NFAT1 deficiency provokes hypertrophic repair of articular cartilage defects and progression of posttraumatic osteoarthritis

Abstract

molecules by a cell-based high-throughput screening (HTS) in human chondrocytes. Methods: To induce cellular senescence, immortalized human chondrocytes (TC28a2) were seeded (5000 cells/well) in 384 well plates, and treated with IL-6 (10 ng/ml) for 24 hours to induce cellular senescence or defective autophagy. Then, chondrocytes were incubated with Prestwick Chemical Library (1280 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 mM for 48 hours. Chondrocytes were incubated for 20 min with 4% formaldehyde and washed 3 times with PBS. Then, nuclei was stained with Hoechst 33342 (10 mM), while b-galactosidase subcellular structures and autophagy vacuoles were stained by using Imagene Green C12FDG substrate (10 mM) and Cyto-ID® (1 mM), respectively. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Image analysis was performed by Harmony software. Relative intensity of C12FDG in cytoplasm and number of autophagosomes per area of cytoplasm were determined to quantitate b-galactosidase activity and autophagy flux respectively. Low signal of senescence was found for chloroquine (30 mM) and used as reference for anti-senescence activity. High signal of autophagy flux was found for Rapamycin (5 mM) and used as reference for proautophagy activity. Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated bgalactosidase activity. From the total number of 1280 small molecules, 299 compounds with anti-senescence effects were identified by HTS. A secondary screening with the 299 compounds was performed for "cherry picking". Finally, 216 compounds were confirmed to have antisenescence activity. The anti-senescence compounds were analyzed by monitoring autophagy flux. 38 compounds with both anti-senescence and pro-autophagy effects were selected. For compound validation, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. LC3 reporter chondrocytes can be used to determine autophagy activation by color changes upon stimulation. The preliminary results indicate a subset of compounds selectively induced LC3-associated autophagy activation. Conclusions: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.

The Induced Pluripotent Stem Cells in Articular Cartilage Regeneration and Disease Modelling: Are We Ready for Their Clinical Use?

“…These conditions were recently used and modified by different teams of researchers, as discussed below. Another aspect concerns obtaining chondrocytes without exhibiting hypertrophy or terminal maturation over time, leading to ossification and degradation of the repaired cartilage tissue and, consequently, an advance of the disease [100,101]. The following part of this review will focus on differentiation protocols of iPS cells into chondrogenic populations using directed methods or through an embryoid bodies (EBs) step and additional stimuli to improve the formation of cells of a specific germ layer, methods using a cell-scaffold combination, and methods using physical/chemical agents during differentiation (Figure 1 and Table 2).…”

Rejuvenated Stem/Progenitor Cells for Cartilage Repair Using the Pluripotent Stem Cell Technology

“…Thus, attempts to manipulate local milieu for the endogenous or exogenous therapeutic cells using known biologics have led to significant benefits in regeneration of hyaline cartilage in repair sites, which are however mostly based on relatively short-term (4–12 weeks) observations. Therefore, some of the regenerated hyaline cartilaginous tissues may not be permanent cartilage in that in a longer term, chondrocytes in the repair site may die or be committed to endochondral ossification: i.e., hypertrophic differentiation, terminal maturation and mineralization, leading to the degradation of repair tissue [ 50 ] and the formation of osteophytes [ 51 ]. Interestingly, death of superficial chondrocytes protects articular cartilage from joint destabilization-induced degradation [ 52 ], but loss of maturation arrest in living articular chondrocytes is associated with the onset of osteoarthritis [ 53 ].…”