Thalidomide blocking of particle-induced TNFα release in vitro

 Tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6), pleiotropic cytokines with osteotropic activities, are produced by multiple cells in the skeletal tissue, including macrophages and osteoblasts. They are thought to be pivotally involved in pathological bone resorption, such as that seen with aseptic loosening. Thalidomide is reported to have antiinflammatory, immunomodulatory effects in a number of inflammatory diseases. We investigated the effect of thalidomide on titanium (Ti) particle-induced TNFα and IL-6 production by both human macrophage U937 and osteoblast MG-63 cell lines. They were stimulated with 1 × 10⁷ Ti particles/ml and treated simultaneously with or without various concentrations of thalidomide (from 2.5 ng/ml to 25 μg/ml) for 24, 48, or 72 h. Cell viability and proliferation were measured. TNFα and IL-6 in the supernatant of the culture media were also analyzed with an enzyme-linked immunosorbent assay. We found that with a concentration of thalidomide of less than 2.5 μg/ml the viability of the two cell lines did not differ significantly from that of controls treated simultaneously with 1 × 10⁷ Ti particles/ml. Cell proliferation was inhibited to some extent when they were treated with thalidomide 2.5 μg/ml co-cultured with 1 × 10⁷ Ti particles/ml. Thalidomide treatment was found to inhibit TNFα production in a dose-dependent manner in human macrophages exposed to Ti particles. At the clinically achievable drug dose of 2.5 μg/ml, 34.4% TNFα inhibition occurs. Thalidomide had no effect on IL-6 secretion in these cultures. These data support the idea that thalidomide may have potential for treating prosthetic loosening in humans. Received: February 13, 2002 / Accepted: August 1, 2002 Acknowledgments. The authors thank Guangrong Sun, Patricia Redecha, and Bruce Rapuano for their technical help and assistance. Offprint requests to: M.P.G. Bostrom     

Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro.

C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.     

Low IGF-I in synovial fluid and serum in patients with aseptic prosthesis loosening

Background We have previously shown that proliferation in primary cultures of human osteoblast-like cells is lower after exposure to synovial fluid from patients with aseptic prosthesis loosening than after exposure to synovial fluid from patients with osteoarthrosis.

Materials and methods Insulin-like growth factors (IGF) I and II and IGF binding proteins (IGFBP) 3–6, were measured with radioimmunoassy in synovial fluid and in serum from patients with aseptic prosthesis loosening or osteoarthrosis. Proliferation in osteoblast-like MG-63 cells was studied with the CyQUANT assay.

Results IGF-I and IGFBP-4 concentrations were lower whereas the concentration of IGFBP-6 was higher in synovial fluids from patients with prosthesis loosening than in synovial fluid from patients with osteoarthrosis. IGF-I concentrations in serum from patients with prosthesis loosening were also lower than in the osteoarthrosis group, and were even below the normal range in most cases (72%). Synovial fluid from patients with aseptic loosening had a weaker stimulatory effect on MG63 osteoblast-like cell proliferation than synovial fluid from patients with osteoarthrosis, but there was no difference between the two groups when a human IGF-I antibody was added.

Interpretation Low levels of IGF-I in synovial fluid possibly result from low serum levelsand may be a mechanism leading to aseptic prosthesis loosening.     

Low IGF-I in synovial fluid and serum in patients with aseptic prosthesis loosening

Background We have previously shown that proliferation in primary cultures of human osteoblast-like cells is lower after exposure to synovial fluid from patients with aseptic prosthesis loosening than after exposure to synovial fluid from patients with osteoarthrosis.

Materials and methods Insulin-like growth factors (IGF) I and II and IGF binding proteins (IGFBP) 3-6, were measured with radioimmunoassy in synovial fluid and in serum from patients with aseptic prosthesis loosening or osteoarthrosis. Proliferation in osteoblast-like MG-63 cells was studied with the CyQUANT assay.

Results IGF-I and IGFBP-4 concentrations were lower whereas the concentration of IGFBP-6 was higher in synovial fluids from patients with prosthesis loosening than in synovial fluid from patients with osteoarthrosis. IGF-I concentrations in serum from patients with prosthesis loosening were also lower than in the osteoarthrosis group, and were even below the normal range in most cases (72%). Synovial fluid from patients with aseptic loosening had a weaker stimulatory effect on MG63 osteoblast-like cell proliferation than synovial fluid from patients with osteoarthrosis, but there was no difference between the two groups when a human IGF-I antibody was added.

Interpretation Low levels of IGF-I in synovial fluid possibly result from low serum levelsand may be a mechanism leading to aseptic prosthesis loosening.     

Inflammatory Response to Implant Particulates in a Macrophage/Osteoblast Coculture Model

The purpose of this study was to further define the cellular response to titanium and polymethylmethacrylate (PMMA) particles in aseptic loosening, and to determine if the use of pamidronate may be effective in inhibiting bone resorption associated with this response. Macrophages and osteoblasts were cocultured to simulate the environment around an aseptically loose prosthesis. Macrophages were plated on the bottom of six well plates and osteoblasts were plated on culture dish inserts, and placed into the wells with the macrophages. Incubation of macrophages with PMMA in this system led to release of prostaglandin E (PGE₂), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-6 (IL-6). Incubation with titanium led to release of tumor necrosis factor (TNF) and IL-6. Exposure of calvaria to media from cells exposed to either PMMA or titanium led to release of calcium 45. Incubation of calvaria with pamidronate was able to inhibit release of calcium 45 associated with exposure to the macrophage/osteoblast/particle conditioned medium. Bone resorption at the interface between implant and bone is a consistent feature leading to loosening of orthopedic implants. By inhibiting bone resorption associated with the inflammatory response to implant particulates, pamidronate or other bisphosphonates may have clinical utility in the treatment or prevention or aseptic loosening. Received: 22 December 1995 / Accepted: 3 May 1996     

Cytokine response of human macrophage-like cells after contact with polyethylene and pure titanium particles.

The aim of this study was to establish a human macrophage cell culture system to examine the effect of polyethylene (PE) and titanium particles on cytokine release by macrophage-like cells (MLC) and to quantify this response with respect to the nature and concentration of particles. Human monocytic leukemia cells were differentiated under standard conditions with vitamin D3 and granulocyte macrophage-colony-stimulating factor. Cells were characterized by fluorescence-activated cell-sorter Scan of CD 14 expression analysis as well as a phagocytosis test exploiting fluorescence-labeled particles of bacteria] walls. To achieve a relevant contact between the floating PE particles (approximately 1 microm in size) and MLC, a rotation device was used (15 rotations/min) during incubation. The same was done with the titanium particles. Cell culture supernatants were then analyzed for interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha using the enzyme-linked immunosorbent assay technique in the absence or presence of particles. Rotation of incubated MLC alone did not influence the secretion of TNF-alpha, but it enhanced secretion of IL-1beta and IL-8 about 30-fold compared to background levels. Both PE and titanium particles significantly enhanced MLC cytokine release, the amount of which depended on the concentration of particles. Using 40 X 10(8) PE particles (0.7 x 10(8) titanium particles) and 10(6) MLC, the maximal release of IL-1beta was about 20-fold (7-fold titanium particles) higher than that of the rotating control sample. The stimulation of IL-8 release was 4-fold (3-fold titanium particles) and of TNF-alpha. 300-fold (170-fold titanium particles) compared to controls. MLC were viable (>90% cell survival) at concentrations less than 108 x 10(8) polyethylene particles per 10(6) MLC and 16 x 10(8) titanium particles per 10(6) MLC. Rotation per se as well as exposure to increasing concentrations of PE and titanium particles stimulates cytokine release (TNF-alpha, IL-1beta, IL-8) by macrophages in vitro. This in vitro model resembles the in vivo situation near arthroplasties, where implant particles make contact with inflammatory cells, such as macrophages. Cytokine release by macrophages may impair osteoblast function as well as stimulate bone resorption by osteoclasts and macrophages, thereby causing aseptic loosening of arthroplasties. Our in vitro model provides a reproducible human cell system that might shed light on the pathogenesis of particle disease and might serve as a reproducible in vitro test system for the biocompatibility of foreign materials.     

Impaired release of interleukin-6 from human osteoblastic cells in the uraemic milieu.

BACKGROUND: Osteoblast-derived interleukin-6 (IL-6) affects bone metabolism and is linked with a number of pathological states characterized by increased bone resorption, including osteoporosis and renal osteodystrophy. To examine the possibility that uraemia directly influences the release of this cytokine in bone, we have investigated the effect of human uraemic serum on the release of IL-6 from human osteoblast-like cells. METHODS: Individual serum samples collected from healthy male volunteers or male haemodialysis patients prior to and during a dialysis treatment were assayed for IL-6, interleukin-1beta (IL-1beta) and soluble IL-6 receptor (sIL-6R) using specific enzyme-linked immunosorbent assays. MG-63 and SaOS-2 cells were cultured in media containing pooled sera from both groups and alongside matching charcoal-stripped sera. IL-6 concentrations were determined in harvested cell supernatants after 24 h. In further experiments, media containing individual sera obtained from five patients at regular intervals during their haemodialysis treatment were incubated with MG-63 cells to determine the effects of the dialysis process on IL-6 secretion. RESULTS: Haemodialysis patients had significantly higher (n = 10, P < 0.001) circulating concentrations of IL-6 (7.0 +/- 1.6 pg/ml) than normal subjects (0.4 +/- 0.1 pg/ml), but there were no significant differences in the concentrations of either IL-1beta or sIL-6R. These serum concentrations did not change significantly during 80 min of dialysis. IL-6 release by MG-63 cells incubated with charcoal-stripped serum from normal or from uraemic subjects was similar. Incubation with untreated sera from normal subjects increased IL-6 release by approximately 6-fold above the charcoal-stripped control, whereas sera from uraemic subjects increased IL-6 release by only approximately 2- to 3-fold (normal vs uraemic of 6878 +/- 595 and 2579 +/- 169 pg/ml, respectively, P < 0.001). Similar results were seen with SaOS-2 cells. Haemodialysis did not restore the capacity of uraemic serum to augment IL-6 release to the same degree as normal serum. CONCLUSIONS: These data show that the augmentation of IL-6 release from human osteoblastic cells after incubation with normal serum is greater than after uraemic serum. This may indicate the presence of an inhibitor of IL-6 release in uraemic serum that is involved in the deranged bone turnover of uraemic patients.     

Effects of Roughness, Fibronectin and Vitronectin on Attachment, Spreading, and Proliferation of Human Osteoblast-Like Cells (Saos-2) on Titanium Surfaces

The influence of surface roughness and the presence of adhesion molecules in the culture medium were studied regarding cell adhesion, shape, and proliferation of osteoblast-like cells grown on two types of titanium disk. Type I disks were acid etched and type II disks were sandblasted and acid etched. Surface roughness was determined by contact profilometry and scanning electron microscopy. Chemical composition and oxide thickness of the superficial titanium layer were established with energy dispersive X-ray spectrometry, electron spectroscopy for chemical analysis and auger electron spectroscopy. Titanium release in the culture medium was assessed by inductively coupled plasma-optical emission spectrometry. Osteoblast-like cells (Saos-2) were cultured on both types of titanium disks (1) in standard conditions (DMEM culture medium supplemented with fetal calf serum), (FCS), (2) with the culture medium alone (DMEM alone), (3) in the presence of fibronectin or vitronectin (DMEM supplemented with fibronectin or vitronectin). Cultures were also performed in the presence of monoclonal anti-integrin (β₁, α_v) to test the cell adhesion molecules involved in the cell binding to the titanium surface. We found that sandblasting does not modify the chemical surface composition and that titanium represents only 5–6% (in the atom percentage) of surface elements. Release of titanium in the culture medium was found to increase from 24 to 72 hours. In the absence of FCS, fibronectin, or vitronectin, cells appeared scanty and packed in clusters. On the contrary, cells cultured in the presence of FCS, fibronectin, or vitronectin were flattened with large and thin cytoplasmic expansions. The addition of anti β₁ or α_v integrin subunit monoclonal antibody in the culture medium decreased adhesion and spreading of cells, particularly in the presence of fibronectin. Cell proliferation was significantly higher on culture plastic than on both types of disks, but was increased on rough but not on smooth surfaces. These results indicate that a high surface roughness and presence of fibronectin or vitronectin are critical elements for adhesion, spreading, and proliferation of cells on titanium surfaces. Received: 14 November 1997 / Accepted: 1 November 1998     

The Role of Osteoblast Density and Endogenous Interleukin-6 Production in Osteoclast Formation From the Hemopoietic Stem Cell Line FDCP-MIX C2GM in Coculture With Primary Osteoblasts

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C₂GM was shown to be strongly dependent on osteoblast density. In cocultures of C₂GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 × 10⁴ cells/cm²), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 × 10⁴ cells/cm²). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear. Received: 26 June 1997 / Accepted: 14 November 1997     

Biological responses of human mesenchymal stem cells to titanium wear debris particles

Wear debris-induced osteolysis is a major cause of orthopedic implant aseptic loosening, and various cell types, including macrophages, monocytes, osteoblasts, and osteoclasts, are involved. We recently showed that mesenchymal stem/osteoprogenitor cells (MSCs) are another target, and that endocytosis of titanium (Ti) particles causes reduced MSC proliferation and osteogenic differentiation. Here we investigated the mechanistic aspects of the endocytosis-mediated responses of MSCs to Ti particulates. Dose-dependent effects were observed on cell viability, with doses >300 Ti particles/cell resulting in drastic cell death. To maintain cell viability and analyze particle-induced effects, doses <300 particles/cell were used. Increased production of interleukin-8 (IL-8), but not IL-6, was observed in treated MSCs, while levels of TGF-β, IL-1β, and TNF-α were undetectable in treated or control cells, suggesting MSCs as a likely major producer of IL-8 in the periprosthetic zone. Disruptions in cytoskeletal and adherens junction organization were also observed in Ti particles-treated MSCs. However, neither IL-8 and IL-6 treatment nor conditioned medium from Ti particle-treated MSCs failed to affect MSC osteogenic differentiation. Among other Ti particle-induced cytokines, only GM-CSF appeared to mimic the effects of reduced cell viability and osteogenesis. Taken together, these results strongly suggest that MSCs play both responder and initiator roles in mediating the osteolytic effects of the presence of wear debris particles in periprosthetic zones.     

ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1)

Extracellular ATP, and to a lesser extent adenosine, an ATP metabolite, stimulated cell proliferation in osteoblast-like cells (MC3T3-E1). ATP increased cytosolic Ca²⁺ due to Ca²⁺ mobilization from intracellular storage in the same concentration range of the nucleotide as that effective for DNA synthesis, suggesting the mediation of the phospholipase C/Ca²⁺ system in the mitogenic action. Since adenosine induced no Ca²⁺ mobilization, P₂-purinergic receptor appears to be associated with ATP actions. The growth-promoting effect of ATP was not inhibited by H7, a protein kinase C inhibitor, and indomethacin, a cyclooxygenase inhibitor, indicating no involvement of activation of protein kinase C and production of prostaglandins in ATP-induced mitogenic signals. Either ATP or adenosine remarkably and synergistically potentiated platelet derived growth factor-induced DNA synthesis. These findings suggest that extracellular ATP and adenosine may play a physiological role in the regulation of bone formation. Received: 17 January 1995 / Accepted: 21 March 1995     

Titanium particles up-regulate the activity of matrix metalloproteinase-2 in human synovial cells

Purpose

Wear debris particle-induced osteolysis and subsequent aseptic loosening is one of the major causes of failure of total joint replacement. The purpose of this study was to investigate the effect of titanium implant material and inflammatory cytokines on human synovial cells and the development to osteolysis and aseptic loosening.

Methods

This study investigated the effect of titanium implant material on the ECM-degraded MMP-2 in human synovial cells and analyzed the contribution of synovial cells in osteolysis and aseptic loosening.

Results

When human synovial cells are exposed to titanium materials, MMP-2 activity is induced by 1.72 ± 0.14-fold with Ti disc and 3.95 ± 0.10-fold with Ti particles, compared with that of the controls, respectively. Inflammatory cytokines TNFα and IL-1β are also shown to induce MMP-2 activity by 3.65 ± 0.28-fold and 6.76 ± 0.28-fold, respectively. A combination of Ti particles and cytokines induces MMP-2 activities to a higher level (10.54 ± 0.45-fold). Inhibitors of various signal pathways involved in MMP-2 reverse Ti particle-induced MMP-2 activities.

Conclusions

Synovial cells surrounding the bone–prosthesis interface may contribute to production of MMP-2, and NFκB inhibitors may be explored as potential therapeutics to alleviate wear debris-induced osteolysis and aseptic loosening.     

Macrophage exposure to particulate titanium induces phosphorylation of the protein tyrosine kinase lyn and the phospholipases Cgamma-1 and Cgamma-2.

A frequent long-term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris production, synovial macrophage activation, inflammatory mediator release, and osteolysis about the implant-bone or cement-bone interface. To elucidate the mechanisms of particle-induced macrophage activation and mediator production, we studied early signal transduction events using J774A.1 macrophages and 3 microm titanium particles. Treating macrophages with herbimycin A or genistein, two inhibitors of protein tyrosine kinases (PTKs), inhibited titanium phagocytosis as well as secretion of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin-E2 (PGE2) in a dose-dependent manner. Both processes therefore depend on a PTK signaling cascade. Specifically, macrophage exposure to titanium-induced phosphorylation of multiple proteins including the Src kinase Lyn and phospholipase Cgamma-1 and Cgamma-2. Phosphorylation peaked within 2 min and returned to baseline within 45 min. Similar but not identical phosphorylation patterns were obtained when cells were stimulated with titanium preincubated with serum or albumin, suggesting distinct signal transduction pathways dependent on particle coating.     

Different Behavior of Human Osteoblast-Like Cells Isolated from Normal and Heterotopic Bone In Vitro

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM β-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3–6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells. Received: 29 March 1996 / Accepted: 21 May 1997