RESUMEN
AIM: This ex vivo study aimed to compare protein expression of advanced glycation end-products (AGE) and receptor (RAGE), and the levels of selected genes associated with inflammation and collagen within dental pulp tissue from patients with type 2 (T2D) diabetes and non-T2D. METHODOLOGY: Noncarious extracted permanent molar teeth from patients with well-controlled T2D (n = 19) and non-T2D (controls) (n = 19) were collected and compared. The coronal pulp was examined using immunohistochemistry (IHC) (n = 10 per group) for anti-AGE and anti-RAGE. Quantitative PCR (n = 9 per group) was used to analyse the gene expression levels of NFKB, S100A12 and COLIA1. Data analyses were performed between the groups using GraphPad Prism using Pearson correlation, Shapiro-Wilk and Mann-Whitney U-tests, and multiple regression using SPSS. RESULTS: AGEs were distributed diffusely throughout the pulp extracellular matrix associated with collagen fibres and were present on several cell types. RAGE was expressed at the pulp-dentine interface and was observed on odontoblasts, immune cells, endothelial cells and fibroblasts. Semi-quantitative analysis of IHC samples showed significantly increased expression of AGE (p < .0001) and RAGE (p = .02) in T2D samples compared with controls. The expression of NFKB (p < .0001), S100A12 (p < .0001) and COLIA1 (p = .01) genes were significantly higher in the T2D pulp, and multivariate logistic regression analysis showed that these findings were not affected by age. CONCLUSION: T2D may exert a similar glycation response in the dental pulp to other body sites. This could occur through activation of NF-κB pathways with a concomitant increase in genes associated with inflammation and collagen.
RESUMEN
AIM: The aim of this study was to investigate the effect of type 2 diabetes (T2D) on clinically normal dental pulp tissue by using special stains and immunohistochemistry (IHC) to determine the morphology of the coronal pulp and distribution of immune markers in non-T2D and T2D groups. METHODOLOGY: Ethics approval for this in vitro pilot study was obtained from the University of Otago Human Ethics Committee (16/069). Twenty extracted permanent molar teeth diagnosed as having clinically normal pulp status were collected. Ten teeth were from participants with well-controlled T2D and ten from participants without diabetes (non-T2D). Each tooth was sectioned transversely at the cemento-enamel junction before the crowns were decalcified and embedded in paraffin. Sections were stained with haematoxylin and eosin, Massons trichrome, and van Gieson stains for histological and morphological evaluation. IHC using anti-CD4, anti-CD68 and anti-CD83 and anti-IL1ß, anti-IL6, anti-IL17, anti-TNF-α, anti-TLR2, anti-TLR4 and anti-FOXP3 identified proteins of interest. Qualitative and semi-quantitative analyses evaluated the morphology of the dental pulp and protein expression. Data analyses were performed with GraphPad Prism, using Student's t-test and multiple regression using SPSS at p < .05. RESULTS: Special stains demonstrated morphological differences in the T2D dental pulp compared with non-T2D. Qualitative analysis indicated that the pulp in the T2D samples was consistently less cellular, less vascular, showed evidence of thickened blood vessel walls, increased pulp calcification and collagen deposition. Semi-quantitative analysis of IHC samples showed the T2D pulp had significantly increased expression of macrophage and dendritic cell markers CD68 (p < .001) and CD83 (p = .04), and there was significantly greater expression of inflammatory cytokines IL1ß (p = .01), IL6 (p < .0001), IL17 (p < .0001) and TNF-α (p = .01). T2D samples showed a significant increase in markers of innate inflammation, TLR2 (p < .001) and TLR4 (p < .001) and decreased expression of regulatory T-cell marker, FOXP3 (p = .01). Multiple regression showed that age-corrected differences were statistically significant. CONCLUSION: Preliminary findings suggest that T2D may exert a similar response in the pulp to complications in other body sites. Hyperglycaemia is associated with changes in the morphology of the clinically normal dental pulp with altered immune cell and cytokine expression.