Extracellular calcium influences chondrocyte differentiation and synthesis of extracellular matrix. Previously, calcium concentrations ranging from 0.1mM to 2mM have been used in vitro and these ...studies indicated that low calcium concentrations were generally favorable for chondrocyte culture. Our objective was to extend these findings to yet lower calcium concentrations and to comprehensively examine effects on morphology and phenotype in two culture systems.
Serum-free media containing 1mM, 50μM or 15μM of calcium and a serum-containing medium were used to culture chondrocytes in suspension and in monolayer, at high and low inoculation density.
In monolayer, at low and high density, removing serum and decreasing calcium concentration decreased cell spreading and lowered collagen type I expression whereas collagen type II expression remained stable. In suspension, cells aggregated for all media tested; however, aggregates were smaller and looser in the absence of serum.
The serum-free 50μM and 1mM calcium media provide good alternatives to classical media for monolayer culture since both growth and chondrocyte phenotype were maintained. In suspension culture, the serum-free 1mM calcium medium also possesses the beneficial properties of limiting aggregate size while maintaining growth and phenotype.
The study of chondrocyte biology requires culture conditions that maintain cell phenotype. Phenotype is rapidly lost in monolayer but is maintained in 3-dimensional scaffolds, which however, ...experience limited cell proliferation and limited mass transport. In this study, we cultured chondrocytes in aggregates in stirred spinner flask suspension cultures to control aggregate size and promote mass transport. A previously optimized serum-free medium, containing the following growth factors (GFs), epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor, all at 2 ng/mL, was used as a control medium. In addition, two modified media were tested: one containing Pluronic F-68 (PF-68) and the other containing PF-68 with 10 times greater GF concentration (20 ng/mL, medium PF-68/10 x GF). Chondrocytes formed limited-size aggregates within 24 h and exhibited high viability (>95%), and cell concentration doubled in 7 days in the presence of PF-68. Low or no collagen I expression was found for any of the three media, whereas collagen II accumulated between cells, as revealed by a dense immunostaining. Integrin alpha10, a marker of differentiated chondrocytes and chondrogenic cells, was also found to be highly expressed. Aggregates resulting from spinner culture were found to be relevant in vitro models and their use for cartilage repair to be also conceivable.
Sprifermin is a human recombinant fibroblast growth factor 18 (rhFGF18) in clinical development for knee osteoarthritis. Previously, we demonstrated that sprifermin exerts an anabolic effect on ...chondrocytes in 3D culture with cyclic but not permanent exposure. Here, we hypothesized that permanent exposure to sprifermin de-sensitizes the cells. To test this, a combination of Western-blot and cell staining methods was used. We demonstrate that sprifermin is transiently internalized in chondrocytes along with a transient increase in ERK1/2 activation. We also show that sprifermin is intracellularly degraded, probably together with its receptor FGFR3, thus preventing further stimulation. However, incubation without sprifermin re-sensitizes the cells. Finally, we show that sprifermin endocytosis is clathrin- and dynamin-independent and that receptor activation is not necessary for sprifermin's endocytosis. In this study, we link the role of endocytosis to the cell response and elucidate for the first time a de-sensitization phenomenon to a FGF.
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