Journal: International Journal of Oral Science

Article Title: Implantation awakens peri-implant osteogenic potential via Snx5-EGFR axis-mediated mechanical transduction

doi: 10.1038/s41368-025-00423-2

Figure Lengend Snippet: Snx5 deficiency reshapes the transcriptomic profile of BMSCs, enhances basal osteogenesis, but impairs osteogenic responses to mechanical stimulation. a PCA based on transcriptomic profiles of WT and Snx5 -KO BMSCs ( n = 3). b Volcano plot (left) showing upregulated genes (red) and downregulated genes (blue); GO-BP and KEGG signaling pathways enriched among differentially expressed genes (right). c–e TPM values for genes associated with mechanosensation ( c ), extracellular matrix components ( d ), and osteogenesis ( e ) between WT and Snx5 -KO BMSCs. f , g Von kossa and ARS staining of WT and Snx5 -KO BMSCs under static or mechanical tension conditions. h Western blot analysis of Snx5 and Alpl expression and quantification of Alpl levels in WT and Snx5 -KO BMSCs under static or mechanical stimulation conditions ( n = 3). PCA Principal component analysis, WT wild-type, KO knockout, GO-BP GO biological processes, KEGG kyoto encyclopedia of genes and genomes, TPM transcripts per million, ARS alizarin red staining. Data are presented as mean ± SD. ns not significant, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1. Statistical analysis for g and h was performed using two-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: In the Baf-A1 treatment group, 1 nmol/L Baf-A1 (HY-100558, MCE) was incorporated into the osteogenic induction medium, while an equivalent volume of DMSO (D103274, Aladin) was administered to the control cells.

Techniques: Protein-Protein interactions, Staining, Western Blot, Expressing, Knock-Out

Journal: International Journal of Oral Science

Article Title: Implantation awakens peri-implant osteogenic potential via Snx5-EGFR axis-mediated mechanical transduction

doi: 10.1038/s41368-025-00423-2

Figure Lengend Snippet: Snx5 deficiency impairs implant-induced osteogenesis and disrupts the mechanical responsiveness of LepR⁺ cells. a Representative micro-CT reconstructed images of bone architecture in WT and Snx5 -KO mice under Sham or Implant conditions, showing sagittal, horizontal, and coronal views. Yellow areas indicate ROIs, defined as annular zones extending approximately 75 μm to 175 μm outward from the implant surface; white solid lines delineate the defect boundaries in the Sham group. Scale bar: 1 mm. b Quantitative analysis of BV/TV, BS/BV, and Tb.Th ( n = 5). c Immunofluorescence staining and magnified views of peri-defect or peri-implant regions from Sham and Implant groups, showing Runx2 (green), LepR⁺ tdTomato⁺ (red), and DAPI (blue). Yellow dashed boxes indicate zoomed-in areas; white dashed lines outline the boundaries of the defect or implant sites ( n = 3). Scale bars: 100 μm. d Quantification of Runx2⁺ LepR⁺ tdTomato⁺ cells as a percentage of total LepR⁺ tdTomato⁺ cells ( n = 3). e Schematic illustration showing differential responses to implant-induced mechanical cues in WT and Snx5 -KO mice: in WT mice (left), implant placement promotes osteogenic differentiation; in Snx5 -KO mice (right), this process is impaired, resulting in reduced osteogenic response. WT wild-type, KO knockout, Imp implant, ROI regions of interest, BV/TV bone volume fraction, BS/BV bone surface-to-volume ratio, Tb.Th trabecular thickness. Data are presented as mean ± SD. ns not significant, * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1. Statistical analysis for b and d was performed using two-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: In the Baf-A1 treatment group, 1 nmol/L Baf-A1 (HY-100558, MCE) was incorporated into the osteogenic induction medium, while an equivalent volume of DMSO (D103274, Aladin) was administered to the control cells.

Techniques: Micro-CT, Immunofluorescence, Staining, Knock-Out

Journal: International Journal of Oral Science

Article Title: Implantation awakens peri-implant osteogenic potential via Snx5-EGFR axis-mediated mechanical transduction

doi: 10.1038/s41368-025-00423-2

Figure Lengend Snippet: EGF-induced EGFR activation suppresses the enhanced osteogenic phenotype caused by Snx5 deficiency. a Representative sagittal, horizontal, and coronal micro-CT images of bone structure in Snx5 -KO mice under Sham and Imp conditions following EGF treatment ( n = 5). Yellow areas indicate ROIs, defined as annular zones extending approximately 75 μm to 175 μm outward from the implant surface; white solid lines delineate the defect boundaries in the Sham group. Scale bar: 1 mm. b Quantitative analysis of BV/TV, BS/BV, and Tb.Th ( n = 5). c Immunofluorescence staining and magnified views of peri-defect or peri-implant regions in Snx5 -KO mice after EGF treatment, showing Runx2 (green), LepR⁺ tdTomato⁺ (red), and DAPI (blue). Yellow dashed boxes indicate zoomed-in areas; white dashed lines delineate the defect or implant site boundaries ( n = 3). Scale bars: 100 μm. d Quantification of Runx2⁺ LepR⁺ tdTomato⁺ cells as a percentage of total LepR⁺ tdTomato⁺ cells ( n = 3). e Schematic illustration summarizing the regulatory effects of EGF on p-EGFR levels and osteogenic differentiation potential. Imp implant, EGFR epidermal growth factor receptor, KO knockout, ROI regions of interest, BV/TV bone volume fraction, BS/BV bone surface-to-volume ratio, Tb.Th trabecular thickness. Data are presented as mean ± SD. ns not significant. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1. Statistical analysis: b and d were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: In the Baf-A1 treatment group, 1 nmol/L Baf-A1 (HY-100558, MCE) was incorporated into the osteogenic induction medium, while an equivalent volume of DMSO (D103274, Aladin) was administered to the control cells.

Techniques: Activation Assay, Micro-CT, Immunofluorescence, Staining, Knock-Out

Journal: International Journal of Oral Science

Article Title: Implantation awakens peri-implant osteogenic potential via Snx5-EGFR axis-mediated mechanical transduction

doi: 10.1038/s41368-025-00423-2

Figure Lengend Snippet: Inhibition of lysosomal degradation abolishes the enhanced osteogenic phenotype induced by Snx5 deficiency. a Micro-CT reconstructed images of bone architecture in Snx5 -KO mice under Sham and Imp conditions after Baf-A1 treatment ( n = 5). Yellow areas indicate ROIs, defined as annular zones extending approximately 75 μm to 175 μm outward from the implant surface; white solid lines delineate defect boundaries in the Sham group. Scale bar: 1 mm. b Quantitative analysis of BV/TV, BS/BV, and Tb.Th in Snx5 -KO mice under Sham and Imp conditions after Baf-A1 treatment ( n = 5). c Immunofluorescence staining and magnified views of peri-defect or peri-implant regions in Snx5 -KO mice after Baf-A1 treatment, showing Runx2 (green), LepR⁺ tdTomato⁺ (red), and DAPI (blue). Yellow dashed boxes indicate zoomed-in areas; white dashed lines outline defect or implant site boundaries ( n = 3). Scale bars: 100 μm. d Quantification of Runx2⁺ LepR⁺ tdTomato⁺ cells as a percentage of total LepR⁺ tdTomato⁺ cells ( n = 3). e Schematic illustration of the proposed mechanism: in Snx5 -KO cells, EGFR is primarily trafficked into Rab7⁺ late endosomes and degraded in LAMP1⁺ lysosomes. Treatment with the lysosomal inhibitor Baf-A1 blocks this pathway and attenuates the enhanced osteogenic phenotype. Baf-A1 Bafilomycin A1, Imp implant, ROI regions of interest. Data are presented as mean ± SD. ns not significant * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1. Statistical analysis for panels b and d was performed using two-way ANOVA followed by Tukey’s multiple comparisons test

Article Snippet: In the Baf-A1 treatment group, 1 nmol/L Baf-A1 (HY-100558, MCE) was incorporated into the osteogenic induction medium, while an equivalent volume of DMSO (D103274, Aladin) was administered to the control cells.

Techniques: Inhibition, Micro-CT, Immunofluorescence, Staining

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Cultivation and identification of PDLSCs. ( A ) Morphology of PDLSCs. Scale Bar: 200 μm. ( B ) Lipid droplets of PDLSCs after adipogenic induction. Scale bar: 200 μm. ( C ) Mineralized nodules of PDLSCs after osteogenic induction. Scale bar: 500 μm. ( D ) Flow cytometric analyses of PDLSCs. Mesenchymal stem cell markers (CD44, CD105) were positive and hematopoietic stem cell markers (CD34, CD45) were negative in PDLSCs.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques:

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of AS-IV on proliferation and osteogenic differentiation of PDLSCs. ( A ) CCK-8 analysis of AS-IV effects on PDLSCs on day 1, 3, and 5. ( B and C ) ALP activity ( B ) and extracellular matrix mineralization ( C ) quantification of PDLSCs affected by AS-IV. ( D ) ALP staining. Scale bar: 500 μm. ( E ) Alizarin Red staining. Scale bar: 500 μm. ( F – H ) qRT-PCR analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV. ( I – L ) Western blot and semi-quantitative analysis of COL-1 (J), ALP(K), and RUNX-2 ( L ) in PDLSCs cultured with AS-IV. 20 μM AS-IV group had a significant promoting effect. Data are presented as mean ± SD. ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques: CCK-8 Assay, Activity Assay, Staining, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of LY294002 on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and D ) ALP staining( A ) and ALP activity quantification( D ) of PDLSCs affected by LY294002. Scale bar: 500 μm. ( B and E ) Immunofluorescence staining( B ) of COL-1 (green) and DAPI staining (blue) and quantification(E). Scale bar: 50 μm. ( C and F ) Immunofluorescence staining( C ) of eNOS (green) and DAPI staining (blue) and quantification(F). Scale bar: 50 μm. ( G ) NO2- concentration represented the level of NO in cell supernatants. ( H–K ) Western blot and semi-quantitative analysis of COL-1(I), ALP(J), and RUNX-2 ( K ) in PDLSCs cultured with AS-IV and LY294002. ( L–O ) Western blot and semi-quantitative analysis of eNOS(M), iNOS( N ) and p-AKT/AKT ( O ) after addition of AS-IV at different time points. ( P–S ) Western blot and semi-quantitative analysis of eNOS(Q), iNOS( R ) and p-AKT/AKT ( S ) in PDLSCs cultured with AS-IV and LY294002. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Each experiment was repeated five times.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Concentration Assay, Western Blot, Cell Culture

Journal: Drug Design, Development and Therapy

Article Title: Astragaloside IV Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells via Activating PI3K/AKT/eNOS/NO Signaling Pathway: In vitro and in vivo Study

doi: 10.2147/DDDT.S514682

Figure Lengend Snippet: Effect of L-NAME on osteogenic differentiation and pathway-associated factor of PDLSCs following AS-IV incubation. ( A and B ) ALP staining( A ) and ALP activity quantification( B ) of PDLSCs affected by L-NAME. Scale bar: 500 μm. ( C and D ) Immunofluorescence staining( C ) of COL-1 (green) and DAPI staining (blue) and quantification(D). Scale bar: 50 μm. ( E–H ) Western blot and semi-quantitative analysis of COL-1 (F), ALP(G), and RUNX-2 ( H ) in PDLSCs cultured with AS-IV and L-NAME. ( I–K ) Western blot and semi-quantitative analysis of eNOS( J ) and p-AKT/AKT ( K ) in PDLSCs cultured with AS-IV and L-NAME. Data are presented as mean ± SD. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ns: P> 0.05. Each experiment was repeated five times.

Article Snippet: Cells were seeded in 6-well plates at a density of 1×10 5 cells per well and incubated with osteogenic induction medium (MEM with 10% FBS, 50μg/mL ascorbic acid, 0.01μM dexamethasone, and 10mM β-glycerophosphate, Solarbio, Beijing, China) or adipogenic induction medium (MEM with 10% FBS, 500mM isobutyl-methylxanthine, 10 mM insulin, 60mM indomethacin, and 0.5 M hydrocortisone, Solarbio, Beijing, China).

Techniques: Incubation, Staining, Activity Assay, Immunofluorescence, Western Blot, Cell Culture