Abstract
Aim: The aims of this study were – (1) to investigate morphological changes in the coronal dental pulp of teeth with dentine caries from individuals with and without T2D, using histological stains and immunohistochemistry for neuronal cell marker, S100; (2) to evaluate the effects of hyperglycaemia and low oxygen conditions on human dental pulp cell (hDPC) proliferation, metabolic activity, and the expression of neuropeptide- and angiogenesis-related genes in an in vitro T2D cell culture model.
Methodology: Ethical approval was obtained (H21/080). Extracted permanent molars with dentine caries were collected from T2D and non-T2D participants. Teeth were formalin fixed, decalcified using Ethylenediaminetetraacetic acid (EDTA), and processed for histology and immunohistochemistry. In vitro, primary hDPCs were cultured under a range of glucose concentrations (5.5, 12.5, 25 mM) and oxygen tensions (2% O₂, 5% O₂ and 21% O₂) over a range of time-points. Cell counts and metabolic activity were assessed using Crystal Violet and PrestoBlue™ assays. Gene expression analysis for angiogenic and neuropeptide-related genes was performed using quantitative real-time PCR (qPCR). Total RNA was extracted, treated with DNase, and gene expression of angiogenic and neuropeptide genes was analysed using custom TaqMan® qPCR arrays. Data were analysed using one-way ANOVA and Tukey’s post-hoc test (p<.05).
Results: T2D pulps showed increased fibrosis, discrete calcifications, and altered S100 expression, with greater neuronal expression in proximity to blood vessels. Non-T2D pulps demonstrated a more organised architecture. In vitro, 25 mM glucose significantly increased hDPC numbers (p=0.023) and metabolic activity over 72 h (p=0.031) under normoxia (21% O₂). Under low oxygen (5% O₂), proliferation was decreased but metabolic activity remained elevated. VEGFA (Vascular endothelial growth factor) was upregulated at 5.5 mM glucose under 5% O₂ (p=0.025), while FLT1 (Vascular endothelial growth factor receptor-1) and FGF2 (Fibroblast growth factor 2) were significantly downregulated under hyperglycaemia and low oxygen. Inflammatory and neuropeptide gene expression remained stable across all conditions. Cells cultured under lower oxygen tension (2% O₂) showed higher metabolic activity compared with those cultured under normoxia (21% O₂) at each time-point and glucose concentration.
Conclusion: T2D was associated with structural changes in the dental pulp, including fibrosis, calcifications, and altered S100 expression. In vitro, hyperglycaemia and low oxygen showed changes in hDPC metabolic activity and angiogenic gene expression, while neuropeptide gene expression was unchanged. These findings suggest that diabetic pulps may have a reduced healing capacity, which could impact the prognosis of vital pulp treatment while pulp calcifications may create difficulties for accessing canals for root canal treatment.