Critical-sized bone defects remain challenging in the clinical setting. Autologous bone grafting remains preferred by clinicians. However, the use of autologous tissue is associated with donor-site morbidity and limited accessibility to the graft tissue. Advances in the development of synthetic bone substitutes focus on improving their osteoinductive properties. Whereas osteoinductivity has been demonstrated with ceramics, it is still a challenge in case of polymeric composites. One of the approaches to improve the regenerative properties of biomaterials, without changing their synthetic character, is the addition of inorganic ions with known osteogenic and angiogenic properties. We have previously reported that the use of a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the TCP particles can enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) (3). To further support the regenerative properties of these scaffolds, inorganic ions with known angiogenic properties, copper or cobalt, were added to the coating solution.

β-TCP particles were immersed in a zinc and copper or zinc and cobalt solution with a concentration of 15 or 45 mM. 3D porous scaffolds composed of 1000PEOT70PBT30 and pure or coated β-TCP were additively manufactured by 3D fibre deposition. The osteogenic and angiogenic properties of the fabricated scaffolds were tested in vitro through culture with hMSCs and human umbilical vein endothelial cells, respectively. The materials were further evaluated through ectopic implantation in an in vivo mini-pig model. The early expression of relevant osteogenic gene markers (collagen-1, osteocalcin) of hMSCs was upregulated in the presence of lower concentration of inorganic ions. Further analysis will focus on the evaluation of ectopic bone formation and vascularisation of these scaffolds after implantation in a mini-pig ectopic intramuscular model.

Background

The CoCrMo large bearings had shown a high failure rate, because of metal ion and particle release. Alumina matrix composite (AMC) ball heads have shown to mitigate such phenomena. The aim of this study was to investigate the leaching properties of AMC clinically as well as experimentally.

Background

Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). ARMD describes numerous symptoms in patients such as pain, osteolysis and soft tissue damage. Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines e.g. interleukin-8 (IL-8). This study investigates whether TLR4-specific antagonists inhibit the inflammatory response to cobalt using IL-8 gene expression and protein secretion as a marker of TLR4 activation.

Introduction

The anatomic dual mobility (ADM) technology utilized a monoblock cobalt chromium acetabular component. However, design limitations conferred difficulties controlling orientation during component insertion and inability to confirm full implant seating; the solution resulted in the creation of the modular dual mobility (MDM). The modular implant combines a standard titanium acetabular component and a cobalt chromium liner insert. Due to the metal-on-metal interface on MDM implants, fretting and corrosion releasing metal ions like previous metal-on-metal THA implants, were a concern. This study prospectively reviewed metal ions (cobalt, chromium and titanium) on patients who were at least 1 year post MDM implantation and compared them to patients with an ADM implant and evaluated radiographic seating of the components.

Introduction

It is well-known that wear debris generated by metal-on-metal hip replacements leads to aseptic loosening. This process starts in the local tissue where an inflammatory reaction is induced, followed by an periprosthetic osteolysis. MOM bearings generate particles as well as ions. The influence of both in human bodies is still the subject of debate. For instance hypersensitivity and high blood metal ion levels are under discussion for systemic reactions or pseudotumors around the hip replacement as a local reaction. The exact biopathologic mechanism is still unknown. The aim of this study was to investigate the impact of local injected metal ions and metal particles.

Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles.

Wear induces osteolysis leading to periprosthetic bone loss and TJA loosening. Inflammatory immune cells can form an aggressive interface membrane activating osteoclasts. The current study shows the effect of metal particles and ions triggering cellular responses.

Blood samples from primary and revision TJA were analysed for systemic inflammation. PBMCs were cultured on different implant materials. Cellular response was monitored by qRT-PCR.

Furthermore, cells were exposed to increasing concentrations of metal particles (10-7 and 10–8 particles/ml) and CoCl2 (50 µM and 100 µM). Cellular response was measured using WST-1 reduction, MitoSox-fluorescence and TUNEL-staining. Cobalt ion influx into osteoblasts was measured using FURA2-staining, cellular effects for HIF-1alpha and qRT-PCR.

No inflammatory parameters were detected in patients' blood from primary and revision TJA. Short inflammatory reaction of their PBMCs was observed in in vitro culture on ceramic implants, whereas there was no such reaction to other tested implant martials. In MM6 and Jurkat cells only metal ions induced oxidative stress but did not significantly reduce cell viability. An increase in HIF1-alpha was observed in tissue containing large amounts of metal wear in comparison to plastic wear containing tissues and OA synovial tissue without wear particles. Cobalt ions were stored by osteoblasts via a calcium channel inducing hypoxia. This effect could be blocked using a TRPM blocking agent.

Ceramic induces a short inflammatory response that may induce periprosthetic inflammation. Ionic Cobalt induces oxidative stress and hypoxia. Ionic metal exerts a more intense reaction on cells than particles.

Introduction

Metal on metal bearings are used especially in hip resurfacing. On the one hand, small bone preserving implants can be used. On the other hand recent studies found a variety of local and systemic side effects, for instance the appearance of pseudotumors, that are explained by pathologic biological reaction of the metal wear debris. The detailed mechanisms are still not understood until now. Thus it was the aim of this study to investigate the local reaction of metal wear particles and metal ions in a murine model. The hypothesis was that mainly metal ions provoke adverse histopathological reactions in vivo.

Background:

Potential systemic toxicity of metal ions from metal-on-metal hip arthroplasties (MoMHA) is concerning. High blood cobalt (Co) levels have been associated with neurological, cardiac and thyroid dysfunctions.

The world-wide experience with metallic implants provides the evidence for biocompatibility of modern cobalt- and titanium-based alloys. However, a corrosion process leading to release of ions has to be taken into account, and controversial data are available about the ‘endogenous’ metal exposure resulting from implants.

Adverse tissue reactions, cytotoxicity and toxic/ sensitizing effects of corrosion products on the immune system, as well as an involvement of metal ions in the pathogenesis of prosthesis loosening, are the main undesired effects. Moreover, metal ions could be responsible of long-term toxic effects, such as genotoxicity and carcinogenicity.

The aim of our study was to analyze the levels of serum metal ions in a large series of patients who underwent total hip and knee replacement (THR and TKR, respectively); both stable and loosened implants were considered. Furthermore, a group of subjects with fracture fixation devices was evaluated.

A consecutive series of 471 individuals (193 M; 278 F) was enrolled in the study, including 151 with hip prosthesis loosening (Group A: 52 M; 99 F; median age 67; median follow up 90 months) and 100 patients with stable hip prosthesis (Group B: 44 M; 56 F; median age 59; median follow up 34 months). Group A and B comprised implants with different coupling, i.e. ceramic-on-ceramic (alumina), metal-on-metal and metal-on-polyethylene, and different stem alloy (TiAlV-THR and CoCrMo-THR). 20 patients with knee prosthesis loosening (Group C: 3 M; 17 F; median age 67; median follow up 25 months), and 12 subjects with failed fracture fixation devices (Group D: 6 M; 6 F; median age 35; median follow up 12 months) were enrolled, too. Osteoarthritis was the most frequent disease that led to joint replacement (59%), followed by hip dysplasia (19%), and trauma (13%). Ion reference ranges were obtained from 188 subjects (88 M; 100 F; median age 52), including 56 healthy subjects and 132 candidates to primary THR or TKR. Serum samples were analyzed for chromium (Cr), cobalt (Co), molybdenum (Mo), nickel (Ni), aluminum (Al), titanium (Ti) and vanadium (V) content, in relation with the presence in the implant alloy, using a graphite furnace atomic absorption spectrometer (GFAAS), equipped with double background correction Deuterium/ Zeeman, autosampler and pyrolytic carbon-coated graphite tubes (Unica Solaar 939 QZ, Cambridge, UK). The highest value found for subjects with no implant was considered the upper reference limit for each element.

Group A (loosened THR) and B (well-fixed THR) patients, compared with controls, showed different behaviour depending on the prosthesis coupling and stem composition.

Metal-on-PE coupling and CoCrMo/Ni stem: a significant increase of Cr and Co serum values was observed, in both groups, even if the increase in loosened implants was higher than in the stable ones. Mo and Ni concentration did not show a significant variation.

A serum Co, Cr increase was demonstrated in Group C patients, too, who had a failed knee prosthesis, and a Cr, Ni increase was observed in Group D patients with stainless steel fracture fixation devices.

Metal corrosion leading to ion release needs further consideration, because it may enhance the inflammatory reaction, depress the immune system, and facilitate peri-prosthetic bacterial growth. Especially metal-on-metal coupling introduces additional biologic risks associated with increased degradation products of prosthetic materials. Otherwise, histiocytic/giant-cell reaction and peri-prosthetic osteolysis induced by polyethylene particles represent the main problem in knee and hip joint implants with metal/PE coupling, and the systemic effects due to the ion release become negligible. On the contrary, ceramic-on-ceramic coupling represents a good alternative, as demonstrated by the absence of metal release in stable implants, even if, in case of loosening, a serum ion increase is observed, probably due to the fretting at the head/neck connection.

Also, metal corrosion of fixation devices is a concern; consequently, it could be appropriate to remove the implants, as early as their function is accomplished.

In conclusion, ion profile should be carefully monitored and the epidemiological survey implemented, in order to establish the tolerance values in patients with implants, especially in young patients, where a long-term ‘endogenous’ exposure has to be faced. In particular, concerning systemic toxic effects, the ‘gene expression profiling’, through the use of microarray technology, could contribute to an improved understanding of the biological responses to metal ions released from orthopedic implants. In such a way a real risk-to-benefit ratio for the patient could be established.

Finally, ion monitoring, as demonstrated by the different serum ion levels in stable and loosened implants, could allow to detect early signs of failure, when radiographic and clinical data are unclear, and to avoid the need of revision using a proper treatment.

Metal-on-metal (MoM) bearing technology, made of cobalt-chromium (Co-Cr) alloys, is being used in anticipation of extending the durability of hip replacements. Increasingly, concern has been expressed that long term exposure to Co2+ and Cr3+ could cause DNA damage and immune dysfunction; specifically a reduction in the circulating number of CD8+ cytotoxic cells. More recently, we reported that Co2+ and Cr3+ affected the differentiation of osteoclast precursors into bone-resorbing osteoclasts. Despite these observations the effects of metal ions on osteoblast activity have been poorly investigated. The aim of the current study was to elucidate the effects of various metal ions on osteoblast activity in vitro.

Cells of the human osteosarcoma cell line SaOS-2 were cultured in the presence of 0, 1, 10 and 100 μM Co2+ and Cr3+. The morphology, viability, cytokine release (TNFalpha, IL-1beta, IL-6, LIGHT, MIP-1alpha and VEGF) and alkaline phosphatase activity were investigated after 24h and 48h in contact with metal ions. Finally the capacity of SaOS-2 to produce and mineralize a new bone matrix was assessed by the Alizarin red method. All experiments were repeated at least 5 times and the differences between each were determined using non-parametric Mann-Whitney test.

Compared to untreated cultures, although the morphology looked normal after 48h, the viability indicated that Co2+ and Cr3+ ions at high concentrations induced some significant and irreversible damages to the osteoblast cells. Interestingly, any of the cytokines investigated were released in contact with metal ions after 24h or 48h. The alkaline phosphatase activity was significantly increased by low concentrations of Co2+ and decreased by high concentrations of Cr3+ after 24h and 48h. Moreover, the degree of mineralization of a new bone matrix in vitro was significantly reduced when the SaOS-2 cells were exposed to high concentrations of Cr3+, but significantly increased when they were exposed to Co2+.

Our results indicated that irreversible damages are caused to the cells as soon as 24h with high concentrations of metal ions. For osteoblasts cells, Co2+ appeared to be less toxic than Cr3+ at high concentrations.

This study was supported by Furlong Research Charitable Foundation

Introduction. The usage of metal-metal bearings in young patients has rekindled the debate about the potential adverse effects of mutagenecity on offspring born to them. This question could be answered in part if it was known whether metal ions are transferred to the developing foetus. One recent study seems to suggest that such transfer does not occur [Brodner et al, J Arthroplasty2004; 19 Suppl(3) p102–107]. Unfortunately the instrument used there was not sensitive enough (5 out of 6 analyses were below the limit of detection), leaving the question of transplacental metal ion transfer unanswered. The present study uses a more powerful analytic technique.

Methods. After informed consent, whole blood specimens were obtained at the time of delivery from five patients who had undergone a Birmingham Hip Resurfacing and from their babies’ umbilical cords. High resolution plasma mass spectrometry (HRICPMS) was used for analysis.

Results. Cobalt and chromium ions have been detected in all the specimens obtained so far. The cord blood cobalt levels were lower than the mothers blood levels in all the specimens. A similar relationship was found in all but one individual chromium measurement. The mean (±95% CI) of the two groups are shown in figure 1.

Discussion and Conclusion. The present study shows that with the use of whole blood specimens rather than serum; and the use of a better analytic method than previously used in the only other study on the topic, metal ions can be detected in all specimens of patients with metal-metal devices and in the cord blood of babies born to them.

There is sufficient evidence in this study to prove that metal ions do cross the placenta. There is therefore a continuing need for vigilance on the possible effects on the offspring born to patients with metal-metal devices.

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.

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

In an effort to understand the role of metal ion analysis and how it relates to revision surgery and implant wear, four revised MOM cases were reviewed. The first case was revised for acute infection and is representative of the low bearing wear predicted by MOM simulator studies. Two of the four cases had apparent anterior subluxation as a result of hip hyperextension occurring with long stride gaits. The last case is a true hypersensitivity response to CoCr ions.

All four MOM prostheses were implanted by one surgeon and revised by the same surgeon approximately 6–8 years postoperatively. The implants had been positioned satisfactorily with inclination angles 45°–55° and anteversion angles 28°–42°.

Patient A (76 y/o female) with bilateral MOM hip replacements, was revised at approximately 8 years due to infection and had moderately elevated ions at the time of revision surgery (Co = 5, Cr = 2.3, Ti = 4). Only the femoral head was retrieved in this case. Retrieval analysis identified a well defined main-wear zone and one polar stripe. The CMM indicated there was minimal wear overall (form factor = 11 μm).

Patient B (33 y/o male) with bilateral MOM hip replacements, was revised at approximately 8 years due to pain, popping/catching sensations, and elevated ions (Co = 33, Cr = 17, Ti = 90). Intraoperatively, the implant was observed subluxing superiorly from the acetabular cup with anterior rotation of the leg. Both the femoral head and acetabular cup were retrieved. Retrieval analysis identified a well defined main-wear zone and multi-directional polar stripe formations similar to those reported on dislocated implants (Figure 1)[McPherson 2012, 2013]. The CMM indicated that overall wear was significant (form factor > 100 μm).

Patient C (77 y/o female) was revised at approximately 6 years due to pain, suspected implant loosening, osteolytic cysts determined by CT, and highly elevated ions (co = 164, Cr = 45, Ti = 33). Intraoperatively, there was evidence of wear including darkly stained tissue and osteolytic cysts. Both the femoral head and acetabular cup were retrieved. Retrieval analysis identified a well defined main-wear zone and one polar stripe. CMM indicated considerable wear (head form factor > 200, cup form factor >300).

Patient D (45 y/o female) was revised at approximately 6 years due to pain, apparent reactive response joint effusion, and moderately elevated ions (Co = 5, Cr = 6, Ti = 71). Only the femoral head was retrieved. Retrieval analysis identified a well defined main-wear zone and one polar stripe. Minimal wear was indicated by CMM (form factor = 21).

These four cases demonstrate distinct failure models of MOM hips and their respective metal ion results. Due to the diversity of patient location, a variety of clinical labs were utilized for this patient population. Caution should be used in interpreting metal ion analysis, as there are still no standards.

Figure 1: Retrieval analysis of stripe wear identified on femoral head from patient B.

Figure 2: Femoral head from patient C showing broader polar stripe associated with anterior subluxation in comparison to narrow polar stripe found on femoral head from patient A.

The Conserve® Plus (Wright Medical Technology Inc., Arlington, TN) was introduced clinically in the United States in 1996. A study of the serum cobalt and chromium ion levels was started in 2000 in our center to monitor the metal ion levels over time as part of an FDA clinical trial.

Thirteen male and five female patients received this resurfacing for idiopathic osteoarthritis (14), post-traumatic degenerative changes (3) or developmental dysplasia (1). Fourteen received a unilateral implant but four subsequently received a contralateral device from 52 to 86 months post-op. Four patients had bilateral resurfacings done in a one-stage procedure. All surgeries were performed by the senior author. None of these patients had known exposure to cobalt or chromium, kidney disease or other metal implants elsewhere in their bodies. Each prospectively provided blood samples and then yearly thereafter to measure cobalt and chromium levels for up to 11 years. Metal levels were measured using atomic absorption spectrophotometry and inductively coupled plasma mass spectrometry by a specialized trace element analysis laboratory. Acetabular component position was evaluated using Einzel-Bild-Röentgen-Analysis (EBRA) software. Contact patch to rim (CPR) distance was computed as described by Langton et al JBJS Br 91: 2009. A mixed model linear regression analysis was performed to evaluate long term trends, and multivariate analysis was performed to examine effects of implant and patient covariates on the metal ion levels.

One bilateral patient underwent revision for femoral loosening, all other patients were clinically well-functioning at the time of last follow-up (ave 89 mos). The median pre-operative Co was below the detection limit (d.l) of 0.3μg/L and the median pre-operative Cr was 0.069μg/L (d.l. 03μg/L). Metal levels increased within the first year then decreased and stabilized (fig 1). For unilaterals over all time intervals, the median Co was 1.06 μg/L, while the median Cr was 1.58 μg/L. For bilaterals, the mean post-operative Co was 2.80 μg/L, while the mean Cr was 5.80 μg/L. Generally, Cr levels were higher at all time points than Co. Bilateral patients had Co values 1.96 times greater on average than the unilateral patients (p<0.001). None of the possible covariates studied (femoral size, cup abduction angle, cup anteversion, CPR distance, activity, BMI and testing method) were related to the assay values.

The results of this study have shown that serum metal levels in well functioning implants can be low and do not increase over time. These are among the lowest levels reported for resurfacing devices and comparable to levels reported for well functioning small diameter metal-on-metal total hips. The study is limited due to the relatively small sample size and limited range of values for the covariates studied. However, it included patients who were active, female or bilateral and we collected ion levels up to 11 years. We now recommend that patients who have well-oriented Conserve Plus components with stable radiographic interfaces and no incidences of unexplained pain or hip noises be scheduled for follow-up every 2–3 years, rather than annually.

Joint replacement failure is usually caused by the formation of wear debris resulting in aseptic loosening. Particulate metal and soluble metal ions from orthopaedic alloys (cobalt chromium or vanadium titanium aluminium) that are used in medical prostheses can accumulate in tissues and blood leading to increased chromosome aberrations in bone marrow and peripheral blood lymphocytes. This paper demonstrates that two of the metals used in orthopaedic prostheses, chromium and vanadium can produce delayed as well as immediate effects on the chromosomes of human fibroblasts in vitro. Fibroblasts were exposed to metal ions for only 24 hours and were then expanded over 30 population doublings involving ten passages. The initial increase of chromosomal aberrations, micronuclei formation and cell loss due to lethal mutations persisted over multiple population doublings, thereby demonstrating genomic instability. Differences were seen in the reactions of normal human fibroblasts and those infected with a retrovirus carrying the cDNA encoding hTERT that rendered the normal human fibroblasts telomerase-positive and replicatively immortal. This suggests that chromosomal instability caused by metal ions is influenced by telomere length or telomerase activity. Formerly this syndrome of genomic instability has been demonstrated in two forms following irradiation. One type is non-clonal and involves the appearance of lethal aberrations that cannot have been carried by the surviving cells. The other type is clonal and the aberrations are not lethal. These may arise as a result of complex rearrangements occurring at a high rate post-insult in surviving cells. The consequences of genomic instability are not yet known but it is possible that the increase of chromosomal aberrations that have been previously observed in human patients could be due to immediate and delayed expression of cellular damage after exposure to orthopaedic metals.

Introduction: Despite a resurgence in cobalt-chromium metal-on-metal arthroplasty and hip resurfacing, the potential toxicity of cobalt ions in the periprosthetic area remains a cause for concern. Cytotoxic effects have been demonstrated in macrophages with cobalt ions inducing apoptosis and TNF-α secretion. A similar cytotoxic effect has been demonstrated in osteoblast-like cells. However, these studies assessed the acute cellular response to cobalt ions over 48 hours. To date, the effect on osteoblasts of chronic exposure to cobalt ions is unknown.

Aim: In this study we investigated the effect on osteoblasts of chronic exposure to cobalt ions. Specifically we investigated the chemokine response and effect on osteoblast function. We also investigated for a change in osteoblast phenotype to a less differentiated mesenchymal cell type.

Methods. Primary human osteoblasts were cultured and treated with cobalt (10ppm) over 21 days. Secreted chemokines (IL-8, MCP-1, TNF-α) were assayed using enzyme-linked immunosorbent assays (ELISA). Osteoblast function was assessed via alkaline phosphatase activity and calcium deposition. For a change in osteoblast phenotype, osteoblast gene expression was assessed using real time PCR. Immunoflourescent cell staining of actin filaments was used to examine for a change in osteoblast morphology.

Results: Chemokine (IL-8) secretion by osteoblasts was significantly increased after 7 days of stimulation with cobalt ions. In parallel with this, osteoblast function was also significantly inhibited as demonstrated by reduced alkaline phosphatase activity and calcium deposition. Regarding osteoblast phenotype, FSP-1, CTGF and TGF-β gene expression were upregulated after 7 days exposure indicating a transition in osteoblast phenotype to a less differentiated mesenchymal cell type. Immunoflourescent staining of actin filaments also showed a change in osteoblast morphology. Taken together, these data demonstrate cobalt ions induce a change in the osteoblast phenotype to that of a mesenchymal cell type. This is the first study to investigate osteoblast plasticity in the context of periprosthetic osteolysis.

Conclusion: After prolonged exposure to cobalt ions, IL-8 chemokine secretion is increased which attracts neutrophils to the periprosthetic area. Furthermore, osteoblasts no longer function as osteogenic cells as demonstrated by a decrease in osteoblast alkaline phosphatase activity and calcium deposition. Instead, they undergo transition to a mesenchymal cell type as demonstrated by an increase in the expression of genes associated with a mesenchymal cell lineage. Instead of secreting osteoid matrix the new cell type secretes unmineralized collagen. Cobalt ions are not benign and may play an important role in periprosthetic osteolysis by inducing osteoblasts to undergo transition to a less differentiated mesenchymal cell type.

Introduction: Despite a resurgence in cobalt-chromium metal-on-metal arthroplasty, the potential toxicity of metal ions in the periprosthetic area remains a cause for concern. Studies to date have assessed the acute effect of cobalt ions on osteoblasts over 48 hours. The aim of our study was to determine the response of osteoblasts to cobalt ions over a prolonged period of exposure.

Methods. Primary human osteoblasts were cultured and treated with cobalt (10ppm) over 21 days. Osteoblast function was assessed via alkaline phosphatase activity and calcium deposition. ELISA were used to assess chemokine (IL-8, MCP-1 and TNF-α) secretion. Osteoblast gene expression was assessed using microarray analysis and real time PCR. Immunoflourescent cell staining of actin filaments was used to examine osteoblast morphology.

Results: Chemokine (IL-8) secretion by osteoblasts was significantly increased after 10 days of stimulation with cobalt ions. In parallel with this, osteoblast function was also significantly inhibited as demonstrated by reduced alkaline phosphatase activity and calcium deposition. Regarding osteoblast phenotype, FSP-1, CTGF and TGF-β gene expression were upregulated indicating a transition in osteoblast phenotype. Immunoflourescent staining of actin filaments also showed a change in osteoblast morphology. Taken together, these data show cobalt ions induce a change in the osteoblast phenotype to that of a mesenchymal cell type.

Conclusion: After 10 days of treatment with cobalt ions, osteoblasts no longer function as osteogenic cells. they undergo transition to a mesenchymal cell type. Furthermore, IL-8 secretion is increased which attracts neutrophils to the periprosthetic area thereby contributing to the inflammatory response that characterises osteolysis.

INTRODUCTION

Systemic levels of metal ions are surrogate markers of in-vivo wear of metal-on-metal hip resurfacings (MoMHRA). The wear-related generation of metal ions is associated with component size and positioning but also with design specific features such as coverage angle, clearance, metallurgy and surface technology.