Collagen X Antibody - #DF13214

Fig. 1 CCO exhibits characteristics of articular cartilage and delays the process of ossification. (A) SO, staining of the CEP. CEP had a quasi-spherical shape. (B) DAPI staining. CEP decellularization of nuclei. (C) DNA content, GAG content, and Total collagen content measurement. CEP was decellularized, preserving a significant amount of GAG and collagen. (D) Cell viability and cytotoxicity assay using Calcein/PI at D1, D3, and D7, with live cells shown in green and dead cells in red. CEP exhibits good biocompatibility. (E) CCK-8 assay at D1, D3, and D7. CEP can promote the proliferation of BMSCs. (F) HE and AB staining of CO and CCO on days 7, 14, and 21. CCO can generate a greater amount of cartilage matrix. (G) Immunofluorescence images for COL2, ACAN in CO and CCO at days 14, with blue representing DAPI and red representing the target proteins. The cartilage markers COL2 and ACAN are expressed at higher levels in CCO. (H)Immunohistochemical images for PRG4 in CO and CCO at days 14 and Immunofluorescence images for COLX in CO and CCO at days 21 with blue representing DAPI and red representing the target proteins. CCO exhibits high expressions of the articular cartilage-specific marker PRG4 and low expressions of the hypertrophic cartilage marker COLX. (I) The mRNA expression of cartilage genes SOX9, COL2, and ACAN, as well as the hypertrophic cartilage gene COLX, was assessed in CO and CCO at 0, 7, 14, and 21 days. Both CCO and CO reached peak cartilage expression in 14 days, after which the expression of hypertrophy-related genes increased. However, CCO demonstrated superior cartilage formation compared to CO, with a delayed hypertrophic process. All experiments were repeated three times. ns p > 0.05, *0.01 

Fig. 5. Evaluation of hBMSC differentiation induced by DWJH/CD56+Exos and sustained release of CD56+Exos in DWJH/CD56+Exos. (A) Histochemical identification using Alcian Blue staining was performed to assess chondrogenic induction of hBMSC by DWJH/CD56+Exos. Scale bar: 100 μm. (E) Statistical analysis was carried out based on the results obtained in A. (B) Immunofluorescence staining for chondrogenic marker SOX9 and (F) quantification of positive cells. (n = 3). Scale bar: 50 μm. (C) Western blot of SOX9 in hBMSC treated with DWJH, CD56+Exos, or DWJH/CD56+Exos. (F) Quantification of SOX9, COL2, and COLX relative protein expression between different groups in control, DWJH, CD56+Exos, and DWJH/CD56+Exos groups. n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3Histological and immunochemistry staining of pellets with semiquantitative analysis. A H–E and Safranin-O staining of pellets and partial enlargement in five groups. B Immunochemistry staining of COLII and COLX of pellets and partial enlargement in five groups. C–E Semiquantitative analysis of AOI of each staining (n = 4). Significant difference symbols: *p < 0.05, **p < 0.01 compared to CCs group, #p < 0.05, ##p < 0.01 compared to hWJMSCs group

Fig. 4 C-kit drives FLS EMT signaling (A) Validation of several shC-kit efficiency by Western blot (n = 3). (B-C) The expression of EMT makers (B) and Transwell invasive assay (C) in OA FLS with pCDNA-Ctrl, pCDNA-C-kit, shCtrl, or shC-kit transfection (n = 3, bar = 100 μm). (D) Morphological observation of DAPI (blue) and vimentin staining (green). Pseudopodia are marked by white arrowheads (bar = 20 μm). (E) The expression of inflammation-related markers (IL-6, IL-8, and MMP13) in FLSs (n = 3). (F) Illustration of co-culture of lenti-virus infected OA-FLS and chondrocytes. (G-H) After co-culture for 7 days, assessment of MMP13 (G) and Runx2 (H) of the chondrocytes (n = 5, bar = 100 μm)

Figure 2 Paracrine effect of SDSCs toward CCs in indirect coculture. A) Schematic of conditioned medium coculture experimental outline. In light blue: CCs; In yellow: SDSCs; In dark blue: CCs pellet. B) Macroscopic appearance of pellets. C) RT-PCR analysis for chondrogenesis and hypertrophic-related gene expression after 3 weeks of culture. D) H&E staining. E) Alcian blue staining. F, G) Safranin-O staining. H) Biochemical evaluation of GAG content and GAG/DNA ratio of pellets. I, J) Immunohistochemistry analysis of type II collagen. K, L) Immunohistochemistry analysis of type X collagen. M) Western blot analysis of type X collagen. Crtl: Control group. CM: SDSCs-conditioned medium group. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2 Paracrine effect of SDSCs toward CCs in indirect coculture. A) Schematic of conditioned medium coculture experimental outline. In light blue: CCs; In yellow: SDSCs; In dark blue: CCs pellet. B) Macroscopic appearance of pellets. C) RT-PCR analysis for chondrogenesis and hypertrophic-related gene expression after 3 weeks of culture. D) H&E staining. E) Alcian blue staining. F, G) Safranin-O staining. H) Biochemical evaluation of GAG content and GAG/DNA ratio of pellets. I, J) Immunohistochemistry analysis of type II collagen. K, L) Immunohistochemistry analysis of type X collagen. M) Western blot analysis of type X collagen. Crtl: Control group. CM: SDSCs-conditioned medium group. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2.| Paracrine effect of SDSCs toward CCs in indirect coculture.M) Western blot analysis of type X collagen. Crtl: Control group. CM: SDSCs-conditioned medium group. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

FIGURE 3 Phenotypic expression of AUCs during in vitro expansion. (A) Alcian staining, and (B) COL II immunofluorescence staining of human and goats at P1 and P3. Gene quantitative analyses of (C) SOX9, (D) Aggrecan, and (E) Col II in human and goat at P1 and P3. (F–I) Analysis of protein and gene expression levels of COL I and COL X in human and goat at P1 and P3. Data were analyzed using t tests (the relative mRNA levels were compared within the species, instead of comparing between the species). Columns with different letters indicate statistical significance (p < 0.05). Abbreviations: AUCs, auricular chondrocytes; Col II, type II collage; Col I, type I collage; Col X, type X collage. Scale bar = 50 μm.