Abstract
Type 2 diabetes (T2D) is an international health burden. Globally, it is the most common chronic disease and its incidence is increasing. Type 2 diabetes mellitus is a metabolic proinflammatory disorder characterised by chronic hyperglycaemia resulting in an altered immune response and delayed healing. Patients with T2D are common in general dental practice. Oral complications of T2D are well recognised, particularly associated with periodontal disease but the influence of hyperglycaemia on the dental pulp is unclear.
Inflammation and immune responses of the dental pulp are similar to those in other connective tissue in the body and are mediated by several cellular and molecular factors to minimise harmful effects induced by the irritating factors. Hyperglycaemia affects body tissues through a non-enzymatic process known as glycation and the accumulation of irreversible advanced glycation end products (AGEs). AGEs accumulation is responsible for diabetic complications such as thickened connective tissue and increased tissue inflammation. AGEs exert noxious effects on tissues through an advanced glycation end-product receptor (RAGE).
Normal tissue and a regulated inflammatory response contribute to healing. The histological, immunological and inflammatory changes in human clinically normal dental pulp in T2D patients are limited. Improved knowledge and understanding of these changes may assist clinicians in planning care to manage patients with T2D diagnosed with pulp disease.
Hypothesis & Objectives
The hypothesis of this thesis is that T2D affects the clinically normal dental pulp by altering histological, immunological, and inflammatory responses. To test this hypothesis, the current study has three main objectives. The first is to evaluate the histological, immunological and inflammatory changes in the clinically normal dental pulp of patients with T2D. The second objective is to evaluate the glycation process in the dental pulp by evaluating AGEs and their receptors RAGE, Galectin-3 (Gal-3), and the related inflammatory response. The third is to establish an in vitro diabetic model and evaluate the effect of high glucose concentration on human dental pulp cell (hDPCs) behaviour and gene expression.
Methods
Ethical approval for this study was gained from the University of Otago Human Ethics Committee (Health) (Project Reference H16/069 and Project Reference H18/077) and Māori consultation was entered with the Ngāi Tahu Research Committee. Clinically normal (healthy) and extracted permanent teeth were collected from T2D (n=20) and non-diabetic (n=20) participants for the first and second objectives.
To achieve the first and second objectives, histological staining, immunohistochemistry (IHC), immunofluorescence (IF), double immunofluorescence (DIF) and quantitative polymerase chain reaction (qPCR) were used. Following extraction, teeth from T2D (n=10) and non-diabetic (n=10) participants were cut transversely below the cementoenamel junction (CEJ), formalin fixed, decalcified in 10% ethylenediaminetetraacetic acid (EDTA), and paraffin embedded. Sections were stained with haematoxylin and eosin (H&E), Massons trichrome, Van Gieson (VG) and silver reticulin stains for histological evaluation. Other sections were used for IHC using anti-TLR2, anti-TLR4, anti- CD4, anti-CD68, anti-CD83, anti-FOXP3, anti-interleukin (IL)1!, anti-IL6, anti-tumour necrosis factor (TNF)-", anti- AGEs, and anti-RAGE. Three sections were used for IF using anti-AGEs and other five sections were used for DIF using anti-RAGE with anti-CD4 and anti-vimentin.
Remaining teeth from T2D (n=10) and non-diabetic (n=14) participants were used for gene expression analyses. Immediately after extraction teeth were sectioned transversely below the CEJ, and the coronal pulp was removed for ribonucleic acid (RNA) extraction. Messenger RNA (mRNA) levels for AGE, RAGE, S100A12 and NF-#B were determined using TaqMan assays.
For the cell culture experiments, coronal pulp tissue was excavated from mature unerupted third molar teeth extracted from healthy adults (n=4) and cell lines generated using the explant method. An in vitro diabetic model by using different glucose concentrations mimicking blood glucose concentrations was used to evaluate the effect of normal and high glucose on viability and gene expression of hDPCs at 1, 3 and 5-day time-points.
Data Analysis
Histological slides and IHC samples were scanned by an Aperio Scanscope CS2 image capture device, analysed under light microscopy and digitised with ImageScope. Data analysis was performed qualitatively and semi-quantitatively (number of positive cells/image area) to assess the histological changes and the protein expression in the specimens.
The DIF sections for RAGE, vimentin and CD4 were viewed and images were taken using an EVOS M5000 inverted fluorescent microscope. The slides were examined, and the individual and merged proteins in non-T2D and T2D dental pulp were qualitatively identified. The staining was examined, and proteins were qualitatively identified, and merged in non-T2D and T2D dental pulp.
The mRNA expression analysis was performed using comparative quantification cycle (Cq) values. The fold difference (FD) for each gene was calculated between the two groups. Data analyses were performed with GraphPad Prism®, using a Student’s t- tests at P-value <0.05.
The viability data analysis in cell culture was performed using GraphPad Prism and Excel software for Mac OS Catalina (Version 10.15.4). The data are presented as mean and standard deviation (SD). A Student’s t-test was used and the differences between the groups were considered significant when P-value <0.05.
The gene expression in cell culture was performed by using the comparative Cq method to calculate the difference in the mRNA expression between the 5.5mM and 25mM D-glucose concentrations at the different time-points.
Results
Histological changes were observed in normal dental pulp of participants with T2D compared to healthy controls. T2D resulted in a dental pulp that was less cellular, less vascular, evidence of thickened blood vessel walls, increased pulp calcification, increased collagen and decreased elastin deposition. The positive cell count/area showed increased expression of CD68 (P<0.001), CD83 (P=0.04), and decreased expression of FOXP3 (P=0.01) in T2D dental pulp compared with non-T2D samples. The positive cell count/area analysis also showed that the cytokines were significantly increased in T2D (IL1! (P=0.01), IL6 (P<0.0001), IL17 (P<0.0001) and TNF-" (P=0.01)). The glycation process was significantly increased in the dental pulp of T2D as evidenced by increased IHC expression of AGE (P<0.0001) and RAGE (P=0.02). The qPCR results showed that the expression of RAGE, S100A12, NF-#B and COL1A1 mRNA genes was significantly increased in the dental pulp of T2D compared with the non-T2D samples (P<0.0001), while no significant changes were detected for Gal-3 mRNA expression. The glycation process was also assessed by IF and DIF which showed increased accumulation of AGE in the extracellular matrix (ECM) and around blood vessel walls. DIF showed co-localisation of RAGE and vimentin and co-localisation of RAGE and CD4, indicating RAGE was expressed on fibroblasts and CD4+ve cells.
The results of the study using an in vitro diabetic cell culture model showed that high glucose concentration was associated with a reduced viability rate of hDPCs with increasing time. Furthermore, the used highest glucose concentration resulted in increased expression of collagen type III (COL3A1) and increased mineralisation due to the increased expression of ALP over time. The cell culture mRNA showed that high glucose resulted in increased gene expression of RAGE and decreased expression of Gal-3.
Conclusions
T2D leads to changes in dental pulp morphology, altered immunosurveillance and increased cytokine expression in the dental pulp. The glycation process was clearly evident in the pulp from T2D patients and may be responsible for the changes observed. These changes in clinically normal dental pulp in T2D patients may influence the healing response following pulpal injury and may affect the treatment plan and treatment outcome for these patients.