Mechanical injury and cytokines cause loss of cartilage integrity and upregulate proteins associated with catabolism, immunity, inflammation, and repair.

The objectives of this study were to perform a quantitative comparison of proteins released from cartilage explants in response to treatment with IL-1beta, TNF-alpha, or mechanical compression injury in vitro and to interpret this release in the context of anabolic-catabolic shifts known to occur in cartilage in response to these insults in vitro and their implications in vivo. Bovine calf cartilage explants from 6-12 animals were subjected to injurious compression, TNF-alpha (100 ng/ml), IL-1beta (10 ng/ml), or no treatment and cultured for 5 days in equal volumes of medium. The pooled medium from each of these four conditions was labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time-of-flight instrument.

Modulation of lubricin biosynthesis and tissue surface properties following cartilage mechanical injury.

Objective: To evaluate the effects of injurious compression on the biosynthesis of lubricin at different depths within articular cartilage and to examine alterations in structure and function of the articular surface following mechanical injury.

Methods: Bovine cartilage explants were subdivided into level 1, with intact articular surface, and level 2, containing middle and deep zone cartilage. Following mechanical injury, lubricin messenger RNA (mRNA) levels were monitored by quantitative reverse transcriptase-polymerase chain reaction, and soluble or cartilage-associated lubricin protein was analyzed by Western blotting and immunohistochemistry.

A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins.

Microfabricated regular sieving structures hold great promise as an alternative to gels to improve the speed and resolution of biomolecule separation. In contrast to disordered porous gel networks, these regular structures also provide well defined environments ideal for the study of molecular dynamics in confining spaces. However, the use of regular sieving structures has, to date, been limited to the separation of long DNA molecules, however separation of smaller, physiologically relevant macromolecules, such as proteins, still remains a challenge.

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis-liquid chromatography tandem mass spectrometry analysis of bovine cartilage tissue response to mechanical compression injury and the inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta.

Objective: To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), by characterizing proteins lost to the medium from cartilage explant culture.

Methods: Cartilage explants from young bovine stifle joints were treated with 10 ng/ml of IL-1beta or 100 ng/ml of TNFalpha or were subjected to uniaxial, radially-unconfined injurious compression (50% strain; 100%/second strain rate) and were then cultured for 5 days. Pooled media were subjected to gel-based separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and analysis by liquid chromatography tandem mass spectrometry, and the data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, expression levels, and matrix protein proteolysis.

Continuous-flow pI-based sorting of proteins and peptides in a microfluidic chip using diffusion potential.

Efficient sample preparation tools are the key to measuring molecular signals in a complex biological system. A novel continuous-flow isoelectric point (pI)-based sorting technique has been developed for proteins and peptides in a microfluidic chip format. It can sort biomolecules at a relatively high flow rate of up to 10 microL/min and does not require carrier ampholytes, which can create molecular backgrounds for subsequent analysis.

Million-fold preconcentration of proteins and peptides by nanofluidic filter.

We have developed a highly efficient microfluidic sample preconcentration device based on the electrokinetic trapping mechanism enabled by nanofluidic filters. The device, fabricated by standard photolithography and etching techniques, generates an extended space charge region within a microchannel, which was used to both collect and trap the molecules efficiently. The electrokinetic trapping and collection can be maintained for several hours, and concentration factors as high as 10(6)-10(8) have been demonstrated.