osteogenic induction medium om  (Procell Inc)

Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Different concentrations of magnesium ions can affect the osteogenic properties of MC3T3-E1 cells. (A-D) Following the addition of magnesium ions at different concentrations, MC3T3-E1 cells were subjected to ALP staining (A-B) and ARS staining (C-D). Subsequently, the staining results were quantitatively analyzed. (E) RT-qPCR was employed to assess the impact of different magnesium ion concentrations on the mRNA expression of osteogenesis - related genes in cells. (F-G) Western blotting was utilized to determine the influence of different magnesium ion concentrations on the expression of osteogenesis - related proteins in cells. (Data are presented as mean ± SD from three independent experiments (n = 3). Statistical differences were analyzed using one-way ANOVA. Post-hoc pairwise comparisons were conducted using the LSD test.).

Article Snippet: The basal osteogenic induction medium (OM) was prepared by supplementing α-MEM (#PM150421; Procell) with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid-2-phosphate.

Techniques: Staining, Quantitative RT-PCR, Expressing, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: Magnesium ions facilitate osteogenic differentiation and intervertebral fusion via m6A methylation of RhoA mRNA

doi: 10.1016/j.jot.2026.101056

Figure Lengend Snippet: Magnesium ions upregulate METTL3 expression, enhancing m6A modification on RhoA mRNA. The m6A reader YTHDF1 recognizes and binds to the modified sites, promoting RhoA translation. This activates the RhoA/ROCK signaling pathway, ultimately driving osteogenic differentiation, bone remodeling, and intervertebral fusion.

Article Snippet: The basal osteogenic induction medium (OM) was prepared by supplementing α-MEM (#PM150421; Procell) with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid-2-phosphate.

Techniques: Expressing, Modification

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: GDF15 secretion was significantly upregulated during the osteogenic differentiation of hDPSCs. (a) Morphology of primary hDPSCs. Scale bars (white): 100 μm. Morphology of passage 3 hDPSCs at 24 h of culture. Scale bars (black): 250 μm. P0 = Passage 0; P3 = Passage three. (b) Colony-forming assay to assess the self-renewal ability of hDPSCs. Scale bars: 250 μm. (c) The CCK-8 assay was employed to assess the proliferation of hDPSCs. (d) Proliferation of hDPSCs detected by crystal violet staining. Scale bars: 250 μm. (e) ALP staining of hDPSCs grown in OM for a week. Scale bars: 250 μm. (f) ARS staining of 21-day-cultured hDPSCs in OM and quantitative analysis of mineralized nodule deposition. Scale bars: 250 μm. (g) Flow cytometry demonstrated that the hDPSCs highly expressed CD105, CD90, and CD73; and lowly expressed CD45, CD19, and CD14. (h) During induction, intracellular mRNA levels of Gdf15 and osteogenic-specific genes ( Alp, Runx2, Osx, Ocn , and D spp ) were elevated. (i) During induction, osteogenic-specific proteins (ALP, RUNX2, OPN, DMP1, and DSPP) were upregulated and intracellular GDF15 protein was reduced in hDPSCs. Relative quantitative analysis of gray scale values of protein bands. The internal control was GAPDH. (j) Increased GDF15 secretion was detected by ELISA after osteogenic induction. Data were displayed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00001).

Article Snippet: Upon reaching 80% confluence, GM was replaced with osteogenic induction medium (OM), where OM was made by adding 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Solarbio, Beijing, China) and 100 nM dexamethasone (Solarbio, Beijing, China) to GM.

Techniques: CCK-8 Assay, Staining, Cell Culture, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: G df15 overexpression promoted osteogenic differentiation of hDPSCs in vitro , while Gdf15 knockdown suppressed it. (a) Images of GFP-positive hDPSCs were observed using fluorescence microscopy. Scale bars: 200 μm. GFP, Green Fluorescent Protein. (b) Relative mRNA expression of Gdf15 in the NC and G df15 overexpression ( Gdf15 ) groups. (c) Protein levels of G DF15 in the NC and Gdf15 groups. (d) Relative mRNA expression of G df15 in Gdf15 overexpressing hDPSCs after 7, 14, and 21 days of incubation in the OM. (e) ALP staining of the G df15 and NC groups on day 7 of osteogenic differentiation. (f) ARS staining and quantitative analysis of overexpressed hDPSCs were performed on days 7, 14, and 21 of osteogenic differentiation. (g) Osteogenic-specific genes ( Alp, Col1a1, Runx2, Osx , and Dmp1 ) and their relative mRNA levels in hDPSCs was examined by qPCR on days 7, 14, and 21 of the induced differentiation. (h) Relative mRNA expression of Gdf15 in the siNC and G df15 knockdown ( siGdf15 ) groups. (i) GDF15 protein levels in siNC and siGdf15 groups. (j) Relative mRNA expression of Gdf15 in siRNA-transfected hDPSCs after 7, 14, and 21 days of incubation in the OM. (k) ALP staining in the siNC and siGDF15 groups on day 7 of induced differentiation. (l) ARS staining and quantitative analysis were performed on hDPSCs transfected with siRNA on days 7, 14, and 21 of induced differentiation. (m) Relative mRNA levels of Alp, Col1a1, Runx2, Osx , and Dmp1 in siRNA-transfected hDPSCs on days 7, 14, and 21 of induced differentiation. Scale bars: 250 μm (ALP and ARS staining); Mean ± SD was employed to express all data (* p < 0.05, ** p < 0.01, *** p < 0.0001, **** p < 0.00001).

Article Snippet: Upon reaching 80% confluence, GM was replaced with osteogenic induction medium (OM), where OM was made by adding 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Solarbio, Beijing, China) and 100 nM dexamethasone (Solarbio, Beijing, China) to GM.

Techniques: Over Expression, In Vitro, Knockdown, Fluorescence, Microscopy, Expressing, Incubation, Staining, Transfection

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: rhGDF15 stimulated the osteogenic differentiation of hDPSCs in vitro . (a) ALP staining was conducted after treating hDPSCs for a duration of 7 days. (b) Typical pictures of ARS staining on day 21 of induction. (c) Relative quantitative analysis of ARS staining. (d) Expression of osteogenic-specific proteins (COL1A1, RUNX2, DMP1, and DSPP) on day 7 of osteogenic differentiation. (e) Grayscale analysis of the protein bands. (f) Relative mRNA levels of osteogenic-specific genes ( Alp, Col1a1, Runx2, Osx, Ocn, Dmp1 , and Dspp ) on days 7, 14, and 21 of induction. (g) Results of ALP staining of hDPSCs cultivated in OM containing 20 ng/mL rhGDF15 at 3, 5, and 7 days. (h) ARS staining images of hDPSCs cultured with OM containing 20 ng/mL rhGDF15 at 7, 14, and 21 days. (i) Relative quantitative analysis of ARS staining. (j) Expression of osteogenic-specific proteins (COL1A1, RUNX2, OPN, DMP1, and DSPP) in hDPSCs was detected by WB on days 7, 14, and 21 after stimulation by 20 ng/mL rhGDF15 and grayscale analysis of protein bands. Scale bars: 250 μm. Data were displayed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Upon reaching 80% confluence, GM was replaced with osteogenic induction medium (OM), where OM was made by adding 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Solarbio, Beijing, China) and 100 nM dexamethasone (Solarbio, Beijing, China) to GM.

Techniques: In Vitro, Staining, Expressing, Cell Culture

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: rhGDF15 promoted bone formation and bone repair in vivo . (a) Schematic diagram depicting the experimental workflow to assess the impact of rhGDF15 on bone formation. (b and c) Typical pictures of Masson and H&E staining of bone formation areas in the implant. Scale bars: 200 μm. Black or green boxes indicate enlarged areas. Scale bars: 100 μm. The arrow indicates the bone tissue in the implants. (d and e) Quantitative evaluation of Masson and H&E staining. (f) Typical photos of immunohistochemical staining for OPN, DMP1, and DSPP in the bone-forming region of the implant. Scale bars: 50 μm. Arrows indicate the osteogenic regions within the implants. (g) Diagrammatic representation of calvarial defect model in rats. (h) Schematic diagram of the experimental grouping of calvarial defects. Eight weeks after transplantation, the defect area was reconstructed using micro-CT analysis. The yellow circle indicated a 5 mm defect area. (i) BV/TV, Tb.N, and Tb.Sp was qualitatively measured. (j and l) Representative pictures of Masson and H&E staining of the area of new bone formation. Scale bars: 500 μm. Magnified images (gray squares) were captured at the center and edges of the calvarial defect. Scale bars: 250 μm. The gray dashed lines indicate the boundaries of the defect area. Irregular areas circled by black or green dashed lines indicate new bone creation in the area of the defect. Arrows indicate new bone or the host bone. (k) Percentage of new bone area in defect region. (m) Immunofluorescence staining to visualize the expression of DMP1 and OPN in the region of new bone formation. Scale bars: 100 μm. (n) The immunofluorescence staining of DMP1 and OPN was quantified. Mean ± SD was employed to express all data (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Upon reaching 80% confluence, GM was replaced with osteogenic induction medium (OM), where OM was made by adding 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Solarbio, Beijing, China) and 100 nM dexamethasone (Solarbio, Beijing, China) to GM.

Techniques: In Vivo, Staining, Immunohistochemical staining, Transplantation Assay, Micro-CT, Immunofluorescence, Expressing

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: rhGDF15 activated TGF-β/SMAD signaling pathway in hDPSCs. (a and b) Tgf-βr2 mRNA levels in hDPSCs overexpressing or knockdown of Gdf15 were measured after culturing in OM for 7, 14, and 21 days. (c) Tgf-βr2 mRNA levels in hDPSCs treated with OM containing rhGDF15 were measured after 7, 14, and 21 days of culture. (d) Structural modeling for molecular docking analysis of GDF15 and TGF-βR2. GDF15 and TGF-βR2 are depicted in blue-purple and orange-yellow, respectively. Hydrogen bonds are indicated in yellow. (e) Co-IP assay showing representative protein bands of GDF15 and TGFβ-R2 in hDPSCs. (f) Levels of TGF-β/SMAD signaling pathway-specific proteins after 20 ng/mL rhGDF15 treatment of hDPSCs for the indicated times were tested utilizing WB and grayscale analysis of protein bands. (g) Levels of TGF-β/SMAD signaling pathway-specific proteins after rhGDF15 (0–100 ng/mL) treatment of hDPSCs for 30 min were detected by WB and grayscale analysis of protein bands. (h) Expression of total, plasma, and nuclear proteins of p-SMAD2 and p-SMAD3 in hDPSCs after stimulation with rhGDF15 (20 ng/mL) for 30 min, and grayscale analysis of protein bands. (i) Immunofluorescence co-localization of TGF-β/SMAD signaling proteins in implants in the nude mouse subcutaneous transplantation model. Scale bars: 100 μm. The area of new bone formation is delineated by yellow dashed lines. The white dashed box demarcates the regions selected for high-magnification demonstration of osteogenic areas. Scale bars: 100 μm (enlarged view). Data were displayed as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Upon reaching 80% confluence, GM was replaced with osteogenic induction medium (OM), where OM was made by adding 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Solarbio, Beijing, China) and 100 nM dexamethasone (Solarbio, Beijing, China) to GM.

Techniques: Knockdown, Co-Immunoprecipitation Assay, Expressing, Clinical Proteomics, Immunofluorescence, Transplantation Assay

Journal: Journal of Tissue Engineering

Article Title: GDF15 promotes osteogenic differentiation of human dental pulp stem cells by activating the TGF-β/SMAD signaling pathway

doi: 10.1177/20417314251357752

Figure Lengend Snippet: Activation of the TGF-β/SMAD signaling pathway by GDF15 is partially reversed by the inhibitors. (a) The concentration of the inhibitor was selected based on the analysis of the protein level ratios of phosphorylated to total SMAD2 or SMAD3. (b and c) The protein expression levels and quantitative analysis of p-SMAD2/SMAD2 and p-SMAD3/SMAD3 were assessed in hDPSCs pretreated with inhibitor for 1 h followed by rhGDF15 stimulation for 30 min. (d) Representative images and quantitative analysis of p-SMAD2 or p-SMAD3 immunofluorescence staining. Scale bars: 100 μm. (e and f) The osteogenic effects of inhibitor-treated hDPSCs were analyzed by qPCR. (g) ALP staining following 7 days of treatment. Scale bars: 250 μm. (h) ARS staining following 21 days of treatment and relative quantitative analysis. Scale bars: 250 μm. (i and j) The osteogenic effects of hDPSCs treated with inhibitors for 7 days were evaluated by WB, and the protein bands were analyzed in gray. Mean ± SD was employed to express all data (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Upon reaching 80% confluence, GM was replaced with osteogenic induction medium (OM), where OM was made by adding 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Solarbio, Beijing, China) and 100 nM dexamethasone (Solarbio, Beijing, China) to GM.

Techniques: Activation Assay, Concentration Assay, Expressing, Immunofluorescence, Staining