Phosphate regulates chondrogenesis in a biphasic and maturation-dependent manner

Abstract

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) ATDC5 chondrogenic models treated with ITS+, β-glycerophosphate (βGP), or ITS+/βGP, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Three levels of abundance were found: basal (Pi/DNA < 10 ng/μg), moderate (Pi/DNA=25.3 – 32.3 ng/μg), and high abundance (Pi/DNA > 60 ng/μg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM βGP resulted in moderate abundance in 2D and high in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters also reduced expression of chondrogenic markers. When 3D ITS+/βGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

3D spheroid culture models for chondrocytes using polyethylene glycol-coated microfabricated chip

“…Based on these results, we fabricated a multi-well device allowing for stable spheroid formation from a large number of ATDC5 cells (3 × 10 5 cells/well). The number of ATDC5 cells required for spheroid formation was comparable to a previous report which produced 3D micromass pellet from 2.5 × 10 5 cells (Wu et al 2017). Furthermore, the spheroid formation in our devices showed a high reproducibility sequential process similar to that observed during chondrocyte differentiation (Atsumi et al 1990), it has been used as a suitable in vitro cell culture model to elucidate the mechanisms of chondrogenesis, including the demonstration of the ideal biomimetic microenvironment for chondrogenesis in 3D spheroid cultures.…”

Exogenous Stimulation of Human Intervertebral Disc Cells in 3-Dimensional Alginate Bead Culture With BMP2 and L51P: Cytocompatibility and Effects on Cell Phenotype

“…Continuing stimulation with the OM might decrease chondrogenic differentiation and instead induce matrix mineralization. Wu et al, 34 also observed this when stimulating ATDC5 cells with β-glycerophosphate; they confirmed early chondrogenesis with an upregulation of COL2 and ACAN and the production of a GAGrich matrix within the first 7 days. Beta-glycerophosphate delivery after early chondrogenesis further upregulated hypertrophic markers.…”

“…Beta-glycerophosphate delivery after early chondrogenesis further upregulated hypertrophic markers. 34 GAG content was significantly higher in AFCs and CEPCs stimulated with BM + BMP2 and L51P than in BM control treatment. The same was observed for DNA content.…”

A Synthetic, Closed-Looped Gene Circuit for the Autonomous Regulation of RUNX2 Activity during Chondrogenesis

“…To assess the accumulation of cartilage-specific matrix in both 2D and 3D cultures, the level of sGAG of each sample was quantified using DMMB assay as previously described 25,29 . Specifically, differentiated cell masses from 2D cultures or pellets from 3D cultures were washed with ice-cold PBS before being digested with 1mg/ml proteinase K in 200-500µl ammonium acetate (0.1M) at 50°C for 16 hours.…”

“…6a and Fig. S1): minimum RUNX2 protein expression (D4), just prior to rapid increase in RUNX2 protein expression (D7), peak gene expression of Col10a1 (D14) 25 , and the peak of RUNX2 protein expression (D21). These were compared to cultures that received no Dox or were treated with Dox from day 0 (D0).…”