Fig 2: PACAP and/or mechanical load altered the collagen production of HDC. PACAP 1–38 at 100 nM was applied continuously from day one. Short time, transient MS, was applied on days two and three for 30 min on both days. (A) Collagen production of HDC was determined by 3H-collagen incorporation; (B) RNA expression of collagen type II (Col2a1), collagen type IX (Col9a1) and collagen type X (Col10a1) on day three. GAPDH was used as a control; (D) Protein expression of collagen type II, collagen type IX, collagen type X on day three. Actin was used as a control. For panels (B,D) numbers below signals represent integrated densities of signals determined by ImageJ software. Representative data of three independent experiments are shown; (C,E) Statistical analysis of RT-PCR and Western blot data. All data presented are the averages of at least three different experiments. Statistical analysis was performed by Student’s t-test. All data were normalized on GAPDH/Actin and data are expressed as mean ± SEM. (C, control; P1–38, PACAP 1–38; MS; mechanical stimulus). Statistically significant difference between collagen production rate of cells in cultures that received the loading regime and/or PACAP 1–38 vs. control cultures is marked by asterisk (* p < 0.05) in all panels.

Fig 3: Heterogenous multicellular WNT signaling models.A, B RT-qPCR and GAG/DNA ratios of pellets treated with various WNTs during pellet culture. Different letters are significantly different from each other (p < 0.05). Mean ± SEM. n = 3–4 pellets per group. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. C WNT treatment increased infiltration of off-target cells (pink arrowheads and white dashed lines) into the pellets, decreased COL2A1 staining, but increased COL1A1 (yellow arrowheads) and COL10A1 staining in the pellets. The pellets with C59 treatment exhibited homogenous COL2A1 staining and decreased COL1A1 and COL10A1 staining. Scale bar = 0.2 mm. The experiment was performed twice with similar results. D Heatmap showing distinct expression levels of various WNTs in various cellular subpopulations in d14 TGF-ß3-treated pellets. A total of 2148 cells from the TGF-ß3-treated d14 pellets was analyzed and used to generate the heatmap. E Percentage of the cells expressing WNT3A and its putative receptors in d14 TGF-ß3-treated pellets. F, G Heterogenous multicellular signaling models in d14 TGF-ß3-treated pellets.

Fig 4: Mutant TRPV4 had little effect on chondrogenic matrix production.(A) Wildtype (WT), V620I, and T89I day-28 pellets exhibit similar matrix production shown by staining for sulfated glycosaminoglycans (sGAGs) with Safranin-O and hematoxylin and labeling with immunohistochemistry (IHC) for (B) COL2A1 (C), COL1A1 (D), and COL10A1. Scale bar = 500 µm. Representative images from 3 to 4 differentiations. (E) The sGAG/DNA ratio increased in all three lines from day 14 to 28 of chondrogenesis. There were no differences between lines at each time point. Mean ± standard error of the mean (SEM). n = 11–16 from 3 to 4 independent differentiation experiments. ****p < 0.0001 Statistical significance determined by an ordinary two-way analysis of variance (ANOVA) with Tukey’s post hoc test. (F) There were no differences in the elastic modulus of the matrix at day 28. Mean ± SEM. n = 11–14 from 3 experiments. Statistical significance determined by an ordinary two-way ANOVA with Tukey’s post hoc test.

Fig 5: V620I and T89I exhibited differing effects on gene expression during chondrogenic differentiation.(A) V620I and T89I had increased ACAN gene expression at day 28 compared to wildtype (WT). (B) The three lines had similar COL2A1 expression throughout differentiation. V620I increased expression of (C) SOX9 and (D) TRPV4 throughout chondrogenesis. T89I increased expression of (E) S100B and (F) COL1A1 throughout chondrogenesis. (G) Both mutations decreased COL10A1 gene expression at day 28 compared to WT. (H) There were no differences in FST expression at day 28. Mean ± standard error of the mean (SEM). n = 10–12 from 3 independent differentiation experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Significance determined by one-way analysis of variance (ANOVA) with Tukey’s post hoc test for each time point.