REVIEW

Aseptic loosening
PH Wooley
1,3
and EM Schwarz
2,4
1
Department of Orthopaedic Surgery, Wayne State University School of Medicine, Detroit, MI, USA; and
2
The Center
for Musculoskeletal Research, Department of Orthopaedics, University of Rochester Medical Center, Rochester, NY, USA
Although total joint replacement surgery is one of the most
successful clinical procedures performed today, bone loss
around knee and hip implants (osteolysis), resulting in
aseptic loosening of the prosthesis, remains a major problem
for many patients. Over the last decade much has been
learned about this process, which is caused by wear debris
particles that simulate a local inammatory response and
osteoclastic bone resorption. Aseptic loosening cannot be
prevented or treated by existing nonsurgical methods. Gene
transfer, however, offers novel possibilities. Here, we review
the current state of the eld and the experimental gene
therapy approaches that have been investigated toward a
solution to aseptic loosening of prosthetic implants.
Gene Therapy (2004) 11, 402407. doi:10.1038/sj.gt.3302202
Published online 15 January 2004
Keywords: prosthesis; bone; inammation; osteoclast; osteolysis
Introduction
Degenerative and inammatory arthritis are disabling
conditions with a high incidence in the general popula-
tion. Orthopedic surgical procedures have proven highly
successful in the treatment of severe joint destruction
secondary to degenerative and inammatory arthritis,
and the number of orthopedic surgical procedures
continues to increase annually. Total hip and knee
replacement surgery usual results marked symptomatic
relief with substantial functional improvement. How-
ever, the benets of prosthetic implants must be
considered in the light of a number of possible
complications. While failure due to sepsis, fracture, and
dislocation have become relatively rare, failure second-
ary to aseptic loosening has become increasingly more
important, and appears likely to increase in prevalence.
It is estimated that over 25% of all prosthetic implants
will demonstrate ndings of aseptic loosening,
14
often
leading to the need for surgical revision. Initially, the
term cement disease was coined to characterize aseptic
loosening.
5
However, it is now known that any type of
particulate debris, including that generated by poor
surgical technique, loss of mechanical xation of the
polymethylmethacrylate (PMMA) bone cement, or wear
at the ultra-high molecular polyethylene (UHMWPE)
metal interface may lead to the aseptic loosening process.
Fragmentation of PMMA and abrasion of polyethylene
may act synergistically, since PMMA debris particles
may become entrapped in the articular surface, creating
polyethylene particles from the resulting wear of the
UHMWPE implant.
6
This wear debris appears to
provoke diverse biological effects, including granuloma
formation, inammatory cell inux, bone resorption, and
eventually osteolysis and loss of the prosthesis support.
7
Pathogenesis of aseptic loosening
The precise etiology of aseptic loosening still remains
unclear. The tissue from the areas of osteolysis shows
a synovial-like layer at the cement surface and the
presence of macrophages and foreign-body giant cells
invading the femoral cortices. The appearance of the
periprosthetic membrane at the cement/bone interface
shares some histological characteristics of both rheuma-
toid arthritis (RA) and a foreign body reaction.
8
Micro-
scopic examination of specimens obtained at revision
surgery of failed hip replacements has revealed a varied
cellular composition of the periprosthetic membrane,
with histiocytes, giant cells, lymphocytes, plasma cells
and neutrophils, although quantitative analysis of cell
types present in this tissue shows heterogeneity between
different individuals.
9,10
Areas around the loosened,
cemented stems were frequently characterized as ag-
gressive granulomatous lesions consisting of well-
organized tissue containing histiocyticmonocytic and
broblastic reactive zones. Furthermore, immunohisto-
logical evaluation has revealed the presence of multi-
nucleated giant cells and C3bi-receptor bearing
phagocytic cells.
11
Particles of acrylic cement and shards
of polyethylene appear incorporated into the histiocyte/
macrophage or giant cell population, resulting in foci of
cellular activity within the periprosthetic membrane.
12,13
Phagocytes in the tissue adjoining the implant site engulf
small particles, and since plastics and metal are
impervious to enzymatic destruction, these particles
frustrate the degradative function of these cells.
Repeated phagocytosis of particles results in activated
cells that secrete proinammatory cytokines and proteo-
lytic enzymes, both of which are known to both damage
bone and cartilage, and activate immune cells. In
particular, interleukin 1 (IL-1) and tumor necrosis factor
Correspondence: PH Wooley, Department of Orthopaedic Surgery, Wayne
State University School of Medicine, 4707 St Antoine Blvd, Detroit, MI
48201, USA
3
PW is supported by a VA Merit Award and a grant from OREF
4
ES is supported by NIH PHS Grants AR45971, AR46545
Gene Therapy (2004) 11, 402407
& 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00
www.nature.com/gt
(TNFa) are potent mediators of bone resorption,
1418
and
recently, the immune-activating cytokines PDGF and IL-
11 have also been identied within the periprosthetic
tissue.
19,20
These cytokines provide activation signals to
lymphocytes,
21
and in turn, the lymphocyte-derived
cytokines interleukin 2 (IL-2), interleukin 6 (IL-6) and
gamma interferon (IFN-g) may inuence osteoclastic
activity and bone remodeling.
22
Studies using an in vitro
osteoclast model have suggested that the response to
PMMA may directly contribute to the process of bone
resorption.
23
UHMWPE wear has been signicantly
correlated with frequency and size of osteolytic lesions
24
and may also act to inhibit biosynthetic pathways of
bone and mesenchymal tissue.
25
Macrophages induced
to phagocytose biomaterials (UHMWPE, PMMA and
orthopedic alloys) differentiated into osteoclastic cells
capable of extensive lacunar bone resorption during
bone culture
26,27
and also express high levels of the
osteoclast activating cytokine M-CSF (CSF-1).
28
Both
UHMWPE and titanium (Ti-6-4) particles signicantly
enhanced in vitro macrophage cytokine (TNFa, IL-1, IL-
8) release in a dose-dependent manner,
29
and explant
cultures of periprosthetic membranes and synovial tissue
derived from osteoarthritic patients undergoing revision
for cemented hip implant failure produce IL-1, TNFa,
IL-8, prostaglandin E2 and nitric oxide,
9,3033
suggesting
immuno-inammatory activity within the implanted
joint. The nature of the dominant wear particle may be
critical to the response in an individual patient, since
cobalt chromium (Co-Cr) may result in a dominant TNFa
release over IL-1 or IL-6 production,
34
while Ti-6-4
particles appear to mediate an IL-6 response.
3537
Osteolysis in aseptic loosening
The release of inammatory mediators results in chronic
inammation and tissue damage that deteriorate the
supporting bone, with adverse effects on prosthesis
xation.
38,39
Normally, bone mass is determined by the
delicate balance that exists between formation and loss.
Osteoblastic cells of mesenchymal origin synthesize and
deposit bone matrix and increase bone mass. Osteoclastic
cells are large, multinucleated phagocytes of hemato-
poietic origin that resorb bone upon activation. Recent
advances in bone biology have focused studies on the
pathogenesis of aseptic loosening beyond the inuence
of the classical inammatory cytokines in response to
wear debris. Two important regulators of osteoclast
differentiation and activation have been identied;
RANKL
4042
and osteoprotegerin (OPG).
43
RANKL, also
known as OPGL
44
and ODF,
45
is a TNF-related cytokine
that is produced by marrow stromal cells and osteo-
blasts
41,42
Mice lacking RANKL have defects in osteo-
clastogenesis that leads to severe osteopetrosis.
46
OPG is
a secreted TNF receptor (TNFR)-related protein that
binds to and neutralizes RANKL bioactivity. Mice
lacking OPG develop severe osteoporosis and have
brittle bones that spontaneously fracture.
47
These data
indicate that OPG and RANKL interact as key regulators
of osteoclastogenesis and bone resorption.
41,42
RANKL
binds to RANK (receptor activator of nuclear factor
kappa B),
48
a member of TNF receptor super family,
which is present on osteoclasts and their precursors
49
and initiates osteoclastogenesis after ligation with
RANKL or agonistic anti-RANK antibodies.
4042,48,50
Dougall et al
51
have determined an essential role of
RANK in regulating osteoclastogenesis in RANK gene
knockout (RANK
/
) mice. Li et al
52
reported that
osteoclastogenic effects of RANKL cannot be reproduced
in RANK
/
mice suggesting that RANK serves as the
sole osteoclast receptor for RANKL and controls osteo-
clast development and activation.
5354
In addition, failure
of RANK
/
mice to mount a signicant osteoclastic
response in the presence of experimental inammatory
arthritis further supports the concept that RANK plays a
gate-keeping role in controlling osteoclastogenesis.
46
As part of the inammation process, genes of
cytokines, chemokines, growth factors, cell adhesion
molecules, and some acute-phase proteins will be turned
on and frequently overexpressed. Part of this genetic
regulation is controlled by nuclear factor kappa B (NF-
kB).
5557
It is becoming evident that NF-kB is a key factor
for the development of osteoclastogenesis. Two recent
studies
58,59
found that the double-knock out of NF-kB1
and NF-kB2 in mice resulted in an unexpected pheno-
type. These animals showed an impaired macrophage
functions and failed to generate mature osteoclasts,
which resulted in severe osteopetrosis. Additional
studies suggested that this abnormality occurred as a
result of a defect either during osteoclast precursor cell
differentiation or during the maturation of osteoclasts.
Obviously, this double knockout experiment could
provide a relevant model to study the etiology of
osteoporosis and raise new directions in regulation of
osteoclastogenesis.
58
It is possible that the osteoclasto-
genic activity of several cytokines, such as TNF, IL-1
might be ameliorated by prevention of NF-kB binding to
their respective binding sites of the promoter sequence.
Regulation of NF-kB activation represents a powerful
therapeutic strategy for reducing inammatory tissue
damage, but complete and persistent blockage of NF-kB
activation could lead to immune deciency and cell
death. Glucocorticoids are widely prescribed immuno-
suppressive and anti-inammatory drugs, whose anti-
inammatory effects are achieved either by glucocorti-
coid receptor-mediated interference of NF-kB DNA-
binding activity or by enhanced synthesis of IkB, which
would compromise the nuclear translocation and DNA
binding of NF-kB.
60,61
The liberal use of glucocorticoids,
however, is limited because of their well-known side
effects on endocrine function and metabolism. A more
rational and promising strategy to block NF-kB activa-
tion is the development of compounds that potentially
targets a specic point in the signaling pathway without
inuencing other cellular biological functions.
Animal models
A prerequisite for a therapeutic or gene therapy is the
development of an animal model that recapitulates the
etiology or some salient features of the human disease.
For obvious reasons, the early models of aseptic loosen-
ing involved an implant in the rat
62
and dog.
63
However,
once an implant is integrated into bone to achieve true
xation, clinical aseptic loosening takes at least 5 years.
This shortcoming is also true in the animal models, as
demonstrated by a modied rat model that incorporated
a running wheel for 2 h/day for 5 days/week.
64
Despite
Aseptic loosening
PH Wooley and EM Schwarz
403
Gene Therapy
the increased mechanical load, which contributed to the
development of a periprosthetic membrane, there was no
evidence of implant loosening after 6 months.
Based on the limitations of the implant models,
investigators have decided to focus on the biology of
wear debris-induced osteolysis independent of the
critical mechanical and biomechanical components of
aseptic loosening. To this end, two mouse models have
been developed that permit the use of exquisite
molecular reagents and genetically dened strains,
together with highly quantitative outcome measures of
osteoclast formation and bone resorption. The rst is the
air pouch model in which particulate wear debris is
coimplanted with a bone substrate into a subcutaneous
space.
65
The wear debris stimulates an inammatory
reaction that culminates in the genesis of an erosive
tissue that is similar to a periprosthetic membrane. A
murine calvaria model has also been established that
involved the surgical implantation of wear debris
particles on top of the skull.
66,67
Again, the wear debris
induces an intense inammatory reaction that leads to
osteoclastic formation and osteolysis within 1 week. By
utilizing mice genetically defective in TNF
68
and RANK
69
signaling, the rst in vivo evidence in support of the
proinammatory osteolysis as the causes of aseptic
loosening was generated. Furthermore, similar ndings
from studies in which wild-type mice were treated with
TNFR:Fc
70
and RANK:Fc
69
demonstrated the potential of
a therapeutic agent or gene therapy for aseptic loosening.
Gene therapy
It has been proposed that pharmacological intervention
targeted towards the macrophage may provide a means
to slow the response to wear debris.
71
However,
delivering adequate levels of cell-specic therapy to the
site of periprosthetic inammation, without undesirable
systemic effects, represents a considerable challenge.
Local cytokine inhibition is a potential therapy that may
reduce inammation in the periprosthetic tissue, and
several biological mediators have known been identied
as useful for clinical applications. In particular, IL-1
receptor antagonist protein (IL-1Ra or IRAP) has been
successful in reducing inammation,
72,73
and the anti-
inammatory cytokine IL-10 appears to possess the
capacity to reduce cell-mediated reactions in inamma-
tion.
74,75
The delivery of appropriate doses of proin-
ammatory cytokine inhibitors to the periprosthetic
tissue remains a problem. However, recent advances in
gene therapy techniques
76,77
suggest that viral vectors
may be capable of delivering anti-inammatory cytokine
genes to the periprosthetic tissues, which could control
the local reaction and extend the life of the prosthesis. We
have conducted experiments using the particle-stimu-
lated murine air pouch model to evaluate the potential
for gene therapy to treat inammation provoked by
orthopedic wear debris. In our initial study,
65
retroviral
vectors encoding human IL-1Ra and the human tumor
necrosis factor receptor (TNF-R) were evaluated using
this in vivo model. Air pouches established in BALB/c
mice were injected with PMMA particles to provoke an
inammatory reaction, and transduced with contrakine
genes or control retroviruses. Pouch membranes and
uids were harvested after 48 h, and retroviral insertion
and contrakine gene incorporation were assessed in
DNA from pouch membranes using polymerase chain
reactions (PCR). ELISA assays were conducted to
determine the level of contrakine and inammatory
cytokine production. Image analysis performed on
pouch membrane sections to evaluate thickness of the
air pouch membrane and the magnitude and morphol-
ogy of the cellular inltrate. Positive PCR reactions for
IL-1Ra and neo genes were observed in DNA extracted
from the membrane of retroviral-infected pouches, while
DNA extracted from either inammatory cells present in
pouch uid, or control pouch membranes, remained
negative for either gene. This result suggests that this
replication-decient vector may achieve incorporation at
a local site without widespread infection of the evolving
inammatory inltrate, an important observation when
considering this approach for clinical use. ELISA assays
revealed the presence of human IL-1Ra in pouch uid
from DFG-IRAP-neo transduced mice, but not control
animals. Histological evaluation indicated that the IL-
1Ra gene transfer was associated with markedly
decreased inammation in the model, with resolution
of the edematous phase of the reaction, decreased pouch
uid accumulation, and lowered macrophage inux. The
results suggest that the air pouch model represents a
useful tool to evaluate gene therapy, and demonstrate
that IL-1Ra gene therapy may be an appropriate
therapeutic approach to inammation.
Similar studies have been performed to evaluate the
effects of TNF-R gene therapy in the calvaria model
using a recombinant adenoviral vector.
78
While the
experiments demonstrated the predicted antiresorptive
effects in nude mice, the study was highlighted by two
additional ndings. The rst was that local gene delivery
was not signicantly more efcacious than systemic gene
therapy, and that efcacy was correlated to the concen-
tration of TNFR:Fc/ml in serum. However, the more
important nding was the tremendous inammatory
reaction from the adenoviral vector alone, which induced
an osteolytic response that signicantly exceeded that of
the wear debris when placed directly on the calvaria of
wild-type mice. This nding is consistent with many
previous studies regarding the immunogenicity of
adenoviral vectors and warns against its clinical use.
We have extended this research to evaluate viral IL-10
(vIL-10) in these models.
79,80
In the air pouch study, an
investigation of mRNA expression using real-time PCR
was conducted to determine whether anti-inammatory
gene therapy affects cytokine gene expression. IL-1, IL-6
and TNF gene expression were readily detected by the
technique, and IL-1 was observed to be the predominant
cytokine expressed in response to UHMWPE particle
stimulation. There were only low amounts of IL-4, and
IL-10 expressed, and IL-2, IL-5 and IFNg were not
detectable. The transfer of the viral IL-10 gene signi-
cantly diminished IL-1 mRNA expression at all time
points investigated, and TNFa and IL-6 mRNA levels in
pouch membranes following viral IL-10 or IL-1Ra
transduction was also signicantly decreased. Interest-
ingly, a signicant higher expression of TGF was
detected in the pouches with IL-1Ra gene insertion, but
no effects of IL-1Ra and vIL-10 gene transfer on IL-4 and
mouse IL-10 expression was observed. Histological
assessments revealed that pouches transduced with
vIL-10 or IL-1Ra exhibited a 40% reduction in inam-
Aseptic loosening
PH Wooley and EM Schwarz
404
Gene Therapy
matory cell inltration when compared with nonviral
controls or LacZ-transduced membranes. Further, ester-
ase staining indicated that both vIL-10 and IL-1Ra gene
therapies dramatically decreased macrophage presenta-
tion in the pouch membranes. The calvaria study
corroborated these ndings by independently demon-
strating that gene delivery of vIL-10 inhibits three
processes critically involved in periprosthetic osteolysis
wear debris-induced proinammatory cytokine produc-
tion, osteoclastogenesis, and osteolysis.
Encouraged by our ndings that anti-inammatory
gene therapy appears to have ameliorating effects on
particle-induced inammation, we have recently ex-
tended this approach to the inhibition of osteoclasto-
genesis. There are reports that inammatory osteoclas-
togenesis can be blocked by neutralizing IL-1/TNF
bioactivity both in vivo
8183
and in vitro.
84
An emerging
concept is that RANK may act as a common mediator for
cytokines relevant osteoclastogenesis.
41,52
In supporting
this concept, a recent study
52
demonstrated that no
osteoclastogenesis and bone resorption can be induced in
RANK
/
mice when treated with IL-1, as shown by the
complete absence of TRAP

and cathepsin K

osteo-
clasts in their calvaria. Therefore, the osteoclastogenic
activity of IL-1 is mediated exclusively through RANK.
The interaction between TNF and RANK is complex.
One recent study showed that TNF induced osteoclasto-
genesis needs the assistance of basal amount of RANKL,
perhaps through their synergistic effects on activation of
NF-kB, a signaling pathway essential for osteoclastogen-
esis.
85
Because a small amount of RANKL is sufcient to
synergize with TNF to promote osteoclastogenesis, TNF
may be a more convenient clinical target than RANKL in
arresting inammatory osteoclastogenesis. Another re-
port also demonstrated
52
that the rare appearance of
TRAP

and cathepsin K

osteoclasts on the bone

surface could be induced in the calvaria of RANK
/
mice treated with TNF at high dose (1.0 mg/kg body wt
per day). However, the osteoclast-like cells are occasion-
ally found within the calvaria near the site of adminis-
tration. These data raised the possibility that TNF might
trigger an alternative compensatory pathway leading to
osteoclast formation in the absence of RANK, presum-
ably by activation of either TNFR1 or TNFR2. Since TNF
and RANKL are considerably overlapping in their
signaling pathway,
86
we postulated that during aseptic
loosening, RANKL and TNF might synergistically
orchestrate enhanced osteoclastogenesis via cooperative
mechanisms. To investigate the potential of the RANKL
system as a therapeutic target in aseptic loosening, we
investigated whether an in vivo gene transfer of OPG
using an adeno-associated virus (AAV)-vector exerted
protective effects against orthopedic wear debris-in-
duced bone loss in a murine model of osteolysis.
87
Bone
tissue was implanted into established pouches on BALB/
c mice, followed by the introduction of ultra-high
molecular polyethylene (UHMWPE) particles to provoke
inammation and osteolysis. The viruses encoding hu-
man OPG gene (rAAV-hOPG) or b-galactosidase marker
gene (rAAV-LacZ) were injected into the air pouches, and
the tissue harvested 7 days after viral infection for
histological and molecular analyses. Successful trans-
gene expression was conrmed by the detection of OPG
by ELISA and positive X-gal staining of pouch tissue
(LacZ). Real-time PCR indicated signicant diminish-
ment of mRNA expression of osteoclast markers in OPG-
transduced pouches compared with rAAV-LacZ-trans-
duced pouches. The transduction and expression of OPG
also markedly decreased the gene copies of the biological
receptor for osteoclast differentiation factor (RANK). The
expression of OPG in the bone-implanted pouch reduced
bone calcium release by a mean of 39% compared with
the calcium release in other two groups. Computerized
image analysis revealed that expression of OPG sig-
nicantly protected against bone collagen loss. We also
evaluated the effects of ex vivo
88
and in vivo
89
OPG gene
therapy in the calvaria model of wear debris-induced
osteolysis. Again the results were similar to the data
obtained in the air pouch model and support the
hypothesis that RANKL is the nal effector of osteoclas-
togenesis and bone resorption. These preliminary results
provide encouraging preclinical data for OPG mediated
therapy in aseptic loosening, and overall, the data
suggest that gene therapy may be an appropriate
technique to provide local anti-inammatory and anti-
bone resorptive activity at the prosthesisbone interface.
Future directions
Signicant evidence now exists indicating that aseptic
loosening of orthopedic implants is caused by wear
debris-induce inammation, osteoclastogenesis and sub-
sequent osteolysis around the prosthesis. Additionally,
several preclinical studies have demonstrated the ef-
cacy of contrakine and anti-osteoclastic gene therapy
approaches in small animal models. Future studies
aimed at evaluated these vectors in large animal models
(eg canine) with quantitative longitudinal radiologic
outcome measures and biomechanical testing are war-
ranted.
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