Fig 2: Chondrocytes show sexual dimorphism in gene expression of PCM proteins. (A-B) No difference between male and female was detected in the expression of COL2A1 and ACAN. (C) SOX9 was higher in male chondrocytes. (D-E) COL10A1 and COL11A1 were not dimorphic. (F-H) The PCM genes COL6A1, COL9A1, and DCN were expressed in a sex-dependent manner, although HSPG2 (I) was not dimorphic. (J-K) No difference was observed in ADAMTS4 and ADAMTS5 expression. (L-O) MMP1, MMP9, and MMP13 were similar between sexes, but MMP3 showed a trend for higher expression in females. (P-Q) None of the TIMPs tested were differentially expressed depending on the sex of the cell. Data are presented as mean ± SD of relative quantity to normalizer (geometric mean of 18S and GAPDH). Cells from 6♂ + 6♀ were used in passage 0. PCM = pericellular matrix; MMP = metalloprotease; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs; DCN = decorin; TIMP = tissue inhibitor of metalloproteases; ACAN = agreccan; COL = collagen; SOX9 = SRY (sex determining region Y)-Box 9; HSPG2 = heparan sulfate proteoglycan 2 (perlecan); GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Fig 3: Catabolic function elicits a dimorphic response. Chondrocytes were treated with 10 ng/ml IL-1ß for 24 hours to provoke an inflammatory response and activate the catabolic function. No differences were detected in the expression of COL2A1, ACAN, SOX9 (A-C), COL10A1, and COL11A (D-E). COL6A1 decreased similarly for males and females (F), but COL9A1 and DCN decreased, resulting in the same trend observed for basal conditions (G-H). No dimorphism was found for HSPG2 (I). All proteases tested increased with treatment (J-O), but dimorphism was observed only in ADAMTS4 and MMP1. No significant differences were detected in TIMP1 and TIMP2 expression (P-Q). The pro-inflammatory cytokines IL-1ß and IL-6 increased with treatment; however, no dimorphism was detected (R-S). Data are presented as mean ± SD of relative quantity to normalizer (geometric mean of 18S and GAPDH). Cells from 6? + 6? were used in passage 0. MMP = metalloprotease; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs; DCN = decorin; TIMP = tissue inhibitor of metalloproteases; ACAN = agreccan; COL = collagen; SOX9 = SRY (sex determining region Y)-Box 9; HSPG2 = heparan sulfate proteoglycan 2 (perlecan); GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

Fig 4: Analysis of ECM remodeling in acute senescent proto-myofibroblasts. After 24 h of cultivation, cells were treated for 1 h with 300 µM H2O2 or H2O (control). Relative COL1A1 (A), FN (B) and DCN (C) mRNA expression levels were analyzed after 72 h of cultivation by quantitative real-time PCR and were normalized on GAPDH mRNA expression level. The protein expression levels of collagen I (D), fibronectin (E) and decorin (F) were detected after a cultivation time of 72 + 72 h via immunoblotting and normalized on GAPDH protein expression. Quantification was determined by using ImageJ. Values shown are means ± SEM for three biological and three (A–C) or one (D–F) technical replicate per experiment (n = 3) and calculated relative to the respective control. Mann-Whitney U test: p < 0.0001 (****).

Fig 5: Cell populations selected based on dual expression of two mRNA targets display enhanced levels of CFU-F and capacity for tri-lineage differentiation. (a) CFU-F following dual SNA-selection. Cells were incubated with SPARCL1 Joe-tagged SNAs, and Cy-5 tagged SNAs targetting CD146, CALD1, DCN, COL1A2, FMO3, NANOG, or TF. The positive and negative fluorescent cells were collected and plated for CFU-F. For each SNA combination, each point represents the mean CFU-F count from a different patient, displayed as a percentage of unsorted CFU-F counts. In total, bone marrow samples from 18 patients were tested. Horizontal bars represent median CFU-F values. To assess tri-lineage capacity, cell populations were collected and expanded in vitro. (b) Osteoinduction. Cells were cultured in basal medium with 50 µM ascorbic acid 2-phosphate and 10 nM vitamin D3 for 14 days. Mineralisation is shown using alkaline phosphatase staining. Scale bars = 100 µm, whole well = 500 µm. n = 3. (c) Chondrogenic induction. Cells were cultured in basal medium supplemented with 100 µM ascorbic acid 2-phosphate, 10 ng/mL TGF-B3, 10 µg/mL ITS solution, 10 nM dexamethasone. Alcian blue/Sirius Red staining revealed proteoglycan synthesis (blue denotes proteoglycan deposition, purple indicates collagen deposition). Scale bars = 100 µm. n = 3. (d) Adipogenic induction. Cells were cultured in basal medium with 100 nM dexamethasone, 500 µM IBMX, 3 µg/mL ITS solution, and 1 µM rosiglitazone for 14 days. Oil-Red-O staining indicates lipid droplet formation. Scale bars = 100 µm. Results for each target were obtained from three different patient samples: representative images are shown.