed to prevent pre-osteoclasts from crossing over to the osteocyte chamber. Flow experiments are conducted using a custom-made fluidic pump. Oscillatory fluid flow (OFF) at 1 Hz is applied to the osteocyte chambers for 2 h per flow session. Microfluidic device is maintained at 37°C and 5% CO2 condition during OFF stimulation. Calcium response quantification MLO-Y4 osteocytes are seeded inside osteocyte chambers at 250 k/ml and grown to 80% confluence before imaging. Osteocytes are stained with Fura-2 AM (Ex: 340 nm/380 nm, Em: 510 nm) live calcium indicator 1 h before the experiment. Each channel is imaged separately with a 40X magnification in a 350 μm × 260 μm field of view containing 20–40 cells. Devices are maintained on a heated imaging stage, where baseline intracellular calcium fluctuations are measured before applying OFF stimulation. Intracellular calcium signals captured during flow stimulation are compared to baseline values, and only signals 2-fold or higher are registered as positive calcium response. Response rates are calculated based on the number of positive responses over the total number of cells observed. Average response magnitude is calculated from all positively responding osteocytes. Figure 2 Open in new tabDownload slide Calcium response results from MLO-Y4 osteocytes seeded in the co-culture device. (A) Calcium response rate of osteocytes seeded in the co-culture device. Response rates increased as shear stress increased. Response rate in the 2-Pa osteocyte chamber was similar to the response rate observed from traditional flow chamber devices. (B) Average response magnitude also increased with higher shear stress level, where magnitudes observed in the 2-Pa osteocyte chamber are again similar to values obtained from traditional flow chambers. (C) Representative calcium response curves from the 2-Pa channel in the microfluidic device vs. standard 2-Pa flow chamber, showing consistency in the spatiotemporal response pattern between the two platforms. Figure 2 Open in new tabDownload slide Calcium response results from MLO-Y4 osteocytes seeded in the co-culture device. (A) Calcium response rate of osteocytes seeded in the co-culture device. Response rates increased as shear stress increased. Response rate in the 2-Pa osteocyte chamber was similar to the response rate observed from traditional flow chamber devices. (B) Average response magnitude also increased with higher shear stress level, where magnitudes observed in the 2-Pa osteocyte chamber are again similar to values obtained from traditional flow chambers. (C) Representative calcium response curves from the 2-Pa channel in the microfluidic device vs. standard 2-Pa flow chamber, showing consistency in the spatiotemporal response pattern between the two platforms. ELISA quantification of RANKL expression At 80% confluence, MLO-Y4 cells cultured in the osteocyte chamber are stimulated with OFF for 2 h and incubated within the device for 24 h. All condition media (CM, 16 μl) from each osteocyte chamber is completely extracted and diluted to 100 μl for standard ELISA quantification of RANKL. Briefly, CM from experiment is added to capture an antibody-coated 96-well plate and visualized using secondary antibody bonded to streptavidin-Horseradish peroxidase. Results are read by the SpectraMax i3 plate reader at 450 nm. Osteoclast-osteocyte co-culture experiment MLO-Y4 cells (100 k cells/ml) and RAW264.7 cells (200 k cells/ml) are seeded into respective chambers before beginning the 7-day co-culture period (Figure S1). OFF (1 Hz, 2 h) is applied to the osteocyte chamber every other day beginning from Day 1. Separate growth media in both osteocyte and osteoclast chambers is changed once every 24 h. For zoledronic acid (ZA) experiments, the appropriate concentration of ZA is added to fresh osteoclast growth media before daily media changes. At the end of Day 7, cells in microfluidic devices are fixed and stained for tartrate-resistant acid phosphatase (TRAP) to visualize osteoclast formation. TRAP-positive cells are counted from six random positions within each osteoclast chamber and added together for quantification. Particle flow velocimetry Microfluidic devices are linked up to the custom in-house OFF pump and filled with floating cell bodies. After turning on the pump, videos at 30 fps for 30 s are taken at each channel to capture the flow cell bodies. Random streak lines across each channel at the peak of the oscillatory flow are used to calculate the average peak velocity. Statistics A minimum of three individual experiments were run with a minimum of 2 samples per experimental group. Each microfluidic device consists of 3 samples, one for each fluid shear stress value at 0.5, 1 and 2 Pa. Flow devices have undergone respective OFF stimulation, while no flow groups have consisted of cells seeded within the microfluidic device without OFF stimulation during the entire duration of the experiment. Student’s t-test (two-tail, non-paired for calcium response; two-tail, paired for all other results) was used to test the significance between flow and no flow groups (α = 0.05) using mean values from each experimental trials. The n value represents the total number of device samples from all experiments. Additional two-way ANOVA test was performed on the ZA experiment samples, with α = 0.05 and a computed Fcrit value of 4.35. RESULTS Flow rate validation To validate the experimental flow velocities inside the microfluidic devices, we conducted particle flow velocimetry. We were able to confirm that the average experimental flow velocity is consistent with those intended for the design (Fig. 1B). Flow velocities are significantly different between each of the osteocyte chambers, ensuring that different osteocyte populations are experiencing drastically different fluid flow shear stresses. The results also confirmed that there is no leakage in the setup, which would result in a loss of flow velocity and require manual adjustment to the inlet flow rates of the OFF pump in order to achieve the desired flow velocities within the microfluidic device. Ca2+ response from osteocytes We observed a significant difference in calcium response patterns between the three osteocyte chambers. As can be seen in Fig. 2A, there is a strong correlation between the calcium response rates and the shear stress levels experienced by osteocytes. Furthermore, we saw a similar pattern in Fig. 2B where the average magnitude of the calcium responses significantly increased with shear stress levels. There was, however, no positive calcium response registered in the 0.5-Pa osteocyte chamber throughout our experiments. These results not only confirm that osteocytes seeded in the co-culture device can modulate their response according to the shear stress they sense, but also demonstrated the versatile capability for our microfluidic device to be used as a live imaging platform for osteocyte mechanotransduction studies. Observed spatiotemporal patterns of calcium response curves from the microfluidic devices are very similar to those commonly seen in macroscale flow chambers (Fig. 2C). RANKL released from osteocytes We measured extracellular secretion of RANKL protein from OFF stimulated osteocytes in our devices. From our experiments, we validated that even with a small-volume reservoir in our osteocyte chambers, we could still observe a significant difference in RANKL expression between cells cultured in chambers with varying fluid shear stress. From our results, we saw a decrease in extracellular RANKL levels in chambers with higher fluid shear stress (Fig. 3). This agrees with previously reported observations in the literature [2]. Higher, than expected, variations in raw expression level were observed between different trials. Results are hence normalized using the 0.5-Pa chamber as the baseline expression level. No significant differences were observed between chambers in the no-flow devices, showing that experimental conditions and micro-environment within the device did not alter the RANKL expression levels of osteocytes. Figure 3 Open in new tabDownload slide Quantification of soluble RANKL collected from osteocytes channel. After normalizing to quantities observed in each device’s 0.5-Pa osteocyte chamber, there was a decrease in the level of extracellular RANKL detected when increasing the shear stress level sensed by osteocytes. No differences were seen in the no-flow devices between all three osteocyte chambers. Figure 3 Open in new tabDownload slide Quantification of soluble RANKL collected from osteocytes channel. After normalizing to quantities observed in each device’s 0.5-Pa osteocyte chamber, there was a decrease in the level of extracellular RANKL detected when increasing the shear stress level sensed by osteocytes. No differences were seen in the no-flow devices between all three osteocyte chambers. Osteoclast differentiation in co-culture Within our co-culture experiments, we quantified the osteoclast formation from RAW264.7 cells using TRAP staining as a positive marker for differentiation. In our devices, we observed a significant decrease in the number of TRAP-positive cells in osteoclast chambers adjacent to osteocytes stimulated with higher levels of fluid shear stress (Fig. 4). This agrees with our previous results demonstrating that a decrease in RANKL expression levels from osteocytes would result in lower numbers of differentiated osteoclasts [24]. We saw a significant difference in TRAP-positive cells between the 0.5-Pa chamber and the other two chambers. However, only a decreasing trend was observed between the 1- and 2-Pa chambers without statistical significance. From our results, it seems that the rate of decrease is in a logarithmic relationship with the level of fluid shear stress, and 1-Pa OFF could be a threshold level for the effect of mechanical stimulation on osteoclast differentiation in a co-culture environment. Figure 4 Open in new tabDownload slide Quantification of osteoclast differentiation when co-cultured with osteocytes. After 7-day incubation in the co-culture device with bi-daily OFF stimulation of osteocytes, a significant decrease in osteoclast formation was seen when co-cultured adjacent to osteocytes in the 2-Pa chamber. A decreasing trend was also observed beside the 1-Pa chamber, but not statistically significant. No differences in osteoclast differentiation were observed in devices without the mechanical stimulation of osteocytes. Figure 4 Open in new tabDownload slide Quantification of osteoclast differentiation when co-cultured with osteocytes. After 7-day incubation in the co-culture device with bi-daily OFF stimulation of osteocytes, a significant decrease in osteoclast formation was seen when co-cultured adjacent to osteocytes in the 2-Pa chamber. A decreasing trend was also observed beside the 1-Pa chamber, but not statistically significant. No differences in osteoclast differentiation were observed in devices without the mechanical stimulation of osteocytes. Effect of ZA on osteoclast differentiation in co-culture After establishing our co-culture microfluidic device and validating observable differences in osteoclast differentiation when co-cultured with osteocytes stimulated under different levels of fluid shear stress, we moved on to demonstrate the potential application of our device. One of the key advantages of our microfluidic device is real-time signaling between osteoclast and fluid stimulated osteocytes. Thus, it is an ideal platform to study how drugs can affect osteoclast differentiation when combined with mechanically stimulated osteocytes. We chose to study how ZA, an established bisphosphonate, can change osteoclast differentiation within our device. From our 7-day co-culture experiments, we observed a significant decrease in osteoclast differentiation in all chambers of the no-flow devices when supplemented with 20 μM of ZA (Fig. 5A), demonstrating that the substantial effect ZA has on osteoclast differentiation in a co-culture environment. After applying OFF stimulation to osteocytes in the adjacent microfluidic channel, we observed a compounding effect between mechanical loading and ZA, where a further decrease in osteoclast differentiation was observed when ZA-supplemented osteoclasts were co-cultured with osteocytes stimulated with 1- and 2-Pa fluid shear stress (Fig. 5B). Using a two-way ANOVA test, we confirmed co-dependencies of fluid shear stress and ZA in the 2-Pa channel with P < 0.05 and F > Fcrit. However, there were no significant interactions between the two variables observed in the 1- and 0.5-Pa channels. These results demonstrated that our microfluidic device is capable of not only studying the effect of mechanical loading only on osteocyte-osteoclast signaling but also allows studying of the synergistic effect of mechanical loading and drug effect on osteoclasts within this co-culture environment. Figure 5 Open in new tabDownload slide Osteoclast differentiation under the treatment of ZA. (A) The effect of ZA on osteoclast differentiation was tested in the co-culture device. Under no OFF stimulation, a significant decrease in osteoclast formation was seen when treated with 20-μM ZA compared with control devices. (B) A similar effect was seen in fluid flow stimulated devices, where a significant decrease was seen in devices treated with 20-μM ZA compared with devices with only fluid flow stimulation. A compounding effect is observed between OFF stimulation and ZA, where both the mechanical loading of the osteocytes and the ZA treatment of cells in the device worked together, resulting in the lowest amount of osteoclast formation observed. Significant interaction effect was observed in 2-Pa channel after analysis with a two-way ANOVA test where P < 0.05. Figure 5 Open in new tabDownload slide Osteoclast differentiation under the treatment of ZA. (A) The effect of ZA on osteoclast differentiation was tested in the co-culture device. Under no OFF stimulation, a significant decrease in osteoclast formation was seen when treated with 20-μM ZA compared with control devices. (B) A similar effect was seen in fluid flow stimulated devices, where a significant decrease was seen in devices treated with 20-μM ZA compared with devices with only fluid flow stimulation. A compounding effect is observed between OFF stimulation and ZA, where both the mechanical loading of the osteocytes and the ZA treatment of cells in the device worked together, resulting in the lowest amount of osteoclast formation observed. Significant interaction effect was observed in 2-Pa channel after analysis with a two-way ANOVA test where P < 0.05. DISCUSSION Through our experimental results, we have demonstrated the versatile analytical methods that can be used in our co-culture microfluidic device when studying osteocyte mechanotransduction and intercellular signaling during the bone remodeling process. Due to simple design principles and established fundamental fluid flow phenomenon, our co-culture device can be easily scaled up to larger array systems, considerably increasing the throughput of experiments. To the best of our knowledge, this is the first device that capable of integrating multi-shear stress level and co-culture microenvironment in studying bone remodeling. We have shown that standard analytics techniques used to study osteocytes–osteoclast interaction, such as calcium response, extracellular RANKL expression and TRAP staining, can be easily adapted to the microfluidic device. Furthermore, the device steps away from traditional time-sensitive CM experiments and moves to a real-time intercellular signaling model, a much closer representation of the in vivo conditions. This is shown through our osteoclast differentiation results, where bi-daily fluid flow stimulation of osteocytes still produced a significant difference in osteoclast formation compared with the previous daily dosage of mechanical stimulation commonly used in large-flow chambers. Our microfluidic devices also require significantly lower media volume and cell count, making it ideal for studying large-scale drug screening as well as primary cell interactions. We have demonstrated here that our co-culture device can be used to study other factors in combination with the impact of fluid flow stimulation on osteocyte–osteoclast interaction. Our results have shown that in a co-culture environment, ZA still decreases the differentiation of osteoclasts. Furthermore, this effect can be combined with the reduction in osteoclast formation caused by mechanically stimulated osteocytes. This agrees largely with what is previous reported in the literature, where ZA inhibits osteoclast formation and activity in vitro [27–29]. However, there has been a study where osteocyte-mediated enhancement of osteoclast formation was observed in vitro using CM experiments [30]. Although we added the ZA supplement to our osteoclast differentiation media, due to the diffusion channels between the two cell populations, osteocytes should also experience the effect of the drug. However, we do not see any signs of enhanced osteoclast formation within the device. This could be due to the synergistic drug effect on both the osteoclast and osteocytes. Further investigations are needed to confirm whether ZA is having any effect on the osteocyte-mediated increase in osteoclast formation in our co-culture devices. Within our microfluidic platform, we observed a significant decrease in RANKL expression and osteoclast differentiation when osteocytes are stimulated with fluid shear stress. This is in agreement with previous in vitro studies involving macroscale flow chambers [2, 31, 32], with similar levels of RANKL expression level normalized to cell numbers. However, our device had significantly fewer numbers of TRAP-positive cells compared with traditional macroscale flow chamber-based experiments that utilized well-plates supplemented with conditioned media for osteoclast differentiation [33, 34]. This could be due to the lower volume to surface ratio within the microfluidic device, which limited the amount of nutrients available to the cells. This issue can be resolved in future studies by either implementing perfusion flow for media exchange or by designing devices with considerably higher volume to surface ratios. Furthermore, we saw a large trial-to-trial variation in the collected soluble RANKL protein levels. We believe that this is due to the limited size of our osteocyte chamber, as the media volume extracted per sample is only enough for the minimum volume requirement of commercialized ELISA kits. This is a common issue among similar types of microfluidic devices, making it even more difficult to measure protein concentrations via ELISA with more scaled down systems. However, many researchers are now working on on-chip protein detection methods based on electrochemistry principles [35, 36]. We hope to adopt these novel detection methods in the future to allow for more accurate and persistent measurement of protein concentrations. These new detection techniques will also allow for real-time tracking of expression factors, enabling our device to map variations in signaling molecules during and after the mechanical stimulation of osteocytes. This will create a powerful tool that provides important insight into the bone remodeling process and enable better understanding of the type of synergies that exist in the bi-directional interaction of osteocytes and osteoclasts. Another limitation of our current device in modeling the bone remodeling process is the lack of osteoblasts in our system. As shown in the literature, communication between osteocyte and osteoblast is another crucial process during bone remodeling [37]. These interactions are difficult to model in vitro due to the limitation of the existing MLO-Y4 cell line, as ongoing work is attempting to establish appropriate osteocyte cell lines suitable for studying osteocyte-osteoblast signaling [38, 39]. Future experiments using our microfluidic device can mitigate this issue by using primary osteocytes, but the current design still lacks the capability for a tri-culture system where communications across osteocytes, osteoclasts and osteoblasts can be modeled. Newer iterations of the design can include an adaptation of a second layer to the device, allowing for the culture of osteoblasts while maintaining proper diffusion channels in between all three cell types. These new devices will provide an even more physiological-relevant micro-environment to study bone remodeling in vitro. It will also provide a more comprehensive understanding of the effects of drugs such as zoledronic acid (ZA), and how administering such chemical reagents can affect the interplay of all three bone cells during remodeling. Furthermore, various disease models can be adapted within our microfluidic devices in order to study changes in cell–cell communication during these defects in bone remodeling, and identify potential clinical targets that can be used as treatments. CONCLUSION Here, we have shown the design of a novel co-culture microfluidic platform that can study the effect of various levels of fluid stimulation of osteocytes within the same device. We validated that osteocytes adapt their behavior to different levels of fluid shear stress, and downstream osteoclast differentiation is directly affected by the level of mechanical stimuli sensed by co-cultured osteocytes. Our microfluidic device has the capability to perform drug studies on bone remodeling and delivers a potential platform for large-scale drug screening experiments. We hope to further develop our platform to enable in vitro studies of a tri-culture system for bone remodeling, empowering researchers to gain a better understanding of this phenomenon and find novel clinical solutions to bone diseases. CONFLICT OF INTEREST STATEMENT None declared. References 1. Dallas SL , Prideaux M, Bonewald LF. The osteocyte: an endocrine cell…and more . Endocr Rev 2013 ; 34 : 658 – 90 . doi: 10.1210/er.2012-1026 . Google Scholar Crossref Search ADS PubMed WorldCat 2. You L , Temiyasathit S, Lee P et al. 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TI - Novel in vitro microfluidic platform for osteocyte mechanotransduction studies
JO - Integrative Biology
DO - 10.1093/intbio/zyaa025
DA - 2020-12-30
UR - https://www.deepdyve.com/lp/oxford-university-press/novel-in-vitro-microfluidic-platform-for-osteocyte-mechanotransduction-5MtsvT25lk
SP - 303
EP - 310
VL - 12
IS - 12
DP - DeepDyve
ER -