murine pre osteoblasts  (ATCC)

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Curvature-controlled orientation of cytoskeletal stress fibers on concave-cylindrical surfaces. (a) Representative confocal microscopy images depicting F-actin (magenta) and nuclei (blue) of fibroblasts, mesenchymal stromal cells, osteoblasts, pre-osteoblasts and endothelial cells seeded on flat surfaces. (b) Brass mold used to fabricate the master GeoChip from which GeoChips for use in cell culture are manufactured via sugar candy molding . Photographs show the topographic surface of the brass mold and the candy mold (Scale bar: 2 mm). Scanning electron microscopy (SEM) verified the smoothness of the resulting curved surface (half-cylinder with Ø = 1000 μm, scale bar: 200 μm). (c) Representative confocal microscopy images of cells seeded on concave-cylindrical surfaces with Ø = 100 and 1000 μm. Yellow dashed lines indicate the half-cylinder boundaries. (d-i) Distribution of stress fiber orientation quantified from the F-actin signal of cells on substrates with increasing curvature (average with standard deviation). Cartesian plots include data for fibroblasts (blue), mesenchymal stromal cells (green), osteoblasts (purple), pre-osteoblasts (orange) and endothelial cells (red). The direction 0° - 180° represents the orientation along the cylindrical surface (minimum curvature) and the direction 90° represents the orientation perpendicular to the cylindrical surface (maximum curvature). The substrate curvature experienced in dependency of the orientation is indicated by the red dashed line and red scale. Random orientation is indicated by the black dashed line. Statistical significance via Mann-Whitney test (two sided) with Bonferroni correction, ∗p < 0.05. N ≥ 3 GeoChips/cell type for a total of N ≥ 12 half-cylinders/cell type, 1 donor/cell type. Scale bars 100 μm (unless otherwise stated).

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: Confocal Microscopy, Cell Culture, Electron Microscopy, Standard Deviation, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Incidence of cell spanning on concave-cylindrical surfaces. (a) Lateral view of fibroblasts exposed to cylinders with increasing diameter (decreasing curvature), with spanning cells marked by yellow arrows. (b) Probability of spanning cells in relation to the half-cylinder diameter. (c-e, top to bottom) Representative 3D reconstructed images of fibroblasts, pre-osteoblasts and endothelial cells on concave-cylindrical surfaces with Ø = 100, 200 and 300 μm. Cells were reconstructed in Imaris using the F-actin (magenta, cell surface reconstruction) and nuclei (blue) signal as obtained by confocal microscopy. Half-cylinder contour is indicated by the yellow dashed line. Spanning cells are indicated by yellow arrows in subfigures c-e for clarity. Polar plots on the right depict the percentage of spanning cells and the corresponding angle of cell orientation for fibroblasts (blue), pre-osteoblasts (orange) and endothelial cells (red). The direction 0° - 180° represents the orientation along the cylindrical surface (minimum curvature) and the direction −90° - 90° represents the orientation perpendicular to the cylindrical surface (maximum curvature). (f) Confocal microscopy images of representative cell morphologies for fibroblasts, pre-osteoblasts and endothelial cells depicting F-actin (magenta), nuclei (blue) and focal adhesions via vinculin staining (green). Focal adhesions are indicated by green arrows (example shown on fibroblasts). (g) Cell length quantified as the major axis of an ellipse fitted around the cell. (h) Cell roundness with a value of 1 representing a perfect circle and value of 0 representing a straight line. (i) FSD calculated as the distance between FA clusters (see methods part for detailed description). (j) FA size distribution per cell plotted as the percentage of FAs that fall into the indicated size classes. (k) Representative force vector maps and (l) total cell force quantified via TFM. Statistical significance via Mann-Whitney test (two sided) with Bonferroni correction, ∗p < 0.05. N ≥ 3 GeoChips/cell type for a total of N ≥ 12 half-cylinders/cell type. N ≥ 60 cells/cell type for FA and morphological analysis. 1 donor/cell type. Scale bar 50 μm.

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: Confocal Microscopy, Staining, Plasmid Preparation, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Cell spanning initiates channel closure and subsequent tissue remodeling. (a) Fabrication of full-cylindrical channels with Ø = 250 μm in PDMS substrates by direct molding from a micro-machined brass mold. (b) Degree of channel closure representing the distribution of cells within the channels at the selected points in time during live confocal imaging. A value of 0 indicates that cells are exclusively found at the wall of the channel and a value of 1 indicates cells have completely closed the channel and are homogeneously distributed. (c) Relative degree of alignment of the cell-network within the channels quantified as the maximum value of the orientation distribution for the individual cell types and time points normalized to the highest detected value of all conditions (see also Supplementary Data S2). Higher values indicate a higher degree of alignment along the channel axis. (d-f) Lateral and front view of the PDMS cylindrical channels obtained by live confocal imaging of fibroblasts (blue), pre-osteoblasts (orange) and endothelial cells (red) using CellTracker™ Green ( t = 4, 12, 24 and 48 h after seeding). Open arrows indicate cells spanning perpendicular to the channel axis. Full arrows indicate cells oriented along the direction of the channel axis after channel closure. Channel contour is highlighted by the yellow dashed lines. The surface of the forming tissue is marked by red dashed lines. White dashed lines indicate the z-volume that is shown in the corresponding lateral views. Statistical significance via Mann-Whitney test with Bonferroni correction, ∗p < 0.05. N = 3 cylindrical channels/cell type. 1 donor/cell type. Scale bars 100 μm.

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: Imaging, MANN-WHITNEY

Journal: Bioactive Materials

Article Title: Cell type-specific response to curvature controls tissue growth dynamics in biomaterial pores

doi: 10.1016/j.bioactmat.2026.02.005

Figure Lengend Snippet: Channel closure mechanism can be controlled by substrate curvature using scaffolds with well-defined geometries. (a, left) Schematic representation of the in vitro culture setup with collagen scaffold presenting channels of controlled diameter with Ø ≈ 600 μm, Ø ≈ 350 μm and Ø ≈ 150 μm. Monolayer seeding on one side of the biomaterial facilitates migration of cells from one end of the biomaterial. (a, right) SEM image of the microarchitecture (Scale bar 20 μm) and channels within the biomaterial (Scale bars 100 μm). SEM images correspond to the outermost surface of the scaffold. (b) Comparison of template diameter against resulting channel diameter after cross-linking and sterilization of the biomaterial. (c) Representative images of fibroblasts, pre-osteoblasts and endothelial cells within channels of distinct diameters 7 days after seeding. Cell cytoskeleton (F-actin) is depicted in magenta and nuclei in blue. Yellow arrows indicate the direction (arrow angle) and degree of alignment (vector length) for the corresponding region. Scale bar close-up images: 25 μm. (d, left) Degree of channel closure for the investigated channel diameters and cell types. (d, right) Relative degree of tissue alignment for the different channel diameters and cell types. Tissue alignment ranges from 0 (fully isotropic) to 1 (fully anisotropic, dashed line). Tissue across the channel and relative degree of is calculated in the central 50 % of each channel. Data displayed as average with standard deviation. N = 4 scaffolds/cell type. 1 donor/cell type. Scale bars 200 μm (unless otherwise stated).

Article Snippet: Murine pre-osteoblasts (MC3T3-E1, CRL-2593TM, ATCC) were cultured in alpha modified minimum essential medium with nucleosides (F 0925, Biochrom AG), supplemented with 10 % v/v FBS, 1 % v/v P/S and 1 % v/v GlutaMAX (35050, Gibco®).

Techniques: In Vitro, Migration, Comparison, Plasmid Preparation, Standard Deviation

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: Recombinant MDK protein inhibits osteogenic differentiation in vitro in a dose-dependent manner. (A) Cell viability after treating MC3T3-E1 cells with recombinant MDK protein after 48 h, assessed using the CCK-8 assay. Inter-group comparisons were analyzed by one-way ANOVA. (B) Western blotting analysis of ALP, RUNX2, OSX, and OCN expression levels following MDK treatment (7 days). (C–F) Reverse transcription PCR analysis of mRNA expression levels of Alpl , Runx2 , Sp7 , and Bglap in MC3T3-E1 cells following MDK treatment (7 days). β-actin served as the internal control. Inter-group comparisons were analyzed by one-way ANOVA. (G, H) ALP staining and activity assays were performed after inducing MC3T3-E1 cells with recombinant MDK protein (0–600 ng/mL) for 14 days. Inter-group comparisons were analyzed by one-way ANOVA. (I, J) ARS staining and quantitative analysis were conducted after inducing MC3T3-E1 cells with recombinant MDK protein (0–600 ng/mL) for 21 days. Inter-group comparisons were analyzed by one-way ANOVA. Scale bar, 100 μm ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; “ns” indicates non-significant differences.

Article Snippet: Procell Life Science & Technology Co., Ltd. (Wuhan, China) supplied the pre-osteoblastic cell line MC3T3-E1 Subclone 14.

Techniques: Recombinant, In Vitro, CCK-8 Assay, Western Blot, Expressing, Reverse Transcription, Control, Staining, Activity Assay

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: MDK suppresses osteoblast differentiation via the PI3K/AKT signaling pathway. (A, B) Western blot detection of the effect of recombinant MDK protein on the protein expression of molecules in the PI3K/AKT signaling pathway during the differentiation of MC3T3-E1 to osteoblasts (7 days). Inter-group comparisons were analyzed by a two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (C, D) ALP, RUNX2, and OSX expression levels were detected by Western blotting. MC3T3-E1 cells were pretreated with 30 μM LY294002. Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; “ns” indicates non-significant differences.

Article Snippet: Procell Life Science & Technology Co., Ltd. (Wuhan, China) supplied the pre-osteoblastic cell line MC3T3-E1 Subclone 14.

Techniques: Western Blot, Recombinant, Expressing, Two Tailed Test

Journal: Genes & Diseases

Article Title: Targeting MDK alleviates bone loss via dual regulation of osteogenic differentiation and inflammatory cytokine expression

doi: 10.1016/j.gendis.2025.101931

Figure Lengend Snippet: Recombinant MDK protein triggers the activation of inflammatory cytokines through the NF-κB signaling pathway. (A, B) IL-6, TNFα, and IL-1β expression levels were detected using Western blotting. MC3T3-E1 cells were treated with recombinant MDK protein (600 ng/mL). Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by a two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (C, D) Western blotting analysis of NF-κB signaling pathway molecules in MC3T3-E1 cells treated with recombinant MDK protein for 7 days during osteoblastic differentiation. Inter-group comparisons were analyzed by two-tailed unpaired Student's t -test (for normally distributed data with equal variance). (E, F) IL-6 and IL-1β expression levels were detected using Western blotting. MC3T3-E1 cells were pretreated with 10 μM BAY 11–7082. Osteogenic differentiation was induced for 7 days. Inter-group comparisons were analyzed by one-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: Procell Life Science & Technology Co., Ltd. (Wuhan, China) supplied the pre-osteoblastic cell line MC3T3-E1 Subclone 14.

Techniques: Recombinant, Activation Assay, Expressing, Western Blot, Two Tailed Test

Journal: Dentistry Journal

Article Title: In Vitro Assessment of Osteogenic Modulation and Molecular Responses Induced by Contemporary Endodontic Sealers in MC3T3-E1 Pre-Osteoblasts

doi: 10.3390/dj14030160

Figure Lengend Snippet: ARS staining and analysis of the effect of various endodontic sealers in MC3T3-E1 cells. ( A ) Schematic diagram of the experimental workflow. Sealers (10 μL spots, 3 mm in diameter) were placed at the center of 6-well plates, allowed to set for 24 h in a laminar flow hood, and sterilized by UV irradiation for 30 min before cell seeding. MC3T3-E1 pre-osteoblasts were then cultured and analyzed by ARS staining, qRT-PCR, and Western blotting. ( B ) Representative images of ARS staining of MC3T3-E1 cells cultured with various endodontic sealers: AH26 ® , ZOE, BC, and Sealapex™. ( C ) Quantification of ARS staining intensity using ImageJ (NIH). Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (* p < 0.01, *** p < 0.0001, n.s., not significant).

Article Snippet: Murine MC3T3-E1 Subclone 4 pre-osteoblasts (ATCC CRL-2593TM, ATCC, Manassas, VA, USA) were maintained in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Staining, Irradiation, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Dentistry Journal

Article Title: In Vitro Assessment of Osteogenic Modulation and Molecular Responses Induced by Contemporary Endodontic Sealers in MC3T3-E1 Pre-Osteoblasts

doi: 10.3390/dj14030160

Figure Lengend Snippet: Osteogenic potential of ZOE and BC sealers in MC3T3-E1 cells. ( A ) Representative images of ARS staining of MC3T3-E1 cells cultured with ZOE or BC sealers. ( B ) Quantitative analysis of ARS staining intensity (ImageJ). Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by Student’s t -test (* p < 0.01). ( C ) Representative images of ARS staining of cells cultured with ZOE and BC for 7 and 14 days after osteogenic induction. Scale bars = 100 μm. ( D ) Quantitative analysis of ARS staining intensity at 7 and 14 days after switching to OI medium. Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (** p < 0.001). ( E ) Runx2 mRNA expression in cells cultured under control (no sealer), ZOE, and BC conditions for 1 week after switching to OI medium (qRT-PCR). Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (** p < 0.001). ( F ) Sp7 (Osx) mRNA expression in cells cultured under control (no sealer), ZOE, and BC conditions for 1 week after switching to OI medium (qRT-PCR). Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (** p < 0.001, n.s., not significant).

Article Snippet: Murine MC3T3-E1 Subclone 4 pre-osteoblasts (ATCC CRL-2593TM, ATCC, Manassas, VA, USA) were maintained in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Staining, Cell Culture, Expressing, Control, Quantitative RT-PCR

Journal: Dentistry Journal

Article Title: In Vitro Assessment of Osteogenic Modulation and Molecular Responses Induced by Contemporary Endodontic Sealers in MC3T3-E1 Pre-Osteoblasts

doi: 10.3390/dj14030160

Figure Lengend Snippet: Effects of ZOE and BC sealers on MC3T3-E1 cell viability and MAPK signaling. ( A ) Western blot analysis of phosphorylated and total c-Jun, p38, and Erk in cells cultured with ZOE or BC for 1 week. Gapdh (loading control). Phospho-c-Jun, p38, and Erk band intensities were normalized to their respective total protein levels. ( B ) Representative images of MC3T3-E1 cells cultured in the presence of ZOE or BC for 4 and 5 days. Scale bars: 100 μm (white), 50 μm (black). ( C , D ) Quantification of cell numbers around sealer spots at day 4 ( C ) and day 5 ( D ). The number of cells in five random fields was counted per experimental condition under light microscopy. Data are presented as mean ± SD, and the experiments were repeated three times. Statistical significance was assessed by Student’s t -test (** p < 0.001). ( E , F ) Percentages of viable ( E ) and non-viable ( F ) cells on day 5 determined by trypan blue exclusion. Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by Student’s t -test (** p < 0.001).

Article Snippet: Murine MC3T3-E1 Subclone 4 pre-osteoblasts (ATCC CRL-2593TM, ATCC, Manassas, VA, USA) were maintained in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Western Blot, Cell Culture, Control, Light Microscopy

Journal: Dentistry Journal

Article Title: In Vitro Assessment of Osteogenic Modulation and Molecular Responses Induced by Contemporary Endodontic Sealers in MC3T3-E1 Pre-Osteoblasts

doi: 10.3390/dj14030160

Figure Lengend Snippet: Effects of MAPK inhibition on BC-induced osteogenic differentiation in MC3T3-E1 cells. ( A , B ) qRT-PCR analysis of Runx2 ( A ) and Sp7 ( Osx ) ( B ) expression after 1 week of culture in OI medium under control conditions (no sealer), BC, or BC combined with MAPK inhibitors [U0126 (Erk), SB203580 (p38), SP600125 (Jnk)]. Expression levels were normalized to Gapdh . Data are presented as mean ± SD from triplicate assays, and the experiments were repeated three times. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (* p < 0.01). ( C ) Western blot analysis of Runx2, Sp7 (Osx), p-c-Jun, c-Jun, p-p38, p38, p-Erk, and Erk under the same conditions as ( A ). Gapdh (loading control). ( D ) Representative of ARS staining images of cells cultured with control (no sealer), BC, or BC plus MAPK inhibitors for 2 weeks with OIM. Scale bars = 100 μm. ( E ) Quantification of ARS staining intensity using ImageJ. Data are presented as mean ± SD from 5 fields, and the experiments were repeated three times. Statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test (*** p < 0.0001). ( F ) Proposed schematic model, based on the present findings, illustrating that the BC sealer regulates MAPK signaling pathways (Jnk, p38, Erk) to induce osteogenic differentiation by modulating the expression of Runx2 and Sp7 ( Osx ) in pre-osteoblasts.

Article Snippet: Murine MC3T3-E1 Subclone 4 pre-osteoblasts (ATCC CRL-2593TM, ATCC, Manassas, VA, USA) were maintained in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, Western Blot, Staining, Cell Culture, Protein-Protein interactions

Journal: Clinical Implant Dentistry and Related Research

Article Title: Bone Morphogenetic Protein ( BMP ) 9 Outperforms BMP2 in Osteogenesis and Osseointegration: In Vitro and In Vivo

doi: 10.1111/cid.70135

Figure Lengend Snippet: Comparative effects of BMP9 and BMP2 on osteogenic differentiation and osteoclastogenesis in vitro. (A) Real‐time PCR analysis of key osteogenic genes (Col1, Runx2, ALP, and OCN) in MC3T3‐E1 cells treated with 8 nM of BMP2 or BMP9 for 3, 5, and 7 days. All gene‐expression levels were normalized to GAPDH. (B) Western blot analysis of osteogenic marker proteins in cell lysates harvested after 7 days of treatment with BMP2 or BMP9. GAPDH was used as the loading control. Densitometric quantification of band intensities (integrated density) normalized to GAPDH is shown below the blots and presented as relative protein expression. (C) Western blot showing dose‐dependent p‐Smad1/5/9 in MC3T3‐E1 cells exposed to varying concentrations of BMP2 or BMP9. Phosphorylation was quantified by densitometry and expressed as fold change vs. control after normalization using [(p‐Smad1/5/9)/(total Smad1/5/9)] and further normalized to GAPDH, as shown in the graph below the blots. Asterisks indicate statistical significance for pairwise comparisons between BMP2 and BMP9 at the same concentration (****, p < 0.0001), unless otherwise indicated. (D) ALP activity and representative images of ALP staining in MC3T3‐E1 cultures after 7 days of induction with BMP2 or BMP9. (E) Alizarin Red S staining illustrating mineralized nodule formation after extended culture with BMP2 or BMP9. (F) Representative TRAP‐stained images of RAW 264.7‐derived osteoclasts following treatment with RANKL (3 nM), BMP2 (8 nM), or BMP9 (8 nM) for 5 days. TRAP‐positive multinucleated osteoclasts are indicated by arrows. Scale bar, 20 μm. (G) Quantification of TRAP‐positive multinucleated cells per well. Data are presented as the mean ± SD ( n = 3 independent experiments), and p ‐values were calculated using one‐way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). BMP, bone morphogenetic protein; PCR, polymerase chain reaction; ALP, alkaline phosphatase; Col1, collagen type I; Runx2, runt‐related transcription factor 2; OCN, osteocalcin; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Mouse pre‐osteoblast MC3T3‐E1 (subclone 4; CRL‐2593), murine macrophage RAW 264.7 (TIB‐71) cells (ATCC, Manassas, VA, USA), and human periodontal ligament stem cells (hPDLSCs) were treated with these growth factors for in vitro experiments.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Marker, Control, Expressing, Phospho-proteomics, Concentration Assay, Activity Assay, Staining, Derivative Assay, Polymerase Chain Reaction