Cytotoxic and inflammatory effects of alendronate and zolendronate on human osteoblasts, gingival fibroblasts and osteosarcoma cells

Accepted Manuscript Cytotoxic and inflammatory effects of alendronate and zolendronate on human osteoblasts, gingival fibroblasts and osteosarcoma cells Prof. rer. nat. Yahya Açil, Dr. med. Mia Leena Arndt, Assoc. Prof. Dr. Aydin Gülses, Dr. med. Dr. med. dent. Henning Wieker, Dr. med. dent. Hendrik Naujokat, Dr. med. dent. Mustafa Ayna, Prof. Dr. med. Dr. dent. Jörg Wiltfang PII:

S1010-5182(17)30447-X

DOI:

10.1016/j.jcms.2017.12.015

Reference:

YJCMS 2868

To appear in:

Journal of Cranio-Maxillo-Facial Surgery

Received Date: 22 September 2017 Revised Date:

3 November 2017

Accepted Date: 15 December 2017

Please cite this article as: Açil Y, Arndt ML, Gülses A, Wieker H, Naujokat H, Ayna M, Wiltfang J, Journal of Cranio-Maxillofacial Surgery (2018), doi: 10.1016/j.jcms.2017.12.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Prof. rer. nat. Yahya Açil1 [email protected] Dr. med. Mia Leena Arndt2 [email protected] Assoc. Prof. Dr. Aydin Gülses3 [email protected] (corresponding author)

Dr. med. dent. Hendrik Naujokat5 [email protected] Dr. med. dent. Mustafa Ayna6 [email protected]

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Dr. med. Dr. med. dent. Henning Wieker4 [email protected]

Prof. Dr. med. Dr. dent. Jörg Wiltfang7 [email protected]

Christian Albrechts University, Department of Oral and Maxillofacial Surgery, Kiel-

Germany Private Practice, Düsseldorf- Germany

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6

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1-5,7

This work should be attributed to: Department of Oral and Maxillofacial Surgery (Head: Prof. Dr. Dr. Jörg Wiltfang), University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany Corresponding author: Aydin Gülses Department of Oral and Maxillofacial Surgery

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(Head: Prof. Dr. Dr. Jörg Wiltfang), University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany. e-mail: [email protected] phone: 004915147509544 fax: 0431 500 26164

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The offprints should be sent to: Prof. rer nat Yahya Açil Department of Oral and Maxillofacial Surgery (Head: Prof. Dr. Dr. Jörg Wiltfang), University Hospital

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Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3, 24105 Kiel, Germany. Funding source: none

ACCEPTED MANUSCRIPT Summary INTRODUCTION Bisphosphonates (BPs) are potent inhibitors of osteoclast-mediated bone loss; their specific

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target is the promotion of mature osteoclasts, which play a central role in physiological and pathological bone loss (Gong et al. 2011, Endo et al. 2017). Their additional key property is strong binding to bone due to their high affinity for hydroxyapatite, which results in a

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prolonged storage within bone (Russel 2006, Kos el al. 2015).

From a chemical point of view, BPs are synthetic analogues of pyrophosphates, possessing

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two side chains (R1 and R2) on the carbon atom of BP molecule attached to basic P-C-P structure. The modification to one or both phosphonate groups allows variations in molecular structure and a range of potency which determines the chemical, biological, and cytotoxic effects of the agent (Russel et al 2007, Papapoulos 2008, Sharma et al. 2013). The group

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occupying the R1 position is usually hydroxyl; it enhances the molecules’ affinity to the bone, and the variable group at R2 position determines its antiresorptive action (Russel et al

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2007Papapoulos 2008, Sharma et al. 2013).

Bisphosphonates could be broadly classified as nitrogen-containing and non−nitrogen

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containing BP, depending on the absence or presence of nitrogen in their R2 group. The main pharmacological mechanism of nitrogen-containing BPs such as alendronate (FOS), zoledronate (ZOL), pamidronat and risedronat is suggested to be the inhibition of an enzyme in mevalonic acid metabolism, via inactivation of farnesyl diphosphate synthase (FDPS), which can lead to a decreased activity and apoptosis induction in different cell types (Levy et al. 2007). Bisphosphonate-related osteonecrosis of the jaw (BRONJ), which could be generally defined as necrotic bone exposure to the oral cavity and inflammatory reactions of the surrounding

ACCEPTED MANUSCRIPT soft tissue, is one of the main side effects in patients treated with BP. Since the first description of the condition in 2003 (Marx 2003), many studies have focused on the pathophysiological mechanisms of the condition; however the exact mechanism remains still unclear. The most frequently used agent and that which is responsible for BRONJ is

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suggested to be the ZOL, which is administrated intravenously for the treatment metastatic cancer patients (Mücke et al. 2016). However, there are also numerous reports on BRONJ cases in patients taking oral BP, mainly in the management of postmenopousal osteoporosis.

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cause the condition (Ulmner et al. 2014).

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(Sedqhizadeh et al. 2003). FOS is the most frequently reported per-oral administered agent to

In the literature, there are numerous articles focusing on the effects of BPs on different cells; however, the exact mechanism of their action is not still understood. The aim of this paper was to assess the effects of ZOL and FOS on cell behaviour and expression of inflammatory cytokines of human osteoblast cells, human gingival fibroblast cells and osteogenic sarcoma

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cells.

MATERIALS AND METHODS

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All procedures performed in the present study were approved by the institutional ethical

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committee of Christian Albrechts University, Kiel, Germany (D 402/07- Ethik Kommission der Christian Albrechts Univesitaet zu Kiel-Deutschland), and were conducted in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. Three cell types (human osteoblasts, human gingival fibroblasts and human osteogenic sarcoma cell lines) were used and investigated during a period of 4 weeks. Human cell culture

ACCEPTED MANUSCRIPT Human osteoblasts and fibroblasts were isolated from samples of the iliac crest and of the gingiva, respectively. The osteoblast samples were obtained from healthy patients who had no exposure to BP therapy and were undergoing various reconstructive surgeries at the Department of Oral and Maxillofacial Surgery at Christian Albrechts University-Kiel,

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Germany. Preparation of the tissue samples and further processing was performed as

previously described by Acil et al. (Açil et al. 2000). Fibroblasts from gingival samples were obtained during routine surgical procedures. The test results for osteocalcin and alkaline

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phosphatase were negative, as described previously (Açil et al. 2000). Human osteogenic sarcoma cells (SaOS-2-cells) were purchased from DSZM (Deutsche Sammlung von

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Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). SaOS-2-cells were defined as osteoblast-like cells by the determination of osteoblast markers (biosynthesis of osteocalcin and activity of alkaline phosphatase).

The cells were subcultured and transferred according to description of Simon et al. (Simon et

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al. 2010). Briefly, the cells were plated at a seeding density of 5 × 104 viable cells/ml in medium and grown as a monolayer in a 37°C incubator with a humidified atmosphere of 5% CO2 in air. Trypsin/EDTA solutions (Biochrom GmbH Germany) were

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used for passaging monolayer cultures.

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Bisphosphonates (BP)

In order to relate the scientific research to the clinical processes, the most common drugs associated with BRONJ with different means of application (intravenous and oral), zoledronic acid (ZOL; 200 mg, i.v.; Zometa, Novartis, Nürnberg, Germany) and alendronate sodium (FOS; Fosamax, MSD Sharp & Dohme GmbH, Haar, Germany) were selected. Five different concentrations (2,5 µm; 1,25 µm; 0,625 µm; 0,3125 µm; 0,15625 µm) were added into the medium.

ACCEPTED MANUSCRIPT Cell proliferation test (MTT) Cell cultures with different concentrations of BPs were incubated for 4 weeks. The cell proliferation was evaluated weekly with the Cell Proliferation Kit I (MTT, Roche Diagnostics GmbH, Penzberg, Germany), which measures the active and viable cells. The cell reaction

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produced a water-insoluble formazan salt that was solubilized and ultimately assessed in a flow-through spectrometer. The detection of cell vitality is based on the reduction of a yellowcoloured dye MTT-test to blue–violet Formazan. The absorbance was measured at 550 nm as

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described previously (Simon et al. 2010). The viability and proliferation of the cells were

microscope (Leitz, Wietzlar, Germany).

Fluorescein diacetate (FD) analysis

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documented every week with a digital camera (Nikon E 5000, Osaka, Japan) under light

Cell vitality was assessed by fluorescein diacetate (FD) test and propidium iodide (PI)

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staining). Briefly, the cells were rinsed with PBS and immersed in a FD solution prepared by diluting 30 µl × 1 mg FDA/ml acetone in 10 ml PBS. After incubation for 15 min at 37 °C and in darkness, the FD solution was removed by suctioning and replaced by a PI solution

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prepared by diluting 500 µl × 1 mg/ml PI in 10 ml PBS. After incubation for 2 min. slides were rinsed twice in PBS. The slides were subjected to fluorescence microscopy with

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excitation at 488 nm and detection at 53 nm (FD, green) and 62 nm (PI, red) (Möller et al. 2012).

Detection of cytokines via PCR analysis (Açil et al. 2012) Gene expression of the cyotkines tumor necrosis factor-α (TNF-α) and interleukin (IL) 1β, 4, 6, 10, and 12 were examined via PCR analysis, which involves measurement of cytokine mRNA transcript abundance. This method is relatively straightforward and quantitative, and

ACCEPTED MANUSCRIPT allows the detection of many different cytokines from relatively small sample amounts. For each sample, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as the housekeeping gene (Açil et al. 2012, Bustin 2002). Three independent tests were performed. For the RNA extraction, RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, Germany) was used.

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A photometer (SpectraMax plus, Molecular Devices Corporation, Sunnyvale, CA, USA) was used to determine mRNA chains, and mRNA-appropriate DNA via reverse transcriptase and primer mix with the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) was

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obtained. DNAs were amplified according to the guidelines provided by the manufacturer (Quanti Tect SYBR Green PCR Kit; Quiagen, Hilden, Germany, and LightCycler®). The

Agarose gel electrophoresis

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lowest point of fluorescence at crossing was set to 5%.

Statistical analysis

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In order to evaluate PCR results quantitatively, agarose gel electrophoresis was performed.

Statistical analysis was performed by the Institute of Medical Informatics and Statistics at

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Christian Albrechts University-Kiel by using the Programm “R” (The R Foundation for Statistical Computing, Wien, Austria). MTT-Test was examined via Box-Plot diagrams.

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Minimum, maximum, median, and 1st and 3rd quartiles were determined for all cells and concentrations, respectively. Cytokine expression was examined via PCR analysis and shown in two-plane diagrams considering the concentrations and cytokine expressions.

RESULTS Cell proliferation

ACCEPTED MANUSCRIPT Overall, in the study group, cytotoxic effects increased parallel to the increase in concentration of BPs. Compared to the FOS, ZOL showed stronger cytotoxic effects in all cell types. The MTT test showed that, among osteoblasts under 1,25 µM concentration, the vitality of the cells decreased to 20% and 65% under ZOL and FOS, respectively. Compared

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to the osteoblasts, fibroblasts showed a less distinct cell viability between ZOL and FOS groups. By the concentration of 2,5 µm ZOL, only 20% cells were viable (Figure 1).

Photographic documentation of cell culture (Figures 2 and 3) showed similar results.

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FD tests showed that the proliferation and cell viability of osteoblasts (Figure 4) and

fibroblasts (Figure 5) were negatively affected in a dose-dependent manner under both ZOL

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and FOS administration. However, osteosarcoma cells showed an increase in proliferation under lower doses of BP, whereas the high dose of BP resulted in inhibition (Figure 6).

Expression of inflammatory cytokines

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The relative expression could not always reach the threshold; data regarding all cytokines for each cell could not be collected. Results regarding the expression of IL 1β, 4, and 10 levels showed a wide variation.

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At the 4th week, among the fibroblasts under ZOL concentration of 2,5 µM, a significant increase of IL 1β was observed. The addition of FOS did not result in a proliferative

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distribution of cytokines, either in the osteoblast or in the fibroblast groups. However, in osteosarcoma cells, higher BP concentrations resulted in increase of TNF-α, IL 6 and 12 expression. All cells reacted to ZOL addition with a relatively increased IL 6 expression. The increase of IL 12 expresion following higher doses of ZOL in the osteoblast group was also remarkable.

ACCEPTED MANUSCRIPT Compared to the control group, osteoblasts and fibroblasts presented 70-fold higher IL 6 levels, whereas among osteosarcoma cells, 200-fold higher levels of IL 6 were observed. Figure 7 shows the IL 6 expression levels of the cells. Due to the lack of significance of the results for each group and cytokine, only IL 1β and 6

fibroblasts under ZOL (Figure 9) could be clearly demonstrated.

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DISCUSSION

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could be shown. IL 6 expression of osteoblasts under FOS (Figure 8) and IL 1β expression of

Since the first presentation of BRONJ in 2003 (Marx 2003), the use of BPs has become one of

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the main problems in oral and maxillofacial surgery. In order to evaluate the effects of BPs on various cell types, many in vitro studies have been performed. A literature survey revealed that in-vitro studies focusing on the effects of BPs had study periods of days to 1 week, (Woodward et al. 2005, Basso et al. 2014); however, in the current study, a duration of 4

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weeks was selected in order to define the exact mechanisms of action. ZOL and FOS are frequently prescribed agents which have only one application form; intravenous and per-oral, respectively. It is well known that both agents have been mostly

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involved in BRONJ cases. Whereas ZOL is administered mostly for the treatment of metastatic cancer patients, FOS is used mainly for the prevention of osteoporosis in

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postmenopausal women. Considering their clearly stated indications and application forms, both agents have often been examined in clinical and experimental studies (Ishtiaq et al. 2015).

Following the introduction of the BRONJ cases, another area interest has been the evaluation of plasma concentrations and dose-dependent effects of BPs on various cell types. Skerjanec et al. have stated that, after intravenous administration of ZOL, the peak plasma concentration reached the level of 1 to 10 µM (Skerjanec et al. 2003). Moreover, in bone and saliva samples

ACCEPTED MANUSCRIPT obtained from BRONJ patients, concentrations of 0,4–5 µM of BP were determined (Scheper et al. 2009). Induction of apoptosis following high doses of BP administration has been frequently demonstrated. An inhibitory effect after 10 µM of ZOL was determined by Giuliani et al. (Giuliani et al. 1998). Denoyelle et al. reported that ZOL had no effects on

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tumor cell proliferation at low concentrations (<10  µM) and that cell numbers were

significantly reduced at a concentration of 100 µM (Denoyelle et al. 2003). Açil et al. (Açil et al. 2012) and Simon et al. (Simon et al. 2010) selected the concentrations of 1 µM, 5 µM, 10

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µM, 20 µM in order to determine the cytototoxic levels in a smaller sample size via PCR. Based on previous research by Açil et al. (Açil et al. 2012), definite doses of BPs mimicking

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the plasma concentrations were selected to clearly observe the cell responses without reaching cytototoxic levels.

It has previously been stated that BPs should stimulate the differentiation and proliferation capacity of osteoblasts (Giuliani et al. 1998). Higher concentrations of ZOL have been shown

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to result in inhibition of proliferation, adhesion and cell migration of osteoblasts and to concomitantly cause cytotoxic reactions (Walter et al. 2011). In a recent study by Krüger et al., it was reported that lower concentrations of FOS (5 and 20 µM) enhanced proliferation,

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whereas 100 µM diminished the proliferation of osteoblasts (Krüger et al. 2016). In the current study, cytotoxic effects in osteoblast group increased, especially following ZOL

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administration, which was demonstrated by decrease of cell proliferation in MTT tests. FOS showed its effects with a decrease of 20% in osteoblast proliferation even under low concentrations.

It is obvious that fibroblasts play an important role in wound healing. Ravosa et al. described the effect of ZOL on oral fibroblasts and stated that ZOL blocked growth capacity and downregulated transcription of type I collagen, which is vital for granulation tissue and reepithelization processes (Ravosa et al. 2011). They have also suggested that delayed wound

ACCEPTED MANUSCRIPT healing might be attributed to the induction of cellular apoptosis of these cells. Similar to the results obtained by Açil et al. (Açil et al. 2012), the data expressed herein confirmed the relatively higher cytotoxic effects of ZOL compared to FOS on human fibroblasts. The decrease in cell proliferation was dose dependent in the case of both BPs.

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Osteosarcoma cells differ from osteoblasts and fibroblasts in their reaction to BP

administration. The inhibition of proliferation and toxic effects of BP on sarcoma cells were previously described, although with higher BP concentration levels and shorter study

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durations compared to the current study (Mackie et al. 2001). According to our results, especially in the ZOL group, it might be concluded that osteosarcoma cells could be

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negatively affected only by high concentration levels, whereas lower doses resulted in cell proliferation. Mackie et al. suggested that BPs not only have direct effects on osteosarcoma cell growth and apoptosis, but they may inhibit the activity of osteoclasts via RANKL production and their recruitment at a concentration of 10 µM (Mackie et al. 2001). These

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findings paralleled the results obtained from the present study, which demonstrated that only higher doses could jeopardize the viability of osteosarcoma cells. A survey of the existing literature could reveal some controversies and consistencies

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regarding the changes in pro-inflammatory cytokine levels under BP treatment. Deng et al. evaluated macrophages under FOS treatment and observed increased levels of IL-1β, but no

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differences were found in levels of TNF-α (Deng et al. 2009). Relatively higher IL-1β levels have been also demonstrated in gingival pockets of ZOL-induced BRONJ cases (Tsao et al. 2013). The authors of a recent study also stated that chronic treatment with ZOL could increase pro-inflammatory cytokines (TNF-α and IL-1β) in the periodontium (de Barros Silva et al. 2017). In the current study, among gingival fibroblasts, only the changes in IL-1β expression could be observed. Under higher levels of ZOL (≥2,5 µM), IL-1β increased

ACCEPTED MANUSCRIPT significantly (n x1000). Unfortunately, no data could be provided regarding the osteoblasts and osteosarcoma cells. Naidu et al. evaluated IL-6 levels of osteoblasts in acute phase reactions associated with BP and observed that high concentrations of ZOS were cytotoxic (Naidu et al. 2008). In addition,

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TGF-β increased even as viability decreased, suggesting a mechanism for BP action. The present study confirmed also the increased IL-6 production, especially following ZOL application.

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It is well known that IL-12 induces cytokine production and enhances cytotoxic activity.

Tsagozis et al. demonstrated boosted production of IL-12 in ZOL-pulsed prostate cancer cells

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(Tsagozis et al. 2008). The data expressed herein showed the dose-dependent increase of IL12 production of osteoblasts under ZOL and of osteosarcoma cells under FOS application. The main pitfall of the current study might be attributed to the disadvantages of PCR in the detection of cytokines. Real-time quantitative PCR, which involves measurement of cytokine

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mRNA transcript abundance, is relatively straightforward and quantitative, and allows the detection of many different cytokines from relatively small sample amounts (Amsen et al. 2009). However, it has been suggested that a major disadvantage of the technique is that the

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presence of RNA does not always accurately reflect protein levels, especially IL 4 and IL 10; thus their secretion was regulated at the translational level (Amsen et al. 2009). Moreover, it

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has been also stated that some of these can exert multiple actions and can signal similar responses due to the employment of the same receptor subunits and signaling pathways, such as for IL-4 (Singh et al. 2017). According to the data obtained throughout the current study, detection and identification of the cellular sources of the cytokines require a precise isolation of different cell types, which may be difficult, and the threshold for detection could not be reached due to the small size of the samples and high sensitivity of the technique.

ACCEPTED MANUSCRIPT CONCLUSION The results of the current study indicated the following: •

The proliferation and cell vitality of osteoblasts and fibroblasts were negatively affected in a dose dependent manner under ZOL and FOS administration. However,

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ZOL has a significantly higher cytotoxic effect compared with FOS on osteoblasts and fibroblasts, which also might increase the risk of BRONJ.

Despite the limited data obtained from PCR results, it could be concluded that BP

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•

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osteosarcoma cells showed an increase in proliferation under lower doses of BP.

could result in an increase in IL-1β expression of fibroblasts.

ZOL increased the production of IL-6 in all cell types, whereas FOS increased only

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•

the production of osteosarcoma cells in a dose-dependent manner. An increase in IL-12 was observed at higher doses of ZOL administration in

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osteoblasts and FOS administration in osteosarcoma cells.

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•

ACCEPTED MANUSCRIPT Conflict of interest The authors declare that they have no conflict of interest.

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There was no funding source for this work.

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Funding source

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ACCEPTED MANUSCRIPT Figure 1. MTT tests showing results regarding the concentration-related effects at the 1st and 4th weeks among a) zoledronate and b) aledronate groups. Among osteoblasts under 1,25 µM concentration, the vitality of the cells decreased to 20% and 65% under ZOL and FOS, respectively. Compared to the osteoblasts, fibroblasts showed less distinction of viability

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between ZOL and FOS groups.

Figure 2. Osteoblasts and fibroblasts showed a rarefication in cell number under BP. a)

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Osteoblast control group (4th week). b) osteoblasts under FOS 2,5 µM (4th week). Note the difference between concentrations of ZOL and FOS with similar results. c) Osteoblasts under

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ZOL 0,625 µM (4th week). d) Fibroblast control group (4th week). e) Fibroblasts under FOS 2,5 µm (4th week). f) Fibroblasts under ZOL 2,5 µm (4th week). Scale: 100 µm. Fibroblasts showed less distinction of viability among ZOL and FOS groups.

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Figure 3. Sparse growth of osteosarcoma cells. It has been observed that, under small concentrations, proliferation was induced. Higher concentrations shows the toxic effects of BPs. a) Control group 4th week. b) FOS 0,3125 µM 4th week. c) FOS 2,5 µM 4th week. d)

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Zoledronat 0,15625 µM 1st week. e) Zoledronat 0,15625 µM 4th week. f) ZOL 2,5 µM 4th

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week. Scale: 100 µm.

Figure 4. Effects of ZOL on osteoblasts at the 1st week. a) Control. b) ZOL 0,625 µM. c) ZOL 2,5 µM; no viable cells were seen. The number of the cells was diminished in the 4th week compared with the d) control group in the e) ZOL 1,25 µM group. Scale: 100 µm.

Figure 5. Comparison of fibroblasts under FOS at the 4th week. a) Control. b) FOS 2,5 µM with ZOL shows a stronger effect than ZOL alone. c) ZOL 2,5 µM 3rd week shows that the

ACCEPTED MANUSCRIPT number of nonviable cells was increased. 4th Week results showed a dose-dependent decrease in cell proliferation. d) ZOL 2,5 µM. Scale: 100 µm.

Figure 6. Similar to the photographic documentation of the osteosarcoma cell culture, FD

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showed at first a low growth behaviour. a) Control group at the 1st week showed an increased cell growth. b) Control group at the 4th week. c) FOS 2,5 µM at 4th week and d) ZOL 2,5 µM at 4th week showed their negative effects on the cells. Under small concentrations, positive

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effects of BP on cell could be seen. e) FOS 1,25 µM at 4th week. f) ZOL 0,625 µM at 4th

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week. Scale: 100 µm.

Figure 7. Expression of IL 6 under ZOL was increased in all cells. a) Osteoblasts, zoledronate, 2nd week. b) Fibroblasts, ZOL 4th week. c) Osteosarcoma cells, ZOL,1st week. Under FOS, an increase could be seen only in osteosarcoma cell lines. d) Osteosarcoma cell

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lines, FOS, 4th week.

Figure 8. IL 6 expression of osteoblasts under FOS: 1 + 19 = standard, 2 = negative control, 3

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0,15625 µM.

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= control, 4─6 = 2,5 µM, 7─9 = 1,25 µM, 10─12 = 0,625 µM, 13─15 = 0,3125 µM, 16─18 =

Figure 9. IL 1β expression of fibroblasts under ZOL: 1 + 19 = Standard, 2 = negative control, 3 = control, 4─6 = 2,5 µM, 7─9 = 1,25 µM, 10─12 = 0,625 µM, 13─15 = 0,3125 µM, 16─18 = 0,15625 µM.

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Table 1 Expression of IL 6 under ZOL was increased in all cell s. a) Osteoblasts, Zoledronat, 2nd week b) Fibroblasts, Zoledronat 4th week c) Osteosarkoma cells, Zoledronat,1st week. Under Aledronate, an increase could be seen only in Osteosarcoma cell lines. d) Osteosarcoma cell lines, Alendronat, 4th week.

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