Metal particles and ions are liberated from the articular interface of metal-metal (MM) total hip arthroplasties. To better understand their cellular effect, we analyzed the internalization of these metal particles and ions by macrophages in vitro. Macrophages were exposed to metal particles isolated from MM prostheses cycled in a hip simulator and to metal ions. Cells were processed for transmission electron microscopy analysis. Results reveal the internalization of metal particles and Cr³⁺ ions in specifically localized cytoplasmic areas. This study is the first to reveal that metal particles of clinically relevant size and Cr³⁺ ions are internalized by an apparently active process.

In order to minimize articular interface wear, metal-metal (MM) hip implants have been considered as an alternative to conventional metal-polyethylene bearings. While the local histological effects of the metallic particles and ions appear to be similar to that seen with metal-polyethylene hip replacements (i.e., a foreign-body macrophage response), little is known about the cellular effects of these metal particles and ions.

The purpose of this study was to better understand the cellular effect of metal particles and ions, we analyzed their internalization by macrophages in vitro.

J774 mouse macrophages were exposed to metal particles isolated from serum of MM prostheses cycled in a hip simulator and to Cr³⁺ (CrCl₃) and Co²⁺ (CoCl₂) ions. Cells were then processed for transmission electron microscopy analysis.

Micrographs revealed the internalization of metal particles and Cr³⁺ ions in specifically localized cytoplasmic areas, suggesting that they are phagocytosed via an active pathway. Energy disperse X-ray analysis spectra of macrophages incubated with Cr³⁺ revealed a chromium phosphate composition. The same structure and composition were also observed when Cr³⁺ ions were incubated in culture medium without cells, suggesting that they were formed outside the cells. Co²⁺ ions did not form visibly agglomerated structures.

This study is the first to reveal that metal particles of clinically relevant size are internalized by an apparently active process and that Cr³⁺ ions can be internalized by macrophages after binding to phosphorus or phosphoproteins. Kinetic studies are now necessary to better understand the mechanism of phagocytosis and the ultimate outcome of these particles and ions in macrophages.

The in situ increased production of matrix metalloproteinases (MMPs) has been associated with the development of periprosthetic osteolysis. The aim of the study was to compare the effect of Co²⁺ and Cr³⁺ ions on macrophages matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP (TIMP-1) expression. Using reverse transcription-polymerase chain reaction (RT-PCR), we showed that both Co²⁺ and Cr³⁺ ions induce the expression of MMP-1 and TIMP-1 in a dose-dependent manner. Since MMP-1 and TIMP-1 participate in the extracellular matrix degradation and tissue remodeling, our results suggest that the modulation of MMP-1 and TIMP-1 may contribute to the development of periprosthetic osteolysis.

The in situ increased production of matrix metalloproteinases (MMPs) has been associated with the development of periprosthetic osteolysis. Aseptic loosening due to periprosthetic osteolysis is the major cause of total hip arthroplasty failure. Because of their potential for improved wear performance, there has been a revived interest in metal-metal bearings, made of cobalt-chromium-molybdenum alloys. However, metal particle and ion toxicity remains a major cause for concern.

The aim of the study was to determine the effects of Co²⁺ and Cr³⁺ ions on the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1), two proteins participating in the extracellular matrix degradation and tissue remodeling.

Human U937 macrophages were incubated with Co²⁺ and Cr³⁺ ions. The expression of MMP-1 and TIMP-1 mRNAs was measured by reverse transcription-polymerase chain reaction (RT-PCR) and calculated as the ratio of the house keeping gene GAPDH expression.

Results show that both Co²⁺ and Cr³⁺ ions induced in a dose-dependent manner the expression of PCR products (mRNAs) of MMP-1 (135 bp) and TIMP-1 (328 bp). Co²⁺ ions were more effective in inducing MMP-1 and TIMP-1 expression than Cr³⁺ ions. The induction of MMP-1 and TIMP-1 paralleled the induction of TNF-α mRNA expression.

Our results demonstrate that the expression of MMP-1 and TIMP-1 were up regulated by incubating macrophages with Co²⁺ and Cr³⁺ ions, suggesting that metal ions contribute to tissue damage in the periprosthetic environment and that variations in MMP-1 and TIMP-1 expression may contribute to periprosthetic osteolysis.

Purpose: To develop an improved understanding of the in vivo behavior of intervertebral disc (IVD) cells for determining the phenotype of a differentiated stem cell in tissue engineering applications.

Methods: Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from adult bovine tails while notochordal cells were extracted from fetal bovine intervertebral disc. Ten million cells (of each cell type) in 500 & #61549;l of DMEM were then injected subcutaneously in C57Bl/6 mice. After 2 weeks, the mice were sacrificed and the specimens harvested. They were examined grossly, histologically and by scanning electronic microscopy (SEM) for the evidence of IVD-like structure formation. Proteoglycan was assessed by the GAG assay and PCR for analysis of gene expression. Control tissue (from bovine NP and AF) were directly fixed in glutaraldehyde, without any isolation technique and examined in SEM.

Results: After 2 weeks, SEM examination of specimens from AF and NP closely resembled normal bovine AF and NP. Of special interest here was the finding that some mice injected with cells from the AF developed an organized arrangement of parallel collagen fibres while NP cells injected mice had an amorphous structure with few collagen fibers. The GAG assay showed pro-teoglycan content for each samples, ranging from 3.8 microg to 26 microg. The morphology of the specimens retrieved from notochordal cells injected mice were also amorphous punctuated with thin collagen fibrils.

Conclusions: This study demonstrates that subcutaneous injection of bovine disc cells in mice can result in formation of disc structures similar to those of the bovine IVD. We believe that the cellular communication of the bovine disc cells is maintained in the mouse leading to architectural organization of the collagen fibers with the mouse as a source of nutrients. This technology may be useful in determining the phenotype of a differentiated stem cell for tissue engineering of IVD.

Purpose: Knowledge of factors regulating the turnover, repair, and degeneration of the intervertebral disc (IVD) is lacking. Although type II collagen (CII) fragments accumulate in the degenerative IVD, little is known of how they affect the degenerative process. In this study, the effect of a CII fragment, CII-(245–270), known to be critical in arthritis was investigated on gene expression of proteinases, collagen, and proteoglycan by bovine disc cells to determine its role in matrix turnover.

Methods: Cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) of adult bovine tails were cultured in the absence (control) or presence of the fragment. The fragment CII-(245–270) (US Biological, Massachusetts) was dissolved in culture medium to a final concentration of 1& #956;g/ml. PCR was performed and products were visualized by ethidium bromide staining.

Results: Addition of the CII-(245–270) peptide at 1& #956;g/ml to NP and AF cells enhanced expression of genes for MMP-1, cathepsin K, and aggrecan after 48 hours compared with the control. MMP-13 was also upregulated in the NP. In contrast, the effect in the AF was time dependent. Type II collagen was upregulated throughout the culture time in the NP as opposed to the AF where its expression was enhanced only on day 2.

Conclusions: We have shown that the CII-(245–270) peptide can alter gene expression of proteinases, collagen, and proteoglycan in bovine disc cells. The present study reveals the complex interrelationships of gene expression in the disc that accompany fragmentation of type II collagen. This new information suggests that increased levels of these fragments, in degenerated discs, may stimulate disc breakdown but may also attempt to protect the disc, by unknown mechanisms Funding: Other Education Grant Funding Parties: AO foundation, Switzerland

Recent evidence indicates that link N can stimulate synthesis of proteoglycans and collagen by adult (2–4 years old) bovine disc tails. Here we sought to determine the effect of link N on the accumulation of disc matrix proteins from young (eight to twenty month old) bovine tails. We show that degradation products of link protein generated by matrix metalloproteinases cannot “feed-back” and stimulate matrix assembly of the disc matrix from young bovine tails but may have a regulatory role in cell proliferation. Link N may have value only in stimulating the growth and regeneration of the old damaged intervertebral disc.

To date, there have been no reports on the effect of the amino terminal peptide of link protein (DHLSD-NYTLDHDRAIH) (link N) on disc cells from young (eight to twenty month old) bovine coccygeal discs. Link N is produced when removed by proteolysis from the N-terminus of the link protein of cartilage proteoglycan aggregates. We recently showed that link N can act directly on disc cells from adult (two to four years old) bovine discs to stimulate matrix production (J Cell Biochem, 2003; 88:1202–13).

To examine whether link N can play a role in maintaining the matrix integrity of young bovine disc cells.

Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from fresh grade I discs from young steers, and cultured in pellets at 1 million cell per tube in 1 ml of DMEM-high glucose supplemented with 1% 100X Pen-Strep, 1% ITS, 1 mg/ml BSA, and 50 μg/ml ascorbic acid. Cell pellets were digested and then analysed for sulfated glycosaminoglycan, type II collagen, percent denatured type II collagen, type IX collagen, and DNA content, using specific assays.

A concentration of 100 ng/ml link N significantly increased the DNA content of AF cells. However, link N had no significant effect on proteoglycan, type II and type IX collagen accumulation.

This study demonstrates that link N at a concentration of 10 ng/ml and 100 ng/ml cannot stimulate matrix production but may increase cell division in young bovine disc tails.

Purpose: Articular surface replacement (ASR) is an alternative for young patients considered for hip replacement. The in vivo release of ions from these surfaces has yet to be well evaluated. The purpose of the present study was to compare the concentrations of metal ions in blood of patients with ASR and metal-on-metal (MM) total hip arthroplasty (THA).

Methods: Blood was collected 6 months after implantation time into Sarstedt Monovette® tubes for trace metal analysis from patients having ASR (n=61), 28 mm-head MM THA (n=18), and 36 mm-head MM THA (n=25). The concentrations of cobalt (Co), chromium (Cr), and molybdenum (Mo) were analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS).

Results: The median Co level was not significantly different between the 3 groups (2.35 ppb, 2.00 ppb, and 2.50 ppb for the 28 mm MM THA, 36 mm MM THA, and the ASR patients, respectively). The median Cr level was significantly lower in the 36 mm MM THA patients (0.10 ppb) compared to the 28 mm MM THA (0.15 ppb) and the ASR (0.40 ppb) patients. The median Mo level was significantly lower in the 36 mm MM THA patients (1.30 ppb) compared to the 28 mm MM THA (2.00 ppb) and the ASR (1.50 ppb) patients.

Conclusions: Our results show that the level of ions in 36 mm MM THA patients was lower than in 28 mm MM THA patients. This can be explained by the fact that 28 mm MM bearings are resistant to microseparation during the normal gait cycle, which is theoretically accompanied by a reduction of fluid film lubrication and increased potential for the production of wear debris. Our results also show that the ion levels in patients having ASRs is similar to that observed in 28 mm MM THA patients but higher than in 36 mm MM THA patients. The diametric clearance of ASRs is typically much greater and the potential for a ‘suction fit’ may be less, leading to higher ion production. The concentration of ions in long-term follow-up remains however to be elucidated. Funding: Educational Grant from the Canadian Orthopaedic Foundation

Purpose: One of the major concerns regarding metal-on-metal prostheses is the biological and biochemical activities of chromium (Cr) ions. Previous studies showed that Cr3+ ions form nanostructures in cell culture media and to date, there has been little attempt to understand the nature of implant-derived metal ions in adjacent tissues or in biofluid. The aim of this work was to determine the nature of proteins present in serum involved in the formation of Cr nanostuctures

Methods: RPMI 1640 and DMEM media supplemented with 5% human serum (HS) or 5% foetal bovine serum (FBS) were incubated for 1h at 37°C in the presence of 50 ppm of Cr3+ (CrCl3). Structures were then isolated and separated by SDS-PAGE. Proteins were stained by Coomassie blue and analyzed by liquid chromatography-quadrupole-time of flight-mass spectrometer (LC-Q-Tof-MS). Data were submitted to Mascot software for a search against the NCBI nonredundant database

Results: Results show that Cr-nanostructures can interact with proteins from both human and bovine serums. On SDS-PAGE, the molecular weights of the proteins were between 40 to 90 kDa. The LC-Q-Tof-MS results suggest that Cr-nanostructures are the result of the interaction with numerous proteins present in serum. However, the complete analysis of results demonstrates that only two proteins (in both RPMI and DMEM) are implicated in these nanostructures: albumin and trans-ferrin. For both proteins, at least 40 peptides matched to the complete sequence of the proteins. The ion scores (“peptide identity score”) were between 79 and 108. Ion scores > 45 indicate identity or extensive homology

Conclusions: Human serum contains more than 400 different proteins. Albumin, the major protein of human serum, has been shown to play a scavenger role by binding and transporting injected and ingested Cr. Albumin could also play an immunological role by addressing signals to defense cells, such as macrophages. Trans-ferrin, known as an iron-carrying protein, also plays a scavenger role for Cr. This suggests that the binding of Cr to these proteins may protect cells from the cytotoxic effect of Cr ions. However, the relation with Cr nano-structures in vivo remains to be determined