Download Cellular profile and cytokine production at prosthetic interfaces

Cellular profile and cytokine production at
prosthetic interfaces
STUDY OF TISSUES RETRIEVED FROM REVISED HIP AND
KNEE REPLACEMENTS
S. B. Goodman, P. Huie, Y. Song, D. Schurman, W. Maloney,
S. Woolson, R. Sibley
From Stanford University School of Medicine, California, USA
he tissues surrounding 65 cemented and 36
cementless total joint replacements undergoing
revision were characterised for cell types by
immunohistochemistry and for cytokine expression by
in situ hybridisation.
We identified three distinct groups of revised
implants: loose implants with ballooning radiological
osteolysis, loose implants without osteolysis, and
well-fixed implants. In the cemented series, osteolysis
was associated with increased numbers of
macrophages (p = 0.0006), T-lymphocyte subgroups
(p = 0.03) and IL-1 (p = 0.02) and IL-6 (p = 0.0001)
expression, and in the cementless series with increased
numbers of T-lymphocyte subgroups (p = 0.005) and
increased TNF expression (p = 0.04). For cemented
implants, the histological, histochemical and cytokine
profiles of the interface correlated with the clinical
and radiological grade of loosening and osteolysis.
Our findings suggest that there are different
biological mechanisms of loosening and osteolysis for
cemented and cementless implants. T-lymphocyte
modulation of macrophage function may be an
important interaction at prosthetic interfaces.
T
J Bone Joint Surg [Br] 1998;80-B:531-9.
Received 14 July 1997; Accepted after revision 24 October 1997
Osteolysis is associated with the accumulation of wear
debris from an implant and may occur in the absence of
1-3
general loosening. Prostaglandins, cytokines, metalloproteinases, lysosomal enzymes and other substances are produced by the interface tissue, but their relative importance in
S. B. Goodman, MD, PhD, Associate Professor and Head
Y. Song, MD, Research Associate
D. Schurman, MD, Professor
W. Maloney, MD, Associate Professor
S. Woolson, MD, Professor
Division of Orthopaedic Surgery
P. Huie, MA, Senior Research Associate
R. Sibley, MD, Professor
Department of Pathology
Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5326, USA.
Correspondence should be sent to Dr S. B. Goodman.
©1998 British Editorial Society of Bone and Joint Surgery
0301-620X/98/38158 $2.00
VOL. 80-B, NO. 3, MAY 1998
4-16
the resorption of periprosthetic bone is controversial
An understanding of the cellular and cytokine profiles of
tissues surrounding revised joint prostheses may further
elucidate the biological processes of loosening and osteolysis and suggest preventative or therapeutic methods
which may favourably affect the survival of the implant.
Our aim in this prospective study was to determine the
major cell types and cytokines produced by periprosthetic
tissues, using immunohistochemistry and in situ hybridisation. We hypothesised that tissues surrounding well-fixed
and loose cemented and cementless prostheses, with and
without osteolysis, showed differences in cell type and
cytokine expression, and that these differences defined the
biological milieu of the interface.
Materials and Methods
We examined clinically and radiologically 101 patients who
were having revision of a total joint replacement. The
degree of loosening on the radiographs was graded accord17
18
ing to the criteria of Charnley, Harris and McGann and
19
Engh, Massin and Suthers. At operation if manual torsional or axial loading of the implant produced movement
at the cement-bone, cement-prosthesis or bone-prosthesis
interface, the prosthesis was classified as loose.
Six distinct groups of implants were identified (Table I):
group 1, loose, cemented implants with osteolysis (24);
group 2, loose, cemented implants without osteolysis (29);
group 3, well-fixed cemented implants without radiological
evidence of loosening (12); group 4, loose, cementless
implants with osteolysis (9); group 5, loose, cementless
implants without osteolysis (13); and group 6, well-fixed,
cementless implants without radiological evidence of loosening (14). Radiographs from patients with loose implants
and osteolysis showed one or more areas of ballooning and
radiolucent zones with scalloped edges. Loose implants
without osteolysis showed prosthetic migration, progressive
complete radiolucent lines or radiolucencies at the cementprosthesis interface. Well-fixed prostheses showed none of
the above radiological signs. There were 85 hip replacements and 16 knee replacements. The prostheses were made
of ultra-high-molecular-weight polyethylene, cobalt-chrome
alloy, and/or titanium 6-aluminium 4-vanadium alloy.
Table I gives details of the patients and periods of
531
532
S. B. GOODMAN,
P. HUIE,
Y. SONG,
ET AL
Table I. Details of the six groups of patients
Specimen location‡
Original diagnosis†
Group*
Number of
patients
Mean (±
(yr)
1
2
3
4
5
6
24
29
12
9
13
14
62
67
76
55
59
59
±
±
±
±
±
±
SEM)
age
4
2
2
4
5
4
Hip
OA
ON
Other
Mean (± SEM) time
in situ (mth)
16
21
12
6
9
7
2
0
0
1
1
3
6
8
0
2
3
4
151
132
75
69
57
36
±
±
±
±
±
±
10
12
12
7
11
5
Knee
Ac
Fem
Fem
Tibia
10
17
4
2
3
10
14
8
0
6
9
2
0
0
3
0
0
1
0
4
5
1
1
1
* see text
† OA, osteoarthritis; ON, avascular necrosis; other, hip dysplasia (9), traumatic arthritis (7), ankylosing spondylitis (3), juvenile
rheumatoid arthritis (2), benign tumour (1), and epiphyseal dysplasia (1)
‡ Ac, acetabulum; fem, femoral
Table II. Primary mouse monoclonal antibodies used in the immunohistochemistry studies
Monoclonal
antibody
Specific
antigen
Dilution
Target cell
CD68*
EMB 11
1:800
Human macrophages
CD14†
CD3†
Leu M3
Leu 4
1:20
1:100
Activated human macrophages
Human T cells
CD2‡
CD4†
CD8†
T11
Leu 3A
Leu 2A
1:100
1:100
1:20
A pan T-cell marker found in human E-rosetting positive lymphocytes
Human T helper/inducer cells (cross reacts with monocytes/macrophages)
Human T cytotoxic/suppressor cells
* DAKO Corporation, Carpinteria, California
† Becton Dickinson, Mountain View, California
‡ Coulter Immunology, Hialeah, Florida
implantation for each group.
Group-3 specimens were excised after a shorter time
than group-1 or group-2 specimens (p = 0.03). Cementless
stems were revised after a shorter time than the respective
cemented group (p = 0.05). All prostheses in groups 1, 2, 4
and 5 were revised because of pain that had not been
relieved by conservative measures; at surgery, all of these
were found to be mechanically loose. Group-3 and group-6
specimens were excised from patients whose prosthesis had
been painful, recurrently dislocating or subluxating and/or
showed considerable polyethylene wear. None of these
prostheses was found to be mechanically loose on clinical
assessment at surgery. No prosthesis was shown to be
infected.
After removal of the implant, tissue specimens, approximately 5 mm in diameter, were taken from a representative
and readily accessible portion of the interfacial membrane
using a curette. They were immediately immersed in saline,
transported to the laboratory, and placed in capsules containing optimum cutting temperature media (Miles, Elkhart,
Indiana). The capsules were immediately frozen in liquid
nitrogen and stored at –70°C until processed. Serial 6 m
sections were cut with a cryostat (Cambridge Instruments,
Buffalo, New York) and mounted on microscope slides that
had been prebaked for three hours at 250°C.
Immunohistochemistry. Immunohistochemistry was used
to identify subpopulations of cells with monoclonal antibodies that are specific for antigens of a particular cell type.
Positively stained cells are visualised using a chromagen
that causes a change in the colour of the cell. The slide is
then counterstained so that the cells that have not reacted
20,21
with the monoclonal antibody are visualised.
Thick 6 m frozen sections were fixed in absolute acetone at –20°C overnight. The primary mouse antihuman
monoclonal antibodies are detailed in Table II. The sections
were incubated with 25 l of the primary monoclonal
antibody at room temperature for one hour. The slides were
rinsed and washed for 25 minutes in a phosphate-buffered
saline (PBS; Sigma, St Louis, Missouri) bath. The sections
were then sequentially incubated with 25 l of rabbit antimouse immunoglobulin (Dakopatts a/s Z109, Denmark)
diluted at 1:200 for 15 minutes, 25 l of swine anti-rabbit
immunoglobulin (Dakopatts a/s Z196, Denmark) diluted at
1:25 for 15 minutes, and 25 l of horseradish peroxidase
and rabbit anti-horseradish peroxidase (Dakopatts a/s Z113,
Denmark) diluted at 1:100 for 15 minutes. After each
antibody step, the sections were washed with PBS for 25
minutes. To visualise the positive cells with a brown colour,
the sections were then exposed for ten minutes to diaminobenzidine tetrahydrochloride tablets (Sigma, St Louis, Missouri) in solution with 0.01% H2O2, and 0.3% sodium azide
in 0.05 M Tris buffer at pH 7.6. They were then counterstained with Gill’s haematoxylin No. 3, dehydrated and
mounted.
Monoclonal antibodies were used to identify certain cell
types as shown in Table II. The diluent solution alone was
used as a negative control to assess the presence of endogenous peroxidase. Positive controls included biopsies from
cardiac and renal tissues from the transplantation service.
The different treatments and target cells for identification
THE JOURNAL OF BONE AND JOINT SURGERY
CELLULAR PROFILE AND CYTOKINE PRODUCTION AT PROSTHETIC INTERFACES
533
Table III. Probes examined in the in situ hybridisation studies
Cytokine/growth factor
Function
IL-1 and IL-1
Produced by many cell types including macrophages. IL-1 activates macrophages, neutrophils and endothelial cells,
stimulates fibroblasts and osteoclasts, and induces prostaglandin E2 and collagenase synthesis. IL-1 and IL-1 produce two
distinct genes. They have 25% homology and recognise the same cell-surface receptors
IL-2
Produced by T helper cells. It stimulates growth and proliferation of T and B cells and activated killer T lymphocytes
IL-6
Produced by macrophages, T cells, fibroblasts and other cell types. It activates T and B cells and induces B cells to
differentiate and secrete immunoglobulins
TGF
Produced by T cells, activated macrophages, and other cell types. It stimulates fibroblast growth, extracellular matrix
formation and suppresses T- and B-cell proliferation. TGF also stimulates osteoblasts and inhibits osteoclasts
TNF
Produced by activated lymphocytes, monocytes, macrophages and other cells. It stimulates fibroblasts and granulocytes and
many of the effects are similar to IL-1
IFN
Produced by T cells. It has antiviral activity and enhances activated killer cells
PDGF
Produced by macrophages, platelets, endothelial cells and fibroblasts. It increases class-II antigen expression in macrophages, stimulates osteoclasts to resorb bone, induces collagenase and prostaglandin production, and is chemotactic for
fibroblasts, monocytes and neutrophils
Poly-t
A probe detecting the poly A tail of mRNA (used as a positive control)
EBV
A probe for the Epstein-Barr virus (used as a negative control)
Hybridisation solution
A negative control
are summarised in Table II. A few selected specimens were
processed using the mouse antihuman fibroblast 5B5 monoclonal antibody that is specific for the beta subunit of prolyl4-hydroxylase (Dako-fibroblast; 5B5 Dakopatts a/s M877,
Denmark), and the CD14 antibody for activated macrophages (Anti-leu-M3; Becton Dickinson, California).
In situ hybridisation. Cytokines are proteins that modulate
the activities of cells in many different organs, including
mesenchymal tissues and bone. They have a very short half
life and are therefore difficult to identify. In situ hybridisation is a technique in which single-stranded DNA probes
are synthesised that are complementary to a target sequence
on mRNA that codes for a specific cytokine or growth
22,23
factor.
A reporter enzyme is indirectly attached to the
specific probe-mRNA complex so that cells that contain the
mRNA for a specific cytokine can be visualised. When an
appropriate substrate is then added, there is a colour change
in the cells. The slide is also counterstained so that negatively staining cells may be visualised.
A thorough search in Genbank (Los Alamos, New Mexico) was performed to ensure that the complementary DNA
(cDNA) probes used in the in situ hybridisation studies did
not match any housekeeping gene sequences for common
cellular proteins found in man. Probes of a uniform length
(30 base pairs) and GC:AT (guanine + cytosine: adenine +
thymine) ratio (0.6 to 0.7) were used and the method of
tissue processing was standardised.
We obtained mRNA sequences from Genbank. Selected
sequences were commercially synthesised (Operon, Alameda, California) and biotinylated at the 3' end using biotin11-dUTP-biotin (Sigma, St Louis, Missouri) and DNA
deoxynucleotidylexotransferase (Gibco, Gaithersburg,
Maryland). Table III summarises the different human antisense probes which were used.
The mounted 6 m sections were fixed for ten minutes in
VOL. 80-B, NO. 3, MAY 1998
4% paraformaldehyde in physiological PBS pH 7.8 at room
temperature. The slides were then washed in three changes
of PBS and 10% ethanol, dehydrated in 95% and absolute
ethanol and allowed to dry in air for 30 minutes. Working
hybridisation solutions were prepared with probe concentrations as follows (expressed as ng probe/l hybridisation
solution): PDGF, IL-1, TGF and poly-t, 2.0 ng/l; IL6, 1.5 ng/l; TNF and IL-1, 1.0 ng/l; IFN, 0.5 ng/l;
and IL-2, 0.25 ng/l. The working hybridisation solution
was applied to tissue slices and allowed to incubate at 42°C
for one hour. The slides were washed in 5x saline sodium
citrate and 2x saline sodium citrate. Excess 2x saline
sodium citrate was removed and streptavidin-peroxidase
(Dako, Carpinteria, California) in 0.1 M Tris-saline, pH 7.5
(1:500), was added to each section and incubated for one
hour at room temperature. The slides were washed in 0.1 M
Tris-saline, pH 7.5, three times for five minutes. The colour
reaction was visualised with diaminobenzidine as described
in the immunohistochemistry section. The sections were
counterstained with Gill’s haematoxylin No 3, dehydrated
and mounted.
Assessment. We first examined the stained sections to
determine the general histomorphological features and then
performed a quantitative assessment for each antibody or
probe by calculating the percentage of positively staining
cells using light microscopy and a grid-counting tech21
nique. We classified 50 to 75 cells in each of the four
quadrants of the tissue section which yielded a minimum of
200 cell counts per stain or probe. The first quadrant for
cell counting was chosen randomly on the slide and
labelled the 12 o’clock position; the second, third and
fourth quadrants for counting were then automatically designated according to the 3, 6 and 9 o’clock positions. The
data were analysed using an analysis of variance; intergroup comparisons were made for each antibody or probe
534
S. B. GOODMAN,
P. HUIE,
using the Fisher protected least-significant-difference test
(Statview; Abacus Concepts, Berkeley, California).
The T-lymphocyte monoclonal antibody CD4 was found
to bind very weakly to macrophages which were much larger
and stained very lightly compared with the T-lymphocytes
21
which were much smaller and stained more intensively.
The macrophages were not counted as positive cells.
Results
Gross and histological
Loose prostheses with osteolysis (groups 1 and 4). In all
cases, there was extensive yellow-brown fibrous tissue
surrounding loose cemented implants with osteolysis which
often extended deeply into the surrounding bone (e.g.
through cement drill holes in the acetabulum) and frequently followed bursal pathways further into the surrounding soft tissues. Excision of this tissue was difficult and
multiple-sized curettes were often necessary to extract its
furthest extensions. Histological examination showed that it
was composed of a highly vascularised fibrous tissue; the
surface which was adjacent to the cement layer was occasionally covered by a synovial-like lining layer. The underlying stroma contained sheets of macrophages and
macrophage polykaryons with engulfed cement, polyethyl8,9,24-27
Using polarised light, a
ene and metal particles.
bluish-yellow tissue birefringence was seen in highly cellular areas, which probably corresponded to widespread
submicron polyethylene particles within the tissue. Greenish-grey, bone-cement remnants were present, surrounded
and engulfed by macrophages. Lymphocytes were scattered
throughout the tissue. In some of the cases, the portions of
the pseudomembranous tissue were necrotic and amorphous, with few recognisable cellular elements.
Tissue from loose cementless prostheses with osteolysis
was less plentiful, more fibrous and less cellular. The
bluish-yellow tissue birefringence of polyethylene particles
was present as were phagocytosed polyethylene and metal
debris. In several cases, areas of the tissue were black in
colour and contained large amounts of phagocytosed and
interstitial metal particles.
Loose prostheses without osteolysis (groups 2 and 5). Loose
cemented and cementless components without osteolysis
were surrounded by a more organised fibrous tissue layer
containing a variable number of cells and particulate debris.
In some areas, granuloma-like reactions were noted, but in
others, macrophages were sparse. A synovial-like lining
was occasionally seen. In general, the volume of tissue was
much less than that excised from around loose implants
with osteolysis.
Well-fixed prostheses without osteolysis (groups 3 and 6).
Well-fixed cemented and cementless implants were always
difficult to remove. Little tissue surrounded these implants,
but was often tightly adherent to the prosthesis, implant or
underlying bony bed and primarily fibrous in nature. Scattered macrophages, lymphocytes and particulate debris
Y. SONG,
ET AL
were seen. There was no synovial-like lining layer.
General immunohistochemical and in situ hybridisation. By processing and examining sequential sections
alternately by immunohistochemistry and in situ hybridisation, we were able to show cytokine production by
specific cellular elements (Figs 1 and 2). The fibrous
stromal cells were spindle-shaped, 5B5-positive fibroblasts,
which often stained positively for TGF. Macrophages and
macrophage polykaryons were larger CD68-positive cells
that expressed mRNA for IL-1, IL-6, TNF and PDGF
(Fig. 3). CD-14-positive activated macrophages were a
subset of the CD68-positive macrophages and were occasionally clustered around blood vessels. The CD3-positive
T lymphocytes were small, round, darkly-staining cells
scattered throughout the tissue and occasionally clustered
around capillaries.
Quantitation of immunohistochemistry and in situ
hybridisation. Table IV outlines the percentage of positively staining cells for each of the monoclonal antibodies,
cytokines and growth factors. The following differences
reached statistical significance (p < 0.05) using an analysis
of variance: CD68 (p = 0.011), CD2 (p = 0.001), CD3
(p = 0.0004), CD4 (p = 0.033), CD8 (p = 0.0004), poly-t
(p = 0.011) and IL-6 (0.008). A trend (0.05 < p < 0.1) was
seen with IL-1 (p = 0.055) and IL-1 (p = 0.067).
In the cemented series, when significant differences were
noted among the three groups, group 1 had the highest
number of positive cells for the above cell-surface antigens
and cytokines compared with the cemented groups with
simple loosening without osteolysis and well-fixed prostheses. Thus, osteolysis was associated with increased numbers of macrophages (p = 0.0006), total T lymphocytes
(p = 0.03), cytotoxic/suppresser T lymphocytes (p = 0.02),
IL-1 (p = 0.02) and IL-6 (p = 0.0001). This was not the
case with cementless prostheses; the values for the cellsurface antigens and cytokines for all three cementless
groups were generally comparable. In the cementless
series, osteolysis was associated with increased numbers of
all T-lymphocyte subgroups (p = 0.005), and increased
TNF expression (p = 0.04).
Cemented implants with osteolysis had greater numbers
of IL-6 positive cells (68% ± 3% v 48% ± 7%, p = 0.008),
and fewer fibroblasts (15% ± 5% v 44% ± 7%, p = 0.004)
compared with cementless implants with osteolysis. Wellfixed, cementless implants had more macrophages (56% ±
7% v 37% ± 5%, p = 0.04), total T lymphocytes (28% ± 3%
v 15% ± 5%, p = 0.04 for CD2 and 22% ± 3% v 11% ± 3%,
p = 0.02 for CD3), IL-6 (56% ± 9% v 34% ± 5%, p = 0.07),
and TGF (49% ± 9% v 26% ± 8%, p = 0.06) compared
with well-fixed cemented implants without osteolysis.
There were no other significant intergroup comparisons.
Discussion
Our study is unique because of the large number of
retrieved specimens, the extensive number of cell-surface
THE JOURNAL OF BONE AND JOINT SURGERY
CELLULAR PROFILE AND CYTOKINE PRODUCTION AT PROSTHETIC INTERFACES
535
Fig. 1a
Fig. 1b
Fig. 1c
Fig. 1d
Photomicrographs of sections from cemented prostheses processed by immunohistochemistry and in situ hybridisation. In each column, the row entries
include staining for (a) haematoxylin and eosin, (b) CD68, (c) CD3 and (d) IL-6. A positive cell for a particular monoclonal antibody or cytokine has
brown, darkly staining cytoplasm. The cytoplasm of negative cells does not stain brown. The cell nucleus is darkly staining for both positive and
negative cells. All photomicrographs for (b) to (d) are counterstained with haematoxylin (magnification 300).
Left column. Figure 1a – A loose prosthesis with osteolysis (group 1). There is infiltration of numerous macrophages and giant cells in a fibrovascular
stroma with a large polyethylene fragment in the centre. Figure 1b – Numerous macrophages and giant cells are identified. Figure 1c – T lymphocytes
are seen as small, darkly staining cells. Some large macrophages stain faintly. Figure 1d – There are many darkly staining cells.
Centre column. Figure 1a – A loose prosthesis without osteolysis (group 2). There is a fibrovascular stroma but fewer macrophages and giant cells
compared with group 1. Figure 1b – The macrophages and giant cells are less noticeable compared with group 1. Figure 1c – T lymphocytes are seen.
Figure 1d – IL-6 mRNA is seen in some of the cells.
Right column. Figure 1a – A well-fixed prosthesis (group 3) with primarily fibrous tissue. There are fewer macrophages and giant cells compared with
groups 1 and 2. Figure 1b – Some macrophages are identified. Figure 1c – Few T lymphocytes are seen. Figure 1d – IL-6 mRNA is identified in some
of the cells.
antigens, cytokines and growth factors investigated, and the
inclusion of multiple sequential sections to enable a correlation to be made between cell type and cytokine
production.
The different clinical and radiological groups in our
study were associated with differences in the cellular content and cytokine profile of the retrieved tissue. Cemented
prostheses with osteolysis showed large numbers of macrophages which exhibited mRNA for many cytokines including IL-1, IL-6, TNF, PDGF and TGF. These were
present to some degree in all tissues from loose and wellfixed cemented implants. For cemented implants, the highVOL. 80-B, NO. 3, MAY 1998
est numbers of positive cells for the above cytokines were
found in the group with osteolysis and the lowest numbers
in well-fixed prostheses. For IL-1 and IL-6, two cytokines
known to be associated with bone resorption, the result was
statistically significant. These findings agree with those of
4,8,9,12,13,15,16,21,28-35.
other in vivo and in vitro studies.
IL-1 and IL-6 modulate the function of many cell lines
including monocytes, macrophages, fibroblasts, osteoblasts,
4,13,36-41
osteoclasts, and T and B lymphocytes,
as well as
the effects of IL-2 and TNF; the latter cytokine is also
proinflammatory. Thus, IL-1 and IL-6 have important roles
in modulating immune functions and the growth, differ-
536
S. B. GOODMAN,
P. HUIE,
Y. SONG,
ET AL
Fig. 2a
Fig. 2b
Fig. 2c
Fig. 2d
Photomicrographs of sections from cementless prostheses processed by immunohistochemistry and in situ hybridisation. In each column, the row entries
include staining for (a) haematoxylin and eosin, (b) CD68, (c) CD3 and (d) IL-6. A positive cell for a particular monoclonal antibody or cytokine has
brown, darkly staining cytoplasm. The cytoplasm of negative cells does not stain brown. The cell nucleus is darkly staining for both positive and
negative cells. All photomicrographs for (b) to (d) are counterstained with haematoxylin (magnification 300). Left column. Figure 2a – A loose
prosthesis with osteolysis (group 4). There are an intermediate number of macrophages. Figure 2b – Moderate numbers of macrophages and giant cells
are identified. Figure 2c – There are moderate numbers of T lymphocytes. Figure 2c – An intermediate number of cells stain positively for IL-6. Centre
column. Figure 2a – A loose prosthesis without osteolysis (group 5). There is a large amount of metal debris which is both interstitial and concentrated
within macrophages and giant cells. A fibrovascular stroma is seen. Figure 2b – Macrophages and giant cells are seen many of which contain metal
particles. Figure 2c – Scattered T lymphocytes are seen. Figure 2d – Moderate staining is seen for IL-6 mRNA. Right column. A well-fixed prosthesis
(group 6). Figure 2a – The tissue is primarily fibrous. Figure 2b – Few macrophages are present. Figure 2c – Few T lymphocytes are seen. Figure 2d
– Little IL-6 mRNA is identified.
entiation and remodelling of mesenchymal tissue. They are
intimately associated with the synthesis of collagenase and
prostaglandin E2, which are also known to be involved in
the remodelling of bone. Furthermore, IL-1 and IL-6 are
important in osteoclast-mediated bone resorption since they
stimulate directly the differentiation and maturation of
36-41
TGF is generally an antiosteoclast precursors.
inflammatory, immune-suppressant cytokine, important in
38
the repair and remodelling of mesenchymal tissue. It also
has a significant role in wound healing, facilitates the
formation of the extracellular matrix by osteoblasts and
other cells, and inhibits osteoclast function. Thus, in the
prosthetic bony bed, both pro- and anti-inflammatory sub-
stances are secreted, which, in turn, modulate bone resorption and formation. These events are closely coupled and,
42
in a recent study, active bone formation was the predominant histological feature in bone and periprosthetic
tissues retrieved from both loose and well-fixed implants.
In these studies, osteoclasts as well as macrophages
appeared to have been directly resorbing bone. Thus, the
prosthetic interface provides a complex series of interactions of cells, cytokines and other substances which
modulate bone remodelling.
With cemented implants, differences were also found in
the T-lymphocyte subgroups, suggesting a possible role for
immunological processes in osteolysis, but this is conTHE JOURNAL OF BONE AND JOINT SURGERY
CELLULAR PROFILE AND CYTOKINE PRODUCTION AT PROSTHETIC INTERFACES
Fig. 3a
537
Fig. 3b
Photomicrographs of sections of a loose, cemented prosthesis with osteolysis (group 1). Figure 3a – There is a large number of CD14-positive, activated
macrophages (CD14 monoclonal antibody stain with haematoxylin counterstain 300). Figure 3b – The cells stain positively for platelet-derived growth
factor (in situ hybridisation for PDGF with haematoxylin counterstain 225).
Table IV. The percentage (± SEM) of positively staining cells for each monoclonal antibody, cytokine and growth
factor for the six groups
Group*
Variable
1
CD68
CD2
CD3
CD4
CD8
IL-1
IL-1
IL-2
IL-6
TGF-
TNF
PDGF
Poly-t
IFN
66
20
19
22
14
38
57
6
68
42
35
45
72
32
2
±
±
±
±
±
±
±
±
±
±
±
±
±
±
4
3
3
3
2
5
6
2
3
5
5
5
4
5
58
15
11
17
7
26
38
10
51
38
29
38
49
26
3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
4
2
2
3
1
4
5
3
4
4
4
5
4
4
37
15
11
16
9
16
30
13
34
26
28
29
51
20
4
±
±
±
±
±
±
±
±
±
±
±
±
±
±
5
5
3
4
2
5
7
4
5
8
6
14
9
6
64
29
26
29
17
36
50
4
48
37
44
55
64
24
5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
6
5
5
6
3
9
8
1
7
10
7
9
6
7
53
11
9
9
5
20
45
5
57
37
18
35
59
22
6
±
±
±
±
±
±
±
±
±
±
±
±
±
±
7
2
2
2
1
5
7
3
8
8
5
6
6
7
56
28
22
27
15
28
39
11
56
49
37
57
54
16
p value
±
±
±
±
±
±
±
±
±
±
±
±
±
±
7
3
3
5
2
7
6
6
9
9
8
9
7
7
0.011
0.001
0.0004
0.033
0.0004
0.055
0.067
0.56
0.008
0.39
0.12
0.2
0.011
0.44
* see text
21,32,43-48
troversial and there are studies which advocate
49-54
and refute
a role for T-cell modulation of the macro55,56
phage response to biomaterials.
With cementless prostheses there were fewer differences
in cell number and cytokine profile in the various groups.
Osteolysis was associated with increased TNF expression.
In general, the tissue excised from cementless prostheses
was less plentiful and more fibrous in nature compared with
that from cemented prostheses. Cementless implants with
osteolysis had fewer IL-6-positive macrophages and more
fibroblasts than cemented implants with osteolysis. Our
study corroborates the findings of others who have shown
that with well-fixed cemented implants there may be bone
immediately adjacent to the cement with little or no inter29
vening fibrous tissue in some locations, but with cementless implants, fibrous tissue rather than bone is
30,57
predominant.
This fibrous layer may be a pathway or
24-28,58
conduit for the migration of cells and wear debris.
Thus, despite being primarily fibrous, well-fixed, cementless implants contained more macrophages, total T lymphocytes, IL-6, and TGF compared with well-fixed cemented
VOL. 80-B, NO. 3, MAY 1998
implants. A well-functioning cement mantle appears to
limit the migration of foreign-body and immune cells, and
the production of specific cytokines along the prosthetic
interface. Recent studies have suggested that a circumferentially porous-coated, osseointegrated implant, or one
that is coated with hydroxyapatite, may function in the
58,59
same way as a well-fixed cemented implant.
There were some limitations to our study. We assigned
an implant to a particular group according to established
clinical and radiological criteria, but no special radiographic views were used. This protocol may have underestimated the number of radiolucent lines and the presence
27
of ballooning radiological osteolysis. The tissues from
well-fixed implants cannot be considered as a control
group, because these implants were painful, dislocating, or
showed excessive polyethylene wear. Furthermore, the time
from implantation to revision for well-fixed implants in
both the cemented and cementless groups was shorter
compared with the respective groups with loosening. Thus,
the tissues surrounding well-fixed prostheses were from
prostheses in the early stages of failure, before more
538
S. B. GOODMAN,
P. HUIE,
advanced mechanical and biological processes could produce loosening or osteolysis.
The materials used in the retrieved implants included
contemporary polymers (PMMA and ultra-high-molecularweight polyethylene) and metals (cobalt-chrome alloy and
titanium 6-aluminium 4-vanadium alloy). The composition
of the excised components was not recorded on the data
sheets by all surgeons and therefore no correlations can be
made between implant materials and cellular and cytokine
profiles. There were, however, no distinguishing histomorphological characteristics in the tissues in which the materials were known, which is in agreement with other
14-16
studies.
Tissue sampling bias may have affected our results. In
general, the surgeon chose a representative, accessible
piece of the interface. If osteolysis was present, an attempt
was made to biopsy the osteolytic areas, but such tissue is
known to be somewhat heterogeneous with respect to cell
21
type and biological activity. Thus, the selected piece of
tissue may not have been representative of the biological
activity of the membrane as a whole. It is more likely that
local mechanical, material and biological factors govern the
local characteristics of the interface. Most tissues were
taken from hip replacements (85 specimens) but 16 were
from revised knee arthroplasties. Only in group 3 were
there more knee than hip replacements. The wear debris
from hip and knee replacements is similar qualitatively but
may differ quantitatively. Nevertheless, the histomorphological appearance of the tissues from these locations was
indistinguishable. The histological assessment of the tissues
was performed in a blind fashion and no differences were
found in the histomorphology, cellular profile or cytokine
expression of tissues in the same clinicoradiological group,
whether they were from the hip or knee. Other studies have
shown, however, that differences in cell profile and cytokine production may be dependent on the location of the
60,61
tissue specimen.
The mRNA for cytokines and growth factors was identified using in situ hybridisation. The final protein production
may have been altered by post-transcriptional and posttranslational mechanisms. Recent unpublished data from
our laboratory, however, have shown a high correlation for
IL-6 cytokine production assessed by in situ hybridisation
(which identified the mRNA for IL-6) and immunohistochemistry (which identified IL-6 protein) in five tissues
from revision cases.
Conclusion. For cemented implants, the histological, histochemical and cytokine profiles of the interface correlated
with the clinical and radiological grade of loosening and
osteolysis. Cemented prostheses with osteolysis were associated with increased numbers of macrophages, T-lymphocyte subgroups, and IL-1 and IL-6 expression. Tissues from
cementless prostheses were more uniform with regard to
cytokine production, probably due to the presence of more
fibrous tissue at the interface. Despite this, in the cementless series, osteolysis was associated with increased num-
Y. SONG,
ET AL
bers of T-lymphocyte subgroups and increased TNF
expression. Specific cellular populations and cytokine profiles may be involved in the processes of loosening and
osteolysis. Furthermore, the profiles for cemented and
cementless implants may differ. T-cell modulation of macrophage function may be an important interaction at prosthetic interfaces.
The authors gratefully acknowledge the financial support of Pfizer/Howmedica in helping to carry out this study. They are appreciative of the
efforts of those who participated in the membrane retrieval studies over a
three-year period.
Although none of the authors have received or will receive benefits for
personal or professional use from a commercial party related directly or
indirectly to the subject of this article, benefits have been or will be
received but are directed solely to a research fund, educational institution,
or other non-profit institution with which one or more of the authors is
associated.
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