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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. References 1. Anthony PP, Gie GA, Howie CR, Ling RSM. 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