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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

To assess the ability of sclerostin antibody therapy to blunt the negative effects of polyethylene particles on implant fixation and peri-implant bone structure in a rat implant fixation model.

Methods

Thirty-six adult male rats received intramedullary titanium implants; 12 rats received vehicle injections only (control), and 24 rats received intraarticular injections of lipopolysaccharide-doped polyethylene particles. Twelve of the rats that received particles also received sclerostin antibody treatment. The 3 groups of rats were maintained for 12 weeks in a pathogen-free environment, at which time mechanical, micro–computed tomography, and dynamic and static histomorphometry end points were assessed.

Results

Sclerostin antibody treatment completely blocked the negative effect of the lipopolysaccharide-doped polyethylene particles on implant fixation and peri-implant bone volume by increasing the bone formation rate and depressing bone resorption.

Conclusion

Anabolic agents targeting the Wnt signaling pathway are a promising new alternative for the prevention of periprosthetic osteolysis and aseptic loosening.

The demand for total hip and total knee revisions in the US is projected to increase by 137% and 601%, respectively, between 2005 and 2030 (1). Periprosthetic osteolysis and aseptic loosening are 2 common indications for revision surgery. Wear and corrosion debris shed from the implants induces an inflammatory cytokine cascade that is widely considered to be the pathogenic mechanism for periprosthetic osteolysis. Specifically, these particles cause a local macrophage-mediated inflammatory reaction (2), which subsequently increases the activity of osteoclasts and may also decrease the activity of osteoblasts (3), leading to net bone loss and implant loosening.

Most investigators agree that particulate-induced osteolysis and implant loosening are mainly caused by increased bone resorption. Not surprisingly, anticatabolic agents, such as bisphosphonates (4, 5), and antagonists of cytokines that mediate the biologic process of bone resorption (6, 7) have been studied for the treatment or prevention of osteolysis and implant loosening. However, no clinical trial has demonstrated that bisphosphonates are effective in treating peri-implant osteolysis (8). Thus, there is a need for an alternative therapeutic strategy for the prevention and treatment of particulate-induced implant loosening. The potential of enhancing bone formation as a countermeasure to particle-induced implant loosening has not been investigated.

Modulation of the Wnt pathway is being examined as a novel means to manipulate bone remodeling. Inhibition of the canonical Wnt signaling pathway down-regulates bone formation (9). One of the Wnt signaling pathway inhibitors is sclerostin, which is the product of the SOST gene and is thought to be exclusively expressed by osteocytes in the adult skeleton (10). Sclerostin-null mice have a high bone mass phenotype (11), and this observation has motivated research on the use of neutralizing antibodies to sclerostin as an approach to enhance bone formation. Systemic administration of sclerostin antibody increased the mineralized surface, mineral apposition rate (MAR), bone formation rate (BFR), bone mass, and bone strength in a rat model of postmenopausal osteoporosis (12). Sclerostin antibody enhances fracture repair (13) and implant fixation in rats (14, 15). There is growing evidence that sclerostin antibody also suppresses bone resorption (16).

Therefore, treatment with sclerostin antibody is a potential therapeutic strategy for a variety of bone-related disorders, including particle-induced implant loosening in the setting of arthroplasty. In this study, we investigated the effect of sclerostin antibody for the prevention of particle-induced implant loosening, using an established rat model that was recently validated in our laboratory in terms of several important clinical criteria (17). We hypothesized that sclerostin antibody treatment would prevent implant loosening by accelerating bone formation and inhibiting bone resorption. Briefly, 3 groups of rats that had received bilateral femoral implants were used; each group comprised 12 rats. One group of rats received no particles and no antibody treatment (control), a second group received lipopolysaccharide (LPS)–doped polyethylene particles, and a third group received LPS-doped polyethylene particles plus sclerostin antibody treatment.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Experimental design.

In this Institutional Animal Care and Use Committee–approved study, 36 male Sprague-Dawley rats (400–425 gm; Harlan) received bilateral femoral titanium implants. Beginning on day 1 after surgery, 12 rats received weekly intraarticular injections of particle vehicle in both knees and twice-weekly subcutaneous injections of antibody vehicle (controls), 12 rats received weekly intraarticular injections of 50 μl polyethylene particle suspensions in each knee and twice-weekly subcutaneous injections of vehicle, and 12 rats received weekly intraarticular injections of 50 μl polyethylene particle suspensions in each knee and twice-weekly subcutaneous injections of sclerostin antibody (Scl-AbIII; donated by Amgen and UCB Pharma) at a dose of 25 mg/kg. Calcein (10 mg/kg; Sigma-Aldrich) was injected subcutaneously 10 days and 2 days before the rats were killed (at 12 weeks).

Implants and particles.

Titanium rods (length 15 mm, diameter 1.5 mm; Goodfellow) were sterilized and dual acid etched, as previously described (17). Polyethylene particles with median size of 1.75 μm (Ceridust VP 3610; Clariant) were prepared and doped with LPS, as described previously (17). Each injection had a volume of 50 μl (4.7 × 107 particles). Vehicle was prepared as 6% rat serum in sterile phosphate buffered saline.

Surgery.

All of the rats underwent bilateral intramedullary implantation of the titanium rods, as described previously (17). An implant was placed into the canal with the proximal 10 mm of the implant press fit in the femoral bone marrow cavity to achieve good initial fixation and the distal 5 mm of the implant surrounded by a 0.25-mm gap.

Contact radiography.

Bones were imaged using contact radiography (Faxitron MX-20) at 30 kV for 15 seconds. The radiographs were examined, and each rat was scored as positive or negative for the presence or absence of a radiolucent region around the implant, based on independent examinations by 2 reviewers.

Micro–computed tomography (micro-CT).

The rat femurs were scanned perpendicular to the long axis of the implant. The parameters were as follows: 70 kVp, 114 μA, 300-msec integration time, 30-μm isotropic voxels (Scanco μCT 40). The femurs used for histology were scanned in 10% neutral buffered formalin, while the femurs used for mechanical testing were scanned in saline. Images were processed using a bone Gaussian filter (σ = 1.5, support = 2) and a titanium Gaussian filter (σ = 2, support = 2). Pilot studies showed that these filter settings appeared to reduce the magnitude of metal-induced artifacts better than other settings. The bone volume/total volume (BV/TV), trabecular number, trabecular thickness, trabecular separation (TbSp), connectivity density, structure model index, and bone surface/bone volume were determined using standard nomenclature (18) in the region adjacent to the distal 3 mm of the implant, using the manufacturer's software. The images were segmented at a threshold of 250 arbitrary units (equivalent to a linear attenuation coefficient of 1.99 cm−1). Total volume was defined as the region between the endocortical surface and 48 μm away from the implant surface, to avoid metal-induced artifacts in the images (19). Endocortical contouring was performed manually, and the manufacturer's software was used to exclude the first 48 μm from the implant surface.

Mechanical pull-out testing.

The strength of fixation of the implant with the host bone was measured by a mechanical pull-out test (17). Fixation strength was calculated by dividing the force (N) at the point of failure by the surface area of the implant in contact with tissue (mm2). Energy to yield (Nmm) and energy to failure were calculated as the area under the load-displacement curve until the yield point or failure point, respectively. Stiffness (N/mm) was calculated as the slope of the linear portion of the load-displacement curve before the yield point.

Bone histomorphometry.

Samples were embedded in methylmethacrylate (Technovit 9100; Heraeus Kulzer), and 1-mm–thick slabs were made by cutting perpendicularly to the long axis of the implant (IsoMet 5000; Buehler). The slabs were attached to plastic slides and ground to ∼100-μm–thick sections, followed by polishing with 3-μm polishing paper (Phoenix 4000; Buehler). Sections from 1 mm proximal to the distal growth plate were selected for histomorphometric measurement (OsteoMeasure; OsteoMetrics) under light and fluorescence microscopy (Nikon Eclipse 80i). Dynamic measurements included single-labeled and double-labeled perimeters, interlabel width, and bone perimeter. Static measurements included eroded perimeter and were performed on slides stained with basic fuchsin and toluidine blue (Fisher Scientific). This approach was used instead of measuring the bone surface occupied by osteoclasts, because the presence of the implant dictated that thick sections must be prepared, meaning that the assessment of cell type is not always possible. These indices were used to calculate mineral surface/bone surface (MS/BS; double-labeled surface plus one-half of single-labeled surface per bone surface), MAR, BFR/bone surface (BS), and eroded surface (ES)/BS (20).

Statistical analysis.

SPSS for Windows (version 15) was used for statistical analysis. Nonparametric statistical methods were used, including the Mann-Whitney test to compare the groups with respect to continuous variables, and the chi-square test of association and Fisher's exact test to compare the groups with respect to percentages. Scatterplots and Spearman's correlations were obtained to investigate relationships between noncategorical variables. P values less than 0.05 were considered significant. Data are presented as the mean ± SD.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

A rat model was used in which mechanical, micro-CT, and histologic end points were measured. Briefly, 36 adult male rats had titanium implants placed in the medullary cavities of both distal femurs. Beginning on day 1 after surgery, 12 control rats received weekly intraarticular injections of vehicle in both knees and twice-weekly subcutaneous injections of vehicle, 12 rats received weekly intraarticular injections of LPS-doped polyethylene particle suspensions in each knee and twice-weekly subcutaneous injections of vehicle, and 12 rats received weekly intraarticular injections of LPS-doped polyethylene particles in each knee and twice-weekly subcutaneous injections of sclerostin antibody.

Effect of sclerostin antibody on particle-induced implant loosening.

Treatment with sclerostin antibody prevented LPS-doped polyethylene particle–induced depression of implant fixation strength (Figure 1A). Remarkably, fixation strength in the rats that received the LPS-doped polyethylene particles and sclerostin antibody was higher than that in the control rats that received no particles. Specifically, intraarticular injection of the LPS-doped polyethylene particles induced 40% lower pull-out strength (P = 0.004). Compared with particle-injected rats treated with vehicle, particle-injected rats treated with sclerostin antibody had 2.5-fold higher pull-out strength (P < 0.001). Even compared with control rats (which had no LPS-doped polyethylene particle injections and no antibody treatment), the particle-injected rats treated with sclerostin antibody had 1.5-fold higher pull-out strength (P = 0.001). Similar differences between groups were observed for stiffness and energy to failure, the other 2 measures of mechanical fixation (Table 1).

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Figure 1. Effect of sclerostin (Scl) antibody (Ab) treatment on mechanical fixation strength (A) and peri-implant trabecular bone volume (B). Bars show the mean ± SD. Horizontal lines indicate significant differences between groups (P < 0.05). PE = polyethylene; BV/TV = bone volume/total volume.

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Table 1. Comparisons of mechanical pull-out data, micro-CT bone architecture data, and bone histomorphometry data in the 3 groups of rats*
Variable/groupControlPolyethylene onlyPolyethylene/sclerostin antibody
  • *

    Values are the mean ± SD. Micro-CT = micro–computed tomography.

  • P < 0.01 versus control.

  • P < 0.01 versus control and P < 0.001 versus polyethylene only.

  • §

    P < 0.001 versus polyethylene only.

Strength, N/mm21.32 ± 0.450.79 ± 0.402.00 ± 0.29
Energy to failure, Nmm154 ± 81104 ± 67348 ± 156
Stiffness, N/mm221 ± 127127 ± 89186 ± 114
Bone volume/total volume, %17.5 ± 5.87.6 ± 2.531.2 ± 7.7
Trabecular number, 1/mm1.31 ± 0.340.92 ± 0.182.01 ± 0.32
Trabecular thickness, μm142 ± 20137 ± 19192 ± 26
Trabecular separation, μm869 ± 2161,182 ± 216502 ± 93
Bone surface/bone volume, %19.25 ± 2.0020.04 ± 2.6314.13 ± 2.32
Connective density, 1/mm39.29 ± 3.003.67 ± 2.3611.64 ± 2.63§
Structure model index1.55 ± 0.432.18 ± 0.601.09 ± 0.46
Single-labeled perimeter, mm10.37 ± 2.145.78 ± 2.7711.25 ± 4.06§
Double-labeled perimeter, mm0.80 ± 0.690.44 ± 0.402.33 ± 1.53
Interlabel width, μm8.88 ± 1.256.17 ± 1.2612.48 ± 2.08
Mineralized surface/bone surface, %12.04 ± 2.129.83 ± 4.7817.64 ± 3.25
Mineral apposition rate, μm/day1.11 ± 0.160.77 ± 0.161.56 ± 0.26
Bone formation rate/bone surface, μm3/μm2/day × 10049.53 ± 15.1829.69 ± 19.77102.14 ± 34.47
Eroded surface/bone surface, %10.26 ± 2.7117.10 ± 3.1710.83 ± 1.92§

Role of sclerostin antibody in blocking the negative effect of particles on bone architecture.

Weekly intraarticular injections of LPS-doped polyethylene particles led to a significant decrease in peri-implant trabecular bone volume; this effect was totally abrogated by contemporaneous treatment with sclerostin antibody (Figure 1B). Specifically, particle treatment resulted in a 57% reduction in BV/TV (P < 0.001), while particle-injected rats that were treated with sclerostin antibody had 4.1-fold greater BV/TV (P < 0.001) than particle-injected rats not receiving the antibody. Even compared with control rats, the particle-injected rats that had been treated with sclerostin antibody had 1.8-fold greater BV/TV (P < 0.001). A detailed examination of the 3-dimensional depictions of the peri-implant trabecular bone showed similar positive results (Table 1). These effects on bone architecture were apparent radiographically (Figure 2). Specifically, contact radiography showed that peri-implant radiolucent regions were present around the distal one-third of the implant in 8 of 12 rats in which LPS-doped polyethylene particles had been injected into the knee joint, but treatment with sclerostin antibody reduced the incidence to 2 of 12 rats (75% versus 16.7%; P = 0.036), the same proportion observed in the control rats. Three-dimensional renderings of the trabecular bone surrounding the distal one-third of the implant showed obvious differences that corresponded to the quantitative findings (Figure 2).

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Figure 2. Effect of sclerostin antibody treatment on the bone architecture of control rats (A and D), rats that received intraarticular injections of polyethylene particles and subcutaneous injections of vehicle (B and E), and rats that received intraarticular injections of polyethylene particles and subcutaneous injections of sclerostin antibody (C and F). Representative images show the lateromedial view of contact radiographs obtained on the same radiographic plate (A–C) and 3-dimensional trabecular bone renderings from micro–computed tomography (D–F) in the peri-implant region at the distal end of the femur. Note that the implant is not shown in the 3-dimensional reconstructions. Bars = 1 mm. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Effect of sclerostin antibody treatment on bone formation and bone resorption.

Inspection of micro-CT cross-sections showed that the particle injections led to depressed peri-implant bone volume, but that treatment with sclerostin antibody blocked this effect (Figures 3A–C). Histologically, the injection of particles led to the loss of trabecular bone, but contemporaneous treatment with sclerostin antibody in rats that received particle injections led to increased trabecular bone even in comparison with the control rats that received no particles (Figure 3D–F). Single and double fluorochrome labeling, a direct histologic marker of bone formation, was more apparent and the distance between double labels was greater in particle-injected rats that received sclerostin antibody than in particle-injected rats that did not receive sclerostin antibody and control rats that received neither particles nor sclerostin antibody (Figures 3G–I).

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Figure 3. Effect of sclerostin antibody treatment on bone formation and resorption in control rats (A, D, and G), rats that received intraarticular injections of polyethylene particles and subcutaneous injections of vehicle (B, E, and H), and rats that received intraarticular injections of polyethylene particles and subcutaneous injections of sclerostin antibody (C, F, and I). Micro–computed tomography (micro-CT) cross-sectional images (A–C), corresponding histology images with basic fuchsin and toluidine blue staining (D–F), and high-magnification fluorochrome double- labeled images were obtained from different sections to illustrate the characteristic differences in interlabel width between groups (G–I). Bars in C and F = 500 μm; bar in I = 100 μm.

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The quantitative dynamic histomorphometry data supported these observations (Figure 4). Specifically, compared with control rats, rats that received particle injections plus vehicle had 18% lower MS/BS (P = 0.123), 31% lower MAR (P < 0.001), 40% lower BFR/BS (P = 0.028), and 67% higher ES/BS (P < 0.001). Compared with particle-injected rats treated with vehicle, particle-injected rats treated with sclerostin antibody had 1.8-fold higher MS/BS (P = 0.003), 2.1-fold higher MAR (P < 0.001), 3.4-fold higher BFR/BS (P < 0.001), and 37% lower ES/BS (P < 0.001). Compared with control rats, particle-injected rats that received sclerostin antibody treatment had 1.5-fold higher MS/BS (P = 0.001), 1.4-fold higher MAR (P < 0.001), and 2.1-fold higher BFR/BS (P < 0.001); the difference in ES/BS between these 2 group was not significant (P = 0.375).

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Figure 4. Mineralized surface/bone surface (MS/BS) (A), mineral apposition rate (MAR) (B), bone formation rate (BFR) (C), and eroded surface/bone surface (ES/BS) (D) in peri-implant trabecular bone. Bars show the mean ± SD. Horizontal lines indicate significant differences between groups (P < 0.05). See Figure 1 for other definitions.

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Correlation between the static and dynamic assessments of bone and implant fixation.

The mechanical attachment of the implant to the host bone was significantly correlated with the static and dynamic parameters depicting bone structure and remodeling (Table 2). For example, pull-out strength, energy to failure, and energy to yield were all significantly correlated with MS/BS, MAR, BFR/BS, BV/TV, and the variables describing trabecular architecture. The strongest correlation was between implant fixation strength and TbSp (ρ = −0.716, P < 0.01). Implant fixation strength was also significantly negatively correlated with ES/BS (ρ = −0.402, P = 0.02).

Table 2. Correlations between static and dynamic assessments of bone and implant fixation*
Assessment (no. of rats assessed)Strength, N/mmEnergy to failure, NmmStiffness, N/mm
  • *

    Values are Spearman's correlation coefficients.

  • P < 0.01.

  • P < 0.05.

Bone volume/total volume (36)0.7030.6700.196
Trabecular number (36)0.7020.6590.186
Trabecular thickness (36)0.4680.5070.072
Trabecular separation (36)−0.716−0.651−0.215
Bone surface/bone volume (36)−0.471−0.496−0.087
Connective density (36)0.6300.5280.238
Structure model index (36)−0.609−0.537−0.210
Mineralized surface/bone surface (33)0.4870.5220.048
Mineral apposition rate (33)0.6730.5390.273
Bone formation rate/bone surface (33)0.5900.5360.141
Eroded surface/bone surface (33)−0.402−0.222−0.213

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

To our knowledge, the current study is the first to demonstrate the effects of a bone-forming agent for preventing particle-induced implant loosening. In the control groups, LPS-doped polyethylene particles decreased implant fixation strength and peri-implant bone volume, which is consistent with the findings of an earlier independent study (17). Here, we show that the tissue-level mechanisms included suppressed bone formation and increased bone resorption. Systemic administration of sclerostin antibody completely blocked the local negative effects of the particles on implant fixation by enhancing bone formation and inhibiting bone resorption.

Most studies of the prevention of periprosthetic osteolysis or aseptic loosening have focused on anticatabolic agents, especially bisphosphonates (4, 5, 21–24). Other therapeutic agents that have been assessed in various in vivo models include RANKL antagonists (7, 25, 26), tumor necrosis factor (TNF) antagonists (27, 28), interleukin-1 (IL-1) receptor antagonist, IL-6 and IL-10 (29–32), erythromycin (33), osteogenic protein 1 (OP-1) (34), hydroxymethylglutaryl-coenzyme A reductase inhibitor (35), α-calcitonin (36), substance P (37), and pan-caspase (38). Of these previous studies, only the study with OP-1 included an anabolic agent. Those investigators observed that OP-1 appeared to increase the amount of trabecular bone present but did not significantly change bone formation (34).

The implant pull-out data obtained in the current study demonstrated that the group of rats that received particles only had significantly lower implant fixation strength than both the control group and the group that received particles and sclerostin antibody. This finding indicates that intraarticular injection of the polyethylene particles led to implant loosening, as previously observed in similar models (17, 39). The novel finding is that sclerostin antibody, when given contemporaneously with the polyethylene injections, completely abrogated the negative effects of the particles and prevented implant loosening.

Here, we report the effect of particles on peri-implant bone remodeling dynamics. Our findings provide insight into the tissue-level mechanisms underlying the negative effects of the particles on implant fixation and how sclerostin antibody treatment abrogated those negative effects. A previous study by our group showed that injection of LPS-doped polyethylene particles induced differences in static parameters (17), and we now report that bone mineral apposition and bone formation rates are depressed in the presence of particles, indicating that the particles inhibited the activity of osteoblasts in peri-implant trabecular bone. In addition, the finding of a decreased mineralized surface may indicate depressed osteoblastogenesis in the peri-implant bone microenvironment. These findings are consistent with current knowledge about the effects of particles from in vitro (40) and in vivo (41) work showing suppressed expression of type I collagen by osteoblasts. Interestingly, it was recently reported that particles increase the expression of sclerostin messenger RNA (42), which could also account for depressed bone formation.

Studies have also shown that wear particles stimulate the production of bone-resorptive cytokines (3) and increase the number of osteoclast-like cells (43); this is consistent with our findings of an increase in the eroded bone surface in rats receiving LPS-doped polyethylene particles. This increase in bone resorption was blocked by treatment with sclerostin antibody. Thus, treatment with sclerostin antibody reversed the biologic effects of particles on both sides of the bone remodeling equation (resorption and formation).

The interpretation that changes in bone remodeling dynamics in the peri-implant region account for the beneficial effects of sclerostin antibody treatment on implant fixation is further supported by the correlations of dynamic and static measurements of bone adjacent to the implant with the mechanical end points. Thus, the LPS-doped polyethylene particles induced lower mechanical fixation strength by stimulating bone resorption and inhibiting bone formation, resulting in derangements in the peri-implant bone architecture. The sclerostin antibody treatment strategy used in the current study completely blocked these negative effects of the polyethylene particles.

In the present study, we examined a prevention scenario, because antibody treatment was contemporaneous with administration of the particles. The study does not address the question of whether or not the antibody would have similar effects if the onset of its administration had been delayed. In addition, it is possible that the lack of loosening in the antibody-treated group was attributable to sclerostin antibody–enhanced bone formation around the implant, preventing ingress of particles along the interface and thus removing an important resorption stimulus. If that is the correct interpretation, the study findings are still important, because they would then provide further evidence that early enhancement of peri-implant bone formation is an effective strategy to guard against particle-induced osteolysis (44).

Clinically, periprosthetic osteolysis is a particle-induced inflammatory disease related to net bone loss that often leads to implant loosening. Some inflammatory cytokines, such as IL-1, IL-6, TNF, and RANKL, have been shown to play a key role in this inflammatory reaction (3, 45). Some of these cytokines are also known to participate in rheumatoid arthritis (another bone-related inflammatory disease) and have been targeted for therapy (46). Sclerostin antibody has shown the ability to down-regulate some inflammatory cytokines, such as TNFα (47) and RANKL (48), suggesting a potential effect on reversing inflammation. In addition, many patients with inflammatory bowel disease, such as colitis, have osteopenia (49). One study has shown that sclerostin antibody can prevent bone loss in an animal model of inflammatory bowel disease (50).

The results of this study indicate that sclerostin antibody is effective in preventing particle-induced implant loosening. It remains to be determined whether treatment with this antibody can slow or reverse the progression of established osteolysis.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Sumner had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design.Liu, Virdi, Sena, Sumner.

Acquisition of data.Liu, Virdi, Sena, Sumner.

Analysis and interpretation of data.Liu, Virdi, Sena, Sumner.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Lou Fogg, PhD, for statistical advice and Dana Glock, Julie Brown, and David Karwo for technical assistance. Dr. Thomas Wronski provided guidance on performing the dynamic and static histomorphometry. The Rush University Medical Center MicroCT/Histology Core provided access to equipment.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES