Coupling plowing of cartilage explants with gene expression in models for synovial joints

Articular cartilage undergoes complex loading modalities generally including sliding, rolling and plowing (i.e. the compression by a condyle normally to the tissue surface under simultaneously tangential displacement, thus generating a tractional force due to tissue deformation). Although in in vivo studies it was shown that excessive plowing can lead to osteoarthritis, little quantitative experimental work on this loading modality and its mechanobiological effects is available in the literature. Therefore, a rolling/plowing explant test system has been developed to study the effect on pristine cartilage of plowing at different perpendicular forces. Cartilage strips harvested from bovine nasal septa of 12-months-old calves were subjected for 2h to a plowing-regime with indenter normal force of 50 or 100 N and a sliding speed of 10 mm s(-1). 50 N produced a tractional force of 1.2±0.3N, whereas 100 N generated a tractional force of 8.0±1.4N. Furthermore, quantitative-real-time polymerase chain reaction experiments showed that TIMP-1 was 2.5x up-regulated after 50 N plowing and 2x after 100 N plowing, indicating an ongoing remodeling process. The expression of collagen type-I was not affected after 50 N plowing but it was up-regulated (6.6x) after 100 N plowing, suggesting a possible progression to an injury stage of the cartilage, as previously reported in cartilage of osteoarthritic patients. We conclude that plowing as performed by our mimetic system at the chosen experimental parameters induces changes in gene expression depending on the tractional force, which, in turn, relates to the applied normal force

Immobilization of an Artificial Imine Reductase within Silica Nanoparticles Improves its Performance

Silica nanoparticles equipped with an artificial imine reductase display remarkable activity towards cyclic imine- and NAD + reduction. The method, based on immobilization and protection of streptavidin on silica nanoparticles, shields the biotinylated metal cofactor against deactivation yielding over 46 000 turnovers in pure samples and 4000 turnovers in crude cellular extracts

Inhibition of osteoclast differentiation and bone resorption by N-methylpyrrolidone

Regulation of RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption

Design, construction and validation of a computer controlled system for functional loading of soft tissue

Osteoarthritis is a chronic degenerative disease affecting body joints. Abnormal mechanical loading could be an initiating factor of cartilage damage, by influencing chondrocytes activity. To date, devices performing mechanical studies of viable tissues are mostly uniaxial. In this work, we developed and validated a multi-axial device for static and dynamic mechanical testing of viable soft tissues. The system, named RPETS, is composed of a motor driven indenter, moving vertically and horizontally along the bottom of a tank containing tissue samples and it can apply combined compression, sliding, and rolling on viable samples. Validation studies were performed with standard rubber and bovine nasal cartilage tissue. Static tests demonstrated that the system is comparable to existing uniaxial devices, with a maximum force control error smaller than 0.5N and a positioning resolution of 5μm. Dynamic tests performed with different motion profiles showed that the system can exert a load of 100N with a maximum velocity of 100mm/s maintaining the force control error within 10% of the desired value. Sinusoidal motion frequency can vary between 0.05 and 0.5Hz. In practical tests, viability staining of dynamically loaded cartilage slices showed extents of cell death to depend on the indenter velocity

Partially shielded enzymes capable of processing large protein substrates.

We report the first method of enzyme protection enabling the production of partially shielded enzymes capable of processing substrates as large as proteins. We show that partially shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on antibodies. Moreover, a partially shielded trypsin is shown to outperform its soluble counterpart in terms of proteolytic kinetics. Remarkably, partial enzyme shielding results in a drastic increase in temporal stability of the enzyme