Neovascularization is indispensable to both bone remodeling and the development of chronic inflammation. A pro-angiogenic state in the periprosthetic tissue may augment the inflammatory response to wear debris. To investigate if a pro-angiogenic state exists in the bone–implant interface of aseptically loosened joint prosthesis, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1/KDR were studied by immunohistochemistry. The VEGF-Flk/KDR pathway has been implicated as a key signaling requirement for pathological angiogenesis. The level of vascularization in periprosthetic tissue was semi-quantitatively compared to osteoarthritic (OA) and rheumatoid arthritic (RA) synovium. The level of vascularization in areas of periprosthetic tissue with heavy or low/moderate wear debris were also compared semi-quantitatively by image analysis.

OBJECTIVE:Nitric oxide (NO) produced by cartilage and synovial membranes is implicated in the pathogenesis of osteoarthritis (OA) and inhibitors of NO synthesis may have indications in the treatment or prevention of joint destruction in OA. Because the signaling mechanisms as well as the NOS isoform involved in induction of NO production in human cartilage remain in many parts unclear, the present study was designed to investigate the regulation of inducible NO synthesis in human intact OA cartilage.METHODS:Cartilage specimens were collected from OA patients undergoing knee replacement surgery and studied for iNOS expression and NO production in organ culture to allow intact chondrocyte-matrix interactions. J774 macrophages were used for comparison as a well-documented source of iNOS.RESULTS:OA cartilage expressed iNOS and produced NO in the absence of exogenous cytokines. Addition of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha) or lipopolysaccharide (LPS) into the culture medium enhanced NO production in a dose-and time-dependent manner. Various NOS inhibitors suppressed NO production in the following order of potency: 1400W (novel selective iNOS inhibitor)=L-NIO>L-NMMA>L-NAME. Cycloheximide (an inhibitor of protein synthesis), pyrrolidine dithiocarbamate (PDTC; an NF-kappa B inhibitor) and genistein (an inhibitor of tyrosine protein kinases) inhibited cytokine-induced NO production, while dexamethasone, diaminohydroxypyrimidine (DAHP; an inhibitor of tetrahydrobiopterin synthesis) and PD 98059 (p42/44 MAP kinase inhibitor) had no effect.CONCLUSIONS:The results suggest that NO synthesis in human osteoarthritic cartilage derives from the glucocorticoid-insensitive expression of iNOS. Very similar mechanisms appear to regulate inducible NO synthesis in human osteoarthritic cartilage and J774 macrophages with the exception that dexamethasone inhibited NO production in J774 cells but not in osteoarthritic cartilage.

Patients with long-term orthopedic implants may develop inflammatory reactions due to the accumulation of biomaterial particles both around the implant and in distant organs. The exact impact of these particles on the normal immune cell function still remain relatively unclear. Activation of T-cells following exposure to biomaterial particles is driven by macrophages and requires synergistic signals primed by both antigen presentation and costimulation. The pattern of costimulatory molecule expression (CD80,CD86) was primarily examined using immunohistochemistry on tissue specimens of bone/implant interface membranes taken from sites of bone erosion. Additionally, costimulatory molecule expression was also assessed in the monocytic leukemia cell line U937 following exposure to clinically relevant titanium aluminum vanadium (TiAlV) and stainless steel particles (FeCrNi) cultured in vitro. This study demonstrates the induction and prominent expression of CD86 on almost all macrophage subsets at the bone/implant interface, including fused forms and large multinucleated giant cells (MNGC). In vitro analysis also indicated phagocytosis of metal particles by differentiated U937 caused significant induction of both CD80 and CD86 (p < 0.01), although the expression of CD86 dominated following prolonged exposure. The data presented highlights that CD86 is the predominant costimulatory molecule ligating to the complementary CD28 molecule at the inflammatory lesion of the interface. We propose that the intracellular presence of indigestible implant material, in addition to elevated costimulatory molecule expression, may promote T-cell inflammatory reactions at sites close to and distant from the orthopedic implant.

The immunologic response to prosthetic biomaterial particles is characterized by macrophage-rich inflammatory infiltrate, formation of multinucleated giant cells, and aseptic loosening at the site of arthroplasty. We investigated the in vivo expression and tissue distribution of transforming growth factor alpha, macrophage colony-stimulating factor, and the receptor for colony-stimulating factor-1 at the site of bone erosion in patients with clinically failed orthopaedic implants (n = 30). The: expression was further compared with that detected in the inflamed synovial membranes from patients with rheumatoid arthritis or osteoarthritis (n = 15) and one patient with osteoclastoma (giant cell tumour of bone). Immunostaining of the tissue demonstrated positivity for transforming growth factor alpha within the inflammatory macrophage and multinucleated giant cell infiltrate in the diseased synovial membrane and the bone-implant interface. A comparative analysis between the synovium and retrieval interface membranes (pseudosynovium) revealed a high level of expression of transforming growth factor alpha, with intense membrane staining on multinucleated giant cells in all failed arthroplasties with pseudosynovium. In addition, the frequency, antigenic phenotype, and pattern of transforming growth factor alpha expression on multinucleated giant cells in the interface were markedly similar to those observed for multinucleated giant cells in osteoclastoma. Multinucleated giant cells within the interface lacked the expression of macrophage colony-stimulating factor and colony-stimulating factor-1 receptor, whereas those at the bone surfaces exhibited strong immunoreactivity. The predominant expression of transforming growth factor alpha by multinucleated giant cells in the bone-implant interface and its similarity to osteoclastoma highlight the importance of assessing transforming growth factor alpha as a possible contributor to the development of bone-resorbing giant cells at the site of failed orthopaedic implants.

Osseointegration at the site of orthopedic implants is dependent on the recruitment, attachment, and differentiation of osteogenic cells. Data concerning the effect of a patient's underlying joint disease on the modulation of the cellular activity and the long-term survival of joint prostheses is limited. In this study, immunocytochemistry was used to investigate the osteogenic cell phenotype within the bone-implant interface fibrous membrane in 60 patients with different underlying joint disease. Tissue specimens were removed during revision operations performed at variable times following implantation. The results provided histological evidence of the presence of fibrocartilage tissue and calcified bone within the interface. TGF-beta, metalloproteinases (MMP1 and MMP2) and their inhibitors (TIMP1 and TIMP2) were immunolocalized within fibroblasts, chondrocytes, and osteoblasts throughout the interface, indicating that signals modulating the osteogenic cell phenotype at these sites are highly regulated. Finally, the study identified a significant difference in the histological changes elicited by implant particulate debris in patients of different diagnostic categories. Such observations imply that the activity of the original joint disorder could augment specific cellular activation/immune signals that subsequently affect the degree of the local inflammatory responses to implant wear particles. The negative balance between the rate of bone growth and resorption around the prosthetic joint is central to the pathogenesis of aseptic loosening of implants.

Joint replacement surgery has a wide clinical application as a successful technique. However, the release of biomaterial in particulate form from various implant components has been implicated as a cause of two major clinical complications: 1) bone lysis with or without aseptic loosening, and 2) dissemination of wear particles to distant sites with adverse local or systemic cellular responses. This review focuses on the analysis of the clinical material obtained at the time of revision operations and its value in identifying the pathological processes taking place within the bone-implant microenvironment. Important issues discussed include the incidence of infection, characterization of wear particles, particle-cell interactions, local histopathological changes that lead to the formation of erosive inflammatory lesions next to the bone, sensitivity reactions, tumor formation, and the induction of inflammatory factors and cytokines that can influence the rate of bone resorption versus bone formation.

IL-4 is a mast cell and T cell produced immune cytokine that is important in the regulation of macrophage function. IL-4 has also been implicated in the induction of foreign body giant cell formation. In patients with long-term joint prostheses, a localized granulomatous inflammation develops in periarticular tissues and other organs where phagocytosis of particulate material from various prosthetic components takes place. In this study we used the inflammatory lesions of the bone–implant interface as a model to investigate the possible production, the frequency and the cellular source of IL-4. 40 samples of the interface membrane obtained from 25 patients undergoing revision of clinically failed implants were analyzed by immunohistochemistry. Cryostat sections were labeled with specific monoclonal antibodies to mast cell products: IL-4, tryptase and the receptor c-kit (CD117). The study has identified a significant level of production of IL-4 by mast cells in all the cases analyzed. There was an apparent difference in the number of mast cells in relation to the histological variants of the interface. The increase in the number of mast cells and IL-4 production was more pronounced in cases with heavy macrophage infiltrate than those exhibiting a predominance of giant cells. The findings imply that the recruitment of mast cell and IL-4 expression precede the granulomatous reaction and may have a role in the induction of a number of immunopathological changes related to mast cell activation by biomaterial particles. © 1998 Kluwer Academic Publishers

The response to wear particles from orthopaedic implants can lead to inflammation, osteolytic lesions, and aseptic loosening. To gain an insight into the development of this pathogenetic process, immunohistochemical techniques were used to identify the expression and tissue distribution of the potent cell mitogen epidermal growth factor (EGF), and the epidermal growth factor receptor (EGF-R) at the site of bone erosion in 30 patients with clinically failed orthopaedic implants. The results showed a large proportion of the macrophage subsets (Mφ) which expressed EGF and EGF-R, also contained wear particles, indicating their expression is a consequence of Mφ phagocytosis of implant material. The surface membrane expression of EGF-R on fusing Mφ suggests its presence is fundamental to the formation of bone-resorbing multi-nucleated giant cells, and the development of osteolysis. Additionally, there is increasing evidence of the long-term systemic spread of wear particles and their accumulation at distal sites including lymph nodes, liver, and spleen. Elevated expression of mitogenic factors in response to wear particles may result in deviation from normal cell growth and regulation, resulting in changes to immune cell function. Such potential transformations at distal sites are clinically significant, as alterations to the patient's immune system may result in acute divergence from normal immune cell responses. © 1998 Kluwer Academic Publishers

An attempt was made to assess the local chronic inflammatory response in patients with failed orthopaedic implant that is clinically associated with osteolysis, bone and bone marrow necrosis. The main objective was to analyse the heterogeneity of the macrophage functional subsets in the bone–implant interface membrane and to evaluate their possible role in the development of an erosive inflammatory lesion within the bone. Immunohistology was performed on 21 specimens of the bone–implant interface obtained from 17 patients during revision arthroplasty, and synovial membranes from rheumatoid (RA, n=4), and osteoarthritis (OA, n=4) patients. Three well-characterized monoclonal antibodies (MAb) recognizing antigenic determinants on specific functional subsets of macrophages (Mφ) were used. RFD1 (interdigitating reticulum cells/antigen presenting cells, (APC), RFD7 (mature phagocytic macrophages), and RFD9 epithelioid cells and foreign body giant cells (FBGC). RFD1 was expressed on a variable number of perivascular and synovial lining Mφ in both RA and OA synovia, at a frequency of 25%–40%. In cases with total joint replacements, the interface showed a marked increase in the expression of RFD1 (20%–90%). A considerably greater percentage of RFD1 positive Mφ and FBGC was noted in the interfaces from cases with a high level of detectable metal particulate wear debris (mean 80%, range 60%–90%) than in cases with polyethylene wear debris (mean 30%, range 0%–50%), p 0.0001. RFD7 labelled most tissue Mφ in each group. Immunoreactivity for RFD9 was restricted to FBGC in all cases analysed. The finding of elevated expression of RFD1 on metal-containing Mφ and FBGC in the bone-implant interface suggests an increase in antigen-presenting phenotype and indicates that metal particles have more impact in the induction of immune-mediated responses. Such responses are characterized by sustained cellular hyperreactivity and phenotypic changes in Mφ subsets.

Our aim was to analyse the influence of the size, shape and number of particles on the pathogenesis of osteolysis. We obtained peri-implant tissues from 18 patients having revision surgery for aseptically loosened Freeman total knee replacements (10), Charnley total hip replacements (3) and Imperial College/London Hospital double-cup surface hip replacements (5). The size and shape of the polyethylene particles were characterised using SEM and their concentration was calculated. The results were analysed with reference to the presence of radiological osteolysis. The concentration of polyethylene particles in 6 areas with osteolysis was significantly higher than that in 12 areas without osteolysis. There were no significant differences between the size and shape of the particles in these two groups. We conclude that the most critical factor in the pathogenesis of osteolysis is the concentration of polyethylene particles accumulated in the tissue.

Although nitric oxide (NO) is a well documented effector molecule in rodent macrophages, its significance in human mononuclear phagocytic cells has been controversial. The foreign body inflammatory reaction around loosened joint replacement implants leads to formation of an osteolytic granulomatous pseudo-synovial membrane rich in activated macrophages. We studied 13 specimens of interface membrane tissue collected from revision surgery of aseptically loosened hip and knee prostheses for the presence of inducible NO synthase (iNOS). The presence of iNOS was demonstrated immunohistochemically in 10 of these specimens. Within the tissue this enzyme was confined to macrophages and vascular endothelial cells. iNOS activity was demonstrated biochemically by measuring the calcium-independent generation of citrulline from L-arginine, and the presence of iNOS mRNA was demonstrated using reverse transcriptase polymerase chain reaction. NO synthesis in the interface tissue may be an important factor in the maintenance of the inflammatory and osteolytic processes.

Bone loss induced by the inflammatory response to wear particles is a major cause of long-term failure of total joint replacement. This review describes the cellular reaction occurring in response to these particles and what is currently known about the inflammatory mechanisms contributing to bone resorption.

The aim of this study was to evaluate the relative contribution of polyethylene, metal, and polymethylmethacrylate (cement) particles to the overall bone loss in aseptic loosening. Twenty-four interface tissues with adjacent bone were obtained during 17 revision total joint arthroplasties (11 hips and six knees). Osteoclasts and macrophages were identified immunohistochemically on the bone surface. The length of the bone surface in contact with these cell types was measured and analyzed with reference to the particulate species present within the fibrous interface. The presence of abundant polyethylene particles significantly increased the proportion of the bone surface in contact with macrophages but did not have a significant influence on that of osteoclasts. Osteoclastic bone resorption was significantly more extensive in the presence of metal particles. In contrast, the presence of cement particles did not have a significant influence on macrophage or osteoclast coverage of the bone surface. These results highlight the significance of polyethylene particles in macrophage recruitment and subsequent osteolysis and suggest a different mechanism of bone loss related to metal, namely mediation through osteoclastic activities. The relative contribution of cement particles was negligible and needs reevaluation in light of evidence provided by others.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor with a regulatory effect on the transformation of immature macrophages into multinucleated giant cells (MNGC) that exhibit phenotypic and functional characteristics of osteoclasts (OC). The authors analyzed the bone implant interface membranes harvested from 15 patients with failed total joint replacements for the production and tissue distribution of GM-CSF and interleukin-1 (IL-1). Immunohistology and liquid culture were employed to assess the contribution of these factors in the recruitment of macrophages and the development of bone resorbing MNGC at these sites. This process has been implicated in osteoclastic bone resorption, bone, and bone marrow necrosis adjacent to orthopaedic implants. Histologic assessment of the interface indicated the presence of granuloma and a variable number of MNGC in 11 cases. Four cases showed sites of intramembranous formation of osteoid and mineralized bone that was accompanied by normal bone marrow in two cases. Granulocyte-macrophage colony stimulating factor was expressed by a distinct subset of phagocytic macrophages in the lining layer on the implant side. interleukin-1-positive cells outnumbered those stained for GM-CSF. Stimulation of cultured cells with prosthetic metal particulate material showed marked similarity in the expression of these cytokines to cultures treated with lipopolysaccharide (LPS) or phytohemagglutinin (PHA). The induction of GM-CSF production in the lining layer where small MNGC develop indicates that these cells differentiate locally following the phagocytosis of particulate wear debris. In conclusion, GM-CSF promotes the proliferation and early stages of fusion and development of MNGC responsible for osteolysis at these sites. These results also highlight the capacity of the interface to display both osteogenic and inflammatory characteristics. Collectively, the findings suggest that the local bone marrow could participate in the development of the interface as a source of myeloid cells in addition to the capacity of marrow stroma to generate various osteogenic cells essential for the ingrowth of bone into prosthetic implants.