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Mediates Orthopedic Implant Osteolysis
From the Departments of Orthopedic Surgery*
and
Pathology,
Washington University School of
Medicine, St. Louis, Missouri
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF) is
a potent osteoclastogenic agent while at the same time is the only
soluble moiety known to be c-src inductive suggests that this cytokine
may mediate implant particle-induced osteoclastogenesis. Consistent
with this hypothesis, prosthesis-derived wear particles,
recovered at revision arthroplasty, dose-dependently prompt TNF
secretion by BMMs. Similarly, particulate
polymemthylmethacrylate, the major component of orthopedic
implant cement, induces BMM expression of TNF mRNA and protein
in a time- and dose-dependent manner. Furthermore, failure of
BMMs derived from mice deleted of both the p55 and p75 TNF receptors to
express c-src in response to polymemthylmethacrylate indicates TNF is
an essential mediator of particle induction of this osteoclast specific
protein. To test the hypothesis that TNF mediates implant
osteolysis, we established an in vivo murine
model of this condition that histologically mirrors that of man.
Verifying that TNF is essential to development of particle
osteolysis, mice failing to express both the p55 and p75 TNF
receptors are protected from the profound bone resorption attending
polymemthylmethacrylate particle implantation on calvariae of wild-type
animals. Finally, the protective effect of deletion of both TNF
receptors is recapitulated in mice lacking only the p55 receptor.
Thus, targeting TNF and/or its p55 receptor may arrest wear
particle osteolysis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The osteolytic process attending prosthetic loosening is associated
with formation, at the implant-bone interface, of a cellular
"membrane" containing abundant tissue macrophages, many of which
have ingested implant-derived wear particles such as
PMMA.5-9
Several macrophage-produced
osteoclastogenic cytokines, including interleukins-1 and -6 and tumor
necrosis factor- (TNF) are present in the tissue surrounding failed
implants6,10-12
and thus serve as potential
mediators of wear particle-induced osteolysis. Hence, the most likely
scenario holds that implant wear particles promote an inflammatory
reaction consisting largely of fibroblasts and activated macrophages.
Secretory molecules of the activated cells recruit osteoclasts, which
are ultimately responsible for periprosthetic bone degradation. Thus,
identification of proinflammatory cytokine(s) mediating the
osteoclastogenesis of implant osteolysis is central to prevention of
this complication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four- to 6-week-old lipopolysaccharide-resistant C3H/HeJ mice (Harlan Sprague-Dawley Inc., Indianapolis, IN) were used. Transgenic mice include: 1) mice in which the p55TNF receptor gene has been deleted13 (provided by Dr. Warner Lesslauer (Hoffman-LaRoche)); 2) those in which both the p55TNF and p75TNF receptor genes have been deleted by interbreeding dominant negative p55TNF receptor knockout mice with their counterparts from p75TNF receptor knockout mice (provided by Dr. Mark Moore (Genentech, Inc., South San Francisco, CA)); and 3) their respective wild-type mice.
Particles
Wear particles were isolated from periprosthetic tissue obtained during revision surgery by a technique similar to that described by Campbell et al.14 Briefly, tissue was minced, papain digested at 65°C for 24 hours, and centrifuged on a discontinuous sucrose gradient at 28,000 rpm at 4°C for 4 hours. The topmost layer was washed and placed under 10 ml each of 0.96 g/cm3 and 0.90 g/cm3 isopropanol and recentrifuged at 25°C for 2 hours. Particles recovered at the air/liquid interface were counted in a hemocytometer. FACS analysis documents 90% of recovered particles are 20 µm or less in diameter. Analysis of the particles by Fourier transform infrared spectroscopy demonstrates the presence of peaks coincident with those obtained with commercially prepared polyethylene (now shown). Commercially prepared PMMA particles, in the form of bone cement, were obtained from Wright Medical Technologies (Arlington, TN) with 50% of particles being 10 µm or less in diameter.
Particle Induction of c-src and TNF
Osteoclast precursors, in the form of bone marrow macrophages
(BMMs), were obtained from long bone marrow.15
Cells were grown to confluence in -MEM/10% fetal calf serum
containing 1000 U/ml M-CSF. TNF protein was measured in
conditioned media using a commercial enzyme-linked immunosorbent assay
(ELISA) (Genzyme, Cambridge, MA). Particle-exposed total BMM RNA was
isolated, and TNF mRNA was detected by Northern analysis using a cDNA
probe to bp 389-1180 primers. 18S ribosomal mRNA was used as a loading
control. In a similar manner, BMMs were cultured with particles
(108
particles/ml) for 24 hours, and cell
associated c-src was determined by immunoblot using anti-c-src
monoclonal antibody 327 (provided by Dr. Andrey Shaw, St. Louis,
MO).16
Transfection of J774A.1 Cells
The evening before transfection, 0.5 x 106 J774A.1 cells (ATCC T1B67) were added to each well of 6-well plates ± 2.0 mg/ml PMMA.17 Cells were washed with 1 ml of OptiMEM (Life Technologies, Inc.), and medium was replaced with 0.8 ml of OptiMEM. The TNF promoter/reporter construct, TNF(-1059)chloramphenicol acetyl transferase (CAT) (provided by Dr. Bruce Beutler, Dallas, TX) was co-transfected with a luciferase reporter plasmid, pMB3–675Luc, as a control for transfection efficiency. pMB3–675Luc contains the first 675 bp of the murine integrin ß3 promoter18 and is not significantly affected by PMMA treatment. DNAs (TNF(-1059)CAT, 1.8 µg/well and pMB3–675Luc, 0.2 µg/well) were mixed in 0.1 ml of OptiMEM per well, and in a separate tube, lipofectAMINE (Life Technologies, Inc.) (5 µl/well) was mixed with 0.1 ml of OptiMEM per well. Diluted DNA samples and lipofectAMINE were mixed in equal volumes and incubated at R.T. for 30 minutes after which 0.2 ml of the mixture was added to each well. After 3 hours at 37°C +5% CO2, 1 ml of OptiMEM containing 20% fetal calf serum was added, and cells were incubated at 37°C +5% CO2 for an additional 20 hours. The wells were then washed with 1 ml of phosphate-buffered saline, scraped in 0.5 ml of phosphate-buffered saline, and transferred to 1.5 ml of microfuge tubes. Cells were pelleted in a microfuge at top speed for 30 seconds and resuspended in 50:1 reporter lysis buffer (Promega, Madison, WI). Cells were lysed twice for 20 seconds by vigorous vortexing. Cell debris was removed by microfuging for 1 minute. Twenty µl of extract were assayed for luciferase activity using the luciferase assay kit (Promega) with an Optocomp II luminometer (MGM Instruments). Twenty-five µl of extract were heat inactivated at 60°C for 10 minutes and mixed with 100 µl of 1x reporter lysis buffer containing 3 µl of 14C chloramphenicol (Amersham) and 0.25 mmol/L N-butryl CoA. After 16 hours of incubation at 37°C, CAT activity was determined by xylene extraction and liquid scintillation counting.
Murine Model of Particle-Induced Osteolysis
Fifteen to 20 g B6X127 p55-/-,p75-/-TNFr or p55-/-, p75+/+TNFr mice and wild-type littermates were anesthetized and the scalp incised longitudinally to expose the external cranial periosteum. The periosteum was elevated off the external cortex of the calvarium by sharp dissection. Thirty mg of commercially prepared PMMA particles were placed directly on the surface of the bone. Sham operation consisted of the entire procedure without particle implantation. The wound was then closed with small staples. Animals were sacrificed at 7 days, and the calvaria was excised, fixed, and decalcified in EDTA. Sagittal histological sections of temporal bones, approximately 2 to 3 mm lateral of the midsagittal suture, were stained for tartrate-resistant acid phosphatase (TRAP) activity. Both cortical porosity as an index of net osteolysis and the number of osteoclasts serve as our analytical target.
Statistics
Data are expressed as mean ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-src expression is a specific marker of commitment to the
osteoclast phenotype.19
Thus, our first efforts
to identify the cytokine mediating implant osteolysis were aimed at
determining if prosthetic wear particles induce c-src in marrow
macrophages, cells capable of acting as either mononuclear phagocytes
or osteoclast precursors.20
To this
end, pure populations of murine BMMs derived from lipopolysaccharide
resistant mice were isolated15
and maintained
with or without authentic wear particles extracted from periprosthetic
tissue recovered from patients with failed implants undergoing revision
surgery.14
After 24 hours, the cells were lysed,
and c-src content of equal amounts of protein were determined by
immunoblot. BMMs exposed to patient-derived particles contain
substantially more c-src protein than do nonparticle-exposed BMMs
(Figure 1)
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We have established that TNF is unique among a panel of
osteoclastogenic cytokines in its capacity to induce c-src expression
by mouse osteoclast precursors.19
We also find
that, like the human condition,21
TNF prompts
osteoclastogenesis in cultured murine marrow (data not shown). Because
TNF and wear particles isolated from patients undergoing revision
surgery both induce c-src expression, we reasoned particle-enhanced
synthesis of the proto-oncogene might be a TNF-mediated event.
Therefore, BMMs were exposed for 24 hours to increasing amounts of
recovered wear debris. Measurement of TNF by ELISA (Genzyme, Cambridge,
MA) within culture medium documents secreted cytokine parallels
particle number (Figure 2)
.
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. In a similar experiment, performed
with time, TNF mRNA substantially escalates within 3 hours of PMMA
exposure after which it progressively declines and, by 24 hours,
mirrors nonparticle-treated cells (Figure 4)
. TNF, secreted under the influence of
PMMA, is detectable by 4 hours and, in contrast to the short-lived
message, remains elevated after 24 hours of particle exposure.
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To determine whether particle-induced TNF reflects transactivation
of the TNF gene, we maintained the murine macrophage line J774A.1
overnight with or without PMMA. The cells were then transfected with a
CAT reporter construct (TNF(-1059)CAT) containing the full length
murine TNF promoter22
(provided by Dr. Bruce
Beutler, Dallas TX) and normalized with co-transfected luciferase
construct. After 23 hours, the transfectants were lysed and the lysate
assayed for CAT activity. Pre-exposure of J774A.1 macrophages to PMMA
particles induces the transfected TNF promoter almost fivefold (Figure 5)
. Thus, PMMA particles enhance
macrophage TNF expression by a mechanism involving gene transcription.
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We hypothesized that particle induction of macrophage
c-src is mediated by TNF. To further test the supposition, we turned to
a genetically engineered mouse in which both the p55 and p75 TNF
receptors are deleted (p55-/-
p75-/-TNFr).13
Wild type
(p55+/+p75+/+TNFr) or TNF receptor deleted
(p55-/-p75-/-TNFr) BMMs were cultured with
PMMA particles. After 24 hours the cells were lysed, and equal amounts
of protein probed by immunoblot for c-src content. BMMs from wild-type
mice exposed to PMMA express c-src in a particle-dependent fashion,
whereas the proto-oncogene is minimal and unaltered in
p55-/-p75-/-TNFr BMMs regardless of
particle number (Figure 6)
. Because
p55-/-p75-/-TNFr mice cannot respond to
TNF, the cytokine is essential for PMMA-induced c-src expression
in vitro.
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Our in vitro findings suggest that TNF may be
pivotal to implant osteolysis. To address this issue we developed an
in vivo murine model involving placement of PMMA particles
beneath the external calvarial periosteum and in direct contact with
the sagittal aspects of both temporal bones. After 7 days, histological
sections of temporal bones, 2 to 3 mm lateral to the midsagittal
suture, were stained for TRAP activity, a feature characteristic of
osteoclasts. Calvariae of wild-type mice
(p55+/+,p75+/+TNFr)
exposed to PMMA particles generate an inflammatory, supracranial
response consisting of a fibrous membrane with abundant mononuclear
cells, many of which contain phagocytosed cement particles (Figure 7)
. Importantly, the PMMA-induced
inflammatory membrane contains numerous juxtacortical osteoclasts,
which within 1 week, have resorbed the width of the calvarial bone.
Thus, the histological lesion induced by supracalvarial implantation of
PMMA mimics tissue recovered at revision arthroplasty for prosthetic
loosening.23
Also, similar to the human
situation, numerous cells within the murine lesion contain
immunoreactive TNF (not shown).
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, the lesion developing in
p55-/-p75+/+TNFr mice
recapitulates that appearing in mice lacking both receptors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The osteoclast is a member of the monocyte/macrophage family.24 Its ability to resorb bone depends on expression of the c-src proto-oncogene.25 c-src-/- mice, although abundant in osteoclasts, develop osteopetrosis, a family of sclerotic diseases reflecting dysfunctional bone resorption.26 These observations, taken with the rescue of c-src-/- animals by marrow transplantation26 or overexpression of transfected c-src cDNA,27 indicate that the proto-oncogene is essential for development of normal osteoclasts, specifically regulating their capacity to polarize and resorb bone.25,26 The central role c-src plays in osteoclast function is consistent with the fact that its expression, by macrophages, parallels and is specific to osteoclast differentiation.19,20
We find that particles derived from periprosthetic tissue obtained at revision arthroplasty, as well as PMMA, induce c-src in BMMs, indicating commitment of these cells to the osteoclast phenotype. This observation is in keeping with the capacity of PMMA to prompt osteoclast differentiation by mouse monocytes in vitro.28 Furthermore, macrophages isolated from PMMA-induced foreign body granulomas become osteoclasts when cultured ex vivo.29
Delineating the cytokines responsible for wear particle-induced osteolysis is central to defining the pathogenetic mechanisms of orthopedic implant loosening. We have shown that of a number of inflammatory cytokines, only TNF enhances c-src expression by BMMs.19 In fact, TNF is the only soluble factor known to induce the proto-oncogene. Because TNF is a major product of activated macrophages, we reasoned that BMMs may play a dual role in implant osteolysis, namely as the host defense cell responding to the particle load and as osteoclast precursors. Taken with the capacity of PMMA particles to induce macrophage expression of TNF via transactivation of its gene, these data led us to hypothesize that particle induction of c-src, and ultimately osteoclastogenesis, is mediated by the cytokine. Our most compelling in vitro data supporting this contention is the failure of PMMA to induce c-src in BMMs lacking both TNFrs. Ultimate proof that TNF is central to implant osteolysis comes, however, from development of a in vivo murine model that mimics the human lesion.23 Mice lacking both TNFrs develop an inflammatory response to implanted particles but fail to generate osteoclasts and thus are protected from osteolysis. This experiment establishes the central role played by TNF in the pathogenesis of implant osteolysis.
c-src induction by TNF in BMMs, as well as endotoxin-induced osteoclastogenesis, is mediated by the type-1 or p55 TNFr.19 In contrast, our data indicate that the type-2 or p75 TNFr does not transmit osteoclastogenic signals (unpublished data). These observations prompted us to hypothesize that p55-/-p75+/+TNFr mice, like double receptor deleted animals, would be protected against particle-induced osteoclastogenesis and bone loss. The fact that such is the case indicates that therapy targeting TNF or its p55 receptor may be effective in preventing orthopedic implant loosening. Such strategies are extant in treating conditions such as AIDS and rheumatoid arthritis.30,31 In this regard, we find thalidomide, which accelerates degradation of the mRNA of the cytokine,30 blocks lipopolysaccharide induced c-src, an event mediated by TNF.19 Furthermore, the anti-osteoclastogenic cytokine interleukin-4 blunts particle induction of the TNF gene.32 Therefore, these two agents present themselves as possible inhibitors of implant osteolysis.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from the Orthopedic Research and Education Foundation (K. D. Merkel), the National Institutes of Health Grants #DE05413, #AR32788 (S. L. Teitelbaum), #AR42404 (F. P. Ross), #AR08335 (K. P. McHugh), #AR02035 (K. D. Merkel), and grants from the Barnes-Jewish Hospital Foundation (K. D. Merkel, Y. Abu-Amer).
Accepted for publication October 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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