Fig 1: Isolation and osteogenic differentiation of BMSCs (A) Normal and osteoporotic BMSCs were isolated from normal and osteoporotic bone marrow tissues, respectively (the scale bar is 50 μm). (B) Flow cytometry was performed to identify isolated BMSCs detecting CD34, CD45, CD73, CD90, and CD105. (C) Normal and osteoporotic BMSCs were induced towards osteogenic differentiation for 21 days, and the protein levels of ALP, OCN, Runx2, and Osterix were examined using Immunoblotting on days 0, 7, and 14 of osteogenic induction (C); ALP staining was performed on day 0, 7, and 14 of osteogenic induction (D); the formation of mineralized nodules were examined using Alizarin red staining on days 0 and 21 of osteogenic induction (E); the expression of miR-4739 was examined using qRT-PCR on day 0, 3, and 7 of osteogenic induction in normal BMSCs and osteoporotic BMSCs (F). n=3, **p < 0.01 compared to OP-BMSC. aa p<0.01 compared between 0 days OP-BMSC and 3 days OP-BMSC; bb p < 0.01 compared between 0 days normal-BMSC and 3 days normal-BMSC; cc p<0.01 compared between 0 days OP-BMSC and 14 days OP-BMSC; dd p < 0.01 compared between 0 days normal-BMSC and 14 days normal-BMSC.
Fig 2: ALP expressions between the groups. (A) Control group, (B) Group 1, (C) Group 2, (D) Group 3 and (E) Group 4, for 4-week and (F) Control group, (G) Group 1, (H) Group 2, (I) Group 3 and (J) Group 4, for 8-week, arrows indicate immunopositive cells. Streptavidin biotin peroxidase method, scale bars = 50 µm.
Fig 3: Expression of RUNX2, Osterix, ALP and OCN in LF and OLF tissues. Reverse transcription-quantitative PCR results for the mRNA expression levels of (A) RUNX2, (B) Osterix, (C) ALP and (D) OCN in the LF and OLF groups. Immunohistochemistry images of (E) RUNX2, (F) Osterix, (G) ALP and (H) OCN. Quantification of the protein expression levels of (I) RUNX2, (J) Osterix, (K) ALP and (L) OCN in the LF group and OLF group. Magnification, ×400. The red arrows represent the typical cells, which were magnified and shown in the upper right-hand corner (magnification, × 1,600). N=8. **P<0.01, ***P<0.001 vs. LF samples. OLF, ossification of ligamentum flavum; LF, ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin.
Fig 4: Osteogenic differentiation of BMSCs growing on scaffolds. The mRNA expression of bone tissue specific markers, including Runx2 (A), ALP (B), Osterix (C), Osteocalcin (D), RANKL (E) were evaluated during the in vitro culturing period of up to 28 days to assess osteogenic differentiation of BMSCs by RT-qPCR at different time points. All data were normalized to the mRNA expression of the corresponding marker at day 0 (cell seeding day). Data indicate the mean relative values calculated from six independent experiments (±S.E.). Statistically significant differences with P < 0.05 were considered significant (*P < 0.05). F, shows a representative protein expression (as indicated) of cell lysates cultured on PCL scaffolds only, PCL scaffolds with poly-lysine coating, PCL scaffolds with HAp, PCL scaffolds with HAp and 30 mg/ml collagen coating, PCL scaffolds with 10 mg/ml collagen coating, PCL scaffolds with 30 mg/ml collagen coating after 28 days of cell culturing.
Fig 5: Effect of the Indian hedgehog signaling pathway inhibitor Cpn on the expression levels of RUNX2, Osterix, ALP and OCN in OLF cells. mRNA expression levels of (A) RUNX2, (B) Osterix, (C) ALP and (D) OCN. Protein expression levels of (E) RUNX2, (F) Osterix, (G) ALP (H) and OCN were significantly decreased in the stretch + Cpn group compared with cyclic stretch alone. (I) Representative images of ALP staining. Magnification, ×400. (J) ALP activity was significantly reduced in the Cpn + stretch group compared with cyclic stretch alone. *P<0.05, **P<0.01, ***P<0.001. Data are presented as the mean ± SD. N=3. OLF, ossification of ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; Cpn, cyclopamine.
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