Journal: International Journal of Molecular Sciences

Article Title: Preliminary Preclinical Evaluation of Innovative Bone Scaffolds Composed of Natural Sources–Whey Protein Isolate and Pearl Powder

doi: 10.3390/ijms26167939

Figure Lengend Snippet: Qualitative assessment of osteogenic differentiation of normal human osteoblasts cultured on control biomaterials–polystyrene and tested biomaterials composed of WPI without or with pearl powder (WPI/P0 and WPI/P2.5; WPI/P5; WPI/P7.5; WPI/P10, respectively). After 21 days of incubation, cells were incubated with primary anti-collagen I antibody or primary anti-osteocalcin antibody, followed by staining with specified secondary antibody-conjugated with Alexa Fluor 488 and additionally with Hoechst 33342. Then, the cells were observed using a confocal laser scanning microscope (CLSM). Cell nuclei = blue fluorescence; collagen or osteocalcin = green fluorescence. Magnification = 200× or 400×, bar scale = 70 or 30 μm.

Article Snippet: After incubation, the cells were stained with primary rabbit/IgG polyclonal anti-collagen I antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), diluted 1:100 in 0.1% bovine serum albumin, BSA (Merck, Warsaw, Poland), or primary rabbit/IgG polyclonal anti-osteocalcin antibody (Bioss, ThermoFisher Scientific, Waltham, MA, USA), diluted 1:100 in 0.1% BSA (Merck, Warsaw, Poland), followed by staining with secondary goat polyclonal anti-rabbit IgG (H + L) antibody-conjugated with AlexaFluor ® 488 (Abcam, Cambridge, UK) , and diluted 1:1000 in PBS (Merck, Warsaw, Poland).

Techniques: Cell Culture, Control, Incubation, Staining, Laser-Scanning Microscopy, Fluorescence

Journal: Bioactive Materials

Article Title: Ribbon-shaped microgels as bioinks for 3D bioprinting of anisotropic tissue structures

doi: 10.1016/j.bioactmat.2025.12.040

Figure Lengend Snippet: μRB bioinks modulate MSC morphology, osteogenesis and bone formation in a stiffness-dependent manner. (A) Live/dead staining of MSCs after extrusion (Day 0) or after 28 days of culture in osteogenic medium. Green: live cells, Red: dead cells. (B) Metabolic activity of MSCs measured by PrestoBlue assay at day 0 and day 14 after bioprinting (n = 6 per group). (C) DNA content per scaffold measured by PicoGreen assay at day 28 (n = 3 per group). (D) Alizarin red S (ARS) staining for mineralized bone matrix, (E) Aniline Blue staining for total collagen, and (F) immunostaining of Osteocalcin (OCN), a mature bone marker, at day 14 and day 21 of osteogenesis (n = 4 per group). (G–I) Quantification of ARS and Aniline Blue percent positive area and OCN mean fluorescence intensity (MFI). Scale bar = 100 μm. Values are presented as mean ± S.D. and p-values were determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.005, ∗∗∗∗p ≤ 0.001.

Article Snippet: Primary antibody for osteocalcin (1:200, 23418-1-AP, Proteintech), GFP (1:100, 50430-2-AP, Proteintech) was diluted in 1 % BSA with 0.1 % Triton X-100 and incubated overnight at 4 °C.

Techniques: Staining, Activity Assay, Prestoblue Assay, Picogreen Assay, Immunostaining, Marker, Fluorescence

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: Immunohistochemical analysis followed established protocols[ ] using primary antibodies against osteocalcin (OCN) (Bioss bs-4917R) and DSPP (Bioss bs10316R).

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: Immunohistochemical analysis followed established protocols[ ] using primary antibodies against osteocalcin (OCN) (Bioss bs-4917R) and DSPP (Bioss bs10316R).

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