Developmental exposure to estrogens has been shown to affect a number of organ systems, including long and short bones. Epigenetic effects of DES exposure have been shown to affect the third generation of progeny. Furthermore, recent studies have shown that environmental exposure to estrogen-like compounds is much higher than originally anticipated. This study aims to discover the effect of in utero exposure to a well-known estrogen agonist, diethylstilbestrol (DES), on lumbar bone, intervertebral disc (IVD), and articular cartilage. Femoral bone was studied to determine the specificity of the effect. C57bl/6n pregnant mice were dosed orally with vehicle (peanut oil) or 0.1, 1.0 and 10 g/kg/day of DES on gestational days 11–14. Male and female pups were allowed to mature without further treatment until 3 months of age, at which point they were divided into swim and sedentary groups. After sacrifice, bone mineral density (BMD), bone mineral content (BMC), bone area (BA), and trabecular bone area (TBA) of the lumbar vertebrae and femur were measured using a PIXImus Bone Densitometer System (GE Medical Systems). Glycosaminoglycan (GAG) content (proteoglycan) was measured by the DMMB assay. Histological analysis of proteoglycan was performed with Safranin O staining. Intervertebral disc height was measured using NDP software (Leeds, UK). Statistical analysis was performed using analysis of variance (ANOVA) followed by Fisher's Protected Least Significant Difference (PLSD). A p-value of < 0.05 was considered statistically significant.Purpose
Method
Metal-on-metal (MoM) articulations in total hip replacement (THR) have become an attractive option for young, active patients. Short-term reports have demonstrated elevated systemic metal ion levels in the blood and urine. Disseminated concentrations of cobalt and chromium have raised concern regarding cellular toxicity, chromosomal damage and adverse local soft tissue reactions. Long-term studies are required to support the increased use of MoM bearings in younger patients given their potential deleterious effects. The purpose of the current study was to report the seven to 13 year clinical, radiographic, and metal ion results in patients following MoM THR. We prospectively followed 165 patients (196 hips) after second-generation MoM THR between July 1997 and November 2003. Functional outcome was measured using the Harris Hip Score (HHS) and the University of California Los Angeles (UCLA) Activity Score. Radiographic analysis was performed using Einzel-Bild-Roentgen-Analyse (EBRA) by two of the authors blinded to the study. Cobalt and chromium metal ions were measured from whole blood and analyzed using inductively coupled plasma-mass spectrometry as previously described.Purpose
Method
Metal-on-metal (MoM) articulations in total hip replacement (THR) have become an attractive option for young, active patients. Short-term reports have demonstrated elevated systemic metal ion levels in the blood and urine. Disseminated concentrations of cobalt and chromium have raised concern regarding cellular toxicity, chromosomal damage and adverse local soft tissue reactions. Long-term studies are required to support the increased use of MoM bearings in younger patients given their potential deleterious effects. The purpose of the current study was to report the 7–13 year clinical, radiographic, and metal ion results in patients following MoM THR. We prospectively followed 165 patients (196 hips) after second-generation MoM THR between July 1997 and November 2003. Functional outcome was measured using the Harris Hip Score (HHS) and the University of California Los Angeles (UCLA) Activity Score. Radiographic analysis was performed using Einzel-Bild-Roentgen-Analyse (EBRA) by two of the authors blinded to the study. Cobalt and chromium metal ions were measured from whole blood and analyzed using inductively coupled plasma-mass spectrometry.Introduction
Methods
A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results.Purpose
Method
Whilst it is known that oxidative stress can cause early degenerative changes observed in experimental osteoarthritis and that a major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients express type X collagen, a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification), little is known whether the expression of type X collagen in MSCs from OA patients can be related to oxidative stress or inflammatory reactions that occur during this disease. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Bone marrow aspirates were processed essentially as previously described. Briefly, non-adherent cells were discarded after 72h of culture and the adherent ones were expanded for 2–3 passages. MSCs from normal donor (control) were obtained from Lonza. Cells were then lysed and protein expression was detected by Western blot using specific antibodies directed against type X collagen, as well as the antioxidant enzymes Mn-superoxide dismutase (MnSOD), catalase (CAT) and glutathione peroxidase-1 (GPx-1) and inflammation related proteins cyclooxygenase-1 (COX-1) and intercellular adhesion molecule-1 (ICAM-1). GAPDH was used as a housekeeping gene and served to normalize the results. Correlations between the expressions of the different proteins were realized using the correlation Z test with StatView (SAS Institute).Purpose
Method
Co and Cr concentrations were measured in both the seminal plasma and in the blood of patients by inductively coupled plasma-mass spectroscopy (ICP-MS).
Patients having metal-on-polyethylene THA or resurfacing without pain (Control group), Patients having MOM THA or resurfacing with high levels of metal ions (cobalt and chromium) and having pain Patients having MOM THA or resurfacing with high levels of metal ions but having no pain and Patients having MOM THA or resurfacing with low levels of metal ions and having no pain. Operated hips were evaluated with MRI by one musculoskeletal radiologist who was blinded to the radiographic findings and clinical symptoms. All images were assessed for the presence of a juxtaarticular or periprosthetic abnormalities, including fluid collections, soft tissue masses, osseous abnormalities, and patterns of contrast enhancement of lesions.
Hip surface replacement 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. In the present study, we compared the concentrations of metal ions in blood of patients with hip surface replacement and metal-on-metal (MM) total hip arthroplasty (THA). Blood was collected six months and one year after implantation time into Sarstedt Monovette® tubes for trace metal analysis from patients having Articular Surface Replacement (ASR®, DePuy Orthopaedics; 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). Since metal ions are potent inducers of oxidative stress, total antioxidant, peroxide, and nitrotyrosine levels (oxidative stress markers) were also measured in plasma of the patients. The median Co and Cr levels progressively and significantly increased in the three groups during the first year post-operation (compared to patients without hip bearings (n=25)). After six months, the levels of Co and Cr were significantly higher in patients with ASR and 28 mm MM THA than in patients with 36 mm MM THA. There was no difference after one year. The level of activity, as measured by the UCLA activity score, was higher in ASR patients than in 28 and 36 mm MM THA after one year. No differences were observed for Mo levels in these patients when compared to our control group. There was no increase of oxidative stress marker levels in patients with ASR and 36 mm MM THA and no correlation between the concentrations of Co and Cr ions and the levels of oxidative stress markers. Our results show that, at one year post-operation, the concentration of ions in patients with ASRs is similar than those in patients with MM THAs. Moreover, results suggest that metal ions liberated from MM bearings do not induce damage to macromolecules by oxidative stress in plasma of patients. Longer follow-ups are still required to characterise the concentration of ions in ASR and to determine conclusively the effects of elevated circulating ions.
Although the etiology of low back pain is unclear, it is believed that intervertebral disc (IVD) degeneration plays a major role. In the present study, we sought to determine if bovine IVD cells maintain their phenotype in a mouse subcutaneous injection model, while embedded or not in biocompatible matrices. Nucleus pulposus (NP) cells were isolated from adult bovine tails. Ten million cells were resuspended either in 500 ƒÝl of DMEM or in a negatively (alginate) or positively (chitosan) charged matrix. The mixtures were then injected subcutaneously in Balc/c nude mice. After two weeks, the mice were sacrificed and the implants harvested. The implants were examined histologically with a hematoxylin and eosin stain. The implant size was measured and the cells were counted. Proteoglycan was assessed by the GAG assay. The expression of type I and II collagens, aggrecan, and CD24 genes was analyzed by reverse transcription ¡V polymerase chain reaction (RT-PCR). Histologic evaluation confirms the presence of cells in all NP implants. The presence of alginate increased the implant size, the number of cells in the implants, and to a lesser extent, the proteoglycan content, compared to implants formed with cells injected alone. However, chitosan had no effect on the implant size, the number of cells and the aggrecan content. NP implants expressed the same pattern of genes as the native NP tissue (i.e. type I and II collagens, aggrecan, and CD24). The presence of alginate did not affect this expression pattern whereas chitosan decreased slightly their expression. After injection in mice, bovine NP cells appeared to retain their native phenotype. The RT-PCR analysis revealed that NP cells expressed aggrecan, type I and type II collagens as well as CD24, a specific marker for the NP phenotype. Also, NP cells can be embedded in matrices to produce NP-like features in vivo. In conclusion, we have developed a simple mouse subcutaneous injection model that recreates the features of the native IVD and avoids the need to use a disc degeneration model.
The aim of this study was to analyze in human macrophages the effects of Co2+ and Cr3+ ions on the activity of caspase-8 and caspase-3, initiator and executioner of apoptosis, respectively. Caspase-3 and -8 activities were measured by colorimetric assays. Results show that Co2+ ions induced caspase-3 activity in a time-dependent manner. Co2+ had no effect on caspase-8 activity. The activation of caspase-3 by Cr3+ was time-dependent while caspase-8 activity reached a maximum after eight hours and decreased thereafter. Since caspase-8 is primarily activated by membrane-associated events, our results suggest that Cr3+ interacts with cell membrane components to induce macrophage apoptosis, whereas Co2+ seems to stimulate apoptosis most likely through intracellularly located mechanisms. Because of their potential for improved wear performance, there has been a revived interest in metal-metal bearings, made of cobalt-chromium-molybdenum alloys, as an alternative to the use of conventional metal-polyethylene bearings. However, metal ion toxicity remains a major cause for concern. Previous studies suggested that both cobalt (Co2+) and chromium (Cr3+) ions induce macrophage apoptosis. The interest in apoptosis lies in the fact that it offers specific targets for therapeutic intervention. The aim of this study was to analyze the effects in human macrophages of Co2+ and Cr3+ ions on the activity of caspase-8 and caspase-3, initiator and executioner of apoptosis, respectively. U937 human macrophages were exposed to 0–10 ppm Co2+ (CoCl2) and 0–500 ppm Cr3+ (CrCl3). Caspase-3 and caspase-8 activities were measured by colorimetric assays based on the recognition of specific amino acid sequences (DEVD and IETD, respectively). Results show that Co2+ ions induced caspase-3 activity with a significant increase after four hour incubation and a maximal 2.65-fold increase reached after twenty-four hour with 10 ppm. Co2+ had no effect on caspase-8 activity. Cr3+ ions significantly stimulated caspase-3 activity after four hours with a maximal 1.75-fold stimulation reached after twenty-four hours, reaching only 50% of that observed with Co2+. Caspase-8 activity was significantly increased after two hours incubation, peaking at eight hours with a 2.2-fold increase, and decreasing thereafter. Since caspase-8 is primarily activated by membrane-associated events, our results suggest that Cr3+ interacts with cell membrane components to induce macrophage apoptosis. On the other hand, Co2+ seems to stimulate caspase-3 activity and apoptosis most likely through intracellularly located mechanisms.
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 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 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 The 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.
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 ( 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.