Fig 1: Schematic diagram of the mechanism by which PLGA/pDA-loaded hADSC-EVs affect the osteogenic differentiation of hPDLSCs. PLGA/pDA-loaded hADSC-EVs may promote the osteogenic differentiation of hPDLSCs by delivering CGRP, thereby promoting the repair of alveolar bone defects.
Fig 2: hPDLSCs uptake hADSC-EVs to increase the CGRP expression. (a) Venn diagram of the significantly highly expressed genes in the GSE53929 dataset and the top 100 genes related to hPDLSC osteogenic differentiation obtained from the GeneCards database. (b) Interaction network of candidate genes. (c) CGRP expression in the GSE53929 dataset. (d) The morphology of single colony forming unit of hPDLSCs (scale bar = 100 µm). (e) Immunofluorescence staining of vimentin and cytokeratin proteins in hPDLSCs (scale bar = 50 µm). (f) Osteogenic differentiation of hPDLSCs determined by ALP staining and ARS staining (scale bar = 50 µm). (g) Adipogenic differentiation of hPDLSCs determined by Oil Red O staining (scale bar = 50 µm). (h) Chondrogenic differentiation ability of hPDLSCs determined by Alcian blue staining (scale bar = 50 µm). (i) Fluorescence microscopic images of hADSC-EVs (red dots) labeled by PKH26 internalized by hPDLSCs. hPDLSCs were stained with phalloidin Alexa Fluor (green), and Hoescht (blue) was used for nuclear counterstaining (scale bar = 25 µm). (j) Western blot analysis of CGRP protein in hPDLSCs cocultured with hADSC-EVs. *p < 0.05, compared with the control samples or control hPDLSCs, N = 3.
Fig 3: Salivary and plasma NGF, CGRP, BDNF, glutamate and substance P concentration in 20 healthy individuals matched for age and gender (Fig. 2A–H). Large variations were observed between different collection methods. Several isoforms was detected for NGF, CGRP and BDNF. The isoform pattern showed significant variation in both expression and chemiluminescence levels between different collection methods (Friedman; P < 0.05). All stimulated saliva samples, whether chemically or mechanically stimulated, showed significant higher expression of total-NGF and total-CGRP compared to unstimulated saliva samples and plasma. Higher concentration of glutamate was found in stimulated whole saliva comparing to other salivary collection methods. However, the plasma levels of glutamate and substance P were significantly higher in comparison with the levels detected in saliva.
Fig 4: The effects of SRT1720 and EX527 on CGRP and c-Fos expression levels in the TNC. A, B, C Compared with the Sham group, the expression of CGRP and c-Fos increased in NTG group. The CGRP and c-Fos levels in the NTG + SRT1720 group were significantly reduced compared with that in the NTG + vehicle group. The CGRP and c-Fos expression levels were higher in the NTG + EX527 group than that in the NTG + vehicle group. D, E, F The average immunofluorescence intensity of CGRP and the relative number of c-Fos-positive cells both were both higher in the NTG group than in the Sham group. Compared with the NTG + vehicle group, the fluorescence intensity of CGRP and number of c-Fos-positive cells were markedly decreased in the NTG + SRT1720 group. The fluorescence intensity of CGRP and the number of c-Fos-positive cells in the NTG + EX527 group were significantly higher than that in the NTG + vehicle group. There was no significant difference between the NTG and NTG + vehicle groups. (n = 5–8 in each group; scale bar = 20/100 µm; **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group; ##p < 0.01, ###p < 0.001 and ####p < 0.0001 compared with the NTG + NC groups)
Fig 5: Recurrent NTG injection induced hyperalgesia and upregulated the expression of CGRP, c-Fos. A The periorbital and paw mechanical threshold and thermal withdrawal latency during the injection of NTG. B, C The protein expression of CGRP and c-Fos in the TNC. D Counting of c-Fos was restricted to laminae I and II of the TNC region. E, F Immunofluorescence staining images of CGRP and c-Fos in the TNC. (n = 6–8 in each group; scale bar = 20/100 µm; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with the Sham group)
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