Fig 2: Cell-to-cell communication analysis revealing stromal cells differentiate towards ossification by interacting with immune cells. (A) Cellphone DB analysis showing many interactions between immune cells and stromal cells 3, 4, 5. (B) Human degenerative NP cells were treated with TNF-a for 24 hours to explore the expression of ossification markers (COL1A1 and RUNX2) by immunofluorescence staining. PBS was used as a negative control. Scale bar, 500 µm. n=5, * vs control, p<0.05. (C) Human degenerative NP cells were treated with CD74 to explore the expression of ossification markers (COL1A1 and RUNX2) by immunofluorescence staining. PBS was used as a negative control. Scale bar, 500 µm. n=5, * vs control, p<0.05. (D) Human degenerative NP cells were treated with CCL-3 and CCL-2 to explore the expression of ossification markers (COL1A1 and RUNX2) by immunofluorescence staining. PBS was used as a negative control. Scale bar, 250 µm. n=5, * vs control, p<0.05. (E) Human degenerative NP cells were treated with TNF-a and MAPK inhibitor (U0126) to explore the expression of ossification markers (COL1A1 and RUNX2) by immunofluorescence staining. PBS was used as a negative control. Scale bar, 500 µm. n=5, * vs control, p<0.05. ** p<0.01; *** p<0.001.

Fig 3: Inhibition of TNF-a in the established rat disc degeneration model. (A) The expression of TNF-a in the established rat disc degeneration model. (B)The expression of ossification markers (COL1A1 and RUNX2) after inhibiting the role of TNF-a by administration of infliximab. (C) HE staining of intervertebral disc after inhibiting the role of TNF-a by administration of infliximab. (D) Safranin O-fast green staining of intervertebral disc after inhibiting the role of TNF-a by administration of infliximab. (E) X rays of rat coccyx disc after inhibiting the role of TNF-a by administration of infliximab. (F) MRI imaging of rat coccyx disc after inhibiting the role of TNF-a by administration of infliximab. AFP, annulus fibrous puncture. *p<0.05.

Fig 4: Wildtype (WT) chondrocytes are more sensitive to BMP4 treatment.(A) WT chondrocytes treated with BMP4 developed a hypertrophic phenotype with enlarged lacunae, which was not present in the mutant cell lines or other conditions, as shown by Safranin-O and hematoxylin staining. Scale bar = 500 µm. Representative images from 2 experiments. (B) Cell diameter was significantly increased in the WT with BMP4 treatment compared to all other groups indicating a hypertrophic phenotype. Mean ± standard error of the mean (SEM). n = 249–304 cells from 2 pellets. Different letters indicate statistical significance (p < 0.05) between groups as determined by Kruskal–Wallis test with multiple comparisons since data was not normally distributed. (C) Western blot shows that WT had a stronger increased production of ALPL, COL10A1, IHH, RUNX2, and RUNX2-9 in response to BMP4 treatment than the mutants. (D) Principle component analysis (PCA) of bulk RNA-seq reveals an increased sensitivity to BMP4 (and TGFß3 + BMP4) treatment in WT human-induced pluripotent stem cell (hiPSC)-derived chondrocytes compared to V620I and T89I. n = 3–4 samples. Figure 5—source data 1.ALPL western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 2.COL10A1 western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 3.IHH western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 4.MMP13 western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 5.RUNX2 western blot: the full raw unedited gel with and without the bands labeled. Figure 5—source data 6.GAPDH western blot: the full raw unedited gel with and without the bands labeled.