Bone is able to adapt its composition and structure in order to suit its mechanical environment.Osteocytes,bone cells embedded in the calcified matrix,are believed to be the mechanosensors and responsible for orchestr...Bone is able to adapt its composition and structure in order to suit its mechanical environment.Osteocytes,bone cells embedded in the calcified matrix,are believed to be the mechanosensors and responsible for orchestrating the bone remodeling process[1].However,detailed cellular and molecular mechanism underlying osteocyte mechanobiology is not well understood.Further,how osteocytes communicate with other cell populations under mechanical loading is unclear.Recently,we developed several microfluidic platforms to address these questions.In this talk,osteocyte intracellular response under mechanical loading in the microfluidic environment will first be presented.Next,inter cell-population communications under mechanical loading and its implication in bone disorder management such as bone metastasis prevention will be discussed.1.Study osteocyte response to mechanical loading in a microfluidic environment Current research has focused on observing bone cell mechanotransduction under different simulated physiological conditions(e.g.,shear stress,strain,pressure,etc.)using macro-scale devices.However,these devices often require large sample volumes,low through-put,extensive setup protocol,as well as very limited designs only suitable for general cell culture[2].On the other hand,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.Recent work on co-culture platform has demonstrated the feasibility of building more complex microfluidic devices for osteocyte mechanotransduction studies,while maintaining its biological relevance[3].However,there lacks a robust system where multi-physiological flow conditions are applied to bone cells to study their intercellular communication.We aim to fulfill this gap by designing and fabricating a multi-shear stress,co-culture platform to study interaction between osteocytes and other bone cells when exposed to an array of physical cues.The project will rely on standard microfluidic principles in designing devices that utilize changing geometric parameters to induce different flow rates that are directly proportional to the levels of shear stress.All channels within the same device will share a common inlet,while adjusting the resistance of each individual channel will result in a different flow rate.Devices are fabricated using PDMS,and bonded to glass slides of equal sizes.MLO-Y4 osteocyte like cells seeded in the device are stimulated with oscillatory fluid flow with a custom in-house pump.Significant differences in RANKL levels are observed between channels,demonstrating that proper cellular response to flow can be elicit from each distinct shear stress channels as designed.Furthermore,we aim to pair these multi-shear stress channels with corresponding culturing chambers connected through perfusion pores.Through perfusion between the multi-shear stress channels and culturing chambers,different cell population can communicate to each other as they are stimulated by varying levels of shear stress.Using this platform,we will be able to mimic the interaction between osteocytes and other bone cells in vitro.Due to the advantage of using microfluidic devices,various analytical methods can be used on-chip to determine cellular response,such as staining for biomarkers and differentiation factors.2.Microfluidic platform for investigation of mechanoregulation of breast cancer bone metastasis Approximately 70%of advanced breast cancer patients experience bone metastasis.Breast cancer cells(BCC)that extravasate across the endothelium to the bone reduce bone quality by disrupting the healthy bone remodeling balance.Exercise,a common cancer intervention strategy,can regulate bone remodeling,thus potentially affect BCC metastasis to bone through signals released by mechanical loaded osteocytes.Our recent in vitro studies showed that mechanically stimulatedosteocytes can regulate BCC migration via endothelial cells[5].However,a more physiologically relevant platform is needed to better investigate the mechanisms leading to interactions between BCC and bone microenvironment under mechanical loading.We present here a novel in vitro microfluidic tri-culture lumen system for studying mechanical regulation of breast cancer metastasis in bone.In this study,highly metastatic MDA-MB-231 human BCCs were cultured inside a cylindrical lumen lined with human umbilical vein endothelial cells(HUVECs),which is adjacent to a population of either static or mechanically-stimulated osteocyte-like MLO-Y4 cells.Physiologically relevant oscillatory fluid flow(OFF)(1 Pa,1 Hz)was produced by a custom pump to mechanically load the MLO-Y4 cells.Soluble factors were diffused through hydrogel-filled side channels to enable inter-cell population communication between MLO-Y4 cells and BCCs over 3 days.BCC extravasation distance and percentage were measured and normalized to the acellular control with MLO-Y4 media only.Paired t-tests(n=5)were used for statistical analysis and the Holm-Bonferroni method was applied for multiple comparison analysis.Statistical significance was taken at P<0.05.Photolithography and soft lithography were used to fabricate silicon SU-8 master and PDMS replicates,respectively.A HUVEC lumen was successfully cultured in the PDMS microfluidic device.Extravasation distance was significantly decreased in the flowed osteocytes(33.6%of control)compared to static osteocytes(108.0%of control),while the extravasation percentage showed a non-significant decreasing trend between the flow(58%of control)and static(106.3%of control)osteocytes.In summary,we developed the first microfluidic platform allowing the integration of physiologically relevant bone fluid stimulation and real-time intercellular signaling between different cell populations in vitro.Using this platform,the significantly reduced extravasation distance was found in the group where conditioned medium from osteocytes exposed to flow.We speculate this could be due to regulation of matrix metallopeptidase 9(MMP-9)used by cancer cells to degrade the surrounding matrix during extravasation.Future work with this platform will determine the key mechanisms involved in osteocyte regulation of BCC metastasis.展开更多
文摘Bone is able to adapt its composition and structure in order to suit its mechanical environment.Osteocytes,bone cells embedded in the calcified matrix,are believed to be the mechanosensors and responsible for orchestrating the bone remodeling process[1].However,detailed cellular and molecular mechanism underlying osteocyte mechanobiology is not well understood.Further,how osteocytes communicate with other cell populations under mechanical loading is unclear.Recently,we developed several microfluidic platforms to address these questions.In this talk,osteocyte intracellular response under mechanical loading in the microfluidic environment will first be presented.Next,inter cell-population communications under mechanical loading and its implication in bone disorder management such as bone metastasis prevention will be discussed.1.Study osteocyte response to mechanical loading in a microfluidic environment Current research has focused on observing bone cell mechanotransduction under different simulated physiological conditions(e.g.,shear stress,strain,pressure,etc.)using macro-scale devices.However,these devices often require large sample volumes,low through-put,extensive setup protocol,as well as very limited designs only suitable for general cell culture[2].On the other hand,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.Recent work on co-culture platform has demonstrated the feasibility of building more complex microfluidic devices for osteocyte mechanotransduction studies,while maintaining its biological relevance[3].However,there lacks a robust system where multi-physiological flow conditions are applied to bone cells to study their intercellular communication.We aim to fulfill this gap by designing and fabricating a multi-shear stress,co-culture platform to study interaction between osteocytes and other bone cells when exposed to an array of physical cues.The project will rely on standard microfluidic principles in designing devices that utilize changing geometric parameters to induce different flow rates that are directly proportional to the levels of shear stress.All channels within the same device will share a common inlet,while adjusting the resistance of each individual channel will result in a different flow rate.Devices are fabricated using PDMS,and bonded to glass slides of equal sizes.MLO-Y4 osteocyte like cells seeded in the device are stimulated with oscillatory fluid flow with a custom in-house pump.Significant differences in RANKL levels are observed between channels,demonstrating that proper cellular response to flow can be elicit from each distinct shear stress channels as designed.Furthermore,we aim to pair these multi-shear stress channels with corresponding culturing chambers connected through perfusion pores.Through perfusion between the multi-shear stress channels and culturing chambers,different cell population can communicate to each other as they are stimulated by varying levels of shear stress.Using this platform,we will be able to mimic the interaction between osteocytes and other bone cells in vitro.Due to the advantage of using microfluidic devices,various analytical methods can be used on-chip to determine cellular response,such as staining for biomarkers and differentiation factors.2.Microfluidic platform for investigation of mechanoregulation of breast cancer bone metastasis Approximately 70%of advanced breast cancer patients experience bone metastasis.Breast cancer cells(BCC)that extravasate across the endothelium to the bone reduce bone quality by disrupting the healthy bone remodeling balance.Exercise,a common cancer intervention strategy,can regulate bone remodeling,thus potentially affect BCC metastasis to bone through signals released by mechanical loaded osteocytes.Our recent in vitro studies showed that mechanically stimulatedosteocytes can regulate BCC migration via endothelial cells[5].However,a more physiologically relevant platform is needed to better investigate the mechanisms leading to interactions between BCC and bone microenvironment under mechanical loading.We present here a novel in vitro microfluidic tri-culture lumen system for studying mechanical regulation of breast cancer metastasis in bone.In this study,highly metastatic MDA-MB-231 human BCCs were cultured inside a cylindrical lumen lined with human umbilical vein endothelial cells(HUVECs),which is adjacent to a population of either static or mechanically-stimulated osteocyte-like MLO-Y4 cells.Physiologically relevant oscillatory fluid flow(OFF)(1 Pa,1 Hz)was produced by a custom pump to mechanically load the MLO-Y4 cells.Soluble factors were diffused through hydrogel-filled side channels to enable inter-cell population communication between MLO-Y4 cells and BCCs over 3 days.BCC extravasation distance and percentage were measured and normalized to the acellular control with MLO-Y4 media only.Paired t-tests(n=5)were used for statistical analysis and the Holm-Bonferroni method was applied for multiple comparison analysis.Statistical significance was taken at P<0.05.Photolithography and soft lithography were used to fabricate silicon SU-8 master and PDMS replicates,respectively.A HUVEC lumen was successfully cultured in the PDMS microfluidic device.Extravasation distance was significantly decreased in the flowed osteocytes(33.6%of control)compared to static osteocytes(108.0%of control),while the extravasation percentage showed a non-significant decreasing trend between the flow(58%of control)and static(106.3%of control)osteocytes.In summary,we developed the first microfluidic platform allowing the integration of physiologically relevant bone fluid stimulation and real-time intercellular signaling between different cell populations in vitro.Using this platform,the significantly reduced extravasation distance was found in the group where conditioned medium from osteocytes exposed to flow.We speculate this could be due to regulation of matrix metallopeptidase 9(MMP-9)used by cancer cells to degrade the surrounding matrix during extravasation.Future work with this platform will determine the key mechanisms involved in osteocyte regulation of BCC metastasis.
