High glucose improves healing of periodontal wound by inhibiting proliferation and osteogenetic differentiation of human PDL cells

Abstract

Periodontal ligament (PDL) cells play an important role in wound healing of periodontal tissues. Response of PDL cells' cellular activity to high‐glucose concentration levels may be the key in understanding the relationship between periodontal disease and diabetes mellitus. We studied the effect of high‐glucose medium on proliferation of PDL cells in vitro. PDL cells were cultured for 1, 4, 7, 10, 14 and 17 days in normal (1100 mg/l) glucose or in high (4500 mg/l) glucose medium. The 3‐(4,5‐dimethylithiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay for proliferation was performed. In order to evaluate the osteogenetic differentiation of human PDL cells, the cells were induced with normal‐ or high‐glucose medium for 1, 7, 14, 21 and 28 days. The results indicated that high glucose significantly inhibited proliferation of PDL cells. Concerning the mineralised nodule formation, the percentage of calcified area to total culture dish of PDL cells in high glucose level was lower than that in normal glucose medium. The increase in alkaline phosphatase activity and collagen expression could be observed in high‐glucose‐containing osteogenetic factor. In conclusion, high glucose improves healing of periodontal wound by inhibiting proliferation and differentiation of PDL cells, which could explain for delayed periodontal regeneration and healing in diabetic patients.

Introduction

The term ‘diabetes mellitus’ describes a group of disorders characterised by elevated levels of glucose in the blood and abnormalities of carbohydrate, fat and protein metabolism. Delayed wound healing is a typical feature of patients with diabetes. It has been established that periodontal diseases are more prevalent and of greater severity in diabetic patients than in non‐diabetic patients 1, 2, 3, 4, 5.

Human periodontal ligament (PDL) cells play an important role in wound healing and tissue regeneration. Although direct mechanisms by which glucose levels affect the health of periodontal tissues are not clear, these studies imply that abnormal glucose levels may influence the wound healing of periodontal disease as in other diabetic complications such as retinopathy, neuropathy, nephropathy and micro‐ and macro‐vasculopathy 6, 7, 8.

Based on these observations, we hypothesised that high glucose might be involved in regulation of cellular activity of human PDL cells. To test this hypothesis, we observed the effects of high glucose on the proliferation and osteogenetic differentiation of human PDL cells.

Materials and methods

Primary culture of human PDL cells

Human PDL cells were isolated from healthy PDLs of first premolar tooth of individuals undergoing tooth extraction for orthodontic treatment in accordance with the method as described previously 9. All subjects were in good general health with healthy periodontal condition. The probing depths were < 3 mm and there was no loss of periodontal attachment level. Healthy periodontal tissue was removed from the centre of the root surface with a surgical scalpel. The tissue was minced and then transferred to culture bottles. The explants were cultured in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, UT) supplemented with 20% fetal bovine serum (FBS) (Hyclone), 100 units/ml penicillin G and 100 µg/ml streptomycin and the medium was changed every 3 days. Cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. When the cells growing out from the explants had reached confluence, they were separated by treatment of 0·25% trypsin and cultured on culture plastic dishes until the cells were fully confluent. The cells were then trypsinised at 1:3 split ratios. The PDL cells from forth‐sixth passages were used. The study protocol was approved by the Institutional Review Board of Wuhan University School of Stomatology.

Proliferation assay

Cell proliferation was determined by the 3‐(4,5‐dimethylithiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay 10. Briefly, cells were seeded into 100 µl culture medium in 96‐well plates at 5 × 10³ cells/well. After 24 hours of culturing, the cell culture medium was removed and each well was filled with 200 µl fresh DMEM with 2% FBS and incubated for 24 hours at 37 °C. After 24 h, the medium was changed with the normal‐glucose (1100 mg/l) medium and high‐glucose medium (4500 mg/l of glucose), respectively, supplemented with 10% FBS. During each experimental period, the culture media were changed every 3 days. On 1, 4, 7, 10, 14 and 17 days, 10 µl of pre‐warmed (37 °C) MTT solution was added to each well and cultured for 4 hours. Then the reaction was stopped by addition of 100 µl of dimethyl sulfoxide to each well and incubated at 37 °C for 4 hours. The optical density of the solution in each well was measured at a wavelength of 570 nm using an enzyme‐linked immunosorbent assay (ELISA) plate reader (Bio‐Tek Instruments, Winooski, VT).

Bone nodule formation assay

Briefly, PDL cells were seeded at a density of 1 × 10⁵ cells per well in six‐well plates. Until cells were 80% confluent, each well was filled with 200 µl fresh DMEM with 2% FBS and incubated for 24 hours at 37 °C and then 50 mg/ml ascorbic acid, 10 mM of ß‐glycerophosphate and 100 nM of dexamethasone were added 11. The media were changed every 3 days. The mineralised nodule formation was detected at days 1, 7, 14, 21 and 28 using Alizarin Red‐S staining. Cells were rinsed three times with phosphate buffered saline (PBS) followed by being fixed with 95% ethanol for 10 minutes at room temperature. After rinsed with water three times, cells were stained with 40 mM Alizarin Red‐S (pH 4·2) for 30 min at 37 °C and then extensively rinsed with water. To determine mineralisation, the percentages of calcified areas to total areas per well and per visual field (randomly selected three per well) were analysed using an image pro‐plus program (Media Cybernetics, Inc., Bethesda, MD).

Alkaline phosphatase activity assay

Human PDL cells were rinsed twice by ice‐cold PBS and scraped down. The total protein was extracted with M‐PER^® Mammalian Protein Extraction Reagent (Thermo fisher, Waltham, MA) according to the manual instructions. The activity of alkaline phosphatase (ALP) was measured by the ALP kit (Jiancheng, Nanjing, China) according to the manual instructions. After incubating at 37 °C for 15 min, the reaction was stopped and the plate was read in an ELISA reader (Bio‐Tek Instruments) at 520 nm.

Statistical analysis

All experiments were performed at three times. Data were presented as mean ± SD. Statistical analysis was performed with Student's t‐test. Values were considered significantly different if P < 0·05.

Results

Proliferation assay

In Figure 1, the proliferation ability of human PDL cells are shown. On the first day, the MTT assay indicated no significant differences in the cell proliferation ability between the high‐ and normal‐glucose groups. After cultured for 4, 7, 10, 14 and 17 days, PDL cells in normal glucose medium show significant changes than those in high‐glucose medium (Figure 1).

Figure 1.

Proliferation ability of human periodontal ligament cells. Cells were cultured in normal glucose (1100 mg/ml) or high glucose (4500 mg/ml) medium for 1, 4, 7 10, 14 and 17 days. Data are expressed as means ± SD of four different measure points of three independent experiments. Significances were calculated by Student's t‐test (P < 0·01).

Bone nodule formation

The mineralised nodule formation is the characteristic of osteoblast‐like cells. The percentages of calcified areas to total areas per well and per visual field were analysed. The PDL cells incubated in normal‐ or high‐glucose DMEM, supplemented with a mineralised medium, started to develop the nodules on day 14. But there is significant distinct between both groups, the areas of nodule in the normal glucose with osteogenetic factor mediums were bigger than those in the high glucose with osteogenetic factor mediums (Figure 2).

Figure 2.

Differentiation of human periodontal ligament cells. Cells were cultured with normal or high glucose Dulbecco's modified Eagle's medium supplemented with osteogenetic factor (50 mg/ml ascorbic acid, 10 mm of ß‐glycerophosphate, and 100 nm of dexamethasone) for 1, 7 14 ,21 and 28 days. The arrows represent the Alizarin Red staining, which determine osteogenetic differentiation. There is significant distinct between both groups, the areas of nodule in the normal glucose with osteogenetic factor mediums were bigger than those in the high glucose medium with osteogenetic factor mediums after cells were cultured for 14 days.

ALP activity

After human PDL cells were cultured for 1, 7, 14 and 21 days, ALP activity increased significantly. But at the end of the experimental period (day 28), ALP enzyme activity decreased obviously. There were significant differences between the groups cultured with normal glucose and high glucose supplemented with osteogenetic factor medium after cultured for 7, 14, 21 and 28 days (Figure 3).

Figure 3.

Alkaline phosphatase (ALP) activity. ALP enzyme activity was elevated. There were significant differences between the groups cultured with normal glucose and high glucose supplemented with a osteogenetic factor medium after cultured for 7, 14, 21 and 28 days. Data are expressed as means ± SD of three different measure points of three independent experiments. Significances were calculated by Student's t‐test (P < 0·05).

Collagen expression enhanced

The expression of collagen was significantly increased in the high‐glucose‐treated group compared with the normal glucose group in 1, 7, 14, 21 and 28 days of treatment (Figure 4). However, the collagen expression was decreased significantly both in high‐ and in normal‐glucose groups.

Figure 4.

Collagen expression. (A) Western blot of the collagen expression. (B) Statistical analysis of the collagen expression. Data are expressed as means ± SD of three different measure points of three independent experiments. Significances were calculated by Student's t‐test (P < 0·05).

Discussion

Diabetes is a disorder of chronic hyperglycaemia, and glucose participates in diabetic complications such as atherosclerosis, cardiac dysfunction, nephropathy and periodontal disease 6, 8, 12, 13. Chronic hyperglycaemia can cause functional and structural alterations in various tissues. Few studies have examined the influence of high extracellular glucose on human PDL cell proliferation and differentiation 9, 10, 14, 15, 16.

Human PDL cells play an important role in wound healing and regeneration of periodontal tissues. They can be differentiated into osteoblast‐like and cementoblast‐like cells that can produce and restore bone and cementum 17, 18. Our results demonstrate that high extracellular glucose has a significant effect on human PDL cells after treatment. The proliferative capacity of human PDL cells in high glucose concentration level was lower than in normal glucose. The PDL cells incubated in normal‐ or high‐glucose DMEM, supplemented with a mineralised medium, started to develop the nodules on day 14. There is significant distinct between both the groups, the areas of nodule in the normal glucose with osteogenetic factor mediums were bigger than those in the high glucose with osteogenetic factor mediums. These results are identical with those of the experiments by Kim et al. 6 previously.

However, our results suggested that ALP activity increased significantly after the PDL cells were cultured with osteogenetic factor mediums. There were significant differences between the groups cultured with normal glucose and high glucose supplemented with a osteogenetic factor medium after cultured for 7, 14, 21 and 28 days. But at the end of the experimental period (day 28), ALP enzyme activity decreased significantly. The most likely explanation is that ALP activity is the early parameter of osteogenetic differentiation of cells during the phase of matrix development and downregulated in calcifying osteoblasts 19, 20, 21 and was downregulated in the fully developed mineralising cells. In contrast, the high‐glucose‐treated cells were maintaining a less mature phenotype with high levels of ALP expression. And at the end of the experimental period (day 28), cells gradually decayed for long‐term culture.

In conclusion, cellular activities such as proliferation and differentiation are essential events for wound healing and regenerating periodontal tissues. The decreased cellular activity of PDL cells due to high glucose concentration may compromise healing and periodontal regeneration in diabetic patients. The data provide an explanation for the delayed periodontal regeneration and healing in diabetic patients.