Ozone saline solution counteracts bisphosphonates noxious effects in primary human gingival fibroblasts

Bisphosphonates are effective antiresorptive agents frequently used in the treatment of different bone disorders, as osteoporosis, Paget’s disease and tumours that cause osteolysis. A major concern related to bisphosphonates therapy is represented by osteonecrosis of jaw, a serious, debilitating, and mostly, a therapy-resistant disease, reported as a frequent side effect of bisphosphonates. In the present study were proposed two approaches: 1) to verify the impact of four commercially available bisphosphonates, very frequently used as oral (Fosamax - F and Actonel - A) and injectable (Ossica - O and Zoledronic acid - Z) therapy on primary human gingival fibroblasts - HGF viability and 2) to evaluate the protective effect of an ozone saline solution on HGF cells pretreated with bisphosphonates. Alamar blue cell viability assay was performed to assess the effect of test compounds (1.5; and after An saline solution – 80 was added for 24 h and cell and cell morphology changes determined by means Alamar blue and microscopic


Abstract Background
Bisphosphonates are effective antiresorptive agents frequently used in the treatment of different bone disorders, as osteoporosis, Paget's disease and tumours that cause osteolysis. A major concern related to bisphosphonates therapy is represented by osteonecrosis of jaw, a serious, debilitating, and mostly, a therapy-resistant disease, reported as a frequent side effect of bisphosphonates. In the present study were proposed two approaches: 1) to verify the impact of four commercially available bisphosphonates, very frequently used as oral (Fosamax -F and Actonel -A) and injectable (Ossica -O and Zoledronic acid -Z) therapy on primary human gingival fibroblasts -HGF viability and 2) to evaluate the protective effect of an ozone saline solution on HGF cells pretreated with bisphosphonates.

Methods
Alamar blue cell viability assay was performed to assess the effect of test compounds (1.5; 2.5; 5 and 10 μM) on gingival fibroblasts viability after a 24 h interval. An ozone -O3 saline solution -80 µg/mL was added to bisphosphonates pretreated fibroblasts for 24 h and cell viability and cell morphology changes were determined by the means of Alamar blue test and microscopic images.

Results
Fosamax and Actonel induced a significant reduction of HGF cells viability even at concentrations as low as 2.5 μM (82 and 79.33%) and changes in cells morphology (round and floating cells), effects that were reversed by O3 saline solution administration: an increased cell viability after F and A at 2.5 μM: 147.54 and 120.11%), no changes in cells morphology and an improved confluence. Ossica and Zoledronic acid exerted no cytotoxic effect.

Conclusions
In conclusion, in these experimental conditions, injectable bisphosphonates (O and Z) proved to be safe for HGF cells, whereas oral compounds (F and A) were cytotoxic even at low concentrations, effects that were counteracted by O3 saline solution administration.
Based on these data, ozone saline solution might represent a therapeutic alternative for bisphosphonates noxious effects on oral mucosa cells.

Background
Bisphosphonates are a class of pharmaceutics applied as treatment for several bone disorders and cancers, as: Paget's disease, osteoporosis, multiple myeloma and hypercalcemia of malignancy, that act by suppressing osteoclast differentiation, impairing its activity and leading to early apoptosis. Their activity on osteoclast is also related to adverse events as damaged bone healing and remodeling processes that have as consequence an augmented risk of developing osteonecrosis of jaw, a serious and debilitating condition, in patients subjected to surgical dental procedures as extraction or implant placement [1,2,3,4,5,6]. Since more than fifteen years ago, there were raised notable concerns regarding the liaison between the use of bisphosphonates and the increased risk to develop osteonecrosis of the jaw after invasive dental procedures [7]. A considerable number of studies were conceived to find strategies for dental management of patients at risk to develop osteonecrosis of the jaw following antiresorptive (bisphosphonates) and antiangiogenic compounds use [1,7,8,9,10,11], still, there are no effective therapeutic alternatives to reverse bisphosphonates noxious effects, and to find an appropriate prevention/treatment method for osteonecrosis of the jaw is rather challenging [12].
Ozone (O 3 ) is an unstable gas, naturally produced by atmospheric air with a triatomic molecule and a reduced half-life (40 minutes at 20 ºC), that exerts multiple biological effects, as: antimicrobial (more efficient as compared to chloride), stimulator of blood circulation and immune response, anti-hypoxic, features that recommend it for medicine and dentistry applications [13,14,15]. Among the multiple dental applications of the ozone, there can be stated the following: prevention of dental caries, endodontic treatment, tooth extraction, periodontal pocket disinfection and osseous disinfection, accelerated healing, tissue regeneration, remineralization of tooth surface, teeth whitening, etc [13,16,17]. Moreover, medical ozone formulated as oil suspension or administered as gas insufflations to patients with bisphosphonates-induced osteonecrosis of the jaw lesions proved to be a promising therapeutic option in small (< 2.5 cm -oil suspension) and big lesions (> 2.5 cm -gas insufflations) [18,19].
In the light of the data presented above, the present study was subjected: (i) to assess the impact of orally (sodium risedronate -Actonel -A and alendronate -Fosamax -F) and

Cell viability assessment
To verify the potential toxicity of the test compounds on human primary gingival fibroblasts -HGF it was applied Alamar blue assay. In brief, HGF cells were seeded in 96wells plates (1x10 4 cells/well/ 200 μL) and let to grow until the appropriate confluence was

Ozone impact on human primary gingival fibroblasts
To determine the effect of ozone (O 3 saline solution -80 µg/mL) on cells viability and its impact on cells capacity to recover after test compounds toxicity, HGF cells were stimulated with Actonel and Fosamax (compounds that proved to be cytotoxic) (1.5; 2.5; 5 and 10 µM) for 24 h. The old media was removed, and it was added 100 μL of fresh medium and 100 μL of ozone saline solution/well for 24 h according to the protocol described by Perez et al. [20], followed by application of Alamar blue assay, as described above.

Cell morphology
The effects of test compounds (A, F, O and Z) ± ozone saline solution on cells morphology were assessed by taking pictures before addition of test compounds and after the stimulation period (24 h). The pictures were acquired by using the Olympus IX73 inverted microscope provided with DP74 camera photo and documented with the CellSens V1.15 software (Olympus, Tokyo, Japan).

Statistical analysis
The results obtained were expressed as means ± SD, and the difference between means was compared by one-way ANOVA, using the post-hoc Tukey's and Dunnett's multiple comparison tests and by unpaired t test with Welch's correction (GraphPad Prism v. 6.0 Software, SUA). The difference between groups was considered statistically significant if p < 0.05.

Results
Bisphosphonates decrease human primary gingival fibroblasts -HGF viability In the present study, it was evaluated the effect induced by several bisphosphonates (A, F, O and Z) frequently used as treatment for osteoporosis, on HGF cells viability.
Stimulation for 24 h of HGF cells with different compounds from the bisphosphonates family led to distinct results regarding the percentage of viable cells, as follows: Z and O ( Figure 1A) had no toxic effects on cells viability, moreover a stimulatory effect was noticed; in the case of F ( Figure 1B     In the case of cells stimulated with Zoledronic acid ( Figure 5) and Ossica (Figure 6), it was observed a higher confluence of the cells that presented similar shapes with control cells (spindle shape with a high adherence to the plate), data that are in agreement with cell viability findings ( Figure 1A).  As it can be seen in Figure 7A

Discussions
Bisphosphonates are synthetic analogues of pyrophosphates known to strongly bind to hydroxyapatite (a mineral from bone structure) and modify bone resorption by decreasing bone metabolism and remodeling processes [7]. There were described two different classes of bisphosphonates: non-nitrogen (risedronate) and nitrogen containing (alendronate, ibandonic acid and zoledronic acid) bisphosphonates, that present a disparate mechanism of action, as follows: non-nitrogen compounds are taken up by the osteoclast and metabolized to adenosine triphosphate analogues with noxious effects that induce osteoclast apoptosis or cell death, whereas nitrogen containing bisphosphonates are interiorized by osteoclasts during resorption process and impair the mevalonate pathway (involved in the synthesis of cholesterol) leading to suppression of bone resorption [21]. The side effects of bisphosphonates include irritation of the oesophagus, dysphagia, migraines, intestinal obstruction, joint and bone pain, but most importantly osteonecrosis of the jaws -a main dental adverse event [3,5,10,11]. Osteonecrosis of the jaw has become a major concern in the field of dentistry, since the risk of development to patients under the treatment with these agents that suffer invasive dental procedures is quite elevated and treating this kind of patients requires a complex management of the pathology. The most cases of osteonecrosis of jaw were recorded after administration of orally bisphosphonates -alendronate (the most frequently used worldwide) [2].
Taking into consideration that the category of patients that use bisphosphonates therapy becomes wider day by day (approximatively 75 million people are affected by osteoporosis in Europe, U.S. and Japan) [22], it is important to know the impact of these compounds in the organism, especially, since this kind of medication is for long-term.
In this study, it was verified the effect of several types of bisphosphonates (Fosamax -F, Actonel -A, Ossica -O and Zoledronic acid -Z) on human primary gingival fibroblasts -HGF viability and morphology after a 24 h stimulation.
Human primary gingival fibroblasts were selected for this study based on the following considerations: (i) these cells are the main constituents of the gingival connective tissue and the most abundant residents of oral mucosa; (ii) play key roles in scarless wound healing process by releasing multiple growth factors (transforming growth factor beta -TGF-Beta, connective tissue growth factor -CTGF, and basic fibroblast growth factor -bFGF); and iii) primary cells offer more reliable data as compared to immortalized cell lines [23,24]. In addition, previous studies proved that primary human gingival fibroblasts are valuable and reliable in vitro models for screening toxicity/cytocompatibility of dental materials [23,25,26].
Our results showed that F and A become cytotoxic even at low concentrations as 1.5 µM (see Figure 1B) and also cells morphology was altered (see Figures 3 and 4), whereas O and Z had no cytotoxic effects at the concentrations tested (1.5; 2.5; 5 and 10 µM) (see Figure 1A). The cytotoxic effect of alendronate -F on human primary gingival fibroblasts was also shown by other groups of research [27,28,29], data that are in agreement with these results. Stimulation of human stem cells derived from the gingiva with risedronate in the range of 1-10 µM led to changes in cells morphology and a reduced viability [30], findings that confirm our results.
An in vitro study developed on human umbilical cord vein endothelial cells (HUVEC), human gingival fibroblasts (HGF), human osteogenic cells (HHOB-c) and human oral keratinocytes (HOK) stimulated with 50 µM ibandronate, pamidronate or zoledronate for 24 h showed that bisphosphonates used reduced all cells viability [31]. The results obtained in the present study showed that Z (zoledronic acid) and O (ibandronic acid) are not cytotoxic for primary gingival fibroblasts at concentrations ≤ 10 µM.
Another study conducted by Soydan et al. [28] demonstrated the toxic effects of Alendronate (Fosamax) and PAM (pamidronate) on human gingival fibroblasts, characterized by significant changes in the apoptotic and proliferative indices leading to an in vitro faulty epithelisation of the oral mucosa. The change of these indices is an important factor in the management of delayed healing of the oral mucosa, secondary to surgery in patients under bisphosphonate treatment, and is a problem that reduces the success rate of healing in cases of bisphosphonate-induced maxillary osteonecrosis [14,32].
Looking for new ways to reduce the side effects of bisphosphonates, ozone was described as a viable choice for healing and restoring oral cells viability. Besides the benefits of ozone in dentistry (carious lesions treatment, endodontics, reduction of dental hypersensitivity, periodontics, oral surgery, pedodontics, orthodontics) [15,17], were also mentioned other biological effects, as: (a) antimicrobial activity against aerobic and anaerobic bacteria (especially Staphylococcus aureus), fungi and viruses; (b) stimulator of the circulatory system, increasing haemoglobin synthesis and the production of red blood cells, thereby producing tissue oxygenation; (c) modulator of immune cells, acting as a cytokine, and increasing their phagocytosis and diapedesis; (d) stimulator of angiogenesis as well as the proliferation of fibroblasts; and (e) pain reduction capacity [15,33].
Addition of the ozone saline solution (stock concentration -O 3 saline solution -80 µg/mL; 100 µL/well) to pretreated HGF cells with F and A for 24 h led to an increase of cells viable percentages as compared to the ones stimulated only with F and A (see Figure 8) and it was also noticed an improvement of cells confluence and a cells morphology similar to the control ones (unstimulated cells) (see Figures 9 and 10), what demonstrates that ozone solution in these experimental conditions was able to reverse the toxic effects induced by F and A. Our data are in line with other data from the literature that confirm the protective effects of ozone on gingival fibroblasts [32,34,35].
Moreover, daily application of an ozonated water treatment accelerated the rate of physiological healing. In a study comparing the use of ozonated oil in an experimental group with a control group using antibiotic therapy in the treatment of alveolitis, it was found that patients treated with ozonated oil healed more quickly [36,37]. Ozone was also used in the treatment of avascular osteonecrosis of the maxilla (ONJ). Complete healing of lesions with disappearance of symptoms has been noticed [38,39].

Conclusions
In the view of these findings, it could be stated that oral bisphosphonates -Fosamax and Actonel exert a concentration-dependent toxicity on human primary gingival fibroblasts by decreasing the percentage of viable cells and modifying cells morphology. In contrast, injectable bisphosphonates -Zoledronic acid and Ossica did not affect the viability or HGF cells morphology. Ozone saline solution administration to bisphosphonates -treated cells reversed the toxic effects of test compounds (F and A) and improved cells capacity to recover by stimulating their growth and proliferation. These in vitro results represent a valuable background that endorse the hypothesis that ozone saline solution could be considered a therapeutic alternative for bisphosphonates noxious effects on oral mucosa cells.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. of data, analysis and interpretation of data and critically revision of the manuscript. ADF, RF, DJ, DC contributed to the design of the study and by revising critically the manuscript.        Unpaired t test with Welch's correction was applied to determine the statistical differences in rapport with O3 unstimulated cells (**p<0.01, ***p<0.001 and **** p <0.0001).

Figure 9
The aspect of HGF -human primary gingival fibroblasts in culture stimulated with different concentrations of Fosamax -F (1.5; 2.5; 5 and 10 µM) for 24 h: without (-O3) and with (+ O3) ozone stimulation. Pictures were taken using the 20x objective at a scale bar of 50 µm.

Figure 10
The aspect of HGF -human primary gingival fibroblasts in culture stimulated with different concentrations of Actonel -A (1.5; 2.5; 5 and 10 µM) for 24 h: without (-O3) and with (+ O3) ozone stimulation. Pictures were taken using the 20x objective at a scale bar of 50 µm.