Biochemical alterations in inflammatory reactive chondrocytes: evidence for intercellular network communication.

Chondrocytes are effectively involved in the pathophysiological processes of inflammation in joints. They form cellular processes in the superficial layer of the articular cartilage and form gap junction coupled syncytium to facilitate cell-to-cell communication. However, very little is known about their physiological cellular identity and communication. The aim with the present work is to evaluate the physiological behavior after stimulation with the inflammatory inducers interleukin-1ß and lipopolysaccharide. The cytoskeleton integrity and intracellular Ca2+ release were assessed as indicators of inflammatory state. Cytoskeleton integrity was analyzed through cartilage oligomeric matrix protein and actin labeling with an Alexa 488-conjugated phalloidin probe. Ca2+ responses were assessed through the Ca2+ sensitive fluorophore Fura-2/AM. Western blot analyses of several inflammatory markers were performed. The results show reorganization of the actin filaments. Glutamate, 5-hydoxytryptamine, and ATP evoked intracellular Ca2+ release changed from single peaks to oscillations after inflammatory induction in the chondrocytes. The expression of toll-like receptor 4, the glutamate transporters GLAST and GLT-1, and the matrix metalloproteinase-13 increased. This work demonstrates that chondrocytes are a key part in conditions that lead to inflammation in the cartilage. The inflammatory inducers modulate the cytoskeleton, the Ca2+ signaling, and several inflammatory parameters. In conclusion, our data show that the cellular responses to inflammatory insults from healthy and inflammatory chondrocytes resemble those previously observed in astrocyte and cardiac fibroblasts networks.

Gene expression profiling of peri-implant healing of PLGA-Li+ implants suggests an activated Wnt signaling pathway in vivo.

Bone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li+). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li+, while PLGA without Li+ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li+ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li+, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li+ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li+. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li+ compared with Ctrl. The presence of ß-catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors' knowledge the first report evaluating the effect of a local release of Li+ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li+ in PLGA implants, Li+ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway.

Cell viability and chondrogenic differentiation capability of human mesenchymal stem cells after iron labeling with iron sucrose.

For evaluation of cell therapy strategies using human mesenchymal stem cells (hMSCs), it is important to be able to trace transplanted cells and their distribution in tissues, for example, cartilage, over time. The aim of the study was to determine effects on cell viability, traceability, and chondrogenic differentiation of hMSCs after iron labeling with iron sucrose. hMSCs were collected (seven donors, 13-57 years) from patients undergoing spinal surgery. Two subsets of experiments were performed. (1) Iron labeling of hMSCs: 1 mg/mL of Venofer(®) (iron sucrose) was added (16 h) to cultures. hMSCs were examined for uptake of iron sucrose (Prussian blue staining) and cell viability (flow cytometry). (2) Iron-labeled hMSCs (passage 4) (n=4, pellet mass), 200,000 cells/tube, were cultured (DMEM-HG) with 10 ng/mL TGFß and compared with controls (from each donor). The pellets were harvested at days 7, 14, and 28. Real-time PCR, IHC, and histology were used to evaluate SOX9, ACAN, C6S, and COL2A1 expression. Mean number of cells containing iron deposits was 98.1% and mean cell viability was 92.7% (no significant difference compared with unlabeled control cells). Pellets containing iron-labeled cells expressed COL2A1 on protein level (all time points), in similar levels as controls, and glycosaminoglycan accumulation was observed in iron-labeled pellets (day 14 or day 28). Results were supported by the expression of chondrogenic genes SOX9, ACAN, and COL2A1. The results in vitro indicate that iron sucrose can be used as a cell tracer for evaluation of cellular distribution in vivo after transplantation of MSCs and thus contribute with important knowledge when exploring new treatment strategies for degenerated cartilaginous tissues.

Effects of interleukin-6 and interleukin-1ß on expression of growth differentiation factor-5 and Wnt signaling pathway genes in equine chondrocytes.

OBJECTIVE: To determine the effects of interleukin (IL)-6 and IL-1ß stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. SAMPLE: Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. PROCEDURES: Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1ß for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3ß (GSK-3ß), and ß-catenin proteins in macroscopically normal cartilage samples and OCFs. RESULTS: Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3ß and coiled-coil domain containing 88C increased after 1 hour and expression of ß-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. CONCLUSION AND CLINICAL RELEVANCE: Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator ß-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.

Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach.

Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.