Journal: World Journal of Stem Cells

Article Title: EZH2, via an association with KDM2B, modulates osteogenic differentiation of root apical papillary stem cells

doi: 10.4252/wjsc.v17.i4.103482

Figure Lengend Snippet: Knockdown of enhancer of zeste homolog 2 inhibits steo/dentinogenic differentiation potential of human apical papillary stem cells. A: Quantitative polymerase chain reaction showed that the expression of enhancer of zeste homolog 2 (EZH2) was inhibited in human apical papillary stem cells (hSCAPs); B: Western blot analysis confirmed the knockdown of EZH2 in hSCAPs; C: Knockdown of EZH2 decreased alkaline phosphatase activity in hSCAPs; D and E: Alizarin red staining and quantitative calcium analysis demonstrated that knockdown of EZH2 inhibited mineralization in hSCAPs; F-H: Quantitative polymerase chain reaction showed that knockdown of EZH2 downregulated mRNA expression levels of bone sialoprotein (F), dentin sialophosphoprotein (G), and osteocalcin (H) in hSCAPs. GAPDH and ACTB was used as the internal controls. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed using Student’s t -test. a P ≤ 0.05, b P ≤ 0.01, c P ≤ 0.001. EZH2: Enhancer of zeste homolog 2; BSP: Bone sialoprotein; DSPP: Dentin sialophosphoprotein; OCN: Osteocalcin.

Article Snippet: The primary antibodies used in this study were those against dentin sialophosphoprotein (DSPP) (Cat. No. bs10316R, Bioss, China), bone sialoprotein (BSP) (Cat. No. bs-0026R, Bioss, China), and GAPDH (Cat. No. G8795, Sigma-Aldrich, MA, United States).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay, Staining

Journal: World Journal of Stem Cells

Article Title: EZH2, via an association with KDM2B, modulates osteogenic differentiation of root apical papillary stem cells

doi: 10.4252/wjsc.v17.i4.103482

Figure Lengend Snippet: Overexpression of enhancer of zeste homolog 2 enhances osteo/dentinogenic differentiation potential of human apical papillary stem cells. A: Quantitative polymerase chain reaction showed that enhancer of zeste homolog 2 (EZH2) was overexpressed in human apical papillary stem cells (hSCAPs); B: Western blot analysis confirmed overexpression of EZH2 in hSCAPs; C: Overexpression of EZH2 increased alkaline phosphatase activity in hSCAPs; D and E: Alizarin red staining and quantitative calcium analysis results demonstrated that overexpression of EZH2 enhanced mineralization in hSCAPs; F-H: Quantitative polymerase chain reaction showed that overexpression of EZH2 upregulated mRNA expression levels of bone sialoprotein (F), dentin sialophosphoprotein (G), and osteocalcin (H) in hSCAPs; I: Hematoxylin-eosin staining and quantitative measurement showed that overexpression of EZH2 promoted bone/dentin-like tissue formation. Scale bar = 100 μm (B: Bone/dentin-like tissues; HA: Hydroxyapatite tricalcium carrier; CT: Connective tissue); J: Immunohistochemical staining and quantitative analysis of dentin sialophosphoprotein and bone sialoprotein. GAPDH and ACTB were used as the internal controls. Data are presented as the mean ± SD ( n = 3). Statistical analysis was performed using Student’s t -test. a P ≤ 0.05, b P ≤ 0.01, c P ≤ 0.001. EZH2: Enhancer of zeste homolog 2; BSP: Bone sialoprotein; DSPP: Dentin sialophosphoprotein; OCN: Osteocalcin.

Article Snippet: The primary antibodies used in this study were those against dentin sialophosphoprotein (DSPP) (Cat. No. bs10316R, Bioss, China), bone sialoprotein (BSP) (Cat. No. bs-0026R, Bioss, China), and GAPDH (Cat. No. G8795, Sigma-Aldrich, MA, United States).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Staining, Expressing, Immunohistochemical staining

Journal: Bioactive Materials

Article Title: Engineering fibroblast with reprogramming and spheronization for bone defect repair

doi: 10.1016/j.bioactmat.2025.04.021

Figure Lengend Snippet: Characteristics of iOBs derived from donors with different ages . (A) Alizarin Red staining of osteoblasts derived from bMSCs (31 years), iOBs from 8-, 79-year-old donors and their parent fibroblasts after cultured in osteogenic medium for 14 days. n ≥ 3 independent experiments. Y, young referred to individuals under 65 years of age; O, old referred to individuals aged 65 years and older. Scale bar = 100 μm. (B) mRNA levels for ALP, OCN, SATB2 and IBSP after 14 days culture in osteogenic medium. The Y-bMSC were isolated from donors of 26-,31-,33-year-old.The Y-Fib or Y-iOBs were derived from donors of 8-,38-,39-year-old. The O-Fib or O-iOBs were derived from donors of 67-,70-,74-,75-,79-year-old. Gene expression was shown as log 2 (fold change). n ≥ 3 independent experiments. (C) Cell number of starting fibroblasts and resultant iOBs after 18 days' treating. n = 8 individuals' iOBs. (D) Schema for extent of cell expansion during iOBs conversion from skin fibroblasts. (E) The secretion of COL1 and VEGF in Y-bMSC-OB, iOBs and their parent fibroblasts. Y-bMSC-OBs in immunofluorescence images were derived from 31-year-old donors, and O-iOBs and its parent O-Fib were from 79-year-old donor. n ≥ 3 independent experiments. Scale bar = 50 μm. (F) SA-β-gal activity in Y-bMSC-OB, O-bMSC-OB, Y-iOB from a 39-year-old individual, O-iOB from a 79-year-old donor. n ≥ 3 independent experiments. Scale bar = 100 μm. (G) The secretion of IL-6 in Y-bMSC-OB, O-bMSC-OB, Y-iOBs and O-iOBs. n ≥ 3 independent experiments. (H) mRNA levels for CDKN1A and CDKN2A in osteoblasts derived from bMSCs and iOBs from young or old individuals. Gene expression was shown as log 2 (fold change). n ≥ 3 independent experiments. (A) (B) (E) (F) (G) (H) data were presented as means ± s.d. Significance was determined when samples were compared to Y-bMSC-OB or pairwise comparisons between the groups connected by lines. One-way ANOVA with Bonferroni correction. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The staining reagents, including antibodies, were provided in detail as follows: anti-GFP (1:600; Abcam, ab13970), anti-IBSP(1:300; reactivity with Human, Mouse; Bioss, bs-2668R), anti-OCN (1:100; reactivity with Human, Mouse; Bioss, bs-4917R), anti-Ki67 (1:200; reactivity with Human, Mouse; Invitrogen, 2747806), anti-SP7 (1:150; reactivity with Human; Abcam, ab94744), anti-RUNX2 (1:200; reactivity with Human, Mouse; santacruz, sc-390351), anti-COL1 (1:400; reactivity with Human, Mouse; Bioss, bs-0578R), anti-VEGF (1:50; reactivity with Human, Mouse; santacruz, sc-7269), human-specific anti-VEGF (prediluted; reactivity with Human; Abcam, ab27620), anti-STEM121 (1:250; reactivity with Human specific; TAKARA, Y40410) and anti-CD31 (1:400; reactivity with Human, Mouse; Immunoway, YP0530; or 1: 400; reactivity with Mouse; Servicebio, GB12063), Nrf2 (1:500; reactivity with Human、mouse、rat; Proteintech, 80593-1-RR, Lot:23013625); secondary antibodies (Invitrogen, A21235, A21244; Univ, 703-545-155, 711-165-152; Abcam, AB150159; Servicebio, GB21301).

Techniques: Derivative Assay, Staining, Cell Culture, Isolation, Gene Expression, Immunofluorescence, Activity Assay

Journal: Bioactive Materials

Article Title: Engineering fibroblast with reprogramming and spheronization for bone defect repair

doi: 10.1016/j.bioactmat.2025.04.021

Figure Lengend Snippet: Therapeutic effects of iOB-Sps on bone regeneration (A) 5 mm in the length and 1.5 mm in width femoral bone defect was created by dental drill on the right femur. Each scale of the ruler indicates 0.5 mm. (B) The preparation of iOB-Sps to fit the damaged size. (C) The transplantation of iOB-Sps into bone defect. (D) μCT imaging of the femur transplanted by Fibs, Fib-Sps, bMSCs and iOB-Sps in NOD/SCID mice with femur defect. iOB-Sps were derived from old donors, O = Old. (E) BMD, BS/TV, BV/TV, Tb. N values in bone defect healing. n = 5 independent experiments. Significance was determined when samples were compared to group Neg or pairwise comparisons between the groups connected by lines. (F) Analysis of Tunel staining for iOBs apoptosis in spheroid before and after 28-days transplantation. n = 7 independent experiments in iOB-Sps culture in vitro and n = 5 independent experiments in iOB-Sps transplanted in vivo. (G) Analysis of Ki67 fluorescent staining for iOBs proliferation in spheroid before and after 28-days transplantation. n = 7 independent experiments in iOB-Sps culture in vitro and n = 5 independent experiments in iOB-Sps transplanted in vivo. (H) Counting of iOBs when cultured in vitro spheroid and in vivo post transplantation. (I) Representative images and quantification analysis of GFP and IBSP positive cells in bone defects. n = 7 independent experiments in iOB-Sps culture in vitro and n = 5 independent experiments in iOB-Sps transplanted in vivo. (E)–(I) data were presented as means ± s.d. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. One-way ANOVA with Bonferroni correction in (E); Student t-test in (F)–(I). Scale bar = 10 μm in (F) (G) (I).

Article Snippet: The staining reagents, including antibodies, were provided in detail as follows: anti-GFP (1:600; Abcam, ab13970), anti-IBSP(1:300; reactivity with Human, Mouse; Bioss, bs-2668R), anti-OCN (1:100; reactivity with Human, Mouse; Bioss, bs-4917R), anti-Ki67 (1:200; reactivity with Human, Mouse; Invitrogen, 2747806), anti-SP7 (1:150; reactivity with Human; Abcam, ab94744), anti-RUNX2 (1:200; reactivity with Human, Mouse; santacruz, sc-390351), anti-COL1 (1:400; reactivity with Human, Mouse; Bioss, bs-0578R), anti-VEGF (1:50; reactivity with Human, Mouse; santacruz, sc-7269), human-specific anti-VEGF (prediluted; reactivity with Human; Abcam, ab27620), anti-STEM121 (1:250; reactivity with Human specific; TAKARA, Y40410) and anti-CD31 (1:400; reactivity with Human, Mouse; Immunoway, YP0530; or 1: 400; reactivity with Mouse; Servicebio, GB12063), Nrf2 (1:500; reactivity with Human、mouse、rat; Proteintech, 80593-1-RR, Lot:23013625); secondary antibodies (Invitrogen, A21235, A21244; Univ, 703-545-155, 711-165-152; Abcam, AB150159; Servicebio, GB21301).

Techniques: Transplantation Assay, Imaging, Derivative Assay, TUNEL Assay, Staining, In Vitro, In Vivo, Cell Culture

Journal: Bioactive Materials

Article Title: Engineering fibroblast with reprogramming and spheronization for bone defect repair

doi: 10.1016/j.bioactmat.2025.04.021

Figure Lengend Snippet: Enhancement of endogenous osteogenesis induced by iOB-Sps Biopsies were obtained 4 weeks after iOB-Sps transplantation into mouse femur defects. DAPI nuclear counterstain as indicated in blue, iOB-labeled GFP counterstain as indicated in green. (A) Regeneration of damaged sites shown in a panoramic view of the complete femur and in high-resolution image stained for COL1 (red). Scale bar = 500 μm in complete femur images; Scale bar = 10 μm in high-resolution images. (B) Regeneration of damaged sites shown stained for IBSP (yellow gold). Scale bar = 500 μm in complete femur images; Scale bar = 100 μm in high-resolution images. (C) Magnification of IBSP staining of the boxed area shown in (B). Scale bar = 50 μm. (D) The proportion of COL1 positive area in the total defective area. n = 4 independent experiments in untreated negative group and n = 5 independent experiments in iOB-Sps treatment group. (E) The proportion of IBSP positive area in the total defective area. n = 5 independent experiments. (D) (E) data were presented as means ± s.d. Student t-test. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The staining reagents, including antibodies, were provided in detail as follows: anti-GFP (1:600; Abcam, ab13970), anti-IBSP(1:300; reactivity with Human, Mouse; Bioss, bs-2668R), anti-OCN (1:100; reactivity with Human, Mouse; Bioss, bs-4917R), anti-Ki67 (1:200; reactivity with Human, Mouse; Invitrogen, 2747806), anti-SP7 (1:150; reactivity with Human; Abcam, ab94744), anti-RUNX2 (1:200; reactivity with Human, Mouse; santacruz, sc-390351), anti-COL1 (1:400; reactivity with Human, Mouse; Bioss, bs-0578R), anti-VEGF (1:50; reactivity with Human, Mouse; santacruz, sc-7269), human-specific anti-VEGF (prediluted; reactivity with Human; Abcam, ab27620), anti-STEM121 (1:250; reactivity with Human specific; TAKARA, Y40410) and anti-CD31 (1:400; reactivity with Human, Mouse; Immunoway, YP0530; or 1: 400; reactivity with Mouse; Servicebio, GB12063), Nrf2 (1:500; reactivity with Human、mouse、rat; Proteintech, 80593-1-RR, Lot:23013625); secondary antibodies (Invitrogen, A21235, A21244; Univ, 703-545-155, 711-165-152; Abcam, AB150159; Servicebio, GB21301).

Techniques: Transplantation Assay, Labeling, Staining