Journal: iScience

Article Title: Dental pulp stem cells accelerate wound healing through CCL2-induced M2 macrophages polarization

doi: 10.1016/j.isci.2023.108043

Figure Lengend Snippet: Bioinformatic analysis gene expression profile of BMMSCs and DPSCs (A) Top 20 differentially expressed genes related to immunity between DPSCs and BMMSCs were visualized. (B) qRT-PCR analysis demonstrated that the mRNA expression levels of CCL2 , TGFBR3 , C1S , IL1R1 , IL10RB , IFNAR1 , CD46 , and CD302 were decreased in BMMSCs at passage three compared to those of DPSCs. (C) ELISA analysis showed that the concentration of CCL2 secreted by DPSCs was significantly higher than that of BMMSCs. Values are means ± SDs. Student’s t-tests were used to determine statistical significance. (∗∗p < 0.01; ∗∗∗p < 0.001).

Article Snippet: Under certain conditions, THP-1 macrophages and DPSCs or BMMSCs (2: 1) of passage 4 and DPSCs of passage 8 were cocultured in a transwell system in the presence or absence of either 10 μg/mL C-C motif chemokine ligand 2 (CCL2) neutralising antibody (MAB679; R&D System, Minnesota, USA) for 3 d. And recombinant human CCL2 (10 ng/mL or 15 ng/mL; 300-04; Peprotech, Rocky Hill, USA) were added for 3 d as positive control group.

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: iScience

Article Title: Dental pulp stem cells accelerate wound healing through CCL2-induced M2 macrophages polarization

doi: 10.1016/j.isci.2023.108043

Figure Lengend Snippet: DPSCs-mediated induction of M2 macrophages by CCL2 (A–D) DPSCs could downregulate the mRNA expression of IL1B , IL6 , and TNFA , and upregulate the mRNA expression of ARG1 , and blocking CCL2 could inhibit the DPSCs-mediated decrease of pro-inflammatory cytokines. The presence of neutralizing antibodies against CCL2 did not influence the immunomodulatory function of BMMSCs. (E and F) DPSCs could increase the concentration of IL-10 and decrease the concentration of TNF-α for 3 days, and blocking CCL2 could inhibit DPSCs-mediated induction of M2 macrophages, but not BMMSCs ( ns P > 0.05, ∗p < 0.05, ∗∗p < 0.01). NAB: neutralizing antibody.

Article Snippet: Under certain conditions, THP-1 macrophages and DPSCs or BMMSCs (2: 1) of passage 4 and DPSCs of passage 8 were cocultured in a transwell system in the presence or absence of either 10 μg/mL C-C motif chemokine ligand 2 (CCL2) neutralising antibody (MAB679; R&D System, Minnesota, USA) for 3 d. And recombinant human CCL2 (10 ng/mL or 15 ng/mL; 300-04; Peprotech, Rocky Hill, USA) were added for 3 d as positive control group.

Techniques: Expressing, Blocking Assay, Concentration Assay

Journal: iScience

Article Title: Dental pulp stem cells accelerate wound healing through CCL2-induced M2 macrophages polarization

doi: 10.1016/j.isci.2023.108043

Figure Lengend Snippet: Passage-induced senescence of DPSCs possesses the ability to induce M2 macrophages polarization via CCL2 (A–D) DPSCs (P8) downregulated the mRNA expression of IL1B , IL6 , and TNFA and upregulated the expression of IL10 for 3 days. Blocking CCL2 could inhibit DPSC (P8)-mediated induction of M2 macrophages. (E and F) DPSCs could increase the concentration of IL-10 and decrease the concentration of TNF-α for 3 days, and blocking CCL2 could inhibit DPSCs (P8)-mediated IL-10 secreting. (G and H) ARG1 protein expression was upregulated by cultured with DPSCs (P8) in transwell for 3 days, and specific neutralizing antibodies of CCL2 could weaken this ability of DPSCs (P8). ( ns P > 0.05, ∗p < 0.05, ∗∗p < 0.01). NAB neutralizing antibody.

Article Snippet: Under certain conditions, THP-1 macrophages and DPSCs or BMMSCs (2: 1) of passage 4 and DPSCs of passage 8 were cocultured in a transwell system in the presence or absence of either 10 μg/mL C-C motif chemokine ligand 2 (CCL2) neutralising antibody (MAB679; R&D System, Minnesota, USA) for 3 d. And recombinant human CCL2 (10 ng/mL or 15 ng/mL; 300-04; Peprotech, Rocky Hill, USA) were added for 3 d as positive control group.

Techniques: Expressing, Blocking Assay, Concentration Assay, Cell Culture

Journal: iScience

Article Title: Dental pulp stem cells accelerate wound healing through CCL2-induced M2 macrophages polarization

doi: 10.1016/j.isci.2023.108043

Figure Lengend Snippet: DPSCs-based therapy enhanced mucosa wound healing in mice via CCL2 (A and B) Mice receiving DPSCs infusion displayed accelerated mucosal wound closure compared with the control mice without treatment, and the application of CCL2 neutralizing antibodies inhibited DPSCs-mediated wound repair, but not BMMSCs. (C) HE staining of wounds showed a more organized granulation tissue at the excisional wound site in DPSCs-treated mice compared with other groups. (D and E) Double immunofluorescence staining of CD68 and ARG1 revealed DPSCs and BMMSCs treatment led to an increase in the number of M2 macrophages compared with the control group, and the percentage of M2 macrophages was significantly higher in the DPSCs group than in the BMMSCs group, and the application of neutralizing antibody weakened this function of DPSCs, but not BMMSCs ( ns P > 0.05, ∗p < 0.05, ∗∗p < 0.01). NAB neutralizing antibody.

Article Snippet: Under certain conditions, THP-1 macrophages and DPSCs or BMMSCs (2: 1) of passage 4 and DPSCs of passage 8 were cocultured in a transwell system in the presence or absence of either 10 μg/mL C-C motif chemokine ligand 2 (CCL2) neutralising antibody (MAB679; R&D System, Minnesota, USA) for 3 d. And recombinant human CCL2 (10 ng/mL or 15 ng/mL; 300-04; Peprotech, Rocky Hill, USA) were added for 3 d as positive control group.

Techniques: Control, Staining, Double Immunofluorescence Staining

Journal: iScience

Article Title: Dental pulp stem cells accelerate wound healing through CCL2-induced M2 macrophages polarization

doi: 10.1016/j.isci.2023.108043

Figure Lengend Snippet:

Article Snippet: Under certain conditions, THP-1 macrophages and DPSCs or BMMSCs (2: 1) of passage 4 and DPSCs of passage 8 were cocultured in a transwell system in the presence or absence of either 10 μg/mL C-C motif chemokine ligand 2 (CCL2) neutralising antibody (MAB679; R&D System, Minnesota, USA) for 3 d. And recombinant human CCL2 (10 ng/mL or 15 ng/mL; 300-04; Peprotech, Rocky Hill, USA) were added for 3 d as positive control group.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, TUNEL Assay, Apoptosis Assay, Microarray, Software

Journal: Journal of Orthopaedic Translation

Article Title: Knockdown of asporin affects transforming growth factor-β1-induced matrix synthesis in human intervertebral annulus cells

doi: 10.1016/j.jot.2016.05.011

Figure Lengend Snippet: Transforming growth factor-β1 regulates the expression of asporin in human intervertebral annulus cells. The human intervertebral annulus cells were cultured in a six-well plate. When they reached confluence, culture medium was replaced and cells were treated with transforming growth factor-β1 at the assigned concentrations (5 ng/mL, 10 ng/mL, and 15 ng/mL) and examined at the indicated time points (6 hours, 12 hours, 18 hours, and 24 hours). Expression of asporin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified using real-time polymerase chain reaction. The asporin message is normalised with GAPDH. The data are shown as mean ± standard deviation of triplicate assays.

Article Snippet: After 12 hours, the cells were treated with different concentrations of TGF-β1 (Sigma-Aldrich Corporation; 5 ng/mL, 10 ng/mL, and 15 ng/mL) and examined at different time points (6 hours, 12 hours, 18 hours, and 24 hours).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Orthopaedic Translation

Article Title: Knockdown of asporin affects transforming growth factor-β1-induced matrix synthesis in human intervertebral annulus cells

doi: 10.1016/j.jot.2016.05.011

Figure Lengend Snippet: Knockdown of asporin expression by small interfering RNA (siRNA). siRNA (100nM) targeting asporin or control siRNA (NC siRNA) was transfected into cultured annulus cells. Twenty-four hours later, the cells were treated or untreated with 10-ng/mL transforming growth factor-β1 (TGF-β1) for another 24 hours. (A) The knockdown efficiency was detected using Western blotting and cell lysates were probed with asporin antibody with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as a loading control. (B) The knockdown efficiency was detected with real-time polymerase chain reaction and real-time polymerase chain reaction for asporin mRNA level normalised to that of GAPDH. The data are shown as mean ± standard deviation of triplicate assays. * p < 0.05.

Article Snippet: After 12 hours, the cells were treated with different concentrations of TGF-β1 (Sigma-Aldrich Corporation; 5 ng/mL, 10 ng/mL, and 15 ng/mL) and examined at different time points (6 hours, 12 hours, 18 hours, and 24 hours).

Techniques: Expressing, Small Interfering RNA, Transfection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Orthopaedic Translation

Article Title: Knockdown of asporin affects transforming growth factor-β1-induced matrix synthesis in human intervertebral annulus cells

doi: 10.1016/j.jot.2016.05.011

Figure Lengend Snippet: The effect of asporin on transforming growth factor-β1 (TGF-β1)-induced matrix biosynthesis. The cultured annulus cells were transfected with asporin siRNA or control small interfering RNA (siRNA; 100nM). After 24 hours, the cells were incubated with 10 ng/mL of TGF-β1 for another 24 hours. The expression levels of (A) aggrecan and (B) type II collagen were determined using real-time polymerase chain reaction. The results were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are from triplicate assays. * p < 0.05.

Article Snippet: After 12 hours, the cells were treated with different concentrations of TGF-β1 (Sigma-Aldrich Corporation; 5 ng/mL, 10 ng/mL, and 15 ng/mL) and examined at different time points (6 hours, 12 hours, 18 hours, and 24 hours).

Techniques: Cell Culture, Transfection, Small Interfering RNA, Incubation, Expressing, Real-time Polymerase Chain Reaction