Epiregulin enhances periodontal tissue regeneration by promoting bone marrow mesenchymal stem cell functions under inflammatory niches

Figure 1 Supplementation with epiregulin enhanced the migration, chemotaxis, and osteogenic differentiation of mouse bone marrow stem cells under inflammatory conditions. A and B: Scratch-simulated wound migration assay results (n = 3, one-way analysis of variance [ANOVA]). Scale bar: 500 μm; C and D: Transwell chemotaxis assay results (n = 3, one-way ANOVA). Scale bar: 50 μm; E: Alizarin Red staining; F: Expression of osterix (OSX) and osteocalcin (OCN) was revealed in mouse bone marrow stem cells that were cultured in the inflammatory condition by western blotting. Error bars represent standard deviation. ^aP ≤ 0.05; ^bP ≤ 0.01. EREG: Epiregulin; TNF: Tumor necrosis factor.

Figure 2 Knockdown of epidermal growth factor receptor blocked the positive effects of epiregulin on migration, chemotaxis, and osteogenic potential of mouse bone marrow stem cells under inflammatory conditions. A: Quantitative analysis of quantitative real-time polymerase chain reaction showed that the transcription of epidermal growth factor receptor (Egfr) mRNA was decreased in the EGFR single hairpin RNA (shRNA) group (n = 3, Student’s t-test). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as an internal control; B: Expression of EGFR was revealed in mouse bone marrow stem cells by western blotting. GAPDH was used as the internal control; C and D: Scratch-simulated wound migration assay results (n = 3, one-way analysis of variance [ANOVA]). Scale bar: 500 μm; E and F: Transwell chemotaxis assay results (n = 3, one-way ANOVA). Scale bar: 50 μm; G: Alizarin Red staining; H: Expression of osterix (OSX) and osteocalcin (OCN) was revealed in mouse bone marrow stem cells that were cultured in the inflammatory condition by western blotting. Error bars represent standard deviation. ^aP ≤ 0.05; ^bP ≤ 0.01. EREG: Epiregulin.

Figure 3 Epiregulin activated the epidermal growth factor receptor-extracellular signal-regulated kinase 1/2 signaling pathway to promote migration, chemotaxis, and osteogenic differentiation of mouse bone marrow stem cells under inflammatory conditions. A: Expression of mitogen-activated protein kinase signaling pathway were revealed in mouse bone marrow stem cells (mBMSCs) that were cultured in the inflammatory condition by western blotting. β-actin was used as the internal control; B: Expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and ERK were detected in mBMSCs that were cultured in the inflammation condition by western blotting; C and D: Scratch-simulated wound migration assay results (n = 3, one-way analysis of variance [ANOVA]). Scale bar: 500 μm; E and F: Transwell chemotaxis assay results (n = 3, one-way ANOVA). Scale bar: 100 μm; G: Alizarin red staining; H: Expression of osterix (OSX) and osteocalcin (OCN) were revealed in mBMSCs that were cultured in the inflammatory condition by western blotting. Error bars represent standard deviation (n = 3). ^aP ≤ 0.05; ^bP ≤ 0.01. EGFR: Epidermal growth factor receptor; EREG: Epiregulin; JNK: C-Jun N-terminal kinase; MAPK: Mitogen-activated protein kinase.

Figure 4 Local injection of epiregulin promoted the regeneration of periodontal tissue in a rat model of periodontitis. A: Three-dimensional micro-computed tomography reconstruction of mouse maxillary from the three groups; B: Sagittal-sectional images of maxillary; C and D: Micro-computed tomography measurement of bone formation (n = 5, one-way analysis of variance); E-M: Hematoxylin and eosin staining indicated new periodontal tissue regeneration from coronal-sectional images of maxillary in the blank control group (E), periodontitis group (H), periodontitis + sodium chloride (NaCl) (K) and periodontitis + epiregulin (EREG) (N). Scale bar: 200 μm; hematoxylin and eosin staining indicated Sharpey’s fibers in the blank control group (G), periodontitis group (J), periodontitis + NaCl (M) and periodontitis + EREG (P). Scale bar: 100 μm; hematoxylin and eosin staining indicated inflammatory cells infiltration in the blank control group (F), periodontitis group (I), periodontitis + NaCl (L) and periodontitis + EREG (O). Scale bar: 50 μm; Q: Quantitative analysis of distance between cemento-enamel junction (CEJ) and height of alveolar bone (n = 3, Kruskal-Wallis test). Error bars represent the standard deviation. ^aP ≤ 0.05; ^bP ≤ 0.01. B: Bone; D: Dentin; HAB: Height of alveolar bone.