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Zoledronate Induces Apoptosis in Cells From Fibro-Cellular Membrane of Unicameral Bone Cyst (UBC)

Posted on: Friday, 7 October 2005, 03:00 CDT

By Yu, John; Chang, Seong-Sil; Suratwala, Sanjeev; Chung, Woo-Sik; Et al

Abstract

Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO- 1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular bone cells showed 17.2% of apoptosis, significantly lower than 24.2% of UBC cells (p- value = 0.007). With the various zoledronate concentrations, mean apoptotic fractions of trabecular bone cells was 19.2%, significantly lower than 27.8% of UBC cells (p-value = 0.040). With GGOH co-treatment in various zoledronate concentrations, 15.1% apoptosis was shown in trabecular bone cells, which was not significantly lower than 20.6% of UBC cells (pvalue = 0.076). This data suggests that zoledronate causes apoptosis in both UBC and trabecular bone cells by inhibition of the mevalonate pathway. In addition to the known anti-osteoclastogenic effect of bisphosphonates, the GGOH inhibitory effects of zoledronate were more prominent in UBC cells than trabecular bone cells, indicating their potential therapeutic role in UBC.

2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: Benign bone tumor; Unicameral bone cyst; Bisphosphonate; Apoptosis

Introduction

Unicameral bone cyst (UBC) is a tumor-like condition which occurs in the ends of long bones in skeletally immature patients. UBC is defined as a minimally expansile lucent lesion consisting of a cavity filled with fluid and lined by a fibro-cellular membrane [2]. The etiology of UBC is poorly understood. Various pathogenetic theories have included dysplastic processes, synovial cysts, and abnormalities in the local circulation [6,11,12,21,26]. Although the histomorphologic features of pathology, such as fibrous membrane, macrophages and occasional giant cells are well known, the molecular mechanisms underlying the cystic process have not been clarified. As theories of pathogenesis have evolved, many treatment methods such as steroid injection, bone marrow injection, excision, bone grafting, intramedullary nailing and trephination have been developed [3,5,15,19,25,34-36]. The clinical outcome after various types of treatment have been beneficial but not consistently favorable due to recurrence. Historically, recurrence rates have ranged from 20% to 50% after single treatment using various methods [39]. Recurrence manifests as persistent or recurrent cyst or recurrent pathologic fracture. As UBC may persist as a lucent cyst with thin cortices, anti-resorptive agents, such as bisphosphonates also may be considered a valuable therapeutic option.

Bisphosphonates are widely used anti-resorptive agents that effectively inhibit osteoclastogenesis, inactivate and induce apoptosis of osteoclasts in osteoporosis, metastatic bone cancers, osteogenesis imperfecta, Paget's disease and multi-focal benign bone tumor-like lesions such as polyostotic fibrous dysplasia [7,10, 16,27,30,32,33]. Bisphosphonates are ideally suited for the treatment of some bone diseases because they bind avidly to the bone mineral and thus accumulate in bone at sites of active bone metabolism. Nitrogencontaining bisphosphonates appear to mediate their effects primarily via inhibition of the mevalonate pathway of cholesterol synthesis, resulting in inhibition of protein prenylation [14,37]. Prenylated proteins are required for osteoclast formation and function. Cellular uptake of nitrogen-containing bisphosphonates lead to inhibition of the mevalonate pathway and loss of prenylated proteins, causing loss of osteoclast function and cell death by apoptosis [14]. Apoptosis is a special form of cell death that results from activation of cell death machinery in response to specific molecular signals. In contrast to necrosis, which is associated with inflammation in response to physical or chemical damage to cells, apoptosis does not cause inflammation and is a physiologic process. Apoptosis or programmed cell death is observed in embryonic development, chemotherapy for cancers, degeneration and aging [9,40]. Since UBC is a cystic lesion and its fibro-cellular membrane contains osteoclasts, inhibition of the osteoclastic activity with nitrogen-containing bisphosphonates may be a logical therapeutic approach.

At concentrations that effectively inhibit bone resorption, bisphosphonates do not have adverse effects on bone mineralization or fracture healing [14,18]. Despite these specific bone-protective effects, bisphosphonates have never been used for localized benign osteolytic lesions other than systemic bone disorders, such as osteogenesis imperfecta or polyostotic fibrous dysplasia. The objective of this study was to characterize the in vitro cells from the fibro-cellular membrane of UBC and to determine whether zoledronate could induce apoptosis on UBC cells in vitro.

Material and methods

Reagents

Zoledronic acid is designated chemically as (l-hydroxy-2- imidazol1-yl-phosphonoethyl) phosphonic acid monohydrate (Novartis Pharmaceuticals Ltd., Basel, Switzerland). Stock solutions of zoledronate were prepared in calcium-free phosphate-buffered saline (PBS) according to the manufacturer's instructions. Geranylgeraniol, (2E,6E, K)E)3, 7, 11, 15-tetramethyl hexadecatetra-2,6,10,14-en-l- ol was obtained from Echelon Biosciences (Salt Lake City, UT). For immunoblotting, RANKL antibody was obtained from Oncogene Science (Cambridge, MA), and the antibodies PARP and RUNX2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Dulbecco's modification of Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Cellgro (Herndon, VA). For tissue digestion, collagenase, hyaluronidase, and DNase were purchased from Sigma (St. Louis, MO). For immunohisto/cytochemistry, we used IgG monoclonal antibody for SDF-Ia and RANKL (R&D Systems, Minneapolis, MN), IgM monoclonal antibody for STRO-I (R&D Systems, Minneapolis, MN), IgG monoclonal for CD68 (DAKO Corporation, Carpinteria, CA), the Vectastain kit (Vector Laboratories, Burlingame, CA), HRP-AEC staining kit (R&D Systems, Minneapolis, MN), and biotinylated anti- IgM and IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA).

Preparation of UBC cells and trabecular bone cells

The use of human tissue was approved by the Institutional Review Board. Patient information was not identified during the experiment. The diagnosis of UBC was established by characteristic radiographie appearance and biopsy (Fig. 1). Fresh UBC membranes were obtained at the time of surgery from five patients with UBC in the femur. The trabecular bones were obtained during routine knee and hip replacements, from the Department of Orthopaedic Surgery at New YorkPresbyterian Hospital, Columbia University Medical Center. After washing the specimens with phosphate-buffered saline (Ix PBS) vigorously twice, tissues were freshly minced with scissors in 1 PBS, producing a cell suspension with small fragments of tissue. The suspension was pelleted by centrifugation and the fragments were enzymatically digested in 1 PBS containing 1 mg/ml collagenase, 0.15mg/ml DNAse, and 0.15 mg/ml hyaluronidase for 1 h at 37 C. The suspension was then passed through sterile gauze to remove any undigested fragments, and the cells were either frozen in liquid nitrogen for later use or seeded in 75 cm^sup 2^ flasks with DMEM supplemented with 10% fetal calf serum, 100U/ml penicillin, 100 ug/ ml streptomycin, and 0.1% fungizone (amphotericin B). Cells were grown at 37 C in a humidified atmosphere of 5% CO2 and 95%air. For both the UBC cel\ls and the trabecular bone cells, cell lines between fourth and sixth passages were used since they demonstrated homogenous morphology and stable stromal-cell phenotypes.

Fig. 1. Photomicrograph demonstrating a loose fibro-cellular membrane containing multi-nucleated osteoclast-like giant cells at the bone resorption site (x400; hematoxylin).

Inmnmohixlolcytochemistry of UBC membrane and cells

Immunohisto/cytochemislry (IHC/ICC) was performed using a biotin- avidin-peroxidase kit or HRB-AEC peroxidase kit. Slides were fixed in acetone at -20 C for 10min. The slides were then washed in 1 PBS for 10min. Antibodies raised against monocyte/ macrophage markers (CD68), mesenchymal stromal cell and early osteoblast differentiation markers (SDF-1α and STRO-1), and receptor activator of nuclear factor kappa B ligand (RANKL) were used. CD 68 positive cells are capable of forming osteoclasts [1]. RANKL is a key signaling molecule that triggers osteoclastogenesis in macrophages [28].

The specimens were incubated with their respective primary antibodies overnight at 4 C in a humid chamber. Sequential incubations were then performed with biotinylated secondary antibody following peroxidase-labeled avidin or peroxidase-labeled HRP. Final steps of staining were revealed with DAB chromogen or AEC chromogen according to the manufacturers' instruction. The slides were counterstained with hematoxylin and mounted. UBC tissue as positive control and negative control slides without primary antibodies were treated with the same reagents.

RNA extraction and mini-array analysis

Total RNA from UBC cultures was extracted using RNeasy (Qiagen, Valencia, CA) according to the manufacturer's instructions. First stranded cDNA was synthesized from 1 μg of total RNA using Superscript(TM) First-strand synthesis system for RT-PCR (Gibco, Carlsbad, CA) and Perkin Elmer DNA thermal cycler.

We obtained GEArray Series for human osteogenesis (Bioscience Corporation, Frednck, MD) for detection of osteogenesis-related gene expression using chemiluminescent detection. cDNA probe synthesis was generated using a SuperArray True Labeling-RT kit (Bioscience Corporation, Fredrick, MD) and Biotin16-dUTP (Roche, Indianapolis, IN) on isolated RNA. Equivalent amounts of RNA (0.5 g) were used for each probe synthesis. Membranes were pre-hybridized at 60 C in a solution of GEAhyb hybridization solution (Bioscience Corporation, Fredrick, MD) and denatured salmon sperm (Invitrogen Life Technologies, Cambridge, MA) for 1 h. After pre-hybridization, the probes were hybridized overnight at 60 C with continuous agitation at 5-10 rpm. After overnight hybridization, membranes were washed twice with a washing buffer (100ml 20x SSC and 50ml 20% SDS per liter) and twice again with another, more diluted washing buffer (5 ml 20x SSC and 25ml 20% SDS per liter) for 15min at 60 C. Membranes were then blocked by blocking solution Q (SuperArray kit), and blotted with alkaline phosphatase-conjugated streptavidin.

After subsequent washes with buffers provided by the manufacturer, membranes were blotted with CDP-Star chemiluminescent substrate and exposed at various times to Kodak BioMax light film (Rochester, NY). Images were scanned and data analysis of the arrays was deciphered using ScanAlyze (Esien, Stanford University) version 2.5 computer software. Statistical quantification and imaging was determined using GEArray Analyzer 1.3 (BioLogic, SuperArray Inc.).

In vitro osteoclastogenesis: isolation of monocytes

To determine whether UBC stromal cells are capable of inducing osteoclastogenesis, human monocytes were isolated from fresh whole blood (Manhattan Blood Bank, New York). Peripheral blood mononuclear cells were isolated from heparinized blood of healthy adult volunteers by density gradient centrifugation with Ficoll-Paque. The cells were washed twice in PBS and were resuspended in medium RPMI 1640 supplemented with 2 mmol/1 glutamine and 10% fetal calf serum. Finally, cells were added to 96-well microtiter plates at a density of 2 10^sup 5^ cells per well. After incubation at 37 C for 1 h, the non-adherent cells were removed by repeated vigorous washings. Human monocyte cells were incubated 18 h prior to assay in serum free DMEM medium. Cells were inspected for round healthy morphology. The adherent cells were detached with 2 mM EDTA/ 0.05% trypsin in Hanks balanced salt solution containing 25 mM HEPES per 100mm dish, incubated at 37 C for 10min, and were made to give 1.0 10^sup 6^ cells/ml. Conditioned media from UBC cultures were added to the monocyte culture (50% conditioned media + 50% DMEM) while control groups were kept in 100% DMEM medium. Generation of multi-nucleated osteoclast-like cells (Nuclei > 4) was graded after microscopic examination under 200 magnification. Grade O was less than two multi- nucleated cells/200 power field; grade 1 was less than five multi- nucleated cells/200 power field; and grade 2 was more than five multi-nucleated cells/200 power field. Five different fields were examined to assess the grade.

Flow cytometry after Annexin V and propidium iodide staining

UBC cells were treated with zoledronate at varying concentrations (0, 30, 100 M) when the cell confluency reached approximately 80%) in 6-well plates. Four cell lines from trabecular bone were treated with O, 30, 50, and 100 M of zoledronate, like the UBC cells, to serve as comparative controls. The specific metabolic effect of nitrogen-containing bisphosphonates on osteoclasts comes from the inhibition of farnesyl diphosphate synthase, an important coenzyme in the mevalonate pathway of cholesterol synthesis. This pathway produces isoprenylated precursors, geranylgeraniol (GGOH), and farnesol, needed for posttranslational modification of small GTP- binding proteins. Prenylated GTPases are essentially needed to control cytoskeletal organization, vesicular trafficking, and apoptosis; therefore, their proper functioning is indispensable to normal osteoclast activity [14]. A duplicate set of UBC primary cultures were incubated with GGOH (100 M) 1 h prior to zoledronate treatment. Both the zoledronate only treated cultures and zoledronate co-treated with GGOH cultures were collected after 72 h. The cells were washed with PBS and the washing buffer was also collected. After trypsinization of the cells, the plate was incubated for 2-3 min at 37 C. Once all of the cells detached, 2 ml of PBS buffer was added to wash the trypsinized cells. Each of the cells from the 6-well plate were collected into their proper tubes and spun down at 1200 rpm for 5 min. The cells were resuspended in 190 l of Annexin V binding buffer (AG Scientific). Ten microliters of Annexin V-FITC (AG Scientific) was added to 190 l of cell suspension and incubated in the dark for 15 min. Seven microliters of 20 g/ml propidium iodide (AG Scientific) was added to the cell suspension. After 15 min in the dark condition, an additional 200 l of Annexin V binding buffer was placed into each sample. Labeled cells were then counted on a flow cytometer (FACS Calibur; Becton Dickinson Science, San Jose, CA) within an hour.

Immunobtotting for active cleaved form ofcaspase-3, poly (ADP)- ribose polymerase (PARP), RANKL and RUNX2

Immunoblotting was performed to determine whether UBC cells were of osteoblastic lineage and whether they could elaborate osteoclastogenic factors. The expression of RUNX2, an osteoblast- specific transcription factor, and RANKL produced by osteoblasts and stromal cells associated with osteoclastogenesis, was determined [9]. Moreover, in order to confirm apoptosis after treatment with zoledronate, blotting for an effector molecule of apoptosis was performed. Caspase-3 is a downstream effector of apoptosis and is present as an inactive, non-cleaved form in the cytoplasm.

UBC primary cultures were treated with zoledronate at varying concentrations (0, 30, 100, 300 M) for 30 h. Briefly, cells were lysed using buffer IP (10 mM tris-HCl, pH 7.4, 15OmM NaCl, 1% Triton X-100, 0.25% Nonidet P-40, and 2 m M EDTA), supplemented with a protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Indianapolis, IN). Equivalent protein extracts (10 g) from each sample were subjected to electrophoresis on 4-20% tris-glycine gels. Total protein was quantitated by BCA assay. Proteins were transferred to Immun-Blot PVDF membranes (Biorad, Hercules, CA). After transferring, the membranes were blocked with 5% non-fat milk powder in tris-buffered saline plus Tween 20. The membranes were washed with TBS-T and incubated with mouse monoclonal antibody to PARP (Santa Cruz, CA) diluted to 1:500, rabbit polyclonal antibody to activated caspase-3 (Cell Signaling, Beverly, MA) diluted to 1:500, and mouse monoclonal antibody to RANKL (Oncogene, Cambridge, MA) and Cbfal (Santa Cruz, CA) diluted to 1:500. They were detected by horseradish peroxidase-conjugated secondary antibody to goat anti- mouse IgG.

Statistical analysis

The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) software (version 10; SPSS, Chicago, IL) [13]. Data were expressed as means standard error (SE) of the mean. Difference between each treated groups and controls were analyzed by t-test or Scheffe test in One-Way Anova and p < 0.05 was considered statistically significant [20].

Results

Characterization of UBC cells

The fibre-cellular membrane of UBC contains osteoclast-like giant cells (oct) along the marginal surface of membrane (Fig. 1). The UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. In UBC culture, cells had elongated morphology. During the first and second passages, the cultures contained a mixture of multi-nucleated giant cells, macrophages and stromalcells. After the fourth passage, there were uniformly stained cells with mesenchymal stromal cell marker SDF-1α, and some cells stained positively with RANKL. But there were no cells stained with STRO-1 in UBC cells (Fig. 2). None of the cells stained in negative controls because of the omitted primary antibodies. cDNA mini-array analysis revealed expression of RUNX2 gene (osteoblastic transcription factor) while genes for osteocalcin (genes expressed by mature osteoblasts), alkaline phosphatase (mature osteoblast marker) and SOX-9 (chondrocyte specific transcription factor) were not expressed (Fig. 3). In addition, immunoblotting of proteins extracted from the UBC cells showed the presence of RUNX2, a key transcriptional factor for osteoblastic differentiation [8].

Fig. 2. Immunohisto/cytochemical staining of UBC membrane and cells in vitro. Multi-nuclear cells (oct) show positive staining with CD68 (a marker for macrophage lineage) antibody in UBC tissue. Both UBC tissue and cultured cells display strong positive stain for mesenchymal stromal cell marker SDF-1a. Some cells in tissue and cultured cells show positive stain for RANKL, a known osteoclastogenic factor. All vascular endothelium and some surrounding stromal cells in UBC tissue showed positive staining with STRO-1, but UBC cells are not positive (x100; peroxidase method with Avidin-DAB or HRP-AEC, and hematoxylin counlerstaining).

Fig. 3. cDNA mini-array of UBC stromal cells. The mini-array analysis revealed expression of RUNX2 gene (cbfa-1, osteoblastic transcription factor), while genes for mature osteoblasts (osteocalcin, alkaline phosphatase) or chondrocytes (SOX-9) were not expressed.

UBC stromal cells induce osteoclastogenesis

Culture media collected from UBC cultures started to generate multi-nucleated cells in human monocyte culture within 14 days of incubation suggesting that UBC cells elaborate molecular mediators that may induce osteoclastogenesis. UBC culture media from all the cultures demonstrated grade 2 generation of multinucleated osteoclast-like cells, while control DMEM demonstrated grade 0 (Fig. 4).

Fig. 4. Photomicrograph of human monocytes (x200). Monocytes did not form osteoclasts in control medium (left). Conditioned media collected from UBC cultures induced formation of multi-nucleated osteoclasts (right).

Induction of apoptosis with zoledronate treatment in UBC cultures morphologic changes

After UBC cells were incubated with zoledronate for 72 h, morphologic changes became evident. As shown in Fig. 5, UBC cells that were treated with zoledronate transformed from a spindle-like morphology to a more oval, shrunken shape and detached from the culture wells. This effect was more pronounced at higher doses of zoledronate displaying a dose-dependent effect of the drug.

Quantitative analysis of apoptosis using flow cytometry

Binding of Annexin V to phosphatidylserine of cell plasma membrane characterizes an early and characteristic step in the apoptosis pathway [4]. Annexin V and propidium iodide staining followed by flow cytometry analysis was utilized to confirm and quantify apoptosis after zoledronate treatment. UBC cells and trabecular bone cells were treated with zoledronate in different concentrations (O, 30, 50, 100 M). The cells were treated for 72 h. Using a flow cytometer, labeled cells were counted and the percentage of apoptotic cells were calculated (Fig. 6).

Fig. 5. Zoledronate-mediated apoptotic changes in UBC cultures (x200). UBC stromal cells treated with 30 M of zoledronate (middle column) and 100 M of zoledronate (right column) demonstrated shrinkage of cells and increasing proportion of apoptotic cells in flow cytometry (arrow) in comparison to control (left column).

The baseline apoptotic fraction in the untreated UBC cells was 11.5%. Zoledronate treated UBC cells demonstrated 29.01M) and 41.8% of mean apoptosis at 30 and 100 M, respectively. Zoledronate inhibits farnesyl diphosphate synthase in the mevalonate pathway, which produces isoprenylated precursors GGOH and farnesol. Therefore, a duplicate set of UBC primary cultures were incubated with GGOH (100 M) 1 h prior to zoledronate treatment and the cells were collected after 72 h. The percentage of total apoptosis with only GGOH treatment was 11.1%, similar to the untreated culture (11.5%), indicating that GGOH did not have a harmful effect on UBC cells. But at 30 and 100 M zoledronate supplemented with GGOH 100 M, the mean apoptosis decreased around 10% from those conditions without GGOH, showing 19.9% and 30.4% of apoptosis, respectively.

Fig. 6. The mean apoptotic fractions were plotted by flow cytometric analysis of zoledronate induced apoptosis in UBC cells (0, 30 and 100 M) and trabecular bone cells (0, 30, 50 and 100 M). Cells were treated with zoledronate for 72 h. Each duplicate sets of cells were treated with GGOH 1 h prior to treatment with zoledronate (mean SE, (*) p < 0.05, (**) p < 0.001, p-valus were calculated from pair-wise comparison from the baseline of each cell lines).

The baseline of apoptosis in the trabecular bone cells was 11.1%, similar to that of UBC cells. At 30, 50 and 100 M zoledronate treatment, the apoptotic fraction increased to 19.4%, 25.2%, and 35.4%,, respectively. Interestingly, apoptosis in trabecular bone cells treated with up to 50 M of zoledronate was still lower than that of UBC cells treated with 30 M zoledronate. This suggests that UBC cells are more susceptible to apoptosis than trabecular bone cells when treated with zoledronate. The difference in the apoptotic fraction between these two cell lines was the greatest when the zoledronate concentration ranged between 30 and 50 M. Supplemented with GGOH, the apoptotic fraction in trabecular bone cells was reduced by about 3-5% when compared to apoptosis induced by various concentration of zoledronate alone. However, apoptosis in UBC cells was inhibited by about 7-10% when co-treated with GGOH. This suggests that UBC cells are more susceptible to both bisphosphonates and GGOH than trabecular bone cells.

As for the overall apoptotic fraction in this study, trabecular bone cells had 17.2% of apoptotic cells, significantly lower than the 24.2% seen in UBC cells (pvalue = 0.007). Without GGOH supplementation in various zoledronate concentrations, mean apoptotic fraction in trabecular bone was 19.2%, significantly lower than 27.8% of UBC cells (/rvalue - 0.040). But with GGOH co- treatment in various zoledronate concentrations, 15.0% apoptosis was shown in trabecular bone cells, which was not significantly lower than 20.6% of UBC cells (p-value = 0.076).

Fig. 7. Immunoblot of activated cleaved form of caspase-3 in UBC culture. Activated caspase-3 is displayed and shows a dose- dependent increase with zoledronate treatment, indicating activation of apoptotic process in response to zoledronate.

Supplementation with GGOH inhibits apoptosis induced by zoledronate consistently throughout the various concentrations of zoledronate, confirming that apoptosis induced by zoledronate is mediated via inhibition of the mevalonate pathway of cholesterol synthesis not only in osteoclast precursor but also in stromal cells.

Immunoblotting for detecting apoptosis in UBC cells

To investigate the effect of zoledronate induced apoptosis, UBC cells were incubated for 30 h with zoledronate at different concentrations (0, 30, 100, 300 M). Cell lysates were prepared and immunoblotting were carried out with an antibody recognizing activated caspase-3. On activation, pro-caspase-3 (35 kDa) is cleaved into two fragments (17/19 kDa). The untreated UBC cells (control) did not display the activated caspase-3 band (17/19 kDa). Moreover, cleavage consistent with caspase-3 activation was observed in zoledronate treated cells in a dose-dependent manner (Fig. 7). The cells were treated with zoledronate (0, 30, 100, 300 M) for 72 h and cleavage of PARP was determined by western blot utilizing a monoclonal antibody which recognizes the intact form. RANKL and RUNX2 were present in each of the UBC group and did not show a dose- dependent correlation after treatment with zoledronate.

Discussion

The pathophysiology of the unicameral bone cyst remains unclear and many theories have been proposed. Cohen proposed that the principal etiologic factor is blockage in the drainage of interstitial fluid in a rapidly growing and rapidly remodeling area of cancellous bone [6]. Chigira et al. studied the internal pressure of patients with UBC and found them to be higher as compared to contralateral normal bone marrow pressures [41]. These authors suggested that venous obstruction within the bone appears to be a likely cause of such simple bone cysts. Moreover, Komiya et al. reported on biochemical studies of the cystic fluid, showing that bone resorptive factors (prostaglandins, interleukin-1β, gelatinase) had a synergetic role in bone cyst formation [21].

In this study, we (1) determined the cellular components of the UBC membrane to be of stromal cells of early osteoblastic lineage; (2) demonstrated that UBC cells possess the capacity to induce osteoclastogenesis in vitro and (3) demonstrated that zoledronate induced apoptosis of UBC stromal cells in vitro.

UBC cell lines were obtained by digestion of the fibre-cellular membrane of unicameral bone cysts. Trabecular bone cells were obtained by the digestion of normal human trabecular bones. Immunocytochemistry in UBC cells revealed positive staining for SDF- 1 and RANKL. In the tissue culture, CD68 positive cells or STRO-1 positive cells are omitted throughout the passage. RANKL was also present by immunoblot assay of UBC cells. RANKL stimulates osteoclast differentiation and is produced by osteoblasts and stromal cells in response to a variety of signals [17,22,23]. In addition SDF-1 has been shown to provide the critical signals that govern hematopoietic cell homing to the bone marrow. SDF-1 is constitutiv\ely expressed at high levels within bone by pre- osteoblastic stromal cells and endothelial cells lining the bone vasculature [41]. A recent study found that SDF-1 may be an important signal for the chemotactic recruitment and transmigration of pre-osteoclasts into bone and their subsequent navigation to marrow stromal sites for development into bone-resorptive osteoclasts. Moreover, RANKL and SDF-1 likely play complementary roles in regulating the recruitment, development, and function of boneresorptive osteoclasts [41]. In addition, our data also showed that stromal cells of UBC membrane express similar phenotype in some chemokines/cytokines as trabecular bone cells, including RUNX2, a member of the runt domain family and a known osteoblastic transcription factor. RUNX2 has two distinct functions in bone formation: an essential role in the differentiation of mesenchymal progenitors into osteoblasts and the ability to stimulate hypertrophie chondrocyte differentiation. The former function supports the clinical observation of bone regeneration after various treatments, such as injection of steroid, demineralized bone matrix and bone marrow [15,19,25,35]. Furthermore, our data showed that UBC stromal cells are capable of inducing osteoclastogenesis in vitro. The osteoclast is a tissue-specific macrophage polykaryon created by the differentiation of monocyte/macrophage precursor cells at or near the bone surface [38]. The isolated peripheral blood mononuclear cells formed multi-nucleated osteoclast like cells after being cultured with a mixture of conditioned UBC media and DMEM. Osteoclast precursors (monocytes/ macrophages) fuse in response to the activation of the RANKL/RANK axis. Furthermore, RANKL can activate mature osteoclasts in a dose-dependent manner in vitro, and can lead rapidly to the resorption of bone in vivo by activating pre- existing osteoclasts [38].

As many theories of pathogenesis have evolved, many treatment modalities were developed for UBC. These include injection of steroid, drilling, injection of bone marrow, intramedullary nailing, bone grafting, and filling with demineralized bone matrix [3,5,15,19,25,34-36]. However, recurrence rates range from 20% to 50% after single treatments [39]. Since UBC is a bone destructive lesion, inhibition of the osteoclastic activity with nitrogen- containing bisphosphonates may prove beneficial. Bisphosphonates are the most effective inhibitors of bone resorption and are extensively used for the treatment of systemic or local bone loss, due for example, to post-menopausal osteoporosis or tumor bone disease [31]. All clinically useful bisphosphonates bind to bone and can be incorporated in the bone mineral matrix. During the bone resorptive process, active osteoclasts take up bisphosphonates previously deposited on the bone surface. Nitrogen containing bisphosphonates induce caspase-dependent formation of pyknotic nuclei and cleavage of caspase-3 to form the active species associated with apoptosis in osteoclasts [31]. Consistent with their inhibition of the mevalonate pathway, apoptosis induced by nitrogen containing bisphosphonates are blocked by GGOH, a precursor of geranylgeraniol diphosphate [31]. Our study also showed that GGOH attenuated bisphosphonate induced apoptosis both in UBC cells and trabecular bone cells and UBC cells were more susceptible to the effect of zoledronate. These findings suggest that bisphosphonates act directly on the osteoclast and may also act directly on stromal cells to induce apoptosis and that caspase cleavage is part of the apoptotic pathway [31].

Nitrogen containing bisphosphonates, such as zoledronate, demonstrate (1) inhibition of osteoclastogenesis; (2) protection of host bone from osteoclastic resorption and (3) induction of apoptosis of osteoclasts in osteoporosis, metastatic bone cancers, Paget's disease, and benign bone tumor-like lesions [7,10,16,27,30,32,33]. Moreover, a study by Pan et al., revealed that zoledronate directly affects the proliferation and differentiation of human osteoblast-like cells in vitro (5-25 M), and may enhance bone formation in vivo (0.5 mM) [29]. Our study showed results consistent with these studies results: The difference of apoptosis induced by zoledronate was maximal between these two cell lines when the zoledronate concentration ranged between 30 and 50 M. Even though UBC cells and cells from trabecular bone had similar baseline of apoptosis, UBC cells were more apoptotic than trabecular bone cells on treatment with various concentration of zoledronate. GGOH supplementation inhibited about 5% of apoptosis in trabecular bone cells at various zoledronate concentration, while apoptosis of UBC cells decreased by about 10%. Interestingly, even though UBC and trabecular bone cells in vitro shared similar chemokine/cytokine expression according to ICC staining, UBC cells seemed to be more susceptible to both bisphosphonates and GGOH than trabecular bone cells. This suggests that the cells of UBC would be susceptible to zoledronate adjunct therapy.

Since bisphosphonates can bind to bone in vitro, direct topical application or indirect application via bone grafts or calcium containing bone graft substitutes may be a feasible method for drug delivery. Even though the dosage for intravenous administration of zoledronate is well established in the literature [24], further studies need to be performed to establish the safe dose of zoledronate in vivo when used as an adjunctive treatment.

Acknowledgements

This study was supported by Orthopaedic Research and Education Foundation (FYE) and Musculoskeletal Transplant Foundation (FYE) research grants.

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John Yu a, Seong-Sil Chang a, Sanjeev Suratwala a, Woo-Sik Chung a, Peter Abdelmessieh a, Hahn-Jun Lee a, Jay Yang b, Francis Young- In Lee a,*

a Department of Orthopaedic Surgery, Center for Orthopaedic Research, Columbia University Medical Center, 630 W 168th Street, BB1411, New York, NY 10032, USA

b Department of Anesthesiology, Center for Orthopaedic Research, Columbia University Medical Center, 630 W 168th Street, BB1411, New York, NY 10032, USA

Accepted 15 February 2005

* Corresponding author. Tel.:+1 212305 1515; fax:+1 2123058271.

E-mail address: fll27@columbia.edu (F.Y.-I. Lee).

Copyright Journal of Bone and Joint Surgery, Inc. Sep 2005

Source: Journal of Orthopaedic Research

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