Fig 2: miR-672-5p Represses Smurf1 to Promote Osteoblast Differentiation(A) Stem-loop structure of miR-672 predicted by miR-base (the mature miR-672-5p sequence is shown in green). (B and C) Expression (qPCR, in triplicate) of miR-672-5p in different tissues (B) and cells, i.e., in osteoblasts during proliferation, differentiation, or mineralization (C). Data are mean ± SE. *p < 0.05 and ***p < 0.001 compared with proliferation stage of osteoblasts. (D–F) Effect of transfection of osteoblasts with the mimic (miR-672-5p), inhibitor (anti-miR-672-5p), and miR-C (control) on ALP (alkaline phosphatase) activity (OD, n = 8) (D) and mineralization (OD, n = 4) (E). (F) Representative wells showing alizarin-positive colony (Cfu-ob) formation in osteoblast cell cultures on day 15 in osteogenic medium. Data are mean ± SE. *p < 0.05 and **p < 0.01 compared with miR-C. (G) Identification of miR-672-5p target gene in osteoblasts and computational analysis was performed for the complementarities of miR-672-5p to the 3′ UTR of Smurf1 and (H) schematic presentation of the reporter plasmid used to illustrate the effect of Smurf1 3′ UTR on luciferase activity. CMV, cytomegalovirus promoter; Luc, luciferase; RLuc, renilla luciferase. (I) Effect of miR-672-5p overexpression on a dual luciferase reporter plasmid containing Smurf1 3′ UTR was analyzed. Cells were co-transfected with the WT-pEZX-MT06-Smurf1 or an empty vector (NC, negative control) and miR-672-5p or miR-C. Firefly and renilla luciferases were measured in the cell lysate. Data are mean ± SE of three independent measurements. **p < 0.01 compared with miR-C. (J) Expression (qPCR, in triplicate) of Smurf1 in osteoblasts during proliferation, differentiation, or mineralization. Data are mean ± SE. *p < 0.05 compared with proliferation stage of osteoblasts. (K) Transfection of osteoblasts with the mimic (miR-672-5p) enhanced the release of BMP2 in conditioned medium (BMP2 ELISA). Data are mean ± SE of three independent experiments. *p < 0.05 compared with miR-C. (L) Effect of miR-672-5p or scrambled miR-C on Runx2 promoter activity using a luciferase (Luc) reporter. Data are mean ± SE of three independent experiments. *p < 0.05 compared with miR-C. (M) qPCR (in triplicate) was performed for Smurf1 (a direct target gene of miR-672-5p) or the osteoblast differentiation genes, including Runx2, BMP2, Smad1, and ALP after 48 h of transfection. Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with miR-C. (N) Western blot analysis was done for Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1000), BMP2 (ab14933-Abcam; 1:1000), and Smad1 (ab63356-Abcam; 1:1,000) after 48 h of transfection. β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were horseradish peroxidase (HRP) conjugated (Sigma-Aldrich). (O–S) miR-672-5p represses Smurf1, which promotes the upregulation of Runx2 and osteoblast differentiation. (O) ALP activity (OD, n = 8), (P) Cfu-ob formation, (Q) mineralization (OD, n = 4), and (R) mRNA expression of Runx2, BMP2, Smurf1, ALP, and Smad1 (qPCR, in triplicate), and (S) protein expression of Smurf1 (ab38866-Abcam; 1:1,000), Runx2 (ab76956-Abcam; 1:1000), and BMP2 (ab14933-Abcam; 1:1,000) in either control osteoblasts or transfected with siRNA against Smurf1 or scrambled siRNA (Si-Scr). β-actin (sc-47778 Santa Cruz Biotechnology; 1:500) was taken as a loading control. Secondary antibodies (either anti-rabbit or anti-mouse; 1:10,000) were HRP conjugated (Sigma-Aldrich). Data are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with miR-C or control. Other comparisons, ˆp < 0.05, ˆˆp < 0.01, and ˆˆˆp < 0.001 as shown.

Fig 3: Noggin, BMP2 antagonist, suppresses VSMC calcification by inhibiting Smad1/5/8 signaling. VSMCs pretreated with Noggin (100 ng/ml) or vehicle for 2 h followed by incubation with 10 mM β-GP. a Alizarin Red staining (×100). b Quantification of Alizarin Red staining. c Calcium contents measured and normalized by protein content of cell lysates. mRNA expression of (d) BMP2, (e) Msx2, (f) Runx2, and (g) osteocalcin analyzed by rt-PCR. h Protein expression of BMP2 and phospho-Smad1/5/8 performed by western blot analysis. β-GP beta-glycerophosphate, DMEM Dulbecco’s Modified Eagle’s Medium, DMEMβ-GP DMEM medium containing 10 mM β-GP, DMEMNoggin+β-GP DMEM medium containing 10 mM β-GP and 100 ng/ml Noggin, Msx2 msh homeobox 2, NC DMEM medium, OD optical density, Runx2 Runt-related transcription factor 2

Fig 4: CD133+ USC-Exos can better promote the chondrogenic differentiation of BMSCs than USC-Exos. (A) Identification and characterization of USC-Exos and CD133+ USC-Exos by observing their morphology, measuring particle size distribution, and evaluating the expression of exosomal markers. Scale bar ​= ​100 ​nm. (B) Electron microscopy and confocal microscopy of hydrogels containing exosomes. The exosomes are labeled with PKH26 dye. (C) Uptake of the exosomes, CD133+ USC-Exos significantly promoted the SOX9 expression of BMSCs. Uptake of the green fluorescence dye PKH67-labelled USC-Exos and the red fluorescence dye PKH26-labelled CD133+ USC- Exos into BMSCs. Scale bar ​= ​50 ​μm. (D) Alizarin Red staining and Alcian Blue staining of BMSCs after culturing with USC-Exos and CD133+ USC-Exos. Comparative analysis of Alizarin Red staining and Alcian Blue staining area after BMSCs cultured with PBS, USC-Exos, and CD133+ USC-Exos. (E) The relative expression levels of Runx2, Alp, Agg, and Sox9 genes of BMSCs under different conditions. Bar ​= ​50 ​μm. n ​= ​3 for each group. Data are showed as means ​± ​standard deviation (∗P ​< ​0.05, ∗∗P ​< ​0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: CD133+ USC extraction and identification of three-lineage differentiation ability. (A) Flow cytometry for the isolated cells. Representative histograms demonstrating positive and negative staining of urine-derived cells from a single human. (B) Gating strategy used to sort the CD133+ USC isolation. Representative FACS plots with the percentage of parent gate for CD133+ USC isolation. (C) Morphology of cells isolated from human urine and the CD133+ USC, the proliferation of urine-derived cells was determined by CCK-8 assay. At P4, spindle-shaped or rise-shaped cells were observed. Scale bar ​= ​50 ​μm. The proliferation of urine-derived cells was determined by CCK-8 assay. n ​= ​4 for each group for each time point. (D) The ability of three lineages differentiation between USC and CD133+ USC in vitro. Alizarin Red staining, Oil Red O staining, and Alcian Blue staining of the two groups. The SOX9 and RUNX2 protein expression of the two groups after 7 days of chondrogenic and osteogenic induction. Scale bar ​= ​50 ​μm. n ​= ​4 for each group. (E) Osteogenic gene (Runx2, Alp) and chondrogenic gene (Sox9, Agg) expression compared between the two groups in the osteogenic medium and chondrogenic cultures, respectively. n ​= ​3 for each group. Data are showed as means ​± ​standard deviation (∗P ​< ​0.05, ∗∗P ​< ​0.01). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)