Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis

Abstract Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application. Objective Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved. Methodology hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction. Results Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine–cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1). Conclusions The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization.


Introduction
Diabetes mellitus constitutes a group of metabolic disorders related to hyperglycemia. The adverse effects of diabetes mellitus include damage, destruction and failure of various tissues, including oral and dental tissues. 1 Dental pulp is one of the tissues affected by diabetes. Studies have reported alteration in dental pulp structure, increased inflammation and impaired pulpal healing in the dental pulp of people with diabetes. 2,3 In addition, in vivo studies conducted using animal models have shown that the severity of dental caries and pulpal inflammation were considerably higher in diabetic mice than in non-diabetic controls. 4,5 Hyperglycemia has been considered to be a major factor involved in the pathogenesis of diabetes, as it induces alterations at the cellular level of the affected tissue. Most of the cellular and molecular mechanisms involving inflammatory responses and overproduction of reactive oxygen species (ROS) causing tissue damage are observed in several diabetic complications. 6 Furthermore, hyperglycemia has been shown to interfere with the healing and regeneration of hard tissues. High levels of glucose have also been reported to inhibit the osteogenic differentiation and mineral tissue formation. 5,7 Dental pulp is one of the tissues with reparative and regenerative potential. 8 Although pulp cells have been proved to be useful in tissue engineering and regenerative medicine, the hyperglycemic condition can interfere with its regenerative capacity. Research shows that hyperglycemia exerts a negative effect on pulpal healing by inhibiting dentin bridge formation and increasing pulpal inflammation. 9 Moreover, treating dental pulp cells with high glucose levels was found to inhibit mineralization. 5 Even though a high level of glucose was reported to inhibit the mineralization of pulp cells, gene expression associated with the influence of glucose has never been explored and may be important as basis for future studies to improve the dental therapeutic application in diabetic patient. Dental pulp tissue was collected from non-carious third molar of 3 healthy donors (2 males and 1 female, aged 18-30 years). hDPCs were isolated using the tissue explant technique as described by Gronthos, et al. 11 (2011). Briefly, a diamond fissure bur with constant irrigation was used to cut the groove along the cementoenamel junction, after which the tooth was separated, and pulp tissue was collected. The pulp tissue was cut into 0.5-1 mm 2 fragments, placed in a 60-mm culture dish and then maintained in Dulbecco's Modified Eagle Medium (DMEM); HyClone, Logan, UT, USA) containing 5.5 mM D-glucose supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% penicillin-streptomycin (Pen-Strep, Gibco BRL, Grand Island, NY, USA), under incubation at 37°C in a humidified atmosphere containing 5% CO 2 . The medium was changed every 3 days. Cells between 4 th -6 th passages were used in this study ( Figure S1).

Experimental design
The hDPCs isolated from 3 individuals were used in this study. These cells were seeded at a density of 1 × 10 5 cells/well in a 6-well plate and cultured in DMEM containing 10% FBS and supplemented with 50 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich) and 100 nM dexamethasone (Sigma-Aldrich) to induce cell mineralization (mineralizing medium; MM). Cells were then incubated under 5% CO 2 in a humidified atmosphere at 37°C. This was followed by treating Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis 2021;29:e20201074 3/16 with 2 different glucose concentrations, namely 5.5 mM D-glucose (hereinafter, referred to as low glucose; LG) and 25 mM d-glucose (hereinafter, referred to as high glucose; HG). For each sample, the medium was changed every 3 days. RNA isolation was performed on day 1 and day 14. On day 21, the cells were stained with alizarin red S (Sigma-Aldrich) and then quantified for the detection of mineral deposition ( Figure S1).

RNA isolation
After culturing for 1 and 14 days, cells were washed twice with PBS, and RNA was isolated from each Mineralization-related genes were also validated. The primer sequences used for the selected genes are described in Table S1. The thermocycling conditions consisted of 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing for 30 s (the annealing temperature for each primer is presented in Table S1) and extension at 72°C for 25 s.

Detection and quantification of mineralized nodule formation
This experiment was conducted to detect the effect of glucose on the mineralization of hDPCs. On day 21, the cells were fixed with 10% (v/v) formaldehyde and stained with 40 mM alizarin red S. The alizarin red Scalcium complex was further quantified as described by Gregory, et al. 12 (2004

Microarray analysis
The number of detected probes was shown in Table S2 Tables S3 and S4.

RT-qPCR validation
The DEGs compared between high-glucose and lowglucose conditions on day 1 and day 14 were further selected for qPCR validation based on the selection criteria that the genes were included in the top 15 most significantly up-or down-regulated genes (Table 1) and the genes must also be involved in the top 5 most enriched GO terms ( Figure 3). Eventually, 6 of the genes on day 1 and 6 of the genes on day 14 were selected for further validation. Heat maps of the selected genes based on their expression levels are illustrated in Figure   4A, and mapping of the selected genes according to their involvement in the top 5 most enriched GO terms is shown in Figure 4B.   Figure 8A and B). In addition, quantification of the alizarin red S-calcium complex revealed a significantly higher (alizarin red S) concentration under low-glucose than under high-glucose condition ( Figure   8C). These results clearly revealed an inhibitory effect of the high-glucose environment on hDPC mineralization.  is also considered one of the key factors contributing to its several complications. In this study, we used a glucose concentration of 5.5 mM (100 mg/dl) as an equivalent to normal blood glucose level, whereas a glucose concentration of 25 mM (450 mg/dl) was used to reflect a high blood glucose level (hyperglycemia).
Other study has also reported significant changes osteoblast function when treated with 25 mM glucose. 13 Furthermore, another study used 450 mg/ dl glucose concentration to represent hyperglycemic condition in an animal wound healing model. 14 The results of our study confirmed the negative effect of high levels of glucose. We observed that the high-   Fbln-7 is a member of the fibulin family. It has been reported that Fbln-7 is expressed in pre-odontoblasts and odontoblasts during tooth development. 28 It was also found in predentin and along the dentinal tubules and was suggested to play a role in odontoblast differentiation and dentin matrix mineralization. 28 Interestingly, after 14 days of odontogenic induction in our study, we found a higher expression of Fbln-7 in cells that were exposed to high glucose. This may indicate a delay in the process of dentinogenesis, thereby leading to an impairment in the healing and regenerative process of dental pulp under high-glucose environment.
Hyaluronan synthase (HAS) is an enzyme that ApoC1 is commonly known as a protein that plays a role in lipid metabolism and transport. 31 The role of ApoC1 in mineral matrix formation remains unknown.
It has been found to be associated with diabetes and inflammatory response. One study reported that ApoC1 transgenic mice were protected from obesity and insulin resistance. 32 High-glucose condition was shown to increase ApoC1 expression in retinal ganglion cells. Furthermore, knock down of ApoC1 inhibited the expression of inflammatory cytokines and cell apoptosis. 33 APOC1 was also found to enhance the lipopolysaccharide-induced inflammatory response. 34 As APOC1 is associated with inflammatory response and diabetes, the above-mentioned findings could occur in the dental pulp leading to interference in the regenerative process.
Interleukin-4 (IL-4) is a cytokine that binds to IL- A suitable further study may determine the key protein involved in the pulp regeneration process.
Manipulation of these selected genes may provide a novel insight into the pulp regeneration process in the infected pulp or in dental pulpitis in patients with uncontrolled diabetes and could further increase the rate of success of dental treatment in the future.
Unfortunately, we did not observe any significant changes in the expression level of genes involved in the intracellular glucose metabolism namely; glucose transporter 1. Moreover, dentin formation namely; nestin and collagen 1, and oxidative metabolismrelated enzyme namely; superoxide dismutase also showed no significant different in high glucose condition.
We did not observe any significant changes in the expression level of genes involved in the intracellular glucose metabolism namely; glucose transporter 1. Moreover, dentin formation namely; nestin and collagen 1, and oxidative metabolism-related enzyme namely; superoxide dismutase also showed no significant difference that considered to lack of high glucose condition effect. This may be due to the phase Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis J Appl Oral Sci. 2021;29:e20201074 11/16 of cell differentiation. 16,17 The limitation of this study is that it is an in vitro experiment, which cultured dental pulp-derived cell in a two-dimension culture system under high glucose concentration which is the important characteristic of diabetes. However, the effect of signal transduction pathway, vascularization and hormonal changes were not presented in this study. The experiment could only control the hyperglycemic factor which may not represent the entire condition in diabetic patients.
Another limitation is that osmotic pressure in the medium possibly affected gene expression without affecting in vitro mineralization.
In this study, we controlled the quantity of cell culture by normalizing RNA concentration. However, number of cells should clarify further the possible influence of cell survival/apoptosis on experimental conditions.
The study used pooled RNA to reduce individual heterogeneity, which may not be able to rule out the effect of dominant donor; however, the results of CDK8 and HAS1 from all the 3 samples exhibited a similar trend. In addition to the selected genes, we found other interesting genes that were up-regulated in the high-glucose environment, such as apolipoprotein E, tenascin C and claudin 23. These genes have been reported to be associated with osteoblast functions, hard tissue formation, tissue healing and diabetes. [38][39][40] Therefore, it is necessary to further investigate the role of these genes in pulp cells under hyperglycemic condition.
High glucose level has shown to inhibit the mineralization of dental pulp cells. The results of this study provide basic knowledge about the effect of high glucose on gene expression during the odonto-and osteogenic differentiation of hDPCs. This provides the basic knowledge with potential for future dental therapeutic application.

Conclusion
To our knowledge, this is the first reported gene