Zoledronic acid (1-hydroxy-2-[(1-H-imidazole-1-yl)ethylidene]1-bisphosphonate) was obtained from Novartis Pharmaceuticals (Basel, Switzerland). Bone cement (Simplex P), composed of 40 g of polymethylmethacrylate powder (including 4 g of barium sulfate) and 20 mL of solvent (2.6% of which is polymerization accelerator N,N-dimethyl-p-toluidine and 97.4% of which is methylmethacrylate monomer), was obtained from Stryker (Mahwah, New Jersey). Our in vitro model was adapted and modified from models in previous experimental studies with bone cement24.
Preparation of Bone Cement Samples
Bone cement cylinders were formed under sterile conditions by hand without vacuum, with increasing concentrations of zoledronic acid (0.1, 0.25, 0.5, 0.75, and 1.0 mg in 1.5 cm3 of bone cement), and kept in sterile, identical tubes in 5 mL of distilled water at 37°C until sampling. Each cylinder was 6 mm in diameter and 10 mm thick. At each consecutive twenty-four-hour time point of the test, the distilled water was removed and replaced by fresh distilled water in order to measure the amount of eluted zoledronic acid. The distilled water was subsequently used for cytotoxic assay (MTT) and high-pressure liquid chromatography-ultraviolet spectrometry. During the whole experiment, the samples (three per concentration) were collected daily. At each time point, 5 mL of distilled water was collected: 2.5 mL was frozen, kept at —20°C, and subsequently used for high-pressure liquid chromatography-ultraviolet spectrometry analysis. Another 2.5 mL of collected distilled water was evaporated. Subsequently, dry crystals of zoledronic acid were dissolved in 2.5 mL of culture medium containing giant cell tumor, multiple myeloma, or renal cell carcinoma cell lines.
Giant Cell Tumor Specimens and Primary Culture
Primary cultures of stromal giant cell tumor cells were established from tumor tissue freshly minced in Dulbecco minimum essential medium containing 100 U/mL of penicillin and 100 µg/mL of streptomycin. The resultant suspension, together with small tissue fragments, was then transferred to 25-cm2 flasks for subsequent culture at 37°C in 5% CO2 and 95% air overnight. Half the medium was changed on the second day. The culture was further maintained in Dulbecco minimum essential medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 100 U/mL of penicillin, and 100 µg/mL of streptomycin until confluence. Primary cultures obtained after the ninth passage that represented the proliferating homogenous tumor cell population were used for the MTT assay.
Cultures of Multiple Myeloma and Renal Cell Carcinoma Cell Lines
Human renal cell carcinoma cells (novel cell line RBM1-IT4, courtesy of Dr. K. Weber, Johns Hopkins Hospital, Baltimore, Maryland), established from human renal cell carcinoma in bone25, and a multiple myeloma (NCI-H929) non-adherent cell line purchased from American Type Culture Collection (Manassas, Virginia) were used. The rates of growth did not change with time, and the cells maintained the characteristics of renal cell carcinoma and multiple myeloma after sequential cultures. RBM1-IT4 (renal cell carcinoma) cells were maintained in Dulbecco minimum essential medium:Ham F-12, supplemented with 10% fetal bovine serum with insulin-transferrin (Sigma, St. Louis, Missouri), sodium pyruvate, nonessential amino acids, L-glutamine, 100 units/mL of penicillin-G sodium, and 100 units/mL of streptomycin sulfate. Adherent monolayer cultures were performed in a 75-cm2 flask and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. Multiple myeloma (NCI-H929) cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin-G sodium, 100 units/mL of streptomycin sulfate, 2 mM L-glutamine, and 50 mM mercaptoethanol. Multiple myeloma cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C.
Measurement of Zoledronic Acid Elution from Zoledronic Acid-Loaded Cement Cylinders
The aqueous zoledronic acid concentration was determined by directly injecting the sample solution onto a reversed-phase high-pressure liquid chromatography-ultraviolet spectrometry system26. Chromatographic separation was achieved with a Luna C18, 250 × 4.6-mm, 5-µm column (Phenomenex, Torrance, California) with use of a mobile phase mixture of 10 mM dipotassium hydrogen orthophosphate and 10 mM tetrabutylammonium bisulfate (pH 6.85) and methanol (95:5, v/v). The flow rate was 0.75 mL/min. The column temperature was 25°C, and the wavelength monitored was 220 nm. The injection volume was 25 µL. The calibration range was 1.25 to 10 µg/mL.
In Vitro Cytotoxicity (MTT) Assay
When giant cell tumor, multiple myeloma, and renal cell carcinoma reached 80% to 90% cellular confluence, the confluent culture cells were separated with trypsin-EDTA (Gibco, Grand Island, New York). The separated cells were diluted in culture medium and centrifuged for five minutes at 1500 rpm. The upper part of the solution was removed, the cells were resuspended in culture medium, and the cells were counted with use of a hemocytometer. One hundred microliters of the suspension containing 1.5 × 103 cells was placed in all of the wells of a ninety-six-well plate, and then 100 µL of medium was added. A tetrazolium (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) dye conversion assay (Promega, Madison, Wisconsin) was used to assess the zoledronic acid cytotoxicity for primary stromal giant cell tumor, multiple myeloma, and renal cell carcinoma cells. Tumor cells were harvested at the time of the ninth passage (giant cell tumor), the sixth passage (multiple myeloma), and the twenty-eighth passage (renal cell carcinoma), and five replicates of these cells were plated at a concentration of 1.5 × 103 cells per well in 100 µL of growth medium. The media contained dissolved zoledronic acid (obtained by collection and evaporation of the distilled water) from cylinders mixed with zoledronic acid in various concentrations (0.1, 0.25, 0.5, 0.75, and 1.0 mg/1.5 cm3 of bone cement) in ninety-six-well plates. Plates were incubated for seventy-two hours at 37°C with 5% CO2. Dye was added to wells on day 3 and allowed to react for four hours at 37°C with 5% CO2. An enzyme-linked immunosorbent assay (ELISA) plate reader was used to read dye absorbance at 575 nm. The assay was run in triplicate for each concentration and each time point. Cytotoxicity was calculated on the basis of the number of living cells measured against those in control wells containing tumor cells not exposed to the drug. In vitro cytotoxicity data are plotted as the average and standard error. Control samples for all three cell lines were collected from cylinders without zoledronic acid and prepared for a proliferation assay in the same way as the samples with zoledronic acid.
Statistical Analysis
The cytotoxicity assay results were tested with analysis of variance assessed with the Tukey-Kramer method with use of InStat software (GraphPad Software, San Diego, California) to determine their relationship with zoledronic acid concentrations over time.
Source of Funding
This study was funded through the Minnesota Medical Foundation.
Release of Zoledronic Acid from the Bone Cement Cylinders
The release of zoledronic acid was greatest during the first twenty-four hours for all concentrations and decreased rapidly during the next forty-eight hours to reach a plateau after four days. In addition, the levels of released zoledronic acid correlated with the amount of the agent incorporated into the bone cement. The concentration of zoledronic acid released from the 0.25-mg-concentration block was 1.55 µg/mL on the first day and decreased to 0.28 µg/mL on the second day. The concentrations of zoledronic acid released from the 0.75-mg-concentration block were 4.48 µg/mL on the first day and 0.73 µg/mL on the second day (Fig. 1).
Cytotoxicity of Released Zoledronic Acid for Multiple Myeloma, Renal Cell Carcinoma, and Giant Cell Tumor of Bone
Primary Stromal Giant Cell Tumor Cell Line
There was no significant difference between the results of the cytotoxicity (MTT) assay in the untreated group (cells only) and those in the group with cement only (without zoledronic acid). In all of the groups treated with zoledronic acid, the number of viable cells was significantly decreased, as compared with the control value, on every day of the experiment, beginning on the first day (p < 0.05) (Fig. 2). The groups treated with the 0.75-mg and 1.0-mg concentrations of zoledronic acid showed significant decreases (56.7% and 53.5%, respectively; p < 0.01) in the number of viable cells on the first day as compared with that in the control group. In the 0.1, 0.25, and 0.5-mg treatment groups, there were modest but significant decreases in the number of viable cells on the first day (22.8%, 21.1%, and 25.6%, respectively; p < 0.05). Analysis of all treatment groups showed a significant decrease in the number of viable tumor cells (p < 0.01) on days 1 through 6 as compared with the number on day 35.
Multiple Myeloma Cell Line
There was no significant difference between the results of the cytotoxicity (MTT) assay in the control group (cells only) and the cement group (without zoledronic acid). In all of the zoledronic acid-treatment groups, the number of viable cells was significantly decreased, as compared with the number in the control group, beginning on the first day and up to day 5 (p < 0.05) and that cytotoxic effect decreased significantly after the third day (p > 0.05 for the difference between days 1, 2, and 3 and days 4 through 35) (Fig. 3). The group with the 0.1-mg concentration of zoledronic acid showed a significant difference as compared with the control group on the first day (11.2%) (p < 0.05), but the difference was rather minor as compared with the rest of the treatment groups. In the 1.0-mg treatment group there was a significant decrease in the number of viable cells, as compared with that in the control group, on the first day (29.2%) (p < 0.01) and a rapid decrease in this cytotoxic effect after the fifth day.
Renal Cell Carcinoma (RBM1-IT4) Cell Line
The numbers of cells did not differ significantly between the control group and the cement group (without zoledronic acid). The number of viable cells in all treatment groups, as compared with that in the control group, decreased significantly beginning on the first day and up to day 28; this was most evident in the 0.75-mg group (p < 0.01). A very significant decrease in the number of viable tumor cells was seen on the first and second days as compared with the numbers on days 3 through 35 (p < 0.05) (Fig. 4).
In this report, we have shown that zoledronic acid is released from bone cement and its cytotoxic activity can be demonstrated by MTT assay of cultures of three histologic types: multiple myeloma, renal cell carcinoma from bone, and stromal giant cell tumor cells derived from bone. Previous reports have demonstrated the effectiveness of local drug delivery with use of bone cement. Greco et al. showed that Adriamycin (doxorubicin) and cisplatin released from bone cement can effectively inhibit the proliferation of colon and breast tumor cells22. In addition, our study showed that the heat produced during the polymerization of bone cement did not affect the biological activity of zoledronic acid. Moreover, we did not notice any toxicity related to the bone cement in our cytotoxicity assays on all three cell lines. In contrast, Adriamycin and vincristine can lose their activity during polymerization of bone cement. The temperature produced during this process reaches 70°C. Thus, the negative effect of polymerization and temperatures produced during solidification of bone cement should be kept in mind before one uses other cytotoxic agents for local control of primary and metastatic bone tumors. The exact mechanism by which the cytotoxic agents are released from polymethylmethacrylate has not yet been described, to our knowledge. Some investigators suggested that cytotoxic agents may diffuse across a concentration gradient and are released to the surrounding microenvironment through pores in the polymethylmethacrylate27.
In order to achieve higher tissue concentrations of drug released from bone cement, it is necessary to increase the surface area of the bone cement within bone. Giant cell tumor and metastatic bone cancers are treated with extended curettage and high-speed burring, which allow the incorporation of a greater amount of bone cement, increasing the polymethylmethacrylate surface. Treatment of giant cell tumors with conventional curettage and bone-grafting has been abandoned because of high local recurrence rates up to 50%. In our in vitro study, we evaluated the cytotoxic effect of zoledronic acid using the MTT assay because of its well known ability to demonstrate the susceptibility of tumor cells to anticancer agents. Our investigation showed that bone cement without zoledronic acid did not affect the number of viable tumor cells in any of the three cell lines. We demonstrated that zoledronic acid decreased the number of viable tumor cells in a dose-dependent manner. Moreover, we showed that renal cell carcinoma from bone (RBM1-IT4) and giant cell tumor of bone are more susceptible to zoledronic acid than are human multiple myeloma cells. The variability of the response among the cell lines may be related to the number of cells in the S phase of the cell cycle because the cultures were not synchronized before the MTT assay.
Recent reports have demonstrated that antibiotics and methotrexate do not have any negative effect on the mechanical features of polymethylmethacrylate23,28. Healey et al.29 reported that, with the addition of as much as 2 g of either doxorubicin or pamidronate to Simplex-P cement, the cement retains 87% of its compressive and tensile strength after six months. In addition, it has previously been shown that macrophages, in response to polymethylmethacrylate particles, differentiate into bone-resorbing osteoclasts and that this process is inhibited by a bisphosphonate. These results indicate that, by mixing a bisphosphonate with bone cement, it is possible to locally deliver anticancer drugs and inhibit polymethylmethacrylate-particle-induced bone resorption at the bone-cement surface.
In addition, the mevalonate pathway is involved in several key cellular functions leading to the production of sterols, such as cholesterol essential for membrane formation, and to the post-translational modification by prenylation of proteins such as Ras and other small G proteins, which are important second messengers of growth signals from membrane growth-factor receptors30,31. Prenylation is usually required for translocation of Ras to the cell membrane during its activation. Ras signaling is essential to many cancers, either as part of activated growth receptor pathways or by the acquisition of activating mutations during carcinogenesis32. There is therefore considerable interest in inhibiting the mevalonate pathway to treat cancers. Zoledronic acid is an inhibitor of farnesyl pyrophosphate (FPP) synthase and therefore reduces the amount of both FPP and geranylgeranyl diphosphate (GGPP) available for prenylation of Ras33. Growth inhibitory effects of this agent have been described in cancer cell lines and in tumor-derived cells34,35. This molecular mechanism may explain the antineoplastic activity of this agent in all three cell lines used in our study.
The present in vitro study was conducted to determine the release of zoledronic acid from bone cement and the effectiveness of the eluted agent for giant cell tumor, multiple myeloma, and renal cell carcinoma. To our knowledge, this is the first study demonstrating release of a third generation of bisphosphonates (zoledronic acid) from bone cement. The in vitro decreased viability of all three tumor cell lines exposed to zoledronic acid eluted from bone cement shows that zoledronic acid is not inactivated by the heat and/or polymerization of polymethylmethacrylate.
Only limited conclusions can be drawn about the clinical usage of zoledronic acid because this was an in vitro, not an in vivo, study. This is primarily because there is no appropriate or established animal model for giant cell tumor of bone despite many attempts by investigators to create one. In vivo studies of myeloma and renal cell carcinoma were not done as some in vivo data on these tumors already exist8,36, and these histologic types were included in the vitro study as an adjunct to the giant cell tumor of bone, which was the main focus of this investigation.
We conclude that zoledronic acid is released from bone cement, remains biologically active despite the polymerization of cement, and inhibits in vitro growth of cell lines from giant cell tumor of bone, myeloma, and renal cell carcinoma. This provides a rationale to support the development of clinical treatments involving packing of zoledronic acid-impregnated cement for these tumors.