traction-microscopy-integrated-with-microfluidics-for-chemotactic

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

The fabrication of patterned microstructures within three-dimensional (3D) matrices is a challenging subject in tissue engineering and regenerative medicine. A 3D, free-moving bioprinting system was developed and hydrogels were patterned by varying the process parameters of z-axis moving velocity and ejection velocity. The patterning of hydrogel based microfibers in a 3D matrigel was achieved with dimensions of 4.5 mm length and widths from 79 to 200 μm. Hyaluronan-based hydrogels mixed with fibroblasts (L929), mouse endothelial cells (MS1), or human mesenchymal stem cells (hMSCs) were patterned using a 3D moving axis bioprinter and cell behavior was monitored in culture for up to 16 days. L929 and MS1 cells and hMSCs in patterned hydrogel revealed cell-cell interactions and a morphological dependency on cell types. HMSCs formed spheres through cell aggregation, while L929 cells increased in cellular mass without cell aggregation and MS1 dispersed into the matrix instead of aggregating. The aggregation of hMSCs was attenuated by treatment with Rho kinase (ROCK) inhibitor and cadherin antibody. This reflected the close relationship between cell aggregation and migration with RhoA and cell-cell adhesion molecules. Angiogenic-specific gene expression profiles showed that expression of CD105 decreased to 22% in the ROCK inhibitor group compared to control group. These results showed that cell-based patterns in a 3D matrix are highly dependent on both cell aggregation and migration over time.

Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction.

Regeneration of chronic myocardial infarction (CMI) is one of the challenging issues due to its limited regeneration activity compared to acute or sub-acute stage. In this study, we examined whether combination of stem cell homing factor (SDF-1) and angiogenic peptides (Ac-SDKP) injected with biomimetic hydrogels promote regeneration of cardiac function in a CMI model. We evaluated the regeneration of chronically infarcted myocardium using injectable biomimetic hydrogels containing two therapeutic factors; stromal-derived factor-1 (SDF-1) and Ac-SDKP for stem cell homing and angiogenesis, respectively. Injection of the two therapeutic factors into the infarct region of the left ventricle showed that the biomimetic hydrogels containing two therapeutic factor exhibited significantly improved left ventricle function, increased angiogenesis, decreased infarct size and greatest wall thickness within the infarct region at 4 weeks post-treatment. From these results, it is clear that hydrogels containing two therapeutic factors showed synergistic effects on regeneration in the chronic heart failure model. In conclusion, these results suggest that combination of stem cell homing factor with angiogenic peptides recruit stem cells to the microenvironments, increase the expression of angiogenic genes, enhance the matured vessel formation and improve the cardiac function in chronic MI. 

Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP.

Combinations of angiogenic growth factors have been shown to have synergistic effects on angiogenesis and natural wound healing in various animal models. Each growth factor has unique roles during angiogenesis; vascular endothelial growth factor (VEGF) plays a key role during the initial step of angiogenesis, whereas PDGF functions in the maturation of blood vessels. We used a combination of three angiogenic growth factors to increase angiogenesis in vitro and in vivo. We chose VEGF as a basic factor and added platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) to induce angiogenesis in three in vitro and in vivo models: 3D angiogenesis assay, 3D co-culture, and matrigel plug implantation assay. Cell proliferation was significantly higher in co-cultured cells treated with PDGF + VEGF + FGF than in the control, single, or dual combination groups. mRNA expression of α-smooth muscle actin (α-SMA), von Willebrand factor (vWF), and CD105 was higher in the triple group (PDGF + VEGF + FGF) than in control, single, or dual combination groups. In the PDGF + VEGF + FGF group, the length and number of branches of spheroids was also significantly higher than in the control, single, or dual combination groups. Furthermore, in a nude mouse model, α-SMA expression was significantly higher in the PDGF + VEGF + FGF group than in other groups. In conclusion, the addition of PDGF and FGF to VEGF showed synergistic effects on angiogenesis in vitro and in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1535-1543, 2016.

Combination of three angiogenic growth factors has synergistic effects on sprouting of endothelial cell/mesenchymal stem cell-based spheroids in a 3D matrix.

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity. 

Cellular Contraction and Polarization Drive Collective Cellular Motion.

Hepatocyte growth factor (HGF) induces cell migration and scattering by mechanisms that are thought to tip a local balance of competing physical forces; cell-to-cell and cell-to-substrate forces. In this local process, HGF is known to attenuate local cadherin-dependent adhesion forces for cell-cell junction development and enhance local integrin-dependent contractile forces for pulling neighboring cells apart. Here we use an expanding island of confluent Madin-Darby canine kidney (MDCK) cells as a model system to quantify the collective cell migration. In the absence of HGF, cell trajectories are highly tortuous whereas in the presence of HGF, they become far less so, resembling free expansion of a gas. At the level of cell-to-cell junctions, HGF attenuates the linkage of stress fibers to cell-to-cell junctions with concomitant decrease in intercellular stress. At the level of cell-to-substrate junctions, HGF augments the linkage of stress fibers to cell-to-substrate junctions with no apparent effect on traction. Together, HGF induces both structural changes in the actin-bound junctional protein complex and physical forces spanning multicellular clusters, which further promotes the expansion of confluent cellular layer.

Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer.

Homing of peripheral stem cells is regulated by one of the most representative homing factors, stromal cell-derived factor 1 alpha (SDF-1α), which specifically binds to the plasma membrane receptor CXCR4 of mesenchymal stem cells (MSCs) in order to initiate the signaling pathways that lead to directional migration and homing of stem cells. This complex homing process and directional migration of stem cells have been mimicked on a microfluidic device that is capable of generating a chemokine gradient within the collagen matrix and embedding endothelial cell (EC) monolayers to mimic blood vessels. On the microfluidic device, stem cells showed directional migration toward the higher concentration of SDF-1α, whereas treatment with the CXCR4 antagonist AMD3100 caused loss of directionality of stem cells. Furthermore, inhibition of stem cell's main migratory signaling pathways, Rho-ROCK and Rac pathways, caused blockage of actomyosin and lamellipodia formation, decreasing the migration distance but maintaining directionality. Stem cell homing regulated by SDF-1α caused directional migration of stem cells, while the migratory ability was affected by the activation of migration-related signaling pathways.

Directional migration of mesenchymal stem cells under an SDF-1α gradient on a microfluidic device.

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis.

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

Collective cellular migration plays a central role in development, regeneration, and metastasis. In these processes, mechanical interactions between cells are fundamental but measurement of these interactions is often hampered by technical limitations. To overcome some of these limitations, here we describe a system that integrates microfluidics with traction microscopy (TM). Using this system we can measure simultaneously, and in real time, migration speeds, tractions, and intercellular tension throughout an island of confluent Madin-Darby canine kidney (MDCK) cells. The cell island is exposed to hepatocyte growth factor (HGF) at a controlled gradient of concentrations; HGF is known to elicit epithelial-to-mesenchymal transition (EMT) and cell scattering. As expected, the rate of expansion of the cell island was dependent on the concentration of HGF. Higher concentrations of HGF reduced intercellular tensions, as expected during EMT. A novel finding, however, is that the effects of HGF concentration and its gradient were seen within an island. This integrated experimental system thus provides an integrated tool to better understand cellular forces during collective cellular migration under chemical gradients.

Article	Year
Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction. Journal of bioscience and bioengineering\| PubMed ID: 23562089	2013
Regeneration of chronic myocardial infarction by injectable hydrogels containing stem cell homing factor SDF-1 and angiogenic peptide Ac-SDKP. Biomaterials\| PubMed ID: 24378015	2014
Combination of three angiogenic growth factors has synergistic effects on sprouting of endothelial cell/mesenchymal stem cell-based spheroids in a 3D matrix. Journal of biomedical materials research. Part B, Applied biomaterials\| PubMed ID: 26268584	2016
Cellular Contraction and Polarization Drive Collective Cellular Motion. Biophysical journal\| PubMed ID: 27332131	2016
Homogenizing cellular tension by hepatocyte growth factor in expanding epithelial monolayer. Scientific reports\| PubMed ID: 28374776	2017
Directional migration of mesenchymal stem cells under an SDF-1α gradient on a microfluidic device. PloS one\| PubMed ID: 28886159	2017
Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy. Journal of biomedical optics\| PubMed ID: 29886577	2018
Traction microscopy with integrated microfluidics: responses of the multi-cellular island to gradients of HGF. Lab on a chip\| PubMed ID: 30924490	2019

Hwanseok Jang

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