In Vivo RANK Signaling Blockade Using the Receptor Activator of NF-κB:Fc Effectively Prevents and Ameliorates Wear Debris-Induced Osteolysis via Osteoclast Depletion Without Inhibiting Osteogenesis
Abstract
Prosthesis failure due to wear debris-induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF-κB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)-induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti-treated RANK−/− mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti-treated were still depleted for multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris-induced osteolysis without jeopardizing osteogenesis.
INTRODUCTION
Focal loss of bone caused by osteolysis is a critical etiological component of human diseases such as erosive arthritis, bone metastasis, and osteomyelitis. Another common clinical condition characterized by inflammatory bone loss is aseptic loosening, secondary to osteolysis, which results in prosthetic implant failure. This remains a major orthopedic problem because up to 20% of total hip arthroplasties have evidence of osteolysis within 10 years of surgery.1-3 Presently, there are no therapies with proven efficacy to prevent or interfere with the disease process.4 Implant failure ultimately requires revision arthroplasty, which has a poorer clinical result and a shorter duration of survival, compared with primary total joint replacement (TJR).5, 6
Although many factors contribute to aseptic loosening (e.g., mechanical and biomechanical), several groups have focused on the host response to wear debris, postulating that wear debris-induced osteolysis is the major cause of prosthetic implant failure.7-9 In this model, wear debris generated from the prosthesis is phagocytosed by macrophages and initiates an inflammatory response that leads to the recruitment of activated osteoclasts and osteolysis at the bone-implant interface. Several lines of evidence support this model: (i) up to 109 particles per gram of tissue can be recovered from the inflammatory membrane attached to the failed prosthesis after revision surgery,10 (ii) ingestion of wear debris particles induces cytokine production by mononuclear phagocytes in vitro,11 (iii) high levels of cytokines that are produced by macrophages are found in the fluid and tissue surrounding loose implants,12-14 (iv) conditioned medium from wear debris-stimulated monocytes can stimulate increased bone resorption in vitro,15 and (v) animal models of wear debris-induced osteolysis have documented the importance of cytokines in this process.16, 17
Although many different cytokines contribute to this process, it has been proposed that the final effector molecules that ultimately control this wear debris-induced osteolysis are receptor activator of NF-κB ligand (RANKL; also called osteoprotegerin ligand [OPGL], osteoclast differentiation factor [ODF], and tumor necrosis factor-related activation-induced cytokine [TRANCE]) and OPG or osteoclastogenesis inhibitory factor (OCIF).18-20 RANKL directly stimulates the differentiation of osteoclast progenitors to mature osteoclasts21, 22 by signaling through its membrane RANK.23, 24 Physiologically, RANKL signaling is regulated negatively by the soluble receptor antagonist protein OPG, which inhibits signaling through RANK and induces osteoclast apoptosis.25 RANK signaling is essential for osteoclast differentiation, activation, and survival. Mice deficient in RANKL26, 27 or RANK28, 29 or transgenic mice overexpressing OPG25 or a soluble form of RANK (RANK:Fc)24 have severe osteopetrosis because they do not form osteoclasts.
To study the role of RANK signaling in wear debris-induced osteolysis, we evaluated the effects of RANK:Fc treatment in a mouse calvaria model.30 In these experiments we found that RANK signaling blockade is both effective in preventing wear debris-induced bone resorption and ameliorating established osteolysis. In doing so, RANK:Fc does not inhibit the bone formation that follows bone resorption. Furthermore, the quality of the new bone matrix in RANK:Fc-treated animals is similar to that of placebo controls. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse inflammatory bone loss without jeopardizing osteogenesis.
MATERIALS AND METHODS
Titanium particles
Pure titanium (Ti) particles were obtained from Johnson Mathey Chemicals (Ward Hill, MA, USA) and prepared as previously described.31 Briefly, particles, 1-3 μm in diameter, were suspended in phosphate-buffered saline (PBS) at a concentration of 1 × 108 particles/ml. Particle size was confirmed with a Coulter Channelizer (Coulter Electronics, Hialeah, CA, USA), which determined 90% of the particles to be <5 μm in diameter. A limulus assay (BioWhittaker, Walkersville, MD, USA) was used to show the suspension was free of endotoxin.
RANK:Fc fusion protein and RANK−/− animals
The RANK:Fc used in these studies was provided by Immunex, Inc. (Seattle, WA, USA) and contains the murine extracellular domain of RANK (through Pro213) fused to human immunoglobulin G1 (IgG1) Fc. The RANK:Fc protein was produced in Chinese hamster ovary (CHO) cells as previously described.32 The animals used in this study were propagated from CBAxB6 mice obtained from the Jackson Laboratory (Bar Harbor, ME, USA) or RANK+/− mice that are random C57BL/6 × 129 hybrids derived from the original line.28 All mice were 8 weeks old at the start of the experiments.
In vivo mouse calvaria resorption model
The in vivo mouse calvaria experiments were performed as previously described.17, 30 Briefly, five healthy female 8-week-old CBAxB6 or RANK−/− were used in each group. Mice were anesthetized with 70-80 mg/kg of ketamine and 5-7 mg/kg of xylazine by intraperitoneal (ip) injection. A 1 cm × 1 cm area of calvarial bone was exposed by making a midline sagittal incision over the calvaria, leaving the periosteum intact. Thirty milligrams of Ti particles were spread over the area and the incision was closed. Sham animals received surgery alone. Different doses of RANK:Fc were administered via ip injection on days 0, 2, 4, 6, and 8 (prophylaxis) or on day 5 only (intervention).
At various times after surgery the mice were killed and the calvariae were harvested for decalcified30 or undecalcified17 histology as we have done previously. Osteoclast numbers and resorption surfaces were quantitated as previously described30 from 3-μm sections that were stained for tartrate-resistant acid phosphatase (TRAP) using the Diagnostics Acid Phosphatase Kit (Sigma, St. Louis, MO, USA) or hematoxylin and eosin, respectively. Osteolysis was quantitated by measuring the soft tissue space between the parietal bones (midline suture area [MSA]) as previously described,31 using ScionImage software (Scion Corp., Frederick, MD, USA).
Calcein labeling studies
Calcein labeling studies were done by injecting 20 mg/kg of calcein (Sigma) in PBS, pH 7.3, ip on day 10 and day 16 and harvesting the calvaria on day 17 for undecalcified histology as described previously.17 Unstained 3-μm sections are examined at 60× magnification using fluorescence microscopy and photographed with a digital camera. The mineral apposition rate (MAR) was determined by dividing the mean distance between the double labels by the duration of the labeling period (6 days) and adjusting for oblique cutting by multiplying by π/4. The bone formation rate (BFR) was determined by multiplying the MAR by the fractional labeled surface, which was determined by dividing the sum of the length of the double-labeled surface and one-half the length of the single-labeled surface by a standard length of the bone surface from the edge of the sagittal suture.
Infrared imaging studies
Undecalcified 3-μm sections as described previously were analyzed by infrared (IR) imaging for mineral content (mineral/matrix ratio), mineral maturity (crystallinity), and collagen maturity (cross-links) as described elsewhere.33-35 The IR imaging was performed at the Hospital for Special Surgery Musculoskeletal Core Center (New York, NY, USA). In brief, five 400 μm2 × 400 μm2 areas from each calvarial section (two per animal) adjacent to the implant site were examined using a BioRad “Sting-Ray” system (Cambridge, MA, USA). The instrument consists of a step-scan interferometer interfaced to an microcomputed tomography (MCT) focal plane array detector imaged to the focal plane of an IR microscope. Interferograms are collected simultaneously from each element of the 64 × 64 array to provide 4096 spectra (4 minutes scan time) at a spectral resolution of 8 cm−1. The sample size imaged (400 μm × 400 μm) corresponds to an optimal spatial resolution of approximately 7 μm × 7 μm.33-35 Background imaging spectra were collected under identical conditions from the same BaF2 windows in which the sections were to be placed. The spectrometer was left continuously powered up, to minimize warm-up instabilities, and purged with dry air (Whatman dry-air pump; Whatman, Maidstone, UK), to minimize the water vapor and CO2 interference. After acquisition, the spectra were transferred off-line and zero-corrected for the baseline in the spectral areas of amide I (∼1590-1700 cm−1) and v1, v3 PO4 (∼900-1200 cm−1) using Grams/32 (Galactic Software, Salem, NH, USA). The areas of these two peaks were calculated and exported as ASCII files into Origin (Microcal Software, Inc., Northampton, MA, USA), in which mineral/matrix ratios were calculated as an indication of ash weight.36 Mineral crystallinity was determined based on the 1030/1020 intensity ratio.34 The relative ratio of the two collagen cross-links pyridinoline (Pyr) and dehydrodihydroxylysinonorleucine (deH-DHLNL) was calculated also as described elsewhere.37 All three IR imaging outcomes (mineral/matrix, crystallinity, and Pyr/DHLNL) were expressed as pixel population distribution histograms (how many pixels in an image have a specific value).
Statistical analysis
All values are presented as means ± SEM. Statistical significance was determined using Student's t-test. Values of p < 0.05 were considered significant.
RESULTS
RANK:Fc prevents Ti-induced osteoclastogenesis and bone resorption in vivo
To determine if prophylactic treatment with RANK:Fc could prevent Ti-induced osteolysis in the mouse calvaria model, varying doses of RANK:Fc were administered at the time of Ti implantation on day 0 and every other day until the end of the experiment on day 10. Then, the calvariae were harvested and analyzed by histomorphometry (Table 1). Consistent with our previous studies,31 Ti treatment with placebo resulted in a 3-fold increase in the MSA because of resorption of the perisutural bone. In contrast, RANK:Fc treatment at doses of ≥2 μg significantly inhibited this osteolysis. Statistically significant effects of RANK:Fc on osteoclast numbers and bone resorption surfaces were observed at the 20-μg dose, which roughly is equivalent to 1 mg/kg. At 10 times this dose, the effects observed were similar to that obtained with RANK knockout mice and baseline controls in that the calvaria were completely devoid of osteoclasts and resorption surfaces.
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The antiresorptive effects of RANK:Fc on established osteolysis
The foregoing studies indicate that intermittent RANK:Fc treatment prevents Ti-induced osteolysis. However, a more clinically relevant question is what therapeutic effect does RANK:Fc have on established osteolysis and subsequent bone formation. To test this, we implanted the Ti particles on day 0, gave a single injection of RANK:Fc or placebo on day 5 (at which time our previous studies have shown that significant osteolysis has already taken place), and then harvested the calvaria on day 21 for analysis. We found that osteoclast numbers in vehicle-treated animals continue to rise after the osteolytic response such that even greater numbers of osteoclasts are present in the sagittal suture area (SSA) during the remodeling phase 21 days after Ti implantation in the control animals (Fig. 1B). In contrast, the antiresorptive effects of RANK:Fc treatment were evident in mice given 200 μg on day 5 because very few osteoclasts could be identified in the calvariae of these animals (Figs. 1B and 2B). Interestingly, RANK:Fc treatment had no long-term effect on the MSA of the mice at any dose tested (Fig. 1A), as the assumed enlarged soft tissue space on day 5 of RANK:Fc-treated animals was reduced to that of Ti-treated controls by day 21. This indicates that even under conditions of extensive osteoclast depletion (200 μg), osteoclast/osteoblast uncoupling does not inhibit bone formation and that the resorbed bone is replaced with new woven bone.
RANK:Fc treatment ameliorates Ti-induced osteolysis. Control mice were untreated (baseline) or given Ti surgery and killed on day 5 and day 10 (white bars). Striped bars indicate experimental groups that were killed on day 21 including animals given the Ti surgery and treated with a single ip injection of PBS or the indicated dose of RANK:Fc. The calvariae from these animals were processed for histology and the (A) SSA and (B) number of osteoclasts in the SSA were determined as described in the Materials and Methods section. (A) Of note is that the increase in the SSA seen in animals killed on day 5 and day 10 postsurgery is reduced down to baseline levels in the RANK:Fc-treated mice by day 21. (B) Also, the effects of a single injection of RANK:Fc are sustained long term as seen in the number of osteoclasts in the SSA 16 days later (*p < 0.01); **p < 0.001.
New bone matrix is synthesized on resorption surfaces in the absence of osteoclasts. (A and C) Representative trichrome and (B and D) TRAP-stained calvariae sections (original magnification ×60) from the (A and B) vehicle-treated and (C and D) 0.2-mg RANK:Fc treatment group in Fig. 1 are shown. (C) Of note is the presence of unmineralized new bone matrix, which stains red with trichrome (arrows), at sites of previous resorption in calvaria depleted of osteoclasts.
RANK:Fc-induced inhibition of resorption does not affect osteoblastic bone formation and remodeling
Having established significant prophylactic and therapeutic effects of RANK:Fc in our model, we chose to investigate the quantity and quality of the new bone formed under conditions of osteoclast depletion. Evidence that new bone is formed at sites of resorption as well as on nonresorbed surfaces was obtained from trichrome-stained sections of calvariae that were depleted of osteoclasts by high-dose RANK:Fc administration in the previous study (Fig. 2A). The arrows point to the new unmineralized matrix (osteoid), which stains red with trichrome. To characterize the quality of this newly formed bone, we analyzed the chemical properties of the mineral and matrix using IR imaging (Fig. 3). Three parameters were calculated: mineral/matrix ratio (Figs. 3A and 3B), mineral maturity (crystallinity; Figs. 3C and 3D), and collagen maturity (Figs. 3E and 3F). The mineral/matrix ratio, which correlates with ash weight,36 showed statistically significant differences when the calvarial sections from the PBS and RANK:Fc-treated mice were compared. The maturity (crystallinity) of the mineral (Figs. 3C and 3D) showed no significant differences when experimental and control animals were contrasted. The maturity of the collagen matrix, expressed as the ratio of Pyr/DHLNL collagen cross-links (Figs. 3E and 3F), revealed no significant differences between the two groups.
Biochemical properties of the new bone matrix synthesized in RANK:Fc-treated mice. Mineralized calvariae sections for the (A, C, and E) PBS and (B, D, and F) 0.2 mg RANK:Fc treatment groups in Fig. 1 were analyzed by IR imaging to determine the (A and B) relative mineral/matrix ratio, (C and D) mineral maturity (crystallinity), and (E and F) collagen maturity (cross-linking) in the tissue as described in the Materials and Methods section. We found a 1.6-fold difference in the mineral/matrix ratio between the PBS and RANK:Fc-treated samples (*p < 0.01); however, no differences were found in the crystallinity or the extent of collagen cross-linking between the two groups.
In our final experiment we determined the MAR and BFR in untreated mice and PBS-treated mice and mice treated with 200 μg of RANK:Fc using calcein labeling (Fig. 4 and Table 2). Separate determinations were made for resorbed (yellow arrows) and nonresorbed (white arrows) surfaces. No differences were observed in the MAR or BFR of matrix laid down on nonresorbed surfaces between any of the groups tested. However, significant differences were observed in the amount of matrix laid down on the resorbed surfaces by RANK:Fc-treated animals.
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Calcein labeling of calvariae from untreated and Ti-treated mice given placebo or RANK:Fc. In vivo calcein labeling was performed as described in the Materials and Methods section. Representative fluorescent microphotographs (magnification ×60) of (A) untreated mice, (B) Ti + placebo, and (C) Ti + RANK:Fc-treated (200 μg) mice are shown. The MAR and BFR from these sections were calculated for bone resorption surfaces (yellow arrow) and nonresorption surfaces (white arrow) and presented in Table 2.
DISCUSSION
Effective management of osteolysis requires the reversal of osteoclastic bone resorption and stimulation of osteoblastic bone formation with remodeling. The dominance of RANK signaling in osteolysis has led investigators to propose that this pathway is an excellent target for intervention of osteoclastic bone resorption.18-20 Indeed, researchers have shown that in preclinical models of osteoporosis,25 inflammatory arthritis,38 and hypercalcemia of malignancy-tumor metastasis to bone,32, 39, 40 perturbing RANK signaling effectively ameliorates disease in animals. Consistent with these studies, here, we show that RANK:Fc treatment effectively prevents Ti-induced osteolysis such as that seen in patients with aseptic loosening (Table 1). Abolition of RANK signaling in the RANK−/− animals prevented the formation of resorption pits on the calvariae because of the complete absence of osteoclasts (Table 1). However, quantification of the MSA in these animals was impossible because of separation of the parietal bones during processing. We also show that RANK:Fc treatment ameliorates established osteolysis by eliminating osteoclasts (Fig. 1). In these studies, a single administration of 1 mg/kg of RANK:Fc had long-term effects because calvariae in these mice had reduced osteoclast numbers compared with placebo controls 16 days after treatment. Because the in vivo half-life of RANK:Fc is only 36 h, it is likely that this treatment effectively eliminates osteoclast precursors as well. It is suggested that in this model, high doses of RANK:Fc induce apoptosis of osteoclast progenitors such that animals treated with RANK:Fc are osteoclast-depleted long after the drug has cleared. Alternatively, the absence of RANK signaling in these progenitors has allowed them to differentiate into a cell without osteoclastogenic potential.
Perhaps the most important question to be asked regarding the use of RANK signaling antagonists to inhibit bone resorption is what effects do they have on the coupling of bone formation to bone resorption. To test this, we used the in vivo calvaria model, in which it is known that the primary bone resorption in response to the wear debris peaks around 1 week after surgery and is followed by osteogenesis that fills the resorbed areas with new woven bone.31, 41 Unfortunately, this model does not permit longitudinal studies with the same animal; thus, assumptions based on control mice killed at previous times were made. The first indication that osteogenesis is unaffected by osteoclast depletion with RANK:Fc treatment was the finding that the MSA effectively remodels after the Ti-induced osteolysis (Fig. 1A). Even at the highest dose of RANK:Fc (200 μg), which depleted osteoclast numbers below baseline levels (Fig. 1B), no statistically significant differences were observed in the MSA of these mice compared with placebo. Furthermore, trichrome-stained sections of these osteoclast-depleted calvariae provided direct evidence that new bone matrix is laid down in sites of resorption (Fig. 2A). A qualitative assessment of the molecular structure of this new bone matrix by IR imaging failed to identify any significant differences in either collagen maturity (cross-linking) or mineral maturity (crystallinity; Fig. 3). This is of potential interest because the bisphosphonate antiresorptive drugs significantly alter the crystallinity of new bone matrix (E. P. Paschalis, unpublished data). Although significant differences were observed in the mineral/matrix ratio between the calvaria of RANK:Fc-treated mice and the placebo group, this was expected because of the enhanced and continuous resorption that occurred in the control animals.
Results of osteoclast depletion experiments (Fig. 4 and Table 2) indicated that osteoclasts do not have a significant role in bone formation in this model because no differences in the MAR and BFR on nonresorbed surfaces were found among any of the groups. Again, the significant differences seen in the MAR and BFR between the RANK:Fc and placebo groups were likely because of the increased resorption, thus requiring more bone formation. The MAR and BFR could not be obtained from the baseline animals for resorbed surfaces because in the absence of Ti, these mice do not resorb bone and new bone formation is limited.
Taken together, these studies indicate that in vivo osteoclast depletion via RANK:Fc treatment (1 mg/kg ip) effectively prevents Ti-induced bone resorption and ameliorates established osteolysis. Although these results were somewhat predicted from the knockout mouse studies, other published studies have concluded that osteoclastogenesis and osteoclastic bone resorption can occur independent of RANK signaling and that the absence of RANKL can be compensated for by tumor necrosis factor (TNF).42, 43 Previously, it has been shown by us and others that large amounts of TNF are produced in response to wear debris in this model.16, 17 Here, we find no evidence that this TNF can compensate for RANK signaling. In fact, we found that RANK:Fc is 10- to 100-fold more potent than TNF receptor (TNFR):Fc.44
Another novel finding is that under conditions of osteoclast-osteoblast uncoupling due to osteoclast depletion, bone formation is unaffected. Calcein labeling and IR microscopy studies showed that the RANK:Fc-treated mice have normal MAR and BFR as determined by the quantity and quality of the new bone matrix. As such, these results strongly indicate that this agent may be useful in treating patients with osteolysis; however, more detailed studies are needed to evaluate other bone metabolic processes like fracture healing and the biomechanical properties of the bone synthesized under these conditions.
In vivo, we have observed that systemic delivery of RANK:Fc effectively induces osteoclast apoptosis in the calvariae and long bones but we have no evidence that apoptosis is induced in any other cell type (data not shown). It is important to consider what possible adverse side effects systemic RANK:Fc or OPG might cause because RANK blockade has such potent effects on osteoclast depletion. The most obvious concern is osteopetrosis such as that seen in RANK:Fc24 and OPG25 transgenic mice. It also has been shown that systemic administration of these proteins into skeletally immature animals induced osteopetrosis.25, 32 Interestingly, we have not observed osteopetrosis in adult mice given RANK:Fc systemically over a similar time period (data not shown). Thus, the susceptibility to osteopetrosis after osteoclast depletion via RANK blockade appears to be age/development related and warrants further investigation.
Acknowledgements
The authors thank J. Harvey for assistance with the histology. The IR Imaging Core at the Hospital for Special Surgery is supported by an National Institutes of Health (NIH) grant (AR46121). E.M.S. is supported by the Orthopedic Research and Education Foundation and grants from the NIH (PHS AR45791 and AR44220) and he receives a research grant from Immunex.
