Type 2 diabetes and the clinically normal pulp: An in vitro study

Abstract

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-IL1β, 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 IL1β (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.

Keywords: dental pulp; histomorphology; immune response; immunohistochemistry; type 2 diabetes.

FIGURE 1

Histological images of coronal clinically normal dental pulp from non‐T2D and T2D participants (H&E staining); (a), (c), and (e) representative non‐T2D dental pulp and (b), (d), (f), (g) and (h) representative TR2D dental pulp. The T2D dental pulp shows denser collagen with a notable presence of tertiary dentine. It appears less cellular, has thickened blood vessel walls, and the presence of amorphous calcified stones within the T2D pulp tissue was common. (a and b; 2× magnification, scale bar, 1 mm, (c),(d), (g) and (h); 20× magnification, 100 μm, e and f; 40× scale bar 50 μm). D, dentine; PD, predentine; O, odontoblast; P, central pulp; CFZ, cell free zone; CRZ, cell rich zone; BV, blood vessels; TD, tertiary dentine; S, pulp calcification/stone

FIGURE 2

Special stain images showing the histomorphology of the clinically normal dental pulp from non‐T2D and T2D participants; (a) and (c) representative non‐T2D dental pulp and (b) and (d) representative T2D dental pulp; (a) and (b) shows Masson's trichrome stain; the intensity of blue collagen stain in central region of dental pulp and around thickened blood vessels (black arrows) was increased in T2D group. Endothelial cells lining blood vessels are stained black; (c) and (d) shows Van Gieson stain; collagen staining was intense in T2D dental pulp especially around blood vessels compared with the non‐T2D dental pulp. The presence of neurovascular bundles was less evident in T2D samples. (a–d; 40× scale bar 50 µm). BV, blood vessels; EC, endothelial cells; NVB, neurovascular bundle

FIGURE 3

Line plot graph showing the mean total non‐endothelial cell nuclei and blood vessel count/section in T2D and non‐T2D dental pulp samples using an unpaired Student's t‐test (*p‐value .05). The error bar indicates standard deviation

FIGURE 4

Photomicrograph showing immunolabelling pattern of anti‐TLR2, anti‐TLR4 and anti‐FOXP3 from non‐T2D and T2D participants, and positive and negative control samples. Anti‐TLR2 staining was only very weakly detected in the odontoblast region (a and b) while moderate anti‐TLR4 staining was found (e and f). Strong anti‐FOXP3 staining was observed for non‐T2D samples around blood vessels (i) while in the T2D samples staining was weak (j). Black arrows indicate positive staining. Lymph node tissue was used as the positive control for all antibodies and non‐specific monoclonal IgG was used as a negative control (a–l; 40× magnification, scale bar 50 µm)