a-microfluidic-platform-for-stimulating-chondrocytes-with-dynamic

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

University of Nebraska-Lincoln

Microscopic sessile suspension feeders are a critical component in aquatic ecosystems, acting as an intermediate trophic stage between bacteria and higher eukaryotic taxa. Because they live attached to boundaries, it has long been thought that recirculation of the feeding currents produced by sessile suspension feeders inhibits their ability to access fresh fluid. However, previous models for the feeding flows of these organisms assume that they feed by pushing fluid perpendicular to surfaces they live upon, whereas we observe that sessile suspension feeders often feed at an angle to these boundaries. Using experiments and calculations, we show that living suspension feeders (Vorticella) likely actively regulate the angle that they feed relative to a substratum. We then use theory and simulations to show that angled feeding increases nutrient and particle uptake by reducing the reprocessing of depleted water. This work resolves an open question of how a key class of suspension-feeding organisms escapes physical limitations associated with their sessile lifestyle.

A new angle on microscopic suspension feeders near boundaries.

The stiffness of the extracellular matrix (ECM) plays an important role in controlling cell functions. As an alternative to the ECM, hydrogels of tunable elasticity are widely used for in vitro cell mechanobiology studies. Therefore, characterizing the Young's modulus of the hydrogel substrate is crucial. In this paper, we propose a confocal microscopy indentation method for measuring the elasticity of polyacrylamide gel as a model hydrogel. Our new indentation method is based on three-dimensional imaging of the indented gel using confocal microscopy and automated image processing to measure indentation depth from the three-dimensional image stack. We tested and validated our method by indenting polyacrylamide gels of different rigidities with various sphere indentors and by comparing it with the rheometric method. Our measurements show consistent results regardless of the type of the indentors and agree with rheometric measurements. Therefore, the proposed confocal microscopy indentation method can accurately measure the stiffness of hydrogels. 

Three-Dimensional Confocal Microscopy Indentation Method for Hydrogel Elasticity Measurement.

The elasticity of soft biological materials is a critical property to understand their biomechanical behaviors. Atomic force microscopy (AFM) indentation method has been widely employed to measure the Young's modulus (E) of such materials. Although the accuracy of the method has been recently evaluated based on comparisons with macroscale E measurements, the repeatability of the method has yet to be validated for rigorous biomechanical studies of soft elastic materials. We tested the AFM indentation method using colloidal probes and polyacrylamide (PAAM) gels of E < 20 kPa as a model soft elastic material after having identified optimal trigger force and probe speed. AFM indentations repeated with time intervals show that the method is well repeatable when performed carefully. Compared with the rheometric method and the confocal microscopy indentation method, the AFM indentation method is evaluated to have comparable accuracy and better precision, although these elasticity measurements appear to rely on the compositions of PAAM gels and the length scale of measurement. Therefore, we have confirmed that the AFM indentation method can reliably measure the elasticity of soft elastic materials.

A Validation Study of the Repeatability and Accuracy of Atomic Force Microscopy Indentation Using Polyacrylamide Gels and Colloidal Probes.

 is a protozoan attached to a substrate by a stalk which can contract in less than 10 ms, translating the zooid toward the substrate with a maximum Reynolds number of ∼1. Following contraction, the stalk slowly relaxes, moving the zooid away from the substrate, which results in creeping flow. Although  has long been believed to contract to evade danger, it has been suggested that its stalk may contract to enhance food transport near the substrate. To elucidate how  utilizes its contraction-relaxation cycle, we investigated water flow caused by the cycle, using a computational fluid dynamics model validated with an experimental scale model and particle tracking velocimetry. The simulated flow was visualized and analyzed by tracing virtual particles around the . It is observed that one cycle can displace particles up to ∼190 m with the maximum net vertical displacement of 3-4 m and that the net transport effect becomes more evident over repeated cycles. This transport effect appears to be due to asymmetry of the contraction and relaxation phases of the flow field, and it can be more effective on motile food particles than non-motile ones. Therefore, our  model enabled investigating the fluid dynamics principle and ecological role of the transport effects of 's stalk contraction.

Flow and transport effect caused by the stalk contraction cycle of .

Vorticella convallaria is a sessile protozoan of which the spasmoneme contracts on a millisecond timescale. Because this contraction is induced and powered by the binding of calcium ions (Ca), the spasmoneme showcases Ca-powered cellular motility. Because the isometric tension of V. convallaria increases linearly with its stalk length, it is hypothesized that the contractility of V. convallaria during unhindered contraction depends on the stalk length. In this study, the contractile force and energetics of V. convallaria cells of different stalk lengths were evaluated using a fluid dynamic drag model which accounts for the unsteadiness and finite Reynolds number of the water flow caused by contracting V. convallaria and the wall effect of the no-slip substrate. It was found that the contraction displacement, peak contraction speed, peak contractile force, total mechanical work, and peak power depended on the stalk length. The observed stalk-length-dependencies were simulated using a damped spring model, and the model estimated that the average spring constant of the contracting stalk was 1.34 nN µm. These observed length-dependencies of Vorticella's key contractility parameters reflect the biophysical mechanism of the spasmonemal contraction, and thus they should be considered in developing a theoretical model of the Vorticella spasmoneme.

Stalk-length-dependence of the contractility of Vorticella convallaria.

Hyaline cartilage is a specialized type of connective tissue that lines many moveable joints (articular cartilage) and contributes to bone growth (growth plate cartilage). Hyaline cartilage is composed of a single cell type, the chondrocyte, which produces a unique hydrated matrix to resist compressive stress. Although compressive stress has profound effects on transcriptional networks and matrix biosynthesis in chondrocytes, mechanistic relationships between strain, signal transduction, cell metabolism, and matrix production remain superficial. Here, we describe development and validation of a polydimethylsiloxane (PDMS)-based pneumatic microfluidic cell compression device which generates multiple compression conditions in a single platform. The device contained an array of PDMS balloons of different sizes which were actuated by pressurized air, and the balloons compressed chondrocytes cells in alginate hydrogel constructs. Our characterization and testing of the device showed that the developed platform could compress chondrocytes with various magnitudes simultaneously with negligible effect on cell viability. Also, the device is compatible with live cell imaging to probe early effects of compressive stress, and it can be rapidly dismantled to facilitate molecular studies of compressive stress on transcriptional networks. Therefore, the proposed device will enhance the productivity of chondrocyte mechanobiology studies, and it can be applied to study mechanobiology of other cell types.

Article	Year
A new angle on microscopic suspension feeders near boundaries. Biophysical journal\| PubMed ID: 24138855	2013
Three-Dimensional Confocal Microscopy Indentation Method for Hydrogel Elasticity Measurement. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 26270154	2015
A Validation Study of the Repeatability and Accuracy of Atomic Force Microscopy Indentation Using Polyacrylamide Gels and Colloidal Probes. Journal of biomechanical engineering\| PubMed ID: 28005134	2017
Flow and transport effect caused by the stalk contraction cycle of . Biomicrofluidics\| PubMed ID: 28670352	2017
Stalk-length-dependence of the contractility of Vorticella convallaria. Physical biology\| PubMed ID: 28862154	2017
Pneumatic microfluidic cell compression device for high-throughput study of chondrocyte mechanobiology. Lab on a chip\| PubMed ID: 29897088	2018

Sangjin Ryu

Biography

JoVE Journal Publications

Other Publications