Abstract
A limited number of clinical studies indicate the supportive role of low level laser therapy (LLLT) on medical and/or surgical approaches carried out in treatment modalities for bisphosphonate related necrosis of jaws (BRONJ), the most common side effect of bisphosphonates used to inhibit bone resorption. The purpose of this study was to investigate the effects of LLLT on cell proliferation and alkaline phosphatase (ALP) activity of human osteoblast-like cells (Saos-2) treated with different doses of zoledronate, the most potent bisphosphonate. Saos-2 cells were treated with different concentrations of zoledronate and were irradiated with diode laser (wavelength 808 nm, 10 s, 0.25 or 0.50 W). Cell numbers and ALP activity of the cells were determined. LLLT mildly increased the proliferation rate or ALP activity, while zoledronate reduced both. When applied together, LLLT lessened the detrimental effects of zoledronate and improved cell function and/or proliferation. Based on the results of this study, it was concluded that LLLT has biostimulative effects on Saos-2 cells, even after treatment with zoledronate. LLLT may serve as a useful supportive method for BRONJ treatment through enhancement of healing by osteoblasts.
The bisphosphonate drug family is widely used in clinical practice for the treatment of Paget’s disease of bone, postmenopausal osteoporosis and cancer-related conditions including bone metastases and hypercalcemia of malignancy. Oral bisphosphonates are primarily used in the management of osteoporosis, whereas intravenous (i.v.) forms are most commonly used for malignancy related conditions. Bisphosphonates are synthetic drugs based on a phosphorus–carbon–phosphorus (P–C–P) pattern, structurally related to endogenous pyrophosphates and are resistant to enzymatic and chemical hydrolysis. When given at high doses the bisphosphonates have two essential biological effects: inhibition of calcification, and inhibition of bone resorption. In general, bisphosphonates have a high affinity for hydroxyapatite, remaining unmetabolized for long periods of time. ‘Osteonecrosis’ is a term commonly used to describe death of bone cells, the osteocytes, in the cortical bone and the bone marrow cells including hematopoietic stem cells, bone marrow stromal cells, pre-osteoblasts, mature osteoblasts, lining cells, and osteoclasts. Osteonecrosis also damages bone endothelial cells and vasculature leading to reduction of blood flow to the region. Bisphosphonates, in particular pamidronate and zoledronic acid, have been widely associated with osteonecrosis of the jaw bones (ONJ), a condition with clinical symptoms that may include pain, swelling, the presence of pus, loose teeth, ill-fitting dentures, and fractures. The phenomenon of bisphosphonate-related osteonecrosis of the jaws (BRONJ) was first described by Marx in 2003 as a warning to the dental and medical authorities. Following the first report, many case series and case reports have been published. In 2004, Ruggiero et al. published case series of 63 patients, and in 2006 Dimitrakopoulos et al. published case series of 11 patients.
There are also many in vivo and in vitro studies in the literature that mention the effects of bisphosphonates. Choi et al. treated calvarial defects with pamidronate, a potent bisphosphonate, to evaluate the effect of the drug and observed histopathologically non-healing and necrotic areas in the defects. Naidu et al. showed that bisphosphonates had significant cytotoxic effects, such as reducing cell proliferation and viability in osteoblast cell cultures. Bisphosphonates adversely affect osteoblasts and soft tissue endothelial cells and fibroblasts.
Management of BRONJ varies with the severity of the necrosis. Usually medical and/or surgical procedures are performed, but researchers have focused on additional methods to support the healing. One of these supporting methods is low level laser therapy (LLLT). The biostimulative effects of lasers have been reported in many in vitro experiments. The first laser biostimulation experiment was carried out by Mester and Jászsági-Nagy in 1971. The resulting biological reactions involve the absorption of a light of a specific wavelength by photoacceptor molecules. These energy absorbing molecules are mostly proteins and they are either elements in the mitochondrial cytochrome system or endogenous porphyrins in the cell. The use of LLLT has been shown to have beneficial effects on many different pathological conditions including pain relief, reduction in inflammatory processes, and promotion of tissue healing. Yaakobi et al. reported the curative effects of LLLT in bone regeneration. In vitro laser biostimulation studies have also been carried out with osteoblasts or osteoblast-like cells. Dörtbudak et al. reported that LLLT has stimulatory effects on bone matrix formation in osteoblast cell cultures. Coombe et al. showed that LLLT increases osteoblast cell proliferation, viability, and alkaline phosphatase (ALP) activity. Stein et al. revealed that the initial biostimulatory effects of LLLT on human osteoblast-like cells were enhancement in cell viability, ALP activity and osteopontin and collagen type I mRNA. Periodontal ligament fibroblasts were shown to produce higher amount of ALP on LLLT application.
Considering these reports in the literature, the severity of BRONJ, the treatment difficulties and patient discomfort, additional supportive methods are needed for BRONJ management. Laser therapy, with its ease of application and biostimulatory effects on bone healing, can be considered as a preferable supportive method. The aim of this in vitro study was to investigate the effects of LLLT on cell proliferation and ALP activity of osteoblast-like cells (bone forming cells) treated with zoledronate, the most potent bisphosphonate, in a dose dependent manner.
Materials and methods
Cell culture
Human osteoblast-like cells (Saos-2), were obtained from American Type Culture Collection (ATCC#: HTB-85, Rockville, MD, USA). Saos-2 cells are derived from an osteosarcoma and have similar properties to primary human osteoblasts. They have an osteoblastic phenotype, but are not real osteoblasts so might behave slightly differently.
They were cultured in RPMI 1640 (HyClone) supplemented with 10% foetal bovine serum, 1% penicillin/streptomycine and 1 μg/ml amphotericin B at 37 °C, 95% humidity and 5% CO 2 .
Experimental setup
The Saos-2 cells were seeded at a density of 5 × 10 4 cells/well in 24-well polystyrene tissue culture plates. After 24 h of cultivation the medium was replaced with 1 ml medium supplemented with zoledronate (Zolenat, MN, USA) at different concentrations. The experimental design is given in Fig. 1 . Zoledronate (4 mg/5 ml) was diluted with cell culture medium to obtain concentrations of 1 μM, 10 μM, 100 μM. The wells without zoledronate served as control groups. Following either 24 h (first experimental group) or 48 h (second experimental group) of culture with zoledronate, the cells were irradiated with a 808 nm diode laser (Fotona XD-2, Slovenia) for 10 s at power outputs of either 0.25 W or 0.50 W at continuous wave mode. The medium in the wells was refreshed with medium of the same zoledronate content immediately after the laser treatment. Unirradiated wells were considered as control groups. The laser beam was adjusted to cover the bottom of one culture well (10 cm above the bottom of the culture plate) exactly. All irradiation was carried out under sterile conditions in a vertical laminar flow cabinet. To avoid any influence of possible scattered irradiation, Teflon separators were placed among the wells and the 24-well plates were placed on a black surface when the cells were being irradiated. The lid of the plate was removed and all other wells in the plate were covered leaving only one well uncovered for irradiation. The handpiece of the laser was fixed in place with a holder and the plate was moved to irradiate one well at a time. Cells were analysed for cell proliferation and ALP activity at 48 h after laser irradiation. Each condition was studied in triplicate.
Morphological analysis
Cell morphology was studied using inverted light microscope (Olympus IX 70, Japan) under phase contrast filter to see the effect of zoledronate concentration and laser irradiation.
Cell number and viability
MTS assay was applied to the cells treated with zoledronate only and to the cells of the first and second experimental groups 48 h after laser irradiation. MTS assay (Promega) is a non-radioactive cell proliferation assay where MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is converted to soluble formazan, a coloured product, and this conversion is directly proportional to the number of viable cells. MTS/PES (tetrazolium compound (inner salt; MTS)/electron coupling reagent (phenazine ethosulfate; PES)) reagent (1 ml, 10%) was added to the wells and the plate was incubated for 2 h at 37 °C in the CO 2 incubator. Absorbance of the medium in each well was determined at 490 nm using an Elisa plate reader (Molecular Devices (USA), Model Maxline). Average absorbance values of cell free (blank) wells were subtracted from values of cell seeded wells to obtain the net absorbance values. Cell amount was determined from the MTS calibration curve. Each condition was studied in triplicate.
ALP activity
Activity of ALP produced by the cells was determined using a kinetic colorimetric assay. The cells on the tissue culture polystyrene were washed with cell culture media and detached with 0.3 ml Tris-Triton buffer (10 mM Tris, pH 7.5, 0.1% Triton X-100). The samples were sonicated for 5 min at 25 W on ice. The samples were centrifuged at 2000 rpm for 10 min and diluted 10 times using the same buffer. An aliquot of 10 μl of each supernatant was added to 240 μl of p-nitrophenyl phosphate solution (Randox) at room temperature in the well of a 96-well plate and absorbance of the coloured product, p-nitrophenol, was measured at 405 nm every minute for 5 min with the Elisa plate reader. The slope of the absorbance versus time plot was used to calculate the ALP activity (change of absorbance per minute). The calibration curve of p-nitrophenol at room temperature was used to determine the enzyme activity in units of nmol substrate converted to product per minute. ALP specific activity was determined by dividing the ALP activity by the cell number obtained for that well. Each condition was studied in triplicate.
Statistics
Statistical analysis of the data for cell proliferation and ALP activity was carried out using the Mann–Whitney U -test and the Kruskal–Wallis test to determine equality of population medians among groups. Differences were considered statistically significant at p < 0.05.
Results
Effect of zoledronate on cell morphology and proliferation
Zoledronate was applied to Saos-2 cells at three different concentrations: 1 μM (the concentration of zoledronate in blood plasma when used for chemotherapy), 10 μM and 100 μM. A dose dependent effect of zoledronate was shown by cell morphology and proliferation. Increasing numbers of cells acquired a retracted circular morphology with thin protrusions as the zoledronate concentration increased ( Fig. 2 ).
There was no significant difference between the untreated (0 μM zoledronate) and 1 μM zoledronate treated wells in terms of cell number following 24 h of zoledronate treatment, but the wells treated with 10 and 100 μM zoledronate had significantly lower cell numbers ( Fig. 3 ). At 48 h zoledronate treatment and later, the difference between cell numbers in all groups was significant. The cell proliferation rate was decreased by zoledronate in a dose dependent manner. A plateau was achieved in cell numbers in the group treated with 100 μM zoledronate between 24 and 48 h and cell death occurred thereafter. Cell death was also observed in the group treated with 10 μM zoledronate after 72 h of treatment.
Laser treatment
In contrast to zoledronate treatment, laser application did not cause any change in morphology of the cells (data not shown). Laser treatment was applied to zoledronate treated samples at two different points, 24 h and 48 h following zoledronate addition to the cell culture medium (i.e. at two points when cell death had not been observed in any of the groups). Cells were grown for a further 48 h after laser treatment prior to determination of cell number and ALP activity, to give cells enough time for division; doubling time for Saos-2 cells is 37 h.
Results
Effect of zoledronate on cell morphology and proliferation
Zoledronate was applied to Saos-2 cells at three different concentrations: 1 μM (the concentration of zoledronate in blood plasma when used for chemotherapy), 10 μM and 100 μM. A dose dependent effect of zoledronate was shown by cell morphology and proliferation. Increasing numbers of cells acquired a retracted circular morphology with thin protrusions as the zoledronate concentration increased ( Fig. 2 ).
