A Wnt/ß-catenin negative feedback loop inhibits IL-1-induced MMP expression in human articular chondrocytes.

Objective: Recent animal studies suggest that activation of Wnt/β-catenin signaling in articular chondrocytes might be a driving factor in the pathogenesis of osteoarthritis (OA) by stimulating amongst others the expression of matrix metalloproteinases (MMPs). This study aimed to investigate the role of Wnt/β-catenin signaling in IL-1β-induced MMP expression in human chondrocytes. Methods: Primary cultures of human, mouse and bovine articular chondrocytes as well as human mesenchymal stem cells (hMSCs) and mouse embryonic fibroblasts (MEFs) were used. Multiple strategies for activation and inhibition of signaling pathways were used. Reporter assays and co-immunoprecipitations were used to study the interaction between β-catenin and NF-κB. Results: In contrast to animal chondrocytes, in human chondrocytes Wnt/β-catenin is a potent inhibitor of MMP1, -3 and -13 expression and generic MMP activity both in basal conditions and after IL-1β stimulation. This effect is independent of TCF/LEF transcription factors but is due to an inhibitory protein-protein interaction between β-catenin and NF-κB. Furthermore we show that IL-1β indirectly activates β-catenin signaling by inducing canonical Wnt7B expression and by inhibiting the expression of canonical Wnt antagonists. Conclusion: Our data reveal an unexpected anti-catabolic role of Wnt/β-catenin signaling in human chondrocytes by counteracting NF-κB-mediated MMP expression induced by IL-1β in a negative feedback loop

Distinct Effect of TCF4 on the NFκB Pathway in Human Primary Chondrocytes and the C20/A4 Chondrocyte Cell Line

Objective: Previous studies indicated a difference in crosstalk between canonical WNT pathway and nuclear factor-κB (NFκB) signaling in human and animal chondrocytes. To assess whether the differences found were dependent on cell types used, we tested the effect of WNT modulation on NFκB signaling in human primary articular chondrocytes in comparison with the immortalized human costal chondrocyte cell line C20/A4. Design: We used gene expression analysis to study the effect of WNT modulation on IL1β-induced matrix metalloproteinase (MMP) expression as well as on WNT and NFκB target gene expression. In addition, we tested the involvement of RelA and TCF4 on activation of the WNT and NFκB pathway by TCF/LEF and NFκB reporter experiments, respectively. Results: We found an inhibitory effect of both induction and inhibition of WNT signaling on IL1β-induced MMP mRNA expression in primary chondrocytes, whereas WNT modulation did not affect MMP expression in C20/A4 cells. Furthermore, TCF/LEF and NFκB reporter activation and WNT and NFκB target gene expression were regulated differentially by TCF4 and RelA in a cell type–dependent manner. Additionally, we found significantly higher mRNA and protein expression of TCF4 and RelA in C20/A4 cells in comparison with primary chondrocytes. Conclusions: We conclude that WNT modulation of NFκB is, at least in part, cell type dependent and that the observed differences are likely because of impaired sensitivity of the NFκB pathway in C20/A4 cells to modulations in WNT signaling. This might be caused by higher basal levels of TCF4 and RelA in C20/A4 cells compared to primary chondrocytes

Raman microspectroscopy: A non-invasive analysis tool for monitoring of collagen-containing extracellular matrix formation in a medium-throughput culture system

The three-dimensional environment is known to play an important role in promoting cell–matrix interactions. We have investigated the possibility of using Raman microspectroscopy—which has the great advantage of noninvasive sensing—for in vitro monitoring of extracellular matrix (ECM) formation in a medium-throughput pellet (3D) culture system with soft-litography, agarose-microwell arrays. Chondrocytes were seeded in the agarose microwells in basic or chondrocyte medium. After 3, 7, and 14 days of culture, samples were analyzed for ECM formation by Raman microspectroscopy, histology, and immunofluorescence. ECM formation in the chondrocyte medium-cultured samples was detected by histology and immunofluorescence, and also noninvasively by Raman microspectroscopy. The Raman band of collagen found at 937 cm−1 can be used as a Raman marker for collagen-containing ECM formation over time in the chondrocyte pellets. This culture system can be implemented as a medium-throughput platform for Raman applications and screening microtissue formation, since with these agarose-microwell arrays relatively large numbers of cell pellets could be screened in a short time in situ, without having to transfer the pellets onto microscopic slides. Moreover, in this manner the culture system is suitable for long-term, real-time live-cell measurements

Nanoparticle system for the local delivery of disease modifying osteoarthritic drugs

Purpose: The purpose of this study is to develop the nanoparticles that i) can be injected intra-articularly ii) target to cartilage due to an opposite charge difference with the extracellular cartilaginous matrix and iii) due to their small size can penetrate into the cartilage. In this way retention time in the joint can be prolonged. By releasing disease modifying OA drugs (DMOAD) in the vicinity of chondrocytes such materials may be beneficial for restoring cartilage tissue homeostasis. Here we demonstrate the generation of drug-containing nanoparticles for intra-articular joint therapy. Methods: We have prepared nanoparticles of biodegradable poly ethylene glycol- poly lactic acid PEG-PLA co-block polymers. The hydrophilic PEG and hydrophobic PLA ends of this polymer make it possible to generate micelles that contain drugs. The polymers are functionalized with UV-sensitive acrylate groups that can be stabilized by UV-crosslinking. These drug containing nanoparticles will be used for intra-articular joint injection and release of DMOADs. We have also established co-culture systems in vitro using MSCs and chondrocytes where the effect of these molecules and nanocarriers can be tested. Results: Micelle type nanoparticles using PEG-PLA co-block polymers were prepared. The obtained dexamethasone loaded nanoparticles had diameters of 20-80 nm. These nanoparticles are photo-crosslinked at their hydrophobic cores which provides stability to the structure and resulted in a slight decrease in average particle size . Dexamethasone was successfully encapsulated in these nanoparticles. The current release profiles show initial burst release in the first 8 hours followed by a sustained release over at least 3 days. Conclusions: We have generated nanoparticles that can serve as a carrier system to deliver clinically relevant disease modifying osteoarthritic drugs in a more effective way after intra-articular injection. We are currently investigating the retention of nanoparticles in the joint and are developing strategies to target these particles to cartilag

Cartilage adhesive and mechanical properties of enzymatically crosslinked polysaccharide tyramine conjugate hydrogels

Using a home-built tensile tester, adhesion and mechanical properties of injectable enzymatically crosslinkable hydrogels were determined by placing the hydrogels in between cartilage surfaces. Dextran–tyramine (Dex-TA) and hyaluronic acid–tyramine (HA-TA) conjugates as well as a 50/50 composite material of these polysaccharide conjugates were tested. To integrate the injectable hydrogels with the cartilage tissue, pretreatment of the tissue with a Dex-TA conjugate solution strongly improved the adhesion. Only failure of the crosslinked hydrogel was observed and not at the hydrogel–tissue interface. Moduli of a Dex-TA hydrogel are higher than those of a HA-TA hydrogel, whereas the ultimate strain of the HA-TA hydrogel was at least three times higher. The Dex-TA/HA-TA hydrogel has similar storage and elastic moduli as the Dex-TA gel and also an ultimate strain of ~30%, similarly as found for the HA-TA gel. The controlled biodegradability and gelation time of the Dex-TA/HA-TA hydrogel, the developed method for strong tissue adhesion of the gel particularly in comparison with fibrin glue, makes this material applicable as an injectable hydrogel for tissue regeneration application