Age-related nanostructural and nanomechanical changes of individual human cartilage aggrecan monomers and their glycosaminoglycan side chains.

The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecan was significantly weaker in compression than newborn aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of aggrecan, with direct implications for the effects of aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.

Time-dependent nanomechanics of cartilage.

In this study, atomic force microscopy-based dynamic oscillatory and force-relaxation indentation was employed to quantify the time-dependent nanomechanics of native (untreated) and proteoglycan (PG)-depleted cartilage disks, including indentation modulus E(ind), force-relaxation time constant τ, magnitude of dynamic complex modulus |E(∗)|, phase angle δ between force and indentation depth, storage modulus E', and loss modulus E″. At ∼2 nm dynamic deformation amplitude, |E(∗)| increased significantly with frequency from 0.22 ± 0.02 MPa (1 Hz) to 0.77 ± 0.10 MPa (316 Hz), accompanied by an increase in δ (energy dissipation). At this length scale, the energy dissipation mechanisms were deconvoluted: the dynamic frequency dependence was primarily governed by the fluid-flow-induced poroelasticity, whereas the long-time force relaxation reflected flow-independent viscoelasticity. After PG depletion, the change in the frequency response of |E(∗)| and δ was consistent with an increase in cartilage local hydraulic permeability. Although untreated disks showed only slight dynamic amplitude-dependent behavior, PG-depleted disks showed great amplitude-enhanced energy dissipation, possibly due to additional viscoelastic mechanisms. Hence, in addition to functioning as a primary determinant of cartilage compressive stiffness and hydraulic permeability, the presence of aggrecan minimized the amplitude dependence of |E(∗)| at nanometer-scale deformation.

Optimizing micromixer design for enhancing dielectrophoretic microconcentrator performance.

We present an investigation into optimizing micromixer design for enhancing dielectrophoretic (DEP) microconcentrator performance. DEP-based microconcentrators use the dielectrophoretic force to collect particles on electrodes. Because the DEP force generated by electrodes decays rapidly away from the electrodes, DEP-based microconcentrators are only effective at capturing particles from a limited cross section of the input liquid stream. Adding a mixer can circulate the input liquid, increasing the probability that particles will drift near the electrodes for capture. Because mixers for DEP-based microconcentrators aim to circulate particles, rather than mix two species, design specifications for such mixers may be significantly different from that for conventional mixers. Here we investigated the performance of patterned-groove micromixers on particle trapping efficiency in DEP-based microconcentrators numerically and experimentally. We used modeling software to simulate the particle motion due to various forces on the particle (DEP, hydrodynamic, etc.), allowing us to predict trapping efficiency. We also conducted trapping experiments and measured the capture efficiency of different micromixer configurations, including the slanted groove, staggered herringbone, and herringbone mixers. Finally, we used these analyses to illustrate the design principles of mixers for DEP-based concentrators.

Microfluidic arrays for logarithmically perfused embryonic stem cell culture.

We present a microfluidic device for culturing adherent cells over a logarithmic range of flow rates. The device sets flow rates through four separate cell-culture chambers using syringe-driven flow and a network of fluidic resistances. The design is easy to fabricate with no on-chip valves and is scalable both in the number of culture chambers as well as in the range of applied flow rates. Using particle velocimetry, we have characterized the flow-rate range. We have also demonstrated an extension of the design that combines the logarithmic flow-rate functionality with a logarithmic concentration gradient across the array. Using fluorescence measurements we have verified that a logarithmic concentration gradient was established in the extended device. Compared with static cell culture, both devices enable greater control over the soluble microenvironment by controlling the transport of molecules to and away from the cells. This approach is particularly relevant for cell types such as embryonic stem cells (ESCs) which are especially sensitive to the microenvironment. We have demonstrated for the first time culture of murine ESCs (mESCs) in continuous, logarithmically scaled perfusion for 4 days, with flow rates varying >300x across the array. Cells grown in the slowest flow rate did not proliferate, while colonies grown in higher flow rates exhibited healthy round morphology. We have also demonstrated logarithmically scaled continuous perfusion culture of 3T3 fibroblasts for 3 days, with proliferation at all flow rates except the slowest rate.