Continuously generated H2O2 stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts

Abstract

Numerous studies have shown that hydrogen peroxide (H(2)O(2)) inhibits proliferation and osteoblastic differentiation in bone-like cells. Human periodontal ligament fibroblasts (PLF) are capable of differentiating into osteoblasts and are exposed to oxidative stress during periodontal inflammation. However, the cellular responses of PLF to H(2)O(2) have not been identified. In this study, we examined how H(2)O(2) affects the viability and proliferation of PLF by exposing the cells to glucose oxidase (GO) or direct addition of H(2)O(2). We also explored the effects of GO on the osteoblastic differentiation of PLF and the mechanisms involved. The viability and proliferation in PLF were increased with the addition of 10 mU/ml GO but not by volumes greater than 15 mU/ml or by H(2)O(2) itself. GO-stimulated DNA synthesis was correlated with the increase in cyclin E protein levels in the cells. Osteoblastic differentiation of PLF was also augmented by combined treatment with GO, as evidenced by the increases in alkaline phosphatase activity, mineralization, collagen synthesis, and osteocalcin content in the cells. The inductions of runt-related transcription factor 2 and osterix mRNA and proteins were further increased in PLF incubated in combination with GO compared to those in untreated cells. These results demonstrate that the continuous presence of H(2)O(2) stimulates the proliferation of PLF and augments their potential to differentiate into osteoblasts through the up-regulation of bone-specific transcription factors. Collectively, we suggest that H(2)O(2) may elicit the functions of PLF in maintaining the dimensions of the periodontal ligament and in mediating a balanced metabolism in alveolar bone.

Fig. 1

Continuous and dynamic production of H₂O₂ in the cultures of PLFs exposed to GO. PLFs were exposed to 5, 10, and 20 mU/ml GO (left panel) or 10 mU/ml GO with DAG (10 nM dexamethasone, 50 μM ascorbic acid, and 20 mM β-glycerophosphate) (right panel) and then the culture supernatants were collected at various times intervals after GO exposure during 24 h incubation. The results indicate the mean ± SD from triplicate experiments.

Fig. 2

Viability and proliferation of PLFs are increased by the addition of GO but not by H₂O₂. PLFs were exposed to the indicated GO concentrations (0–50 mU/ml) for 24 h and then processed for the WST-8 assay (A), tritium uptake assay (B), and flow cytometric analysis after PI staining (C). (D) The cells were also exposed to H₂O₂ itself with increasing concentrations (0–2 mM) for 24 h and then processed for the cell viability assay. (E) PLFs were exposed to 10 mU/ml GO in the presence and absence of 500 U/ml catalase for 24 h prior to the WST-8 assay. In the experiment of C, cell cycle progression for each experiment was analyzed using the WinMDI 2.9 program. ^*p < 0.05 and ^***p < 0.001 vs. the untreated control values. ^#p < 0.05 vs. the experimental values.

Fig. 3

The activities of intracellular antioxidant systems depend on the concentration of GO. PLFs were exposed to various GO concentrations (0–20 mU/ml) for 24 h. The activities of SOD and catalase and the levels of reduced GSH were then determined. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. the untreated control values.

Fig. 4

Effects of GO on the proliferation and cell cycle progression of DAG-treated PLFs. PLFs were exposed to the indicated GO concentrations (0–10 mU/ml) in the presence of DAG. After 7 days of incubation, the levels of DNA synthesis and the cell populations in each stage of cell cycle progression were determined by tritium uptake assay (A) and flow cytometric analysis after PI staining (B and C), respectively. The data in B show representative results from three independent experiments. ^*p < 0.05 and ^**p < 0.01 vs. the untreated control values. ^#p < 0.05 vs. DAG treatment alone.

Fig. 5

Effects of GO on the induction of cell cycle regulatory proteins in DAG-treated PLFs. (A) The cells were incubated in the DAG-containing osteogenic medium with and without the indicated doses (0–10 mU/ml) of GO for 7 days and then processed for Western blot analysis using total protein lysates. (B) The values represented are the mean ± SD of three independent experiments, where actin was used as the control protein. ^*p < 0.05 and ^***p < 0.001 vs. the untreated control values. ^##p < 0.01 and ^###p < 0.001 vs. DAG treatment alone.

Fig. 6

GO augmentation of the DAG-mediated increase inALP activity in PLFs. Cells were exposed to the indicated GO concentrations (0–10 mU/ml) in the presence of DAG and processed for the analysis of ALP activity after 3 (A), 7 (B), and 10 days (C) of incubation. (D) PLFs were also incubated in DAG-containing medium with and without 10 mU/ml GO. At various times, ALP activity was determined. ^*p < 0.05 and ^**p < 0.01 vs. the untreated control values.

Fig. 7

Stimulating effect of GO on mineralization in DAG-treated PLFs. (A) Cells were cultured with the DAG-containing osteogenic medium in the presence and absence of GO for 14 days. The resulting mineralization was assessed by Alizarin red staining. Each microscopic image shown is a representative of five separate experiments. (B) Absorbance specific for Alizarin red was measured, and ^**p < 0.01 and ^***p < 0.001 represent significant differences between the cells treated with DAG only and those treated in combination with GO.

Fig. 8

GO treatment increases the contents of collagen and osteocalcin in DAG-treated PLFs. Cells were incubated in the osteogenic medium with and without the indicated GO concentrations (0–10 mU/ml). The cellular levels of collagen (A) and osteocalcin (B) were determined after 10 days of incubation. ^*p < 0.05 vs. the untreated control values.

Fig. 9

Inhibitory effect of catalase on the GO-mediated facilitation of osteogenic differentiation in PLFs. Cells were incubated in DAG-containing osteogenic medium in the presence of 10 mU/ml GO, 500 U/ml SOD, and/or 500 U/ml catalase for 10 days and then processed for the ALP activity (A), collagen synthesis (B), mineralization (C), and osteocalcin content. ^*p < 0.05 and ^**p < 0.01 vs. DAG treatment alone. ^#p < 0.05 and ^###p < 0.001 vs. GO treatment.

Fig. 10

Combined treatment with GO accelerates the expressions of osteogenic transcription factors in DAG-treated PLFs. Cells were incubated in DAG-containing medium with and without the increasing GO concentrations (0–10 mU/ml). The levels of osterix, Runx2, and osteopontin proteins were determined by Western blot analysis using total protein lysates after 3 (A) and 7 days (C) of incubation. The expression patterns of these proteins were analyzed using a densitometer from three independent experiments after normalizing the bands to the level of actin. The values represented in B and D are the mean ± SD corresponding to A and C, respectively. The mRNA levels of Runx2 (E) and osterix (F) in the cells exposed to 10 mU/ml GO and/or 500 U/ml catalase were also analyzed after 7 days of osteoblastic differentiation. ^*p < 0.05, ^**p <0.01, and ^***p < 0.001 vs. DAG treatment only. ^#p < 0.05 vs. GO + DAG treatment.