The Eff ect of Ozone Gel on Bone Matrix Production by Human Osteosarcoma Cell Line Saos-2

Pao-Li Wang1)*, Yoichi Tachi2)*, Kazuya Masuno1), Nobutaka Okusa3) and Yasuhiro Imamura4)
1) Department of Innovation in Dental Education, Osaka Dental University, Osaka, Japan
2) Laboratory of Nutritional Physiology, Tokyo Kasei University, Tokyo, Japan
3) Department of Forensic Dentistry, Osaka Dental University, Osaka, Japan
4) Department of Dental Pharmacology, Matsumoto Dental University, Shiojiri, Japan
(Accepted for publication, April 20, 2018)

Abstract

Ozone is currently being considered as a potential oral antiseptic agent because it is highly antimicrobial and does not induce microbial resistance. In this study, we demonstrated that an optimal dosage of ozone gel enhanced the proliferation, type 1 collagen production, and alkaline phosphatase (ALP) secretion of Saos-2 cells in vitro. Proliferation of Saos-2 cells was assessed by MTT and DNA synthesis assays. Type 1 collagen production and ALP secretion were evaluated using enzymelinked immunosorbent assay (ELISA) and ALP assays. The cells were treated with/without 0.05, 0.5, 5 ppm ozone gel for 24 h. Ozone gel (0.5 ppm) signifi cantly induced the proliferation of Saos-2 cells. At this concentration, ozone gel enhanced type 1 collagen production and ALP secretion. The results indicated that ozone gel controls the cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.

Key words

Ozone gel, ALP, Collagen, Osteoblast, Saos-2 cells

Introduction

Ozone is currently being considered in dentistry as a potential alternative oral antiseptic agent. Its strong antimicrobial eff ect without the development of drug resistance has been previously noted in water purification and food preservation techniques1-3). In dentistry, ozone has been used either in gaseous or aqueous forms for the elimination of pathogens causing caries, in the disinfection of root canals, and as a rinse for avulsed teeth4-8). However, ozone has an unpleasant smell and a short half-life of approximately 40 min9). Ozone also has low water solubility, and therefore, aqueous ozone formulations provide no longterm sterilization effect. On the contrary, ozone gel, which consists of a glycerin solution containing ozone, has a long-term sterilization eff ect. The advantages of ozone gel include a 6-month-long sterilization eff ect, the lack of an unpleasant smell, and no development of bacterial strains manifesting ozone-resistance. Previously, we reported the safety evaluation of ozone for the skin and eye, as well as its antimicrobial eff ects and role in hemostasis using ozone gel10-12). In addition, a number of reports have shown that ozone can ameliorate periodontal diseases13-15). However, the effects of ozone on the functions of cells involved in periodontal disease are yet to be elucidated16-19). Recently, we reported the effects of ozone gel on the production of inflammatory cytokines and type I collagen in human gingival fi broblasts (HGFs) in vitro, and attempted to elucidate the mechanism of action of ozone on periodontal disease20). In this study, we examined effects of ozone gel on type 1 collagen production and alkaline phosphatase (ALP) secretion in the human osteosarcoma cell line Saos-2.

Materials and Methods

Cell cultures

Saos-2 cells (RIKEN BRC Cell Bank, Tokyo, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nissui pharmaceutical Co. Ltd., Tokyo, Japan) with 10% fetal bovine serum (FBS), 100 units/ml penicillin G, and 100 μg/ml streptomycin at 37°C in a 5% CO2 and 95% air humidifi ed incubator.

DNA synthesis and MTT assays

For DNA synthesis, Saos-2 cells (1×104 ) were cultured in DMEM containing 0.5% FBS (0.5% DMEM) for 24 h. The cells were cultured with the ozone gel (VMC Co. Ltd. Tokyo, Japan) or at 0.05, 0.5, and 5 ppm for 2 min, and the culture medium was removed. Then, the cells were washed with 0.5% DMEM and were cultured with 0.5% DMEM containing bromodeoxy uridine (BrdU) for 24 h. The level of DNA synthesis was determined by measuring BrdU-incorporation using the BrdU cell proliferation assay kit (Millipore Tokyo, Japan).

For MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma- Aldrich) assay, cells were cultured with the ozone gel at 0.05, 0.5, and 5 ppm in DMEM containing 10% FBS (10% DMEM) for 2 min, and the culture medium was removed. After washing with 10% DMEM, the cells were cultured with 10% DMEM for 24 h. The subsequent procedures were performed as described elsewhere21).

Enzyme-linked immunosorbent assays (ELISA)

For collagen production, Saos-2 cells (1×104 ) were cultured in DMEM containing 1% FBS (1% DMEM) with the ozone gel (0.5 ppm) for 2 min. The cells were washed and cultured with 1% DMEM. Levels of type I collagen in the media were measured using the biotinylated antitype I collagen antibody (0.2 μg/ml, Rockland). ELISA was performed as described in the user manual of CytoSet kits (Biosource, Tokyo, Japan)22). The cells for collagen production were lysed with 0.5% TritonX-100, and the protein concentration of the cell lysates was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Tokyo, Japan). Collagen production was normalized to the protein content of the cell lysates.

ALP activity

Saos-2 cells (1×104 ) were cultured with the ozone gel (0.5 ppm) for 2 min, and the culture medium was removed. The cells were washed with 0.5% DMEM and cultured with 10% DMEM for 24 h. The cells were lysed with 0.05% TritonX-100 and ALP activity of the lysates was measured using a LabAssay ALP kit (Wako Pure Chemicals Industries, Ltd., Osaka, Japan). The protein concentrations of the cell lysates were also measured using a BCA protein assay kit. ALP activity was normalized to the protein content of the cell lysates.

Statistical analysis

Quantitative data were statistically analyzed using either one-way analysis of variance (ANOVA) followed by Tukey’s test (DNA synthesis and MTT assays) or Student’s t-test (measurement of ALP activity and ELISA for collagen production) using the StatMate software (ATMS). Diff erences were considered to be signifi cant at p < 0.05.

J Hard Tissue Biology Vol. 27(3):195-198, 2018 27(3):195-198, 2018

(a) MTT assay. (b) DNA synthesis assay. Saos-2 seeded at a volume of 3.1 × 104/ cm2 were exposed to media, which contain 0.05, 0.5, or 5 ppm ozone gel (designated as +, ++, and +++) for 24 h. All data were compared with those for cells treated with control medium without ozone gel. Data have been provided as mean ± S.D. (n = 3). **P < 0.01 and ***P < 0.001, analyzed with ANOVA with a Dunnett’s test (vs. none).

Saos-2 cells were seeded at a volume of 3.1 × 104/cm2 and were exposed to media containing 0.5 ppm Ozone gel for 24 h. Data have been provided as mean ± standard deviation (n = 3). ***P < 0.001, analyzed with Student’s t-test (vs. none).

Saos-2 was seeded at 3.1 × 104/cm2 condition, and was exposed to media containing 0.5 ppm ozone gel for 24 h. Data have been provided as mean ± standard deviation (n = 3). **P < 0.01, analyzed with Student’s t-test (vs. none).

Results

Eff ect of ozone gel on proliferation of Saos-2 cells

After 24 h of incubation, the ozone gel treatment increased Saos-2 proliferation in a dose-dependent manner (Fig. 1). Ozone gel at 0.5 ppm signifi cantly induced cell growth compared to the other concentrations, whereas no obvious change was observed with 0.5 ppm ozone gel treatment. Similar to the results of the MTT assay, 0.5 ppm ozone gel eff ectively elevated DNA synthesis in Saos-2 cells, indicating the optimal concentration of ozone gel required for facilitating the proliferation of Saos-2 cells.

Eff ect of ozone gel on type 1 collagen production and ALP secretion by Saos-2 cells

Ozone gel at 0.5 ppm significantly induced cell growth. This, considering the results of the proliferation assays, we further evaluated the effect of ozone gel on collagen 1 production and ALP secretion from Saos-2 cells using 0.5 ppm ozone gel. Addition of ozone gel in the media eff ectively increased type 1 collagen production (Fig. 2) and ALP secretion (Fig. 3) from Saos-2 cells compared to that without ozone gel treatment.

Discussion

The eff ect of ozone gel on bone matrix production by osteoblasts is unclear. The present study showed that while high concentrations of the ozone gel decreased the cell viability index, low concentrations of the gel enhanced cell proliferation. Furthermore, 0.5 ppm ozone gel promoted the secretion of type 1 collagen and ALP, which are highly related to bone formation.

Previously, Oda et al. reported that 10 ppm ozone gel suppresses the proliferation of human gingival fibroblasts23). The present study showed that while cell proliferation was signifi cantly suppressed at high concentrations (5 ppm) of ozone gel, 0.5 ppm ozone gel promoted cell proliferation. In a study on non-gel forms of ozone, Tsujiue et al. reported that ozone water dissolved in phosphate-buff ered saline (PBS) induced cell damage at 3 ppm as visualized by hematoxylin and eosin staining24). A study using alveolar macrophages reported that 0.5 ppm ozone gas causes cellular damage to the monocyte strain THP-125). Ozone gel has the advantage of providing sustained release of ozone, which is contained within glycerin20). On the basis of these observations, we conclude that the ozone gel exhibits pharmacological effects without cytotoxicity at the available low concentrations and by further lowering the effective concentration via its sustained-release eff ect; however, it shows Holmins eff ect, i.e., cytotoxicity above a certain concentration.

Type 1 collagen and ALP are widely known as markers secreted in the early phases of bone formation20). In this study, these initial-phase bone matrix markers were secreted at an optimum concentration of the ozone gel. Currently, moderate levels of active oxygen species are thought to act as an intracellular signaling substance. Reactive oxygen species control intracellular signal transduction by chemically modifying proteins, nucleic acids, and lipids. Among these, participation of stress adaptation response by the Keep 1/Nrf 2 pathway is being actively researched. However, Nrf2 is known to negatively regulate the differentiation of osteoblasts. Therefore, it is highly likely that type 1 collagen secretion and increased expression of ALP observed in this study occurred via routes that did not involve Nrf2. Unfortunately, this study did not investigate the mechanism underlying the upregulation of these early bone matrix markers. Previous reports have identified that oxygen ozone therapy promotes the production of growth factors such as VEGF27). In addition, ozone gas is known to promote the production of FGF and other related cytokines28). VEGF enhances ALP expression in the mouse osteoblast cell line MC3T3-129). Thus, it is possible that bone matrix secretion may have been indirectly promoted via the secretion of these growth factors.

Our study demonstrates that 0.5 ppm ozone gel increases the secretion of osteoblast matrix without showing any signs of cell damage. Our previous reports have elucidated that the same concentration of ozone gel promotes secretion of collagen matrix and suppresses LPS-induced IL-6 and IL-8 expression without exhibiting cytotoxicity to human gingival fi broblasts20). Although it is diffi cult to directly link the results of in vitro and in vivo experiments, our results suggest that ozone gas, ozone water, ozone-olive oil30), and ozone gel may be used as therapeutic agents for periodontal disease. However, such applications will require detailed evaluation, such as assessment of biological safety using animal models and functional studies on its long-term eff ects in terms of cellular damage.

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