Human umbilical cord vein endothelial cells (HUVEC), human gingival fibroblasts (HGF) (Lonza Group AG, Basel, Switzerland), human osteogenic cells (HHOB-c; PromoCell GmbH, Heidelberg, Germany) and human oral keratinocytes (HOK; Provitro, Heidelberg, Germany) were examined. The cells were cultured in an incubator with 5% CO₂ at 37̊C. Cells were passaged at regular intervals depending on their growth characteristics using 0.25% trypsin (Biochrom GmbH, Berlin, Germany).

HOKs were cultured in keratinocyte growth medium, (Provitro GmbH, Berlin, Germany). The medium contained fibroblast growth factor (FGF), epidermal growth factor (EGF), Ca²⁺<0,1 mmol/l and insulin, but without bovine pituitary extract and hydrocortisone.

HGFs were grown in stroma cell growth medium (Lonza Group AG) with 1% penicillin-streptomycin-neomycin antibiotic mixture (PSN), 10% FCS and 500 ng basic FGF in 500 ml medium.

HUVECs were cultured in an endothelial basal medium supplemented with 1 µg/ml hydrocortisone, 12 µg/ml bovine brain extract, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 10 ng/mL EGF and 10% FCS until the third passage.

Osteogenic cells were cultivated in a solution composed of Dulbecco's modified Eagle's medium with 1% PSN, 1% L-glutamine and 10% FCS.

Due to the results of a previous study (16), the cells were incubated with 50 µmol of clodronate, ibandronate, pamidronate or zoledronate for 24 h prior to the cell viability test.

The cells were irradiated via a diode laser with 280 mW, at 670 nm for 60 sec (Periowave; Ondine Biopharma Corp., Toronto, Canada). During irradiation the laser tip was placed in the medium.

The cells were transferred into 24-well dishes for irradiation and bisphosphonate incubation. For each cell line, six different experiments were performed. The first group was the control group with no irradiation or bisphosphonate treatment. The second group underwent irradiation only. The third group had no irradiation but underwent bisphosphonate treatment. The cells in group four were irradiated shortly following incubation with bisphosphonates. The cells in group five were treated vice versa to group four. For the last group, the medium was mixed with bisphosphonates and irradiated prior to placing the osteoblasts into the medium (Fig. 1).

To examine the cell viability of all four cell lines, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay (MTT M5655; Sigma-Aldrich Produktions GmbH, Steinheim, Germany) was performed. Tetrazolium bromide is fermented to formazan by viable cells. Formazan can be measured photometrically following cell lysis at 550 nm. The experiments were performed six times for each of the groups as specified above.

Continuous variables are expressed as mean ± standard error of mean. Comparisons between groups were analyzed by analysis of variance (ANOVA; post hoc test: Tukey). The software SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for calculations. P<0.05 was considered to indicate a statistically significant difference.

For each cell type, the four ANOVAs conducted (one for each bisphosphonate tested) showed significant differences among the control and experimental groups. Each ANOVA yielded a P-value of <0.001.

The exclusive irradiation of keratinocytes increased cell viability significantly as compared to the control group (P=0.015). Cells that were only treated with bisphosphonates had reduced cell viability: Clodronate, P=0.027; and ibandronate, pamidronate and zoledronate, P<0.001. There were no significant differences in the two approaches of combining irradiation and bisphosphonate treatment in different orders. The differences between the cells incubated with bisphosphonate and the cells that were additionally irradiated were significant for all the groups (P<0.001), except for one experimental approach in the clodronate group. In this group, the sequence of irradiation followed by bisphosphonate treatment had no significant difference (P=0.285), but in the reverse sequence the P-value was 0.010. For all the other approaches, the P-values were ≤0.001. There was no significant difference between the cells incubated with bisphosphonates and the cells that were grown in bisphosphonate-containing medium that was irradiated (Fig. 1A).

Although the viability was increased following laser stimulation, there was no significant difference between the control cells and the cells with laser stimulation in three out of four tests. A significant difference was only obtained in the experiments using ibandronate (P<0.001). The addition of 50 µmol bisphosphonate reduced the viability significantly for ibandronate (P=0.001), pamidronate (P=0.004) and zoledronate (P<0.001), but not for clodronate (P=0.336). There was no significant difference between the sequence of bisphosphonate treatment and laser treatment, but there was a significant difference between the cells incubated with bisphosphonates only and the cells that were additionally irradiated. P-values for clodronate and ibandronate were <0.001, and for pamidronate a P-value of 0.093, compared to the cells incubated with bisphosphonates first and irradiated subsequently. The cells treated with pamidronate in the reverse order (irradiation first) had a P-value of 0.039. The cells treated with zoledronate had a significance difference with P=0.001 for the two approaches. The analysis of the cells with irradiation of the cell-free bisphosphonate-containing medium revealed no differences compared to the experimental approach with the incubation of cells with bisphosphonates only [P-values ranging between P=0.349 (ibandronate) and P=1.000 (clodronate)] (Fig. 1B).

The treatment consisting exclusively of radiation and bisphosphonates revealed significant changes in cell viability. Irradiation increased the viability, with P-values between P<0.001 and P=0.002, and bisphosphonate treatment decreased the viability, with P-values between P<0.001 and P=0.004. The combination of irradiation and bisphosphonate treatment increased cell viability significantly, independent of order with P-values between P<0.001 and P=0.008. There was no difference between the cells treated with bisphosphonates and the cells treated with irradiated medium [P-values between P=0.251 (pamidronate) and P=0.981 (zoledronate)] (Fig. 1C).

Low-level laser application increased the viability of cells, but was significant only in the experimental approach with pamidronate (P=0.026), and had a significant tendency with zoledronate (P=0.076). Adding bisphosphonates reduced the cell viability in the ibandronate (P=0.002), pamidronate and zoldedronat-treated assays (P<0.001). Osteoblasts treated with clodronate had no significant reduction in cell viability. The sequence of irradiation and bisphosphonate treatment did not result in significant differences for any of the cell lines. However, the additional irradiation of the cells treated with bisphosphonates increased the cell viability in all the cases, with P-values ranging from P<0.001 (clodronate) to P=0.046 (pamidronate and zoledronate). There was no significant difference between the cells treated with bisphosphonates only and the cells receiving the bisphosphonate medium that had been irradiated, with P-values ranging between P=0.761 (zoledronate) and P=1.000 (clodronate) (Fig. 1D).