Toxicity of cobalt-chromium nanoparticles released from a resurfacing hip implant and cobalt ions on primary human lymphocytes in vitro.

Adverse tissue responses to prostheses wear particles and released ions are important contributors to hip implant failure. In implant-related adverse reactions T-lymphocytes play a prominent role in sustaining the chronic inflammatory response. To further understand the involvement of lymphocytes in metal-on-metal (MoM) implant failure, primary human lymphocytes were isolated and treated with cobalt-chromium (Co-Cr) wear debris and Co ions, individually, and in combination, for 24, 48 and 120 h. There was a significant increase in cell number where debris was present, as measured by the Neutral Red assay. Interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) secretion levels significantly decreased in the presence of metal particles, as measured by ELISA. Interleukin-2 (IL-2) secretion levels were significantly decreased by both debris and Co ions. Flow cytometry analysis showed that the metal nanoparticles induced a significant increase in apoptosis after 48-h exposure. This investigation showed that prolonged exposure (120 h) to metal debris induces lymphocyte proliferation, suggesting that activation of resting lymphocytes may have occurred. Although cytokine production was affected mainly by metal debris, cobalt toxicity may also modulate IL-2 secretion, and even Co ion concentrations below the MHRA guideline levels (7 ppb) may contribute to the impairment of immune regulation in vivo in patients with MoM implants.

405 nm Light exposure of osteoblasts and inactivation of bacterial isolates from arthroplasty patients: potential for new disinfection applications?

Infection rates after arthroplasty surgery are between 1-4 %, rising significantly after revision procedures. To reduce the associated costs of treating these infections, and the patients' post-operative discomfort and trauma, a new preventative method is required. High intensity narrow spectrum (HINS) 405 nm light has bactericidal effects on a wide range of medically important bacteria, and it reduced bacterial bioburden when used as an environmental disinfection method in a Medical Burns Unit. To prove its safety for use for environmental disinfection in orthopaedic theatres during surgery, cultured osteoblasts were exposed to HINS-light of intensities up to 15 mW/cm2 for 1 h (54 J/cm2). Intensities of up to 5 mW/cm2 for 1 h had no effect on cell morphology, activity of alkaline phosphatase, synthesis of collagen or osteocalcin expression, demonstrating that under these conditions this dose is the maximum safe exposure for osteoblasts; after exposure to 15 mW/cm2 all parameters of osteoblast function were significantly decreased. Viability (measured by protein content and Crystal Violet staining) of the osteoblasts was not influenced by exposure to 5 mW/cm2 for at least 2 h. At 5 mW/cm2 HINS-light is an effective bactericide. It killed 98.1 % of Staphylococcus aureus and 83.2 % Staphylococcus epidermis populations seeded on agar surfaces, and is active against both laboratory strains and clinical isolates from infected hip and knee arthroplasties. HINS-light could have potential for development as a method of disinfection to reduce transmission of bacteria during arthroplasty, with wider applications in diverse surgical procedures involving implantation of a medical device.

Cytotoxicity of cobalt chloride in brain cell lines - a comparison between astrocytoma and neuroblastoma cells.

High levels of circulating cobalt ions in blood have been reported to induce systemic reactions in patients with metal-on-metal (MoM) hip implants. We still lack information regarding these adverse effects, which may specifically impact on patients showing adverse neurological symptoms. To investigate this, we used a battery of in vitro viability and proliferation assays to identify toxic cobalt chloride (CoCl2) concentrations in two different brain cell types: SH-SY5Y neuroblastoma and U-373 astrocytoma cells. Cobalt cytotoxicity was characterised by MTT and Neutral Red (NR) assays at concentrations ranging from 0 to 500 µM after 24, 48, and 72 h exposure. MTT and NR showed a dose- and time-dependent toxicity with cobalt decreasing cell viability at high concentrations. IC50s for MTT at 72 h (astrocytes: 333.15 ±â€¯22.88; neurons: 100.01 ±â€¯5.91 µM) and for BrdU proliferation assays (astrocytes: 212.89 ±â€¯9.84; neurons: 88.86 ±â€¯19.03 µM) demonstrate that SH-SY5Y neurons are significantly more vulnerable to cobalt than astrocytes. Increased BrdU and MTT assay sensitivity suggested that DNA synthesis and metabolism disruption were involved in Co toxicity. Intracellular cobalt level measured by ICP-MS was significant after 100 µM treatment. Astrocytes displayed improved resistance to cobalt toxicity and higher uptake, which may reflect their neuroprotective nature. In summary, exposure to high concentrations of extracellular cobalt has deleterious effects in neurons and astrocytes, with neurons showing particular sensitivity.

Development and mechanical characterisation of self-compressed collagen gels.

Collagen gels are considered a promising biomaterial for the manufacturing of tissue engineering scaffolds, however, their mechanical properties often need to be improved to enable them to provide enough mechanical support during the course of tissue regeneration process. In this paper, we present a simple self-compression technique for the improvement of the mechanical properties of collagen gels, identified by the fitting of bespoke biphasic finite element models. Radially-confined highly hydrated gels were allowed to self-compress for 18h, expelling fluid, and which were subsequently subjected to unconfined ramp-hold compression. Gels, initially of 0.2%, 0.3% and 0.4% (w/v) collagen and 13mm thickness, transformed to 2.9±0.2%, 3.2±0.3% and 3.6±0.1% (w/w) collagen and 0.45±0.06mm, 0.69±0.04mm and 0.99±0.07mm thickness. Young's moduli of the compressed gels did not increase with increasing collagen fibril density, whilst zero-strain hydraulic permeability significantly decreased from 51 to 21mm4/Ns. The work demonstrates that biphasic theory, applied to unconfined compression, is a highly appropriate paradigm to mechanically characterise concentrated collagen gels and that confined compression of highly hydrated gels should be further investigated to enhance gel mechanical performance.

The interaction of chromium (VI) with macrophages: depletion of glutathione and inhibition of glutathione reductase.

There are reports of alterations in the number and functions of the cells of the immune system in patients with metal-on-metal (MOM) orthopaedic implants. These effects have been correlated with elevated chromium levels in the patients' blood. We have investigated the interactions of clinically relevant concentrations of Cr VI with macrophages in vitro, and the mechanisms responsible for its toxicity. Cr VI causes a concentration dependent decrease in macrophage viability above 1 microM as measured by the MTT and Neutral Red assays. This falls well within the range of circulating chromium serum concentrations measured in patients with MOM. Intracellular reduced glutathione (GSH) levels fall as a result, and most of the loss (86%) is accounted for by oxidation to the dimer, GSSG. Prior depletion of GSH does not sensitise the cells to Cr VI toxicity, implying that it is not involved in protecting the cells against the effects of Cr VI. During the metabolism of Cr VI, glutathione reductase activity is inhibited. In contrast, the activities of catalase and superoxide dismutase are not significantly altered. Prior inhibition of glutathione reductase activity protects against the toxicity of Cr VI to a significant extent, suggesting that it reduces Cr VI to a toxic metabolite.

Metabolism of troglitazone in hepatocytes isolated from experimentally induced diabetic rats.

Troglitazone (TGZ), the prototype 2,4-thiazolidinedione antidiabetic agent, is associated with hepatotoxicity in patients with Type 2 diabetes. Although the mechanism of toxicity has not been established, alterations in the clearance of TGZ from in-vitro hepatocyte cultures through metabolic conjugation reactions are believed to modulate the toxicity of the compound. In this study, the metabolism of TGZ in freshly isolated hepatocytes from the fat-fed streptozotocin-treated rat model of Type 2 diabetes is described. Biochemical parameters such as cellular reduced glutathione content, content of cytochromes P450 and b5, and the expression of glutathione-S-transferase alpha (subunits Ya and Yc2) were not affected by the induced diabetes. TGZ was metabolized primarily to a sulfonate, a quinone and a glucuronide in both control and experimentally diabetic animals. However, metabolism after induction of diabetes was characterized by a moderate increase in sulfation, a decrease in the elimination half-life of TGZ and the absence of the minor metabolites of TGZ, notably the glutathione adduct of the putative reactive intermediate (m/z = 747 (M + H)+; m/z = 745 (M - H)-).

Osteoblast interactions with calcium phosphate ceramics modified by coating with type I collagen.

Complications associated with the use of autogenous bone in the repair or replacement of tissue lost through injury or disease have driven the search for alternative sources of graft material. Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP), or composites that combine the best properties of both of these materials are among the principal candidates. In this study, we have investigated the in vitro proliferation, morphology, and viability of an immortalized rat osteoblast cell line cultured on HA, TCP, and composites of the two in the ratios 75:25 (H75), 50:50 (H50), and 25:75 (H25) for 28 days. The biocompatibility of each material was examined in the presence and absence of a collagen coating. With the exception of H50, cell proliferation, quantified by carboxyfluorescein fluorescence, was enhanced by collagen coating of all materials for the first 14 days, although at later time points cell numbers were unaffected. It is notable that the collagen coating was least stable on H50, the only material not to show enhancement of cell growth on coating. Confocal laser scanning microscopy confirmed that cell growth was more extensive on coated materials over the first 7-14 days in culture, and the development of cell extensions and bridges across the pores in the materials was observed. Results indicate that collagen coating of calcium phosphate ceramics may also increase their compatibility and osseointegration in vivo.

Inactivation of micro-organisms isolated from infected lower limb arthroplasties using high-intensity narrow-spectrum (HINS) light.

High-intensity narrow-spectrum (HINS) light is a novel violet-blue light inactivation technology which kills bacteria through a photodynamic process, and has been shown to have bactericidal activity against a wide range of species. Specimens from patients with infected hip and knee arthroplasties were collected over a one-year period (1 May 2009 to 30 April 2010). A range of these microbial isolates were tested for sensitivity to HINS-light. During testing, suspensions of the pathogens were exposed to increasing doses of HINS-light (of 123mW/cm(2) irradiance). Non-light exposed control samples were also used. The samples were then plated onto agar plates and incubated at 37°C for 24 hours before enumeration. Complete inactivation (greater than 4-log10 reduction) was achieved for all of the isolates. The typical inactivation curve showed a slow initial reaction followed by a rapid period of inactivation. The doses of HINS-light required ranged between 118 and 2214 J/cm(2). Gram-positive bacteria were generally found to be more susceptible than Gram-negative. As HINS-light uses visible wavelengths, it can be safely used in the presence of patients and staff. This unique feature could lead to its possible use in the prevention of infection during surgery and post-operative dressing changes. Cite this article: Bone Joint J 2015;97-B:283-8.

Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes.

The cytochrome P-450 content of rat hepatocytes declined rapidly over 72 h in culture, due primarily to denaturation to cytochrome P-420. Six different media were investigated for their ability to conserve cytochrome P-450 during culture, and the most successful was a modified Earle's medium. After 72 h culture in this medium, cytochromes P-450 and b5, NADH-cytochrome b5- and NADPH-cytochrome c-reductases were maintained at 40, 100, 35 and 52% of fresh cell values, respectively. Cytochrome P-450 showed differential functional stability during culture with ethoxyresorufin O-deethylation being more stable than either pentoxyphenoxazone O-depentylation or biphenyl 4-hydroxylation. Monooxygenase than did cytochrome P-450 content. This discrepancy was not explained by loss of flavin nucleotides, FMN or FAD.

The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line.

When grown in the standard Dulbecco's medium the human liver derived Hep G2 hepatoma cell line shows only 10-20% of the cytochrome P-450-dependent mixed function oxidase (MFO) activity of freshly isolated human adult hepatocytes. However, the MFO activities and, to a lesser extent, the activities of UDP-glucuronyltransferase and glutathione-S-transferase can be increased by altering the composition of the growth medium. Modified Earle's medium was more effective in this respect than Williams' E medium and increased the O-dealkylations of ethoxyresorufin, benzyloxyresorufin and pentoxyresorufin 50-, 30- and 10-fold, respectively.

Conjugation reactions in hepatocytes isolated from streptozotocin-induced diabetic rats.

The activities of three drug conjugation reactions, glutathione, glucuronic acid and sulphate conjugation and the synthesis of glutathione, have been measured in hepatocytes isolated from streptozotocin-induced male diabetic rats. The intracellular content of reduced glutathione (GSH) was decreased in diabetic rat hepatocytes compared with controls. Following depletion of the intracellular GSH stores with diethylmaleate, the resynthesis of GSH in the presence of 0.5 mM L-methionine, occurred faster in diabetic rat hepatocytes than in those from control rats indicating that the cystathione pathway may be more efficient in the diabetic animals. In contrast, there was no significant difference in the resynthesis of GSH between control and diabetic rat hepatocytes in the presence of L-cysteine. The GSH conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) was deficient in diabetic rat hepatocytes, although only the effect on the former reaction was statistically significant (P less than 0.05). The Vmax for CDNB conjugation was significantly lower (P less than 0.05) in cytosolic fractions prepared from diabetic rat liver than in control rat liver fractions. This was accompanied by an increase in the affinity of the enzyme for CDNB. In contrast, the Vmax and Km for the conjugation of DCNB in cytosolic fractions were unaffected by the induced-diabetes. Glucuronic acid conjugation of both 1-naphthol and phenolphthalein was markedly deficient in diabetic rat hepatocytes. The intracellular concentrations of the cofactor for glucuronidation, UDP-glucuronic acid, were decreased in diabetic rat liver and this was thought to contribute to the defect in glucuronidation. The sulphation of 1-naphthol was not significantly altered by the induced diabetes. Deficiencies in glutathione and glucuronic acid conjugation in streptozotocin-induced diabetic rats may result in an increased susceptibility to xenobiotic induced cytotoxicity.

The toxicity of menadione and mitozantrone in human liver-derived Hep G2 hepatoma cells.

The cytotoxic properties of quinone drugs such as menadione and adriamycin are thought to be mediated through one-electron reduction to semiquinone free radicals. Redox cycling of the semiquinones results in the generation of reactive oxygen species and in oxidative damage. In this study the toxicity of mitozantrone, a novel quinone anticancer drug, was compared with that of menadione in human Hep G2 hepatoma cells. Mitozantrone toxicity in these cells was not mediated by the one-electron reduction pathway. In support of this, inhibition of the enzymes glutathione reductase and catalase, responsible for protecting the cells from oxidative damage, did not affect the response of the Hep G2 cells to mitozantrone, whereas it exacerbated menadione toxicity. In addition, the toxicity of menadione was preceded by depletion of reduced glutathione which was probably due to oxidation of the glutathione. Mitozantrone did not cause glutathione depletion prior to cell death. DT-diaphorase activity and intracellular glutathione were found to protect the cells from the toxicity of both quinones. Inhibition of epoxide hydrolase potentiated mitozantrone toxicity but did not affect that of menadione. Our experiments indicate that mitozantrone toxicity may involve activation to an epoxide intermediate. Both quinone drugs inhibited cytochrome P-450-dependent mixed-function oxidase activity, although menadione was more potent in this respect.

Spermine toxicity and glutathione depletion in BHK-21/C13 cells.

Spermine, a polycationic amine, produced a dose-dependent inhibition of BHK-21/C13 cell growth. This response was not due to the extracellular metabolism of spermine by an amine oxidase found in bovine serum, as the toxicity was observed when the cells were grown in medium supplemented with horse serum. Three indices were used to monitor cell growth, cell number, protein content and [3H]thymidine incorporation into DNA. Spermine (2mM) caused significant reductions in all three measurements after a 6-8 hr exposure. The amine was rapidly taken up into the cells reaching levels 15-16-fold greater than in control cells within 2 hr. There was a rapid loss of intracellular reduced glutathione following exposure to toxic concentrations of spermine, which occurred before any effect on cell growth. Three methods for the determination of intracellular glutathione content were compared in this system. The effect on both cell growth and glutathione was reversible following removal of spermine from the extracellular medium. The possible mechanisms involved in this toxic response are discussed with particular reference to the depletion in intracellular reduced glutathione.

The effects of inducing agents on cytochrome P450 and UDP-glucuronyltransferase activities in human HEPG2 hepatoma cells.

Selective induction in vitro of cytochrome P450-dependent mixed-function oxidase (MFO) and UDP-glucuronyltransferase (GT) activities was observed in the human HepG2 hepatoma cell line. 1,2-Benzanthracene (BA) induced MFO O-dealkylation activities for ethoxyresorufin, methoxyresorufin and benzyloxyresorufin, whereas phenobarbitone (PB) selectively induced pentoxyresorufin O-dealkylation and rifampicin (RIF) selectively induced benzyloxyresorufin O-dealkylation. Antibody inhibition experiments indicated that ethoxyresorufin and methoxyresorufin O-dealkylations were catalysed mainly by the P450 1A subfamily in untreated and BA-induced HepG2 cells, that additional unidentified P450 forms were considerably involved in methoxyresorufin and benzyloxyresorufin O-dealkylations and that the P450 2B subfamily was partially responsible for pentoxyresorufin O-dealkylation in PB-induced cells. Bilirubin GT activity was induced by PB, BA, RIF and dexamethasone, but 1-naphthol, morphine and testosterone GT activities were not induced by any of these treatments.

Mixed function oxidase and UDP-glucuronyltransferase activities in the human Hep G2 hepatoma cell line.

In cultured human hepatoma cells phenolphthalein glucuronidation was increased 3-fold by 2 mM phenobarbitone (PB) in the culture medium but not by 25 microM benz(a)anthracene (BA), while 1-naphthol glucuronidation was not increased by either PB or BA. Ethoxyresorufin O-deethylation (EROD) was increased 15-fold by BA but not by PB, while the O-dealkylations of pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) were increased by either PB or BA. The BROD activity increased by BA was sensitive to inhibition by alpha-naphthoflavone whereas that induced by PB was not. This suggests induction of different cytochrome P-450 isoenzymes. Control Hep G2 cells had similar glucuronide conjugation and cytochrome reductase activities to freshly isolated human adult hepatocytes, but had lower O-dealkylation and elevated microsomal epoxide hydrolase activities.

Human adult hepatocytes in primary monolayer culture. Maintenance of mixed function oxidase and conjugation pathways of drug metabolism.

The stabilities of several drug oxidation and conjugation pathways in human adult hepatocytes have been investigated during 72 hr culture. Cytochrome P-450-dependent mixed function oxidase was measured by the O-dealkylations of ethoxyresorufin (EROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD), which are probes for different isozymes of cytochrome P-450 in the rat. EROD declined to 64% of initial fresh cell values after 72 hr in culture, whereas PROD increased to 162% and BROD remained relatively constant. Addition of phenobarbitone to the culture medium selectively increased PROD to a greater extent than EROD and did not affect BROD. NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase were markedly labile during culture, declining to 32% and 22% of fresh cell values respectively. Epoxide hydrolase (EH) showed a large transient increase (2-5-fold) in enzyme activity 24 hr after culture, declining to fresh cell values by 48 hr. UDP-glucuronyltransferase (GT) activity towards phenolphthalein and 1-naphthol also increased (2-3-fold) during the 72 hr of culture, the greater and more rapid increase being observed with phenolphthalein glucuronidation. Sulphotransferase activity declined rapidly within 24 hr of culture, whereas reduced glutathione (GSH) levels and GSH conjugation were maintained at fresh cell values for 72 hr.

Chondroitin-6-sulphate incorporated into collagen gels for the growth of human keratinocytes: the effect of cross-linking agents and diamines.

This study demonstrates the effect of the glycosaminoglycans, hyaluronic acid and chondroitin-6-sulphate (Ch6SO4), diamines and a carbodiimide cross-linking agent on the growth of human epidermal cells on collagen gels. Ch6SO4 incorporated into collagen gels stimulated cell growth rate, but the effect was found to be inconsistent. We found that approximately 50% of the incorporated Ch6SO4 in the gels leached out into the growth medium after the first 3 d in culture, and this is thought to lead to the inconsistent cell growth response. In order to minimize the elution of Ch6SO4 from the gels and thereby maximize its effect on the growth of the keratinocytes, 1-100 micrograms ml-1 Ch6SO4 was added in the medium. The results showed that Ch6SO4 at these concentrations in the medium did not stimulate the cell growth on either plain collagen gels or gels containing 20% Ch6SO4. As an alternative strategy, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and diamines (putrescine or diaminohexane) were used to immobilize Ch6SO4 onto the collagen gels and to cross-link the gels. The cross-linking process partially prevented the elution of Ch6SO4 from the gels. Interestingly, only putrescine, not diaminohexane, promoted the growth of keratinocytes on the cross-linked plain collagen gels. We proposed to develop an artificial skin substitute containing putrescine as a growth factor for the human epidermal cells.

Investigation into the biological stability of collagen/chondroitin-6-sulphate gels and their contraction by fibroblasts and keratinocytes: the effect of crosslinking agents and diamines.

Artificial skin substitutes based on autologous keratinocytes cultured on collagen-based substrata are being developed for grafting onto patients with severe burns. The properties of the substratum can be manipulated by crosslinking the collagen with the glysocaminoglycan, chondroitin-6-sulphate (Ch6SO4), carbodiimides and polyamines. Biological stability, assessed by resistance to collagenase, was increased by incorporation of Ch6SO4, but crosslinking with the carbodiimides, 1-ethyl-3-(dimethylaminopropyl)carbodiimide and 1,1-carbonyldiimidazole or the polyamines, putrescine or diaminohexane, had little further benefit. Contraction of the collagen gels occurred to a greater extent when seeded with fibroblasts than with keratinocytes. The extent of contraction by either cell type was not influenced by the presence of Ch6SO4 in the gel, but the carbodiimides, and to a lesser extent the polyamines, limited cell-mediated contraction, particularly that mediated by fibroblasts. Optimum substratum composition for artificial skin substitutes will involve a compromise between the desired attributes of biological stability, rate of contraction, mechanical strength, biocompatibility and promotion of cell growth.

Interactions of orthopaedic metals with an immortalized rat osteoblast cell line.

The toxicity of nickel, chromium (III) and (VI), vanadium and aluminium was compared in an immortalized neonatal rat osteoblast cell line using the MTT assay and a novel index of cytotoxicity, alkaline phosphatase (ALP) activity. Where toxicity was observed, ALP was a consistently more sensitive detection method than the MTT assay. The toxicity of the metals increased in the order aluminium < chromium (III) < vanadium < nickel < chromium (VI). alpha-Tocopherol partially prevented nickel-induced toxicity (as assessed by ALP activity), whereas ascorbic acid had no protective effect. Chromium (VI) was more toxic than (III), with significant toxicity observed at 0.5 microM. It is thought that Cr (III) cannot readily penetrate the cell membrane and this may account for the lower toxicity. Aluminium had a stimulatory effect on cell growth at low concentrations (0.5 microM). The combination of immortalized rat osteoblasts and the ALP activity test provides a powerful tool for in vitro testing of orthopaedic materials.

In vitro biocompatibility testing of polymers for orthopaedic implants using cultured fibroblasts and osteoblasts.

The biocompatibility of two polymers for potential use as orthopaedic implant materials in an isoelastic hip prosthesis was investigated. The interactions of polyetheretherketone (PEEK) and epoxy resin polymers (with and without carbon fibre reinforcement) with both fibroblasts and osteoblasts were tested using cell protein, intracellular reduced glutathione (GSH), leakage of lactate dehydrogenase and the MTT assay as indices of cellular cytotoxicity. The epoxy resin polymer was slightly cytotoxic to and inhibited the growth rate of fibroblasts (as assessed by total cell protein), and depleted GSH in both cell types. In contrast, the PEEK material did not display overt signs of cytotoxicity and, in fact, increased osteoblast cell protein content. This suggests that, of these two materials, PEEK would be the one of choice for development of an isoelastic implant and, in view of its stimulatory effect on osteoblast protein content, it may encourage ingrowth of bone around the prosthesis and thus minimize joint loosening.