High-Throughput, Temporal and Dose Dependent, Effect of Vitamins and Minerals on Chondrogenesis

Abstract

Tissue engineered hyaline cartilage is plagued by poor mechanical properties largely due to inadequate type II collagen expression. Of note, commonly used defined chondrogenic media lack 14 vitamins and minerals, some of which are implicated in chondrogenesis. Type II collagen promoter-driven Gaussia luciferase was transfected into ATDC5 cells to create a chondrogenic cell with a secreted-reporter. The reporter cells were used in an aggregate-based chondrogenic culture model to develop a high-throughput analytic platform. This high-throughput platform was used to assess the effect of vitamins and minerals, alone and in combination with TGFβ1, on COL2A1 promoter-driven expression. Significant combinatorial effects between vitamins, minerals, and TGFβ1 in terms of COL2A1 promoter-driven expression and metabolism were discovered. An "optimal" continual supplement of copper and vitamin K in the presence of TGFβ1 gave a 2.5-fold increase in COL2A1 promoter-driven expression over TGFβ1 supplemented media alone in ATDC5 cells.

Keywords: cartilage tissue engineering; defined chondrogenic media; defined media; high-throughput chondrogenesis; high-throughput organoid; in vitro chondrogenesis.

FIGURE 1

Oxygen and cell density response. Dose response curves, normalized to the control of 0 ng/ml TGFβ1 in chondrogenic media, were constructed and show a time dependent increase in luminescence but with similar responses in terms of both cell number and oxygen tension. (A) 20% O₂, 100,000 cells; (B) 5% O₂, 100,000 cells; (C) 20% O₂, 50,000 cells; (D) 5% O₂, 50,000 cells. * = all concentrations significantly greater than basal media, £ = concentrations ≥ 2 ng/ml TGFβ1 significantly greater than basal media (p < 0.05).

FIGURE 2

Time and dose dependent effect of TGFβ1 on chondrogenesis. Dose response curves, normalized to the control of 0 ng/mL in chondrogenic media, were constructed and show a time dependent increase in luminescence but with similar EC50s, a dotted line is shown to show 20 pg/mL in the log scale.

FIGURE 3

Type II collagen promoter-driven expression with various vitamin and mineral combinations in the presence of TGFβ1 on day 21. Vitamins and minerals (biotin, copper, iodine, thyroxine, and zinc) which had been identified as promoting COL2A1 promoter-driven expression in the initial screen were tested in combination at their individually optimized concentrations (3, 670, 0.92, 20, and 840 μg/L, respectively) in the presence of TGFβ1 at 1 ng/mL. The values are normalized to the plate control (basal media). Green circles () indicate values that are significantly greater than those for basal media alone, and the dashed red line indicates the luminescence achieved with TGFβ1 supplementation at 1 ng/mL. An orange triangle () and an * indicate values that were discovered different, with false discovery rate set at 1%.

FIGURE 4

Combinations of chromium, vitamins B12, E, and K in supplemented media at day 21. Vitamins and minerals (chromium, vitamins B12, E, and K) which had been identified as promoting COL2A1 promoter-driven expression in the initial screen were tested alone and in combination in supplemented medias 1 (A) Basal media + TGFβ1; 2 (B) Basal + TGFβ1 + biotin + iodine; 3 (C) Basal + TGFβ1 + copper; 4 (D) Basal + TGFβ1 + iodine + thyroxine + copper. Vitamins and minerals were supplemented at their individually optimized concentration in basal media (2.8 μg/L, 91.4 ng/L, 1.84 μg/L, and 2.2 μg/L, respectively). The values are normalized to the plate control (basal media); the red dashed red line indicates the luminescence achieved with that particular supplemented media alone. Green circles indicate values that are not significantly different to supplemented media (red dashed line), all are greater than basal media. Conditions that were statistically greater than their respective supplemented media are represented by an orange triangle () and an *, black symbols (▼) indicate that those values were lower than the respective supplemented media. n ≥ 3 ± S.D.

FIGURE 5

Day 21 evaluation of COL2A1 promoter-driven expression in vitamin and mineral supplemented TGFβ1 supplemented basal medium. Conditioned media was sampled at day 21 and assessed for luminescence, and normalized against the plate control basal medium. The red dashed line indicates the luminescence of the TGFβ1 supplemented basal medium. The symbol “*” significantly different vs. TGFβ1 supplemented basal medium, two-way ANOVA p < 0.05 Sidak’s multiple comparison test. Blue circles represent each data point.

FIGURE 6

Comparison of Vitamin B12 and TGFβ1 supplemented medium with TGFβ1 supplemented basal medium. Type II collagen promoter-driven luminescence was assessed from the conditioned media and normalized to the plate basal medium control. Lines indicate a linear regression; symbols indicate individual data points. The symbol “*” significant difference vs. TGFβ1 supplemented basal medium; two-way ANOVA p < 0.05 Sidak’s multiple comparison test.

FIGURE 7

Effect of vitamin and mineral addition to TGFβ1 supplemented medium on metabolic activity. Cells were treated with resazurin, media was sampled at day 21 and assessed for fluorescence. All data were normalized against the conditioned media from pellets treated with basal chondrogenic media. The red dashed line indicates the fluorescence of the TGFβ1 supplemented basal medium. An orange triangle () and an * indicate greater than TGFβ1 supplemented basal medium; black triangles (▼) indicate lower vs. TGFβ1 supplemented basal medium, one-way ANOVA p < 0.05 Dunnett’s multiple comparison test. Conditions that were similar to TGFβ1 supplemented basal media are represented by a green circle ().