Abstract
Background: Previous studies of bone resorption around failed joint
replacements have focused on a limited number of cytokines, primarily tumor
necrosis factor-a (TNF-a), interleukin (IL)-1, and IL-6, with use
of enzyme-linked immunosorbent assay and immunohistochemistry techniques. In
this study, we utilized high-throughput protein chips to profile twenty-nine
inflammatory cytokines around failed total joint replacements.
Methods: Peri-implant granulomatous tissues were harvested from
around the failed total hip prostheses of thirteen patients. Synovial lining
capsular tissues from thirteen patients with end-stage degenerative joint
disease were used as controls. After homogenization, twenty-nine cytokines
were quantified with use of high-throughput protein chips.
Results: IL-6 and IL-8 were found consistently in failed joint
replacement tissues, reaffirming their prominent role in osteoclastogenesis
and end-stage bone resorption. High levels of interferon-?-inducible
protein of 10 kDa (IP-10) and monokine induced by interferon-? (MIG),
both chemoattractants of activated Th1 lymphocytes, were also detected.
Soluble intercellular adhesion molecule (sICAM) and transforming growth
factor-ß1 (TGF-ß1) were not detected universally, nor
were TNF-a or IL-1. After a twenty-four-hour organ culture, IL-1ß
levels increased substantially along with those of other mediators. We
measured but did not detect any activators of cytotoxic T-cells,
antibody-producing Bcells, or eosinophils involved in delayed-type
hypersensitivity. Variations from patient to patient were seen across all
cytokines and highlight the unique response of individual patients to their
joint replacements.
Conclusions: In failed total joint replacements in patients with
end-stage osteolysis, IL-6 and IL-8 may be the primary drivers of
osteoclastogenesis. The presence of IP-10 and MIG imply a role for T-cells,
while TGF-ß1 and sICAM may represent a systemic attempt to
modulate the inflammation. TNF-a and IL-1 do not appear to play a major
role in the end stages of the disease.
Clinical Relevance: These results demonstrate that proteomic tools
can provide a foundation for understanding the cytokine-driven osteolysis
cascade and a base from which to identify and evaluate potential targets for
blockage or augmentation.
Total joint replacements are effective treatments for patients with
degenerative joint disease, providing long-term pain relief and restoration of
function1.
Peri-implant bone resorption, manifesting as an osteolytic lesion or resulting
in aseptic loosening of the implant, remains one of the few concerns of this
otherwise superlative
procedure2,3.
Currently, medical or therapeutic opportunities to treat osteolysis are
limited, and revision surgery to replace loose components remains the primary
alternative. To gain a deeper understanding of the cause of osteolysis and
potentially develop pharmaceutical interventions, it is imperative to identify
molecules orchestrating peri-implant bone resorption at the bone-implant
interface.
Numerous investigators have studied granulomatous tissues harvested from
lytic lesions and have demonstrated cellular interaction with wear debris to
be a critical event leading to bone
resorption4-7.
Goldring et al. placed such tissues in organ culture and established their
capacity to release mediators such as prostaglandin E2
(PGE2) and the matrix degrading enzyme
collagenase8. Other
investigators, including ourselves, have demonstrated that granulomatous
tissues release potent proinflammatory mediators such as interleukin
(IL)-1a, IL-1ß, IL-6, IL-8, and tumor necrosis factor-a
(TNF-a), among
others9-13.
These factors are actively involved in recruiting osteoclast precursors to
resorb
bone8,12,14.
Proteolytic enzymes involved in matrix breakdown and degradation such as
stromelysin, gelatinase, and matrix metalloproteinases are also
secreted15. These
early studies portrayed the periprosthetic granulomatous tissues in a
biochemically imbalanced situation: macrophages constantly stimulated by
phagocytosed wear debris and no longer tempered by anti-inflammatory mediators
initiate and maintain local inflammation, leading to bone
resorption12.
Previous studies have used protocols in which tissues were placed in organ
culture for twenty-four to seventy-two hours, and the conditioned media was
assayed with use of enzyme-linked immunosorbent assay
(ELISA)8,9,13.
While these proved reliable, investigators could only study one cytokine,
chemokine, or mediator at a time, and large sample volumes (approximately 200
µL) were needed. These requirements restricted the assortment of mediators
investigated to a handful in each study. Investigators have also used in situ
hybridization and immunohistochemistry to identify mediators in
tissues16-21.
While these techniques have the advantage of localizing individual cytokines
in cells and tissues, they too permit a limited array of assessments in each
study.
Using cDNA microarrays for gene-expression profiling, we recently reported
that macrophages cultured with particulate wear debris are stimulated to
express >500 genes in twenty-four
hours22,23.
This finding emphasized the complexity of the macrophage response to wear
debris and thus the need to more completely define the biological markers
associated with osteolysis and aseptic loosening. The large number of genes
upregulated by macrophage culture also highlights the limitations of
traditional ELISA for identifying the biological mediators of pathologic bone
resorption.
The emerging field of proteomics has spawned a variety of new technologies
to quantify large numbers of proteins simultaneously, in minute biological
samples. Similar in concept to the gene array, the protein chip is a
solid-phase ligand-binding assay system with use of Fab' fragments of
monoclonal antibodies tethered in a spatially optimized manner onto the
surface of microscopically etched silica
posts24,25.
We utilized such high-throughput protein chips to comprehensively define the
inflammatory microenvironment around failed total joint replacements, better
conceptualize the biochemical processes leading to osteolysis and aseptic
loosening, and provide a stronger foundation for identifying therapeutic
targets.
Patients
After institutional review board approval, clinical material was collected
from twenty-six patients. Peri-implant granulomatous tissues were harvested
from thirteen patients undergoing revision surgery for periprosthetic
osteolysis and aseptic loosening of their total hip replacements. The five
male and eight female patients were an average of sixty-three years old, and
the revision was performed an average of seventeen years after implantation.
All but one sample was from around the femoral component, and eight of the
thirteen components had been inserted without cement. According to the medical
records, all primary arthroplasties had been performed for end-stage
degenerative joint disease, with no primary diagnosis of rheumatoid arthritis.
The synovial lining capsular tissues were also harvested at the time of
primary total hip replacement in thirteen patients with end-stage degenerative
joint disease, including three who had developmental dysplasia and a single
patient who had osteonecrosis. These patients had an average age of sixty-six
years, and none of them had rheumatoid arthritis.
Tissue Collection and Sample Preparation
During the surgical procedure, frozen sections and swab cultures were
evaluated in all cases to rule out infection. Representative tissue samples
were collected and immediately frozen in a liquid nitrogen vat in the
operating room and were stored at —76°C. To facilitate protein
extraction, frozen tissue samples were homogenized for 1.5 minutes (1 g/20 mL
T-PER dissociation buffer; Pierce Biochemicals, Rockford, Illinois).
Homogenate samples were centrifuged (at 10,000 g for five minutes) to sediment
cellular debris, and the supernatants were aliquoted and stored at
—76°C until protein chip analyses (see schematic in
Figure 1, a).
Organ Culture
To compare this method with the traditional practice of evaluating samples
after organ culture, we performed both techniques in a subset of samples from
three failed total hip replacements. After tissue samples were snap-frozen in
the operating room, remaining tissues were placed in organ culture as
previously
described12 (see
schematic in Figure 1,
b). Briefly, in a laminar flow hood, tissues were minced in 1 to
2-mm3 pieces, rinsed in sterile phosphate-buffered saline solution,
and approximately 400 mg of tissue was transferred to each well of a
twelve-well culture dish. Samples were placed in organ culture in 2 mL of
Dulbecco's modified Eagle medium (DMEM; BioWhittaker, Walkersville, Maryland)
supplemented with antibiotics, antimycotics, and 10% fetal bovine serum at
37°C in 5% CO2 for twenty-four hours. The medium did not
completely immerse the tissue samples, and parts of the tissues remained at
the air-medium interface. After incubation, spent medium was collected from
each well and aliquots stored at —76°C. Cultured tissue samples were
also collected, frozen, and homogenized as described for other samples.
Protein Chips
Protein profiles of tissue lysate samples were analyzed with use of the
human cytokine chip and associated protein profiling system (Zyomyx, Hayward,
California)24,26.
Each cytokine chip measures twenty-nine human cytokines, chemokines, and
growth factors in a multiplexed assay format (see Appendix). For each run,
twelve chips were processed in parallel on the automated fluidics workstation
(Zyomyx Assay 1200). One chip, containing six flow chambers, was used to
generate a 6-point calibration curve. On each of the remaining chips, five
lysate samples were analyzed in parallel, with the sixth chamber used for
normalization and quality control. Each sample chamber contains 200 etched
silica posts with specific monoclonal antibodies tethered in a predetermined
configuration. The 200 spot features account for five replicate samples for
each analyte, and positive and negative controls.
Protein chips were first activated and blocked. Forty microliters of each
sample was injected into a flow chamber on a chip for a ninety-minute
incubation at room temperature. After washing, the detection solution was
applied for sixty minutes, followed by a final wash. All steps were performed
in the automated microfluidics station. On completion of the multiplexed
assays, chips were scanned on a modified fluorescent scanner equipped with a
532-nm laser (Zyomyx Scanner 100). Laser power and photomultiplier tube (PMT)
voltage adjustments provided the largest dynamic range with minimal feature
saturation.
Protein Chip Data Analysis
Median feature pixel intensities from the features on each chip were
background subtracted, after outliers were removed with use of either Dixon's
Q-test for cytokine features or the box plot for background
features24.
Background signal was determined from the mean intensity of the negative
control features. Feature intensities exceeding the linear range of the
scanner were discarded, and the significance above background was determined
with use of a modified Z-factor parameter calculated at a 90% confidence
interval. Quantification of each analyte concentration, for each sample, was
calculated with use of the 6-point multianalyte calibration curve that was
derived from the calibration chips. Each reported concentration value
represents the average of five replicate measurements from each assay. Final
analyte concentrations were normalized to total protein content of each sample
quantified with use of the Bradford assay (Sigma, St. Louis,
Missouri)27.
Data Analysis
Data from five replicate samples were averaged, and absolute values were
calculated from the 6-point calibration curve for each analyte with an r value
of >0.98. The results varied widely in many cases, and cytokines were
present at either very high or very low levels. This wide variation resulted
in a non-normal data distribution and required use of the appropriate
Mann-Whitney test for significance.
Of the twenty-nine cytokines analyzed, only eight were detected in
sufficient samples to perform statistical analyses and are presented in
Table I. All peri-implant
samples were negative for infection as tested intraoperatively.
Interleukin-6
This stimulator of osteoclastic activity was detected in all osteolysis and
osteoarthritis samples demonstrating its importance in end-stage bone
resorption. Interleukin (IL)-6 levels were higher in capsular tissues
harvested from patients with osteoarthritis compared with peri-implant
granulomas, highlighting the substantial bone destruction accompanying
end-stage degenerative joint disease. Previous studies have also reported
higher IL-6 levels in capsular tissues from patients with
osteoarthritis12.
Interleukin-8
IL-8 is a potent chemotactic factor for acute inflammatory cells such as
neutrophils and T-cells. Released primarily by macrophages, IL-8 was barely
detected in any osteoarthritis samples. In stark contrast, IL-8 levels in
peri-implant samples were 100-fold higher (p = 0.0001)
(Table I).
Interferon-?-Inducible Protein of 10 kDa and Monokine Induced
by Interferon-?
Both of the chemokines interferon-?-inducible protein of 10 kDa
(IP-10) and monokine induced by interferon-? (MIG) are secreted by
monocytes and endothelial cells after induction by interferon-?
(IFN-?), share the same CXCR3 receptor, and are potent chemoattractants
for activated T-cells (Th1) subsequent to antigen presentation. IP-10 was
present in all control tissue samples as well as in peri-implant granuloma
samples, indicating its role in end-stage joint disease. Levels of IP-10 were
more than doubled in peri-implant samples compared with controls (p = 0.020).
Average levels of MIG in patients with failed total hip replacements were also
two times the levels in patients with osteoarthritis, but high variability
precluded significance.
Soluble Intercellular Adhesion Molecule
Intercellular adhesion molecule (ICAM)-1 is an inflammatory mediator
expressed solely on activated monocytes and facilitates transendothelial
migration of leukocytes and activated T-cells. ICAM-1 on antigen-presenting
cells binds integrin lymphocyte function-associated antigen-1 on recruited
CD4+ T-cells to initiate and maintain the immunological synapse, representing
a key first step in the development of an adaptive immune response. The
soluble form of ICAM-1 (sICAM-1) interferes with and disrupts the immune
synapse formation, inhibits T-cell chemotaxis, and consequently suppresses the
adaptive immune response. Very high levels of sICAM-1 were released in all
osteolysis samples, representing nearly a thirty-two-fold increase over
controls (p < 0.0001). This finding suggests that, in the midst of an
intense inflammatory microenvironment, there is an ongoing active effort to
subdue the adaptive immune
response28,29.
The soluble form of ICAM-1 is only released after an enzymatic cleavage of
ICAM-1, and there is no indication that a one-minute tissue homogenization as
performed in this protocol can cleave it. However, it is possible that bound
ICAM-1 could interfere with the assessment in homogenates.
Monocyte Chemotactic Proteins (MCP)-1, MCP-3, and Soluble CD23
The chemokines MCP-1, MCP-3, and sCD23 were found less frequently in
patients. MCP-1 was detected in six of thirteen failed joint replacement
samples and was not detected in any control tissues harvested during primary
arthroplasty. Similarly, MCP-3 was identified in four of thirteen peri-implant
granulomas and in only one control sample. Tissue levels of sCD23 were not
detectable in most samples, but they were present at high levels in two of the
thirteen control samples and at levels that were, on the average, greater than
threefold higher in six of the thirteen implant-associated samples. While high
levels of specific cytokines in some patients hint at pathologic associations,
no generalized correlations with clinical variables could be found.
Transforming Growth Factor-ß1
Transforming growth factor-ß1 (TGF-ß1), an
important growth factor with anti-inflammatory effects, was detected in most
samples from patients with implant failure but in only five of the thirteen
control samples. The TGF-ß1 levels in the samples from
patients with osteolysis were greater than three-fold higher than those in
samples from patients having a primary arthroplasty (p = 0.0038).
Snap-Freezing Samples Compared with Organ Culture
Twenty-one of the twenty-nine assayed cytokines and chemokines were not
detected in most tissue samples. The absence of these mediators permits us to
exclude specific cell populations and their actions in the peri-implant
inflammatory process. The absence of IL-1a, IL-1ß, and TNF-a
was particularly surprising, considering their nearly universal documentation
in the aseptic loosening literature. To confirm that the absence of key
mediators was not due to specimen harvesting or a lack of sensitivity of the
protein chips, we compared the same tissues using both our current methodology
as well as the traditional practice of organ culture and assaying spent
conditioned media.
For a subset of three samples from patients with a failed total hip
replacement, portions of tissues were snap-frozen in the operating room, while
remaining tissues were minced, rinsed, and placed in organ culture according
to previously established
protocols8,9,12.
After a twenty-four-hour culture, the spent conditioned media was harvested
and analyzed (see schematic in Figure
1). Spent media has provided the basis for most previous
investigations of the biochemical potential of peri-implant
granulomas8,9,12,30,31.
Cultured tissues were also harvested, homogenized, and proteins extracted as
before, and analyzed with use of protein chips.
When tissue samples were placed in organ culture, an additional five
cytokines (IL-1ß, IL-2, IL-10, MCP-1, and G-CSF
[granulocyte-colony-stimulating factor]) were found at high levels
(Fig. 2). These additional
cytokines were detected both in the cultured tissue homogenate and in spent
conditioned media. Further, other cytokines detected previously at low levels
(IL-6, IL-8, IP-10, and MIG) increased dramatically, two to twenty-five-fold
higher after placement in organ culture
(Fig. 2). Even after organ
culture, sixteen of twenty-nine mediators were not detected in tissues.
Over the last four decades, investigators have elucidated the biological
underpinnings of peri-implant bone loss leading to osteolysis and aseptic
loosening of total joint replacement. Analyses of tissues from around failed
arthroplasty components have yielded key insights into the mechanisms of
osteolysis. In this study we introduced a novel emerging technology using
high-throughput protein chips to quantify twenty-nine inflammatory cytokines
and chemokines in the peri-implant granuloma. The simultaneous assessment of
important inflammatory molecules allowed us to infer the presence of various
subpopulations of involved cells and hypothesize how their actions are
coordinated, leading to this pathology. The cytokines and chemokines
identified in the present study are mediators secreted primarily by activated
macrophages, and we confirmed their primary role in inciting inflammation at
the bone-implant interface. We also identified chemokines involved in
recruiting activated T-lymphocytes, as well as the transient role of
IL-1ßa. In the peri-implant granuloma, we measured but did not
detect any activators of cytotoxic T-cells (e.g., IL-12), antibody producing
B-cells (e.g., IL-4), or eosinophils (e.g., IL-5) involved in delayed-type
hypersensitivity.
Interrelations Between Molecules of Acute Inflammation
Role of IL-6 and IL-8
Among the proinflammatory cytokines, IL-6 was consistently present in
tissues from patients with osteolysis, in agreement with previous findings
from various
laboratories12,30,32-35.
IL-6 is produced primarily by monocyte-macrophages and fibroblasts, enhancing
the recruitment of osteoclast precursors and promoting
osteoclastogenesis36-39.
IL-6 levels are also elevated in patients with inflammatory joint diseases
such as rheumatoid arthritis and
osteoarthritis37,40-43.
Since tissues and fluids harvested during surgery represent a so-called end
stage of disease, IL-6 is clearly associated with the chronic phase of bone
resorption and joint destruction and probably represents a common biochemical
thread among rheumatoid arthritis, osteoarthritis, and peri-implant bone
loss.
IL-8 was rarely detected in control patients, while it was present at
100-fold higher levels in tissues from patients with failed joint
replacements. IL-8 is produced by several cell types, including monocytes, and
was earlier described as a chemotactic factor for neutrophils and
T-cells44,45.
More recent studies have indicated that IL-8 is importantly involved in
osteoclastogenesis and promotes the differentiation of human peripheral blood
monocytes along the pathway leading to bone-resorbing
osteoclasts46. In
other reports, we find that phenotypic changes cause cancer cells to
overexpress IL-8, correlating with their penchant for bone
metastasis47. Koch
et al. reported that IL-8 is strongly angiogenic for endothelial cells and
accounts for the majority of the angiogenic activity of inflamed rheumatoid
synovial fluid cells and in vitro stimulated
macrophages45.
Thus, it is not surprising that other investigators have reported the presence
of this important chemokine in patients with loose joint
replacements12,44
as well as in patients with rheumatoid
arthritis40,48.
IL-6 and IL-8 therefore appear to form an effective team at the bone-implant
interface, to impel bone resorption on a chronic basis. While IL-8 promotes
persistent angiogenesis, recruits leukocytes, and facilitates differentiation
of macrophages to the osteoclastic lineage, IL-6 facilitates cellular
proliferation, maturation, and stimulation of osteoclasts, leading to bone
resorption.
Role of IL-1ß and TNF-a
During acute inflammation, IL-1ß and TNF-a are potent inducers
of
IL-636,49,50.
Interestingly, neither IL-1ß nor TNF-a was found in tissues
"as harvested" intraoperatively, but levels of IL-1ß surged
after tissues were placed in organ culture. It is likely that transporting
tissues to the laboratory, mincing, and culturing them for twenty-four to
seventy-two hours stimulates the cells with "newly released" wear
and cell debris, inducing release of inflammatory mediators.
The absence of these mediators is not without precedence. For example,
Goodman et al. did not find any IL-1ß in as-harvested osteolytic
tissues9. Glant et
al. also reported that as-harvested tissues did not express mRNA for
IL-1a51.
After tissues were minced and cultured, IL-1a mRNA levels increased
dramatically. Glant et al. rationalized that "cells of the interfacial
membrane retain a high latent capacity" to express IL-1a in
response to changes in the microenvironment, which is apparently triggered
during the period after harvest, manipulation, and organ
culture51,52.
In samples from patients with osteoarthritis, Brenner et al. detected neither
IL-1a nor
TNF-a42.
Similarly in patients with rheumatoid arthritis, Bhardwaj et al. reported that
synovial cells did not release IL-1, but they could be induced to release
copious amounts when placed in
culture37. When
Tanabe et al. did not detect IL-1a, IL-1ß, or TNF-a in serum
of patients with rheumatoid arthritis, they too proposed that these mediators
of acute inflammation may only be elevated during the initiation phase of the
pathologic process and can reappear on exacerbation of the
disease40. Other
investigators have reported the presence of IL-1 and TNF-a in
peri-implant tissues using immunohistochemistry and in situ
hybridization16,18,53.
While we are unable to explain the individual findings, it is possible that
even a brief storage of harvested tissues in saline solution, while
transported to the laboratory, can result in gene expression of inflammatory
mediators.
IL-1a, IL-1ß, and TNF-a are important mediators of acute
inflammation54,55,
and IL-1ß and TNF-a are thus critical initiators of the
inflammatory cascade and best modeled by in vitro cell culture. After
inflammation has been initiated, levels of these markers recede, while other
mediators such as chemokines, IL-6, adhesion molecules, and T-cell chemokines
relay and maintain the chronic inflammatory response. During this chronic
phase, regular signals provided by IL-6 support the recruitment of osteoclast
precursors and maintenance of long-term bone
resorption43.
In a recent finding from a human clinical trial, the TNF-receptor
antagonist etanercept (Enbrel; Amgen, Thousand Oaks, California) was not
effective in treating osteolysis in
patients56. Our
findings that TNF-a is not present in chronic osteolysis support the
results of this clinical study. Our results also affirm that the role of
TNF-a in chronic human osteolysis may be more limited than alluded to in
in vitro and in vivo animal models of wear-mediated
osteolysis57-59.
Role of T-cells, IP-10, and MIG
Evans et al. reported a strong association between a T-cell response and
failed joint
replacements60.
Since then, numerous studies have indicated the presence of T-lymphocytes in
the periprosthetic tissue surrounding failed
implants16,61,62.
High levels of MIG and IP-10 seen in our study also indicate T-cell
recruitment to the peri-implant tissues. Both of these mediators are induced
by interferon-? (IFN-?) stimulation of monocytes, macrophages, and
endothelial cells. MIG and IP-10 bind the same receptor and work in tandem to
recruit T-cells (Th1) and natural killer (NK)
cells63-68.
They are both abundantly elevated in T-cell inflammatory lesions such as
rheumatoid arthritis, psoriatic arthritis, and multiple
sclerosis65,66,69,70.
While the presence of T-cells in the peri-implant granuloma is not
contested, investigators have reported an absolute absence of IL-2,
IFN-?, and activated subtypes of
T-cells20,71,72.
The lack of the immune mediators IL-2, IL-4, IL-5, and IFN-? in our
present study supports these findings, downplaying the role of activated
T-cells in end-stage bone resorption. Collectively, these studies suggest that
macrophage phagocytosis of particles results in an initial antigen
presentation, stimulation of IFN-?, and the consequent release of IP-10
and MIG. However, the absence of immune mediators indicates that a
full-fledged T-cell response is not continued in the chronic stages of
osteolysis and aseptic loosening.
Cytokine Interactions at the Bone-Implant Interface
On the basis of these findings, and with compiled evidence from previous
investigations, we propose the following sequence of events. Wear debris
stimulates macrophages and initiates an acute inflammatory response beginning
with the release of IL-1a, IL-1ß, TNF-a, and IL-8, as well as
chemokines involved in recruiting new macrophages and other cells.
Inflammatory cytokines initiate and sustain maturation of macrophages and
upregulate the next wave of cytokines, including the potent bone-resorbing and
osteoclastogenic cytokine IL-6. Antigen presentation in the context of the
major histocompatibility complex (MHC) and recruitment of T-cells is indicated
by the release of the IFN-?-induced T-cell chemotactic factors, IP-10
and MIG. Consistent with this hypothesis, several studies have described
various T-cell subgroups in peri-implant
tissues16,53.
This suggests that wear debris, or more likely degraded protein in the context
of wear debris, is presented by macrophages as antigen. IFN-? may be
released transiently during preliminary antigen presentation, but it is no
longer detected in chronic osteolysis. The absence of IL-2, IL-4, IL-5, and
IL-10 indicates that neither cytotoxic T-cells nor antibody-producing B-cells
are stimulated. The lack of eotaxin, which is required for recruitment
of eosinophils and basophils, implies that delayed-type hypersensitivity or
allergic reactions do not play a role in these osteolysis cases.
Despite the presumed presentation of antigen, it is not immediately evident
what prevents a full-blown adaptive immune response from being activated.
Soluble ICAM-1 (sICAM-1) was released copiously in our samples and may play a
key role. Elevated levels of sICAM-1 can bind the active domain of ICAM-1 and
lymphocyte function-associated antigen integrin on T-cells, disrupting their
interaction and effectively aborting the maturation of the immunological
synapse and, consequently, the adaptive immune responses. T-cells already
recruited but not involved in the adaptive immune response are nonetheless
capable of enhancing the activation of macrophages.
Issues of Patient Variability
The wide variability in levels of mediators, and the binary response of
specific cytokines (either high secretion or not detected), indicates that
individual patients respond uniquely to their joint replacements. These
results reinforce what clinicians and scientists already know from experience,
which is that the aggressiveness of the osteolytic response depends acutely on
patient-specific variables and results in a unique patient-specific clinical
course. Cytokine levels in patients who are particularly high responders
likely enhance the catabolic activity of adjacent cells, leading to more
aggressive bone resorption. The genetic basis for this differential patient
response is being vigorously pursued and may indicate why only a subset of
patients have particle-mediated bone loss develop. While we have an incomplete
understanding of the source of this patient-specific variable, protein chips
and related technology can provide sophisticated opportunities to identify and
monitor its manifestation in patients.
Limitations of the Study
We used an emerging technology, which provides a very accurate, precise,
and validated profile of the inflammatory environment. Nevertheless, it still
represents a narrow window into the vast world of cytokines and chemokines
associated with failed joint replacements. On the basis of what we have
already learned, additional panels of relevant angiogenic and growth factors
are required to better define the inflammatory milieu at the bone-implant
interface. Characterizing the interplay between cytokines, the expression of
their receptors, and decoy receptors is also an important goal. Such studies
need to be performed in parallel with gene-expression profiling studies.
A drawback of our studies is that no control, healthy tissues were
available to estimate normal levels of these cytokines in joints. We were
forced to rely on synovial lining capsular tissues from patients with
end-stage degenerative joint disease for comparison. As has already been
noted, degenerative joint disease is also an inflammatory disorder associated
with joint destruction and bone resorption. While the heterogeneity of tissue
sampling could be a concern, it accurately represents the bone-implant
interface. These factors notwithstanding, a larger sample was used for our
analysis to obviate the concerns and drawbacks of localized heterogeneity.
Specifically, despite the rigorous methodology, the failure to detect
IL-1ß, TNF-a, and other mediators in the tissues might reflect a
reduced sensitivity to these specific molecules on the protein arrays. Higher
sensitivity arrays are certainly forthcoming. Additionally, in our limited
sample size, we did not identify any correlation among implant-specific
features, such as metal or cement used, to mediator levels. Larger population
studies would be required to determine the role of materials and wear
rates.
In this study, we relied on measuring levels of cytokines in clinical
tissues associated with osteolysis to develop a deeper understanding of the
underlying biochemical processes. While high levels of specific mediators have
been correlated with pathologic processes, they are not necessarily causative
of disease. Other mediators not measured in the present study could play a key
role in osteolysis. Additionally, the biological activity of cytokines is
modulated in vivo by a variety of processes including upregulation of soluble
receptors, the presence of inhibitory proteins, and other antagonistic
processes. Furthermore, cytokines at very low and as yet undetectable tissue
levels might reach important physiological levels in the microenvironment of
the cells. Despite these shortcomings, investigating biochemical mediator
levels allows us to develop a working hypothesis of the underlying biology and
to pinpoint their physiological function and circumstance of release.
A table showing all cytokines studied and their actions is available with
the electronic versions of this article, on our web site at jbjs.org (go to
the article citation and click on "Supplementary Material") and on
our quarterly CD-ROM (call our subscription department, at 781-449-9780, to
order the CD-ROM). ?
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