Abstract
Type 2 Diabetes (T2D) is a chronic metabolic disorder characterised by hyperglycaemia and is associated with micro and macrovascular complications. Vascularity and angiogenesis of the pulp are essential for healing and while international guidelines recommend the use of vital pulp treatment (VPT) for deep caries and some pulp exposures the influence of hyperglycaemia on angiogenesis is unclear. Angiogenesis is a tightly regulated process and occurs via the interaction of angiogenic growth factors and their corresponding receptors. Currently, we have an increasing understanding of pulp angiogenic processes in health, however we need to better understand how systemic disease influences this important process. Understanding of the pulpal response in patients with T2D would inform prognosis and treatment planning.
This study had two aims 1) to investigate the influence of type 2 diabetes (T2D) on the histomorphology and angiogenic protein expression within the coronal pulp chamber; and 2) to evaluate the influence of hyperglycaemia on cell viability, metabolic activity and angiogenic gene expression using an in vitro human dental pulp cells (hDPCs) model to mimic T2D.
In part 1, healthy permanent molars were collected from T2D (n=5) and non-T2D (n=5) participants, decalcified in 10% EDTA and paraffin embedded. Sections were stained with Haematoxylin and Eosin; Verhoeff-Van Gieson stain; and immunohistochemistry (IHC) with anti-CD34 and anti-VEGFR2 was used to evaluate protein expression within the tissues prior to qualitative analysis.
In Part 2, coronal pulp tissue was excavated from extracted healthy permanent molars and the explant culture technique was used to establish primary hDPC lines (n=4). hDPCs were cultured with D-glucose at a range of concentrations (normoglycaemic 5.5mM (control), 12.5mM (prediabetes) or 25mM (T2D)) for 24, 48 and 72 hours (h). Cell viability was evaluated with Prestoblue™ and crystal violet assay. RNA expression levels for the angiogenic-associated genes VEGFA, KDR, FLT1, FGF2, ANGPT1, ANGPT2, TIE1, and TEK were analysed using quantitative real-time polymerase chain reaction (qPCR) and normalised to the reference gene GAPDH. For statistical analysis, student’s t-test, ANOVA and Tukey’s post-hoc tests were used at a significance level of p<.05. (Ethical approval H21/080).
Histological analysis showed that T2D dental pulps were less cellular and less vascular with thickened blood vessel walls. Qualitative analysis of anti-CD34 confirmed reduced vascularity in T2D compared with non-T2D pulps. Immunopositive staining of VEGFR2 was noted in the odontoblast region and scattered throughout the central pulp of non-T2D samples but was not observed in T2D samples. Cell culture studies found that hDPCs cultured with 25 mM D-glucose showed an increase in relative cell count with time when compared with 5.5 mM at 48 h (p=.067) and 72 h (p=.061). At 24 hours, when 25mM and 5.5mM were compared there was a significant difference in relative cell count (p=0.023). Cell metabolic activity was highest at 72 h with the 25mM D-glucose concentration group, compared with the 5.5mM exposure however this difference was not significant (p=.274). There was no significant change in hDPC expression of angiogenic-associated genes over the culture period. However, there was relatively high expression of VEGFA, FGF2, and ANGPT1. TEK, ANGPT2 and TIE1 expression levels fluctuated across glucose concentrations and KDR was undetected.
Within the limitations of this study, T2D may result in morphological changes in the dental pulp. In an unstressed normoxic environment, hyperglycaemia may increase hDPC viability and alter metabolic activity over time but may have limited influence on angiogenic-associated gene expression over 72 hours of culture.