Stimulation of bone formation by zoledronic acid in particle-induced osteolysis

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Biomaterials 26 (2005) 3719–3725 www.elsevier.com/locate/biomaterials

Stimulation of bone formation by zoledronic acid in particle-induced osteolysis$ Christian Wedemeyera, Fabian von Knochb, Andreas Pingsmanna, Gero Hilkenc, Christoph Sprecherd, Guido Saxlera, Frank Henschkee, Franz Lo¨era, Marius von Knocha, a

Department of Orthopaedics, University of Duisburg-Essen, Pattbergstrasse 1-3, D-45239 Essen, Germany b Department of Orthopaedic Surgery, Kantonsspital, 7000 Chur, Switzerland c Central Animal Laboratory, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany d AO Research Institute, Clavadelerstrasse, 7270 Davos, Switzerland e Pathologisches Institut am Johannisstift, Reumontstrasse 28, 33102 Paderborn, Germany Received 27 June 2004; accepted 8 September 2004 Available online 2 November 2004

Abstract We investigated the effect of a single subcutaneous dose of zoledronic acid on particle-induced osteolysis and observed excessive regional new bone formation. We utilized the murine calvarial osteolysis model and polyethylene particles in C57BL/J6 mice. Twenty-eight mice were used, seven per group. Specimens were stained with Giemsa dye. The osteoid tissue area was determined. Bone thickness was measured as an indicator of bone growth. Net bone growth was significantly increased in animals with zoledronic acid treatment: 0.02 mm270.03 mm2 in animals with particle implantation only (group 2), 0.25 mm270.08 mm2 with particle implantation and zoledronic acid treatment directly after surgery (group 3; p ¼ 0:0018), and 0.21 mm270.11 mm2 with particle implantation and zoledronic acid treatment on the fourth postoperative day (group 4; p ¼ 0:0042). The mean bone thickness was 0.2 mm70.04 mm (range 0.17 mm–0.31 mm) in group 1 (sham controls) and 0.16 mm70.02 mm (range 0.14 mm–0.19 mm) in group 2, 0.31 mm70.04 mm (range 0.28 mm–0.39 mm) in group 3, and 0.29 mm70.02 mm (range 0.28 mm–0.34 mm) in group 4. Student’s t-test revealed a statistically significant difference between groups 2 and 3 (p ¼ 0:00042), and groups 2 and 4 (p ¼ 0:0019). In conclusion, our observational study suggests that zoledronic acid may stimulate bone apposition locally in the process of particle-induced osteolysis. r 2004 Elsevier Ltd. All rights reserved. Keywords: Net bone growth; Wear debris; Polyethylene particles; Histomorphometry; Zoledronic acid

1. Introduction Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacements [1,2]. A consensus has emerged that the predominant process is one $ Investigation performed at the University of Duisburg-Essen, Germany. Corresponding author. Tel.: +49 201 4089 2161; fax: +49 201 4089 2722. E-mail address: [email protected] (M. von Knoch).

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.09.026

of cytokine production in response to phagocytosis of implant wear particles resulting in increased proliferation and differentiation of osteoclast precursors into mature osteoclasts [3]. Bisphosphonates are used in the treatment of Paget’s disease, osteoporosis, osteolytic bone disease, and malignant hypercalcemia. The bisphosphonate alendronate prevented implant-induced peri-implant bone loss after total hip arthroplasty in humans [4]. This bisphosphonate has already been proved to prevent particleinduced osteolysis and implant loosening in a canine [5]

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and a rat [6] model. The same drug has been used by Schwarz et al. [7] in a particle-related murine calvarial osteolysis model. Recently, zoledronic acid, a third generation, nitrogen-containing heterocyclic imidazole bisphosphonate, has been found to be a more potent inhibitor of bone resorption than other currently available bisphosphonates [8]. The increased bone mineral density in patients receiving bisphosphonates has been primarily attributed to the inhibition of osteoclasts and the induction of their apoptosis [9,10]. It has been observed that zoledronic acid has a direct regulating effect on proliferation, differentiation, and gene expression in human osteoblasts [11,12]. It has been shown that bisphosphonates are able to induce the recruitment of osteoblasts from among human bone marrow stromal cells [13], stimulate osteoblastic proliferation and maturation [14]. Furthermore, in vivo studies showed enhanced net bone growth into implant porosities [15] and during distraction osteogenesis [16] as well as a pronounced thickening of periprosthetic cortical bone after bisphosphonate treatment [17]. In addition, bisphosphonates may have direct effects on osteoblast function and enhance bone forming activation as shown in previous in vitro studies [18,19]. In a particle-induced murine calvarial osteolysis model we previously showed that bone resorption can be substantially decreased by a single s.c. dose of zoledronic acid [20]. We aimed to describe the in vivo effects of zoledronic acid on osteoblastic bone formation under conditions of polyethylene particle-induced osteolysis. To our knowledge, the stimulation of local osteoid formation in the presence of polyethylene particles after treatment with bisphosphonates has not been shown in vivo to date.

at a dose of approximately 25 mg/kg of body weight directly after surgery, seven animals received Zoledionic acid 4 days after surgery (group 4). This dose regime was in the range of the dosage recommended for application in humans (25 mg/kg of body weight). The group size of seven was determined in a power analysis indicating that for a two-sided test at alpha=0.05 there was 95% power to detect a significant difference in the two groups. In accordance with pertinent studies to be found in specialist literature [7] five animals were determined to give sufficient power. This number was increased to seven. 2.2. Particles Commercially pure UHMWPE polyethylene particles were obtained from the manufacturer (Ceridust VP 3610, Clariant, Gersthofen, Germany). The particle size was described by the manufacturer as 50% of the particles being smaller than 5 mm and 90% being smaller than 9 mm. The particle size was determined using sizeindependent morphological description. The samples were coated with 10 nm gold/palladium (Au/Pd=80/ 20%) for detection in the scanning electron microscope (Hitachi FESEM S-4100, Hitachi, Kyoto, Japan) (Fig. 1). The area and perimeter of approximately 2000 particles were measured with PC-Image software (Version 2.2.03, Foster Findlay Associates Ltd., Newcastle upon Tyne, UK) and calculated in Excel software (Version 2000, Microsoft, New York, USA). The mean particle size (given as Equivalent Circle Diameter) was 1.7471.43 mm (range 0.05–11.06 mm) with more than 34% of the particles smaller than 1 mm. A detailed morphologic particle description has been published recently by our group [21].

2. Materials and methods 2.1. Study design Histological specimens from a recently performed study [20] were reevaluated with regard to new bone formation. A recently established murine calvarial model of UHMWPE particle-induced osteolysis [2] was applied in twenty-eight C57BL/J6 mice in accordance with the official guidelines and following approval by the university’s ethics committee and the local authorities. Animals were randomized to four groups. Animals in group 1 underwent sham surgery only, animals in group 2 were treated with ultra-high molecular weight polyethylene (UHMWPE) particles (about 6  106 particles), animals in group 3 were treated with particles and received additional zoledronic acid (1-hydroxy-2-(1H-imidazol-1-yl) ethylidene]bisphosphonic acid, Novartis Pharma AG, Basle, Switzerland)

Fig. 1. Scanning electron microscope picture of polyethylene particles used (for magnification (5.0 kV) see 30-mm scale at the right lower border of the photograph). 34% of particles were smaller than 1 mm.

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The particles were processed under sterile conditions under a cell culture hood and meticulous care was taken to avoid contamination with endotoxins. The particles were washed in 70% ethanol for 24 h (ethyl alcohol, absolute, 200 proof, for molecular biology, Aldrich Co. Inc., Milwaukee, WI, USA) at room temperature using a rocking device (Thermal Rocker, Lab Line Instruments, In., Melrose Park, IL). This treatment resulted in negative testing for endotoxin using a quantitative Limulus Amebocyte Lysate (LAL) Assay (Charles River, Kent, UK) at the detection level of o0.25 EU/ mL. This static endotoxin test was a commercially available standard kit and was used according to the manufacturer’s recommendations. We used endotoxinfree solutions and materials as provided by the manufacturer. The particles were washed in phosphate-buffered saline (8.5 g NaCl, 1.43 g K2HPO4, 0.25 g KH2PO2 ad 1000 mL aqua dest., pH 7.2–7.4) three times and allowed to dry in a dessicator. After centrifugation (30 min, 3000 rpm, Marathon 21000R, Fisher Scientific/IEC, Needham Heights, MA, USA) a volume of 2.5 mL of particles was resuspended in phosphate-buffered saline to an overall volume of 5 mL and stored at 4 1C. During preparation and storage, ultrasterile tubes were used (screw-cap tube conical, Sarstedt, Inc., Newton, NC, USA). Prior to implantation particles were allowed to dry in a dessicator.

chamber. Complications did not occur in the 28 animals. All wounds healed uneventfully.

2.3. Surgical procedure

2.5. Bone histomorphometry

The surgical procedure was as described previously [20]. Briefly, the mice were anesthetized with 70–80 mg/ kg ketamine (CEVA, Sante animale Ketaminhydrochlorid, Du¨sseldorf, Germany) and 5–7 mg/kg xylazine (CEVA, Sante animale Xylazinhydrochlorid, Du¨sseldorf, Germany) by intraperitoneal injection. During surgery the eyes of the animals were protected using sterile lubricant eye ointment (Bepanthen, Roche, Basle, Switzerland). The skin was scrubbed using Cutasept F (Bode Chemie, Hamburg, Germany). Using a scalpel (No. 15, Aesculap, Tuttlingen, Germany), a 1.0  1.0 cm area of periosteum was exposed by making a 10-mm midline sagittal incision over the calvarium anterior to the line connecting both external ears. In sham controls (group 1) the incision was closed without any further intervention. In groups 2, 3 and 4, the exposed but intact periosteum was covered with 30 mL of dried polyethylene particles (2  108 particles per 1000 mL; dosage established through previous experiments [20]) using a sterile sharp surgical spoon. The incision was closed using single 4-0 Ethilon sutures (Ethicon, Somerville, NJ, USA). The animals were returned to their cages as soon as they had recovered from the anesthesia. Water and food were given ad libidum. Fourteen days after operation, the animals were sacrificed in a CO2

Using a standard high-quality light microscope the specimens were photographed with a digital camera (Coolpix 995, Nikon, Du¨sseldorf, Germany). Each section was digitally photographed at a magnification of  10 with the midline suture in the middle of the field. Histomorphometric measurements were performed with a system consisting of a personal computer and an image analysis software (UTHSCA Image Tool, IT Version 3.0, University of Texas, San Antonio, TX, USA). The midline bone resorption area was determined in Giemsa-stained sections by tracing the area of soft tissue including any bone resorption pits in the midline suture as described previously [7]. In detail, using one microscopic field at a magnification of  10, the regions of interest. Osteoid formation in Giemsa-stained sections was determined by tracing the area of osteoid. In detail, using one microscopic field at a magnification of  10, the osteoid tissue area was encircled and the area was calculated automatically. Bone thickness was measured as an additional indicator of new bone formation. To this purpose, specimens were divided in four 0.5-mm steps to the left side and four measured steps to the right side of the midline suture. Bone thickness was measured successively at these measured steps and at the midline suture. The mean bone

2.4. Specimen retrieval and histological processing After sacrifice, the calvaria were removed by dissecting bone free from the underlying brain tissue and removing an elliptical plate of bone defined by the foramen magnum, auditory canals, and orbits [7]. Retrieved calvaria were processed utilizing undecalcified hard tissue techniques. Specimens were dehydrated by daily changes of ethanol (1 day in 95% and 6 days in 100%) before infiltration with polymethylmethacrylate. Undecalcified calvaria were oriented on edge and embedded in the methylmethacrylate. The embedded tissues were cut in the coronal plane centered over the area of particle deposition using a Reichert–Jung microtome (Model 2065, Heidelberg, Germany). Five-micrometer sections of the regions of interest (sites where polyethylene particles were detected within the calvarial tissue) were stained with Giemsa dye. Giemsa-stained sections were analyzed by transmission light microscopy for evidence of osteoid formation, granulomatous foreign body reaction and bone resorption and for the presence of various cell types including macrophages, foreign-body giant cells, fibroblasts, and osteoclasts.

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thickness for each group was calculated from the means of 9 regional bone thickness measurements determined at the 0.5 mm intervals.

4. Discussion

2.6. Statistical analysis

In our reported experiment a single subcutaneous dose of zoledronic acid was given and shown to be effective in preventing particle-related osteolysis previously [20]. During that experiment we observed that zoledronic acid enhanced the bone formation activity in 14-week-old adult mice which we report here. In keeping with the findings of Reinholz et al. [11], Shanbhag et al. [15], von Knoch et al. [13] our results suggest that osteoblasts exposed to a single dose given subcutaneously are able to increase their bone forming potential, although the mechanisms by which they mediate their actions remain as yet unclear. Animal studies showed that zoledronic acid caused a dosedependent suppression of cancellous bone turnover and resorption and resulted in an increase in the net volume of cancellous bone [15]. Little et al. [16] used a rabbit model to demonstrate generalized bone growth after zoledronic acid treatment and found that a zoledronic acid treatment increased the regenerate volume, mineralization, and tibial strength. To our knowledge, the effect of locally increasing osteoid formation in vivo under conditions where bone is resorbed, e.g. due to wear debris, has not previously been shown.

The results were expressed as means and standard errors of the mean. The data were analyzed by one-way ANOVA, followed by the post hoc paired Student’s two-tailed t-test. All p-values were compared to an avalue of 0.05 to determine significance.

3. Results Postoperatively, wound healing problems or other complications were not observed. All animals survived surgery and were sacrificed after 14 days. Histologically, there was formation of fibrous and granulomatous scar tissue overlying the calvarium (Figs. 2A–D). UHMWPE particle burden without any further intervention (group 2) induced bone resorption to sham control levels (Figs. 2A and B). Additional treatment with Zoledronic acid significantly increased osteoblast’s activity and the osteoid formation (Figs. 2C and D) as quantitatively assessed by histomorphometry. The osteoid formation area was 0 mm2 (range 0 mm–0 mm) in animals without particle implantation (group 1) and 0.0270.03 mm2 (range 0 mm–0.21 mm) in animals with particle implantation only (group 2). The net bone growth area was 0.25 mm270.08 mm2 (range 0.14 mm–0.35 mm) in animals with particle implantation and zoledronic acid treatment immediately after surgery (group 3) and 0.21 mm270.11 mm2 (range 0.07 mm–0.36 mm) in animals with particle implantation and zoledronic acid treatment on the fourth postoperative day (group 4; Fig. 3). One-way ANOVA revealed a statistically significant difference for the bone growth area (po0:05). There was a statistically significant difference in the net bone growth area between groups 2 and 3 (p ¼ 0:0018) and groups 2 and 4 (p ¼ 0:0042). No statistically significant difference in net bone growth area was found between groups 1 and 2 (p ¼ 0:17) and groups 3 and 4 (p ¼ 0:17). The mean bone thickness was 0.2 mm70.04 mm (range 0.17 mm–0.31 mm) in group 1 and 0.16 mm70.02 mm (range 0.14 mm–0.19 mm) in group 2. The bone thickness was 0.31 mm70.04 mm (range 0.28 mm–0.39 mm) in group 3, and 0.29 mm70.02 mm (range 0.28 mm–0.34 mm) in group 4 (Fig. 4). One-way ANOVA revealed a statistically significant difference for the bone thickness (po0:05). There was a statistical significant difference in bone thickness between groups 2 and 3 (p ¼ 0:00042) and groups 2 and 4 (p ¼ 0:0019). There was no statistically significant difference in bone thickness between groups 3 and 4 (p ¼ 0:07).

4.1. New bone formation

4.2. Cranial bone growth Cranial vault sutures are the major sites of bone growth during craniofacial development [22]. It is important to distinguish between a bone growth center and a bone growth site [23,24]. A bone growth center is the epi-/metaphyseal region of a growing long bone, which lengthens the long bone through cartilage proliferation and hypertrophy. Bone growth sites are secondary adapting regions at which bone remodeling takes place without intermediate cartilage formation. In mice, all cranial sutures with the exception of the posterior interfrontal suture remain patent for lifetime. Bone growth site and life-long forming suture cells may explain the bone forming process. This process relies on the production of sufficient new bone cells from the undifferentiated state which are recruited into the bone fronts. These cells lie within the osteogenic layer of cells lining the bone fronts [25,26] and may be activated by zoledronic acid. Another concept suggests that bone structural integrity consists of the removal of bone by osteoclasts and synthesis of new bone in its place by osteoblasts. Resorption and formation are therefore closely linked and interdependent processes. The anatomical structure consists of an osteoclast group in front with a group of osteoblasts and associated tissue like vessels and connective tissue behind [27]. Known of inductive

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Fig. 2. (A–D) Typical calvarial histology (Giemsa staining) of a control mouse calvarium after surgery without particle implantation (group 1) (A), after polyethylene particle implantation (group 2) (B), after particle implantation and a single dose of zoledronic acid immediately after surgery (group 3) (C), and after polyethylene particle implantation and a single dose of zoledronic acid on the fourth postoperative day (group 4) (D). The external cranial surface is at the top of the image and the mid-sagittal sutures or its remnant are located centrally. Magnifications are at original  10.

influences like the growing and expanding neurocranium and the dura mater which are capable of inducing bone growth [28,29] may probably be dismissed in our study design. We did not find any bone formation in the sham group. The mice used were 14-weeks-old and skeletally mature. We hypothesize that zoledronic acid may have the potency to preserve canalicular bone network, is able to induce apoptosis of osteoclasts, prevents bone erosion, and induces an external modeling. As a result there is an increase in bone thickness. 4.3. Bone thickness We used a specific method to determine bone growth. The measured bone thickness showed the course of the

bone specimen and made it possible to compare the calvaria. The bone thickness values underline the net bone growth results. 4.4. Shortcomings The animal model applied here was initially used to study particle-induced osteolysis in the absence of mechanical loading and fluid pressure to investigate the effect of mere particle exposure. It was not specifically designed to examine new bone formation. In detail, this murine experiment was designed to study the effect of a single dose of zoledronic acid on particle induced osteolysis [20]. Additionally, we did not use a specific staining for bone growth. Bone growth was only determined at a single anatomical site in this observa-

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New Bone Formation / mm2

3724 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05

drug, but it may also stimulate bone apposition locally during the process of particle-induced osteolysis.

Acknowledgements 1

2

3

4

Group

Fig. 3. The new bone formation was calculated as described in the Materials and Methods section. The data are given as the mean and the standard error of the mean for each group: after surgery without particle implantation (group 1), after polyethylene particle implantation (group 2), after surgery and a single dose of zoledronic acid immediately after surgery (group 3), and after polyethylene particle implantation and a single dose of zoledronic acid on the fourth postoperative day (group 4). New bone formation area was statistically significant different between groups 2 and 3 (p ¼ 0:0018) and between groups 2 and 4 (p ¼ 0:0042).

Bone Thickness / mm

0.4 0.35 0.3

The study was supported by a grant from Novartis Pharma Germany. Dr. Marius von Knoch was supported by German Research Foundation (DFG) Bonn, Germany (Grant KN 553/1). The authors would like to thank Dr. Ulrike Haus for advice regarding the study design and support in writing the manuscript, Dr. Jonathan Green for advice regarding drug dosage and study design, Dr. Anke Fuchs and Prof. Dr. med. Rainer Ansorg for advice during preparation of the particle suspension, Sylvia Marks for technical assistance and Prof. Dr. Hartmut Sto¨X for advice during histological and immunhistochemical processing of the specimens, Dr. Olaf Dirsch for advice regarding histomorphometry, cand. med. Anja Heckelei for support and control measurements, and Kaye Schreyer for editorial assistance with the manuscript.

0.25 0.2 0.15

References

0.1 0.05 0 Group 1

Group 2

Group 3

Group 4

Fig. 4. Bone thickness was calculated as described in the Materials and Methods section. The data are given as the mean and the standard error of the mean for each group: after surgery without particle implantation (group 1), after polyethylene particle implantation (group 2), after surgery and a single dose of zoledronic acid immediately after surgery (group 3), and after polyethylene particle implantation and a single dose of zoledronic acid on the fourth postoperative day (group 4).

tional study. The effect of zoledronic acid on calvarial bone in the absence of particles remains to be determined. Therefore, our results have to be interpreted with some caution, because they are of an observational nature. Despite these shortcomings, net local bone growth and increased bone thickness was observed to a large extent. The surgical sites and the location of the tissue sections were clearly defined. 4.5. Conclusions In summary, our observational study suggests two important findings: Zoledronic acid treatment increased (a) net calvarial bone growth in vivo in adult mice and (b) osteoid formation in the presence of osteolysisinducing particles. These important findings hold great potential for the treatment of local bone erosion. Zoledronic acid is not only a potent anti-resorptive

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