Cobalt-induced cardiomyopathy - do circulating cobalt levels matter?

Elevated levels of circulating cobalt ions have been linked with a wide range of systemic complications including neurological, endocrine, and cardiovascular symptoms. Case reports of patients with elevated blood cobalt ions have described significant cardiovascular complications including cardiomyopathy. However, correlation between the actual level of circulating cobalt and extent of cardiovascular injury has not previously been performed. This review examines evidence from the literature for a link between elevated blood cobalt levels secondary to metal-on-metal (MoM) hip arthroplasties and cardiomyopathy. Correlation between low, moderate, and high blood cobalt with cardiovascular complications has been considered. Elevated blood cobalt at levels over 250 µg/l have been shown to be a risk factor for developing systemic complications and published case reports document cardiomyopathy, cardiac transplantation, and death in patients with severely elevated blood cobalt ions. However, it is not clear that there is a hard cut-off value and cardiac dysfunction may occur at lower levels. Clinical and laboratory research has found conflicting evidence of cobalt-induced cardiomyopathy in patients with MoM hips. Further work needs to be done to clarify the link between severely elevated blood cobalt ions and cardiomyopathy. Cite this article: Bone Joint Res 2021;10(6):340-347.

Cobalt Administration Causes Reduced Contractility with Parallel Increases in TRPC6 and TRPM7 Transporter Protein Expression in Adult Rat Hearts.

Exposure to circulating cobalt (Co2+) in patients with metal-on-metal orthopaedic hip implants has been linked to cardiotoxicity but the underlying mechanism(s) remain undefined. The aim of the current study was to examine the effects of Co2+ on the heart in vivo and specifically on cardiac fibroblasts in vitro. Adult male rats were treated with CoCl2 (1 mg/kg) for either 7 days or 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure Co2+ uptake into various organs of the body. Co2+ accumulated in the heart over time with significant levels evident after only 7 days of treatment. There was no evidence of cardiac remodelling following Co2+ treatment as assessed by heart weight:body weight and left ventricular weight:body weight. However, a decrease in fractional shortening, as measured using echocardiography, was observed after 28 days of Co2+ treatment. This was accompanied by increased protein expression of the ion transient receptor potential (TRP) channels TRPC6 and TRPM7 as assessed by quantitative immunoblotting of whole cardiac homogenates. Uptake of Co2+ specifically into rat cardiac fibroblasts was measured over 72 h and was shown to dramatically increase with increasing concentrations of applied CoCl2. Expression levels of TRPC6 and TRPM7 proteins were both significantly elevated in these cells following Co2+ treatment. In conclusion, Co2+ rapidly accumulates to significant levels in the heart causing compromised contractility in the absence of any overt cardiac remodelling. TRPC6 and TRPM7 expression levels are significantly altered in the heart following Co2+ treatment and this may contribute to the Co2+-induced cardiotoxicity observed over time.

Human Hepatic HepaRG Cells Maintain an Organotypic Phenotype with High Intrinsic CYP450 Activity/Metabolism and Significantly Outperform Standard HepG2/C3A Cells for Pharmaceutical and Therapeutic Applications.

Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, interdonor functional variability and early phenotypic alterations in PHHs restrict their use, whilst standard cell lines such as HepG2 lack a substantial and variable set of liver-specific functions such as CYP450 activity. Alternatives include the HepG2-derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co-culture model of hepatocytes and cholangiocytes reported to maintain in vivo-like liver-specific functions, including intact Phase I-III drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre-clinical drug testing or therapeutics. Compared with C3As, HepaRG co-cultures exhibit a more organotypic phenotype, including evidence of hepatic polarity with the strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes in HepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α5 ß1 - an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC-MS/MS and HPLC, respectively, revealed that HepaRGs had more intact (Phase I-II) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for the potential pharmaceutical and therapeutic applications.

The effects of 405 nm light on bacterial membrane integrity determined by salt and bile tolerance assays, leakage of UV-absorbing material and SYTOX green labelling.

Bacterial inactivation by 405 nm light is accredited to the photoexcitation of intracellular porphyrin molecules resulting in energy transfer and the generation of reactive oxygen species that impart cellular oxidative damage. The specific mechanism of cellular damage, however, is not fully understood. Previous work has suggested that destruction of nucleic acids may be responsible for inactivation; however, microscopic imaging has suggested membrane damage as a major constituent of cellular inactivation. This study investigates the membrane integrity of Escherichia coli and Staphylococcus aureus exposed to 405 nm light. Results indicated membrane damage to both species, with loss of salt and bile tolerance by S. aureus and E. coli, respectively, consistent with reduced membrane integrity. Increased nucleic acid release was also demonstrated in 405 nm light-exposed cells, with up to 50 % increase in DNA concentration into the extracellular media in the case of both organisms. SYTOX green fluorometric analysis, however, demonstrated contradictory results between the two test species. With E. coli, increasing permeation of SYTOX green was observed following increased exposure, with >500 % increase in fluorescence, whereas no increase was observed with S. aureus. Overall, this study has provided good evidence that 405 nm light exposure causes loss of bacterial membrane integrity in E. coli, but the results with S. aureus are more difficult to explain. Further work is required to gain greater understanding of the inactivation mechanism in different bacterial species, as there are likely to be other targets within the cell that are also impaired by the oxidative damage from photo-generated reactive oxygen species.

An evaluation of Minor Groove Binders as anti-lung cancer therapeutics.

A series of 47 structurally diverse MGBs, derived from the natural product distamycin, was evaluated for anti-lung cancer activity by screening against the melanoma cancer cell line B16-F10. Five compounds have been found to possess significant activity, more so than a standard therapy, Gemcitabine. Moreover, one compound has been found to have an activity around 70-fold that of Gemcitabine and has a favourable selectivity index of greater than 125. Furthermore, initial studies have revealed this compound to be metabolically stable and thus it represents a lead for further optimisation towards a novel treatment for lung cancer.

Cytotoxic responses to 405nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species.

Light at wavelength 405 nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405 nm light at 36 J/cm(2) (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54 J/cm(2)), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405 nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione were observed from 405 nm light treated cells. The mammalian cells were significantly protected from dying at 54 J/cm(2) in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324 J/cm(2). Results therefore suggested that the cytotoxic mechanism of 405 nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage.

Bioinspired Silica Offers a Novel, Green, and Biocompatible Alternative to Traditional Drug Delivery Systems.

Development of drug delivery systems (DDS) is essential in many cases to remedy the limitations of free drug molecules. Silica has been of great interest as a DDS due to being more robust and versatile than other types of DDS (e.g., liposomes). Using ibuprofen as a model drug, we investigated bioinspired silica (BIS) as a new DDS and compared it to mesoporous silica (MS); the latter has received much attention for drug delivery applications. BIS is synthesized under benign conditions without the use of hazardous chemicals, which enables controllable in situ loading of drugs by carefully designing the DDS formulation conditions. Here, we systematically studied these conditions (e.g., chemistry, concentration, and pH) to understand BIS as a DDS and further achieve high loading and release of ibuprofen. Drug loading into BIS could be enhanced (up to 70%) by increasing the concentration of the bioinspired additive. Increasing the silicate concentration increased the release to 50%. Finally, acidic synthesis conditions could raise loading efficiency to 62% while also increasing the total mass of drug released. By identifying ideal formulation conditions for BIS, we produced a DDS that was able to release fivefold more drug per weight of silica when compared with MCM-41. Biocompatibility of BIS was also investigated, and it was found that, although ∼20% of BIS was able to pass through the gut wall into the bloodstream, it was nonhemolytic (∼2% hemolysis at 500 µg mL-1) when compared to MS (10% hemolysis at the same concentration). Overall, for DDS, it is clear that BIS has several advantages over MS (ease of synthesis, controllability, and lack of hazardous chemicals) as well as being less toxic, making BIS a real potentially viable green alternative to DDS.

Effects of CoCr metal wear debris generated from metal-on-metal hip implants and Co ions on human monocyte-like U937 cells.

Hip resurfacing with cobalt-chromium (CoCr) alloy was developed as a surgical alternative to total hip replacement. However, the biological effects of nanoparticles generated by wear at the metal-on-metal articulating surfaces has limited the success of such implants. The aim of this study was to investigate the effects of the combined exposure to CoCr nanoparticles and cobalt ions released from a resurfacing implant on monocytes (U937 cells) and whether these resulted in morphology changes, proliferation alterations, toxicity and cytokine release. The interaction between prior exposure to Co ions and the cellular response to nanoparticulate debris was determined to simulate the situation in patients with metal-on-metal implants receiving a second implant. Effects on U937 cells were mainly seen after 120h of treatment. Prior exposure to Co ions increased the toxic effects induced by the debris, and by Co ions themselves, suggesting the potential for interaction in vivo. Increased TNF-α secretion by resting cells exposed to nanoparticles could contribute to osteolysis processes in vivo, while increased IFN-γ production by activated cells could represent cellular protection against tissue damage. Data suggest that interactions between Co ions and CoCr nanoparticles would occur in vivo, and could threaten the survival of a CoCr metal implant.

CoCr wear particles generated from CoCr alloy metal-on-metal hip replacements, and cobalt ions stimulate apoptosis and expression of general toxicology-related genes in monocyte-like U937 cells.

Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with both FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p<0.05) amounts of Co and Cr ions into the culture medium, and significant (p<0.05) cellular uptake of both ions. There was also an increase (p<0.05) in apoptosis after a 48h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p<0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions+debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one.

Differential sensitivity of osteoblasts and bacterial pathogens to 405-nm light highlighting potential for decontamination applications in orthopedic surgery.

Healthcare associated infections pose a major threat to patients admitted to hospitals and infection rates following orthopedic arthroplasty surgery are as high as 4%. A 405-nm high-intensity narrow spectrum light has been proven to reduce environmental contamination in hospital isolation rooms, and there is potential to develop this technology for application in arthroplasty surgery. Cultured rat osteoblasts were exposed to varying light intensities and it was found that exposures of up to a dose of 36 J/cm2 had no significant effect on cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], function (alkaline phosphatase activity), and proliferation rate (BrdU cell proliferation assay). High irradiance exposures (54 J/cm2) significantly affected the cell viability indicating that the effects of 405-nm light on osteoblasts are dose dependent. Additionally, exposure of a variety of clinically related bacteria to a dose of 36 J/cm2 resulted in up to 100% kill. These results demonstrating the differential sensitivity of osteoblasts and bacteria to 405-nm light are an essential step toward developing the technique for decontamination in orthopedic surgery.

The complex degradation and metabolism of quercetin in rat hepatocyte incubations.

1. The current study demonstrated that there is still new information to be obtained on the chemical and biological transformation of the widely studied flavonoid quercetin. 2. In rat hepatocytes, 35 metabolites of quercetin were observed by using high-resolution mass spectrometry. The metabolites included glucuronides, sulfates, mixed sulfate/glucuronide metabolites and methylated versions of these metabolites. 3. Several metabolites were formed from chemical degradation products of quercetin which were found to form in Krebs-Henseleit (KH) buffer, degradants of quercetin were also formed in the buffer under the conditions used for hepatocyte incubations. 4. The degradants and metabolites of quercetin were characterized by using high-resolution MS(2). It was observed that the glutathione (GSH) conjugates of quercetin formed in large amounts in ammonium bicarbonate solution although the pattern of conjugates formed was different from that observed in hepatocytes suggesting some degree on enzymatic control on GSH conjugate formation in the hepatocyte incubations. 5. GSH conjugates were not formed when GSH was included in incubations of quercetin in KH buffer alone and only small amounts of quercetin degradation occurred. Instead, GSH was extensively converted into GSSG, thus presumably reducing the levels of oxygen in the incubation thus preventing quercetin degradation.

Bioinspired silica as drug delivery systems and their biocompatibility.

Silica nanoparticles have been shown to have great potential as drug delivery systems (DDS), however, their fabrication often involves harsh chemicals and energy intensive laborious methods. This work details the employment of a bioinspired "green" method for the controlled synthesis of silica, use of the products to entrap and release drug molecules and their cytotoxicity in order to develop novel DDS. Bioinspired silica synthesis occurs at pH 7, room temperature and in less than 5 minutes, resulting in a rapid, cheaper and greener route. Drugs were loaded into silica during the silica formation, thus allowing a one step and one pot method for simultaneous silica synthesis and drug loading. We established that the drug release profile can be modulated by synthetic parameters, which can allow design of tailored DDS. A systematic investigation using a two level factorial design was adopted in order to identify the key synthetic parameters and quantify their effects on silica formation, drug loading and drug release. The observation that these new DDS are considerably less cytotoxic than their current counterparts, and exhibit additional benefits such as green synthesis and ease of functionalization, strengthens the argument for their future use in DDS and other biomedical applications.

The abundant dietary constituent ferulic acid forms a wide range of metabolites including a glutathione adduct when incubated with rat hepatocytes.

1. The metabolism of ferulic acid (FA) has been studied in a number of different systems and several metabolites of FA have been characterised. No previous work has been carried out using hepatocytes to characterise the metabolism of FA. 2. A metabolomics approach in combination with high resolution mass spectrometry was used to characterise the metabolites of FA formed in isolated rat hepatocytes. FA was incubated with rat hepatocytes and the metabolites formed were profiled at 30 and 120 minutes. The metabolites were characterised according to their accurate mass at <2 ppm using Fourier transform mass spectrometry (FT-MS). 3. Sixteen metabolites of FA were identified. The most abundant metabolite was the sulphate of FA and this was followed by FA glucuronide and glycine conjugates. A wide range of low level metabolites were produced in the hepatocyte incubations. Novel metabolites resulted from side chain oxidation. 4. In addition, a glutathione (GSH) adduct of FA was formed. Incubation of a solution of FA with GSH also resulted in formation of this adduct indicating that it could be formed purely by a chemical reaction. Thus the metabolism of FA in rat hepatocytes is more complex than previously described.

The effect of ascorbic acid on the distribution of soluble Cr and Co ions in the blood and organs of rats.

Metal ions (Cr and Co) are released from metal orthopaedic implants in situ. We investigated tissue dissemination of Cr III, Cr VI and Co II ions in the body, and determined if administration of ascorbic acid (AA) affected their in vivo distribution using rats as a model system. Organs of rats treated with both Cr (VI) and Co (II) have higher metal ion levels when compared with control levels in the organs of rats without metal treatment. The reduced form of chromium, Cr III, is reported to be relatively impermeant to cell membranes in vitro, and in line with this, Cr III did not distribute into the organs of the rats after administration in vivo. Potent in vitro reduction of Cr (VI) to Cr III by AA was observed in this study. Prior intraperitoneal injection of AA lowered tissue uptake of both Cr VI and Co II, and increased faecal excretion, but not to a significant extent. AA may only be effective in increasing elimination of Cr VI at high concentrations when plasma reduction is saturated, and may be of limited therapeutic use in patients with orthopaedic implants.

Elucidation of the Phase I and Phase II metabolic pathways of (±)-4'-methylmethcathinone (4-MMC) and (±)-4'-(trifluoromethyl)methcathinone (4-TFMMC) in rat liver hepatocytes using LC-MS and LC-MS².

(±)-4'-Methylmethcathinone hydrochloride [(±)-mephedrone, 4-MMC] is a synthetic "legal high", with a classical cathinone structure similar to methcathinone. In this study, the in vitro metabolism of 4-MMC was investigated in Sprague-Dawley rat hepatocytes to characterise the associated Phase I and II metabolites. 4-MMC was incubated with rat liver hepatocytes, and the reaction mixture was analysed on a zwitterionic hydrophilic interaction (ZIC-HILIC) column using LC-MS and LC-MS(2). 4-MMC was metabolised, yielding 17 metabolites. These metabolites were structurally characterised on the basis of accurate mass analyses and LC-MS(2) fragmentation patterns and the major metabolic routes for 4-MMC determined to be via (i) oxidation of the 4'-methyl group and (ii) reduction of the ß-keto moiety. The biotransformation of a modified 4'-trifluoromethyl- derivative (4-TFMMC) has also been studied and shows significant differences in its metabolism compared to 4-MMC. Key pharmacokinetic parameters for both drugs have been calculated [biological half-lives (t(½)) for 4-MMC=61.9 min and for 4-TFMMC=203.8 min] and this data may aid in the understanding of in vivo metabolism and the likely pharmacokinetic effects of chemical/structural modifications within this class of controlled substances.

Collagen-nanofiber hydrogel composites promote contact guidance of human lymphatic microvascular endothelial cells and directed capillary tube formation.

Collagen and fibronectin matrices are known to stimulate migration of microvascular endothelial cells and the process of tubulogenesis, but the physical, chemical, and topographical cues for directed vessel formation have yet to be determined. In this study, growth, migration, elongation, and tube formation of human lymphatic microvascular endothelial cells (LECs) were investigated on electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic-co-D-lactic acid) (PLDL) nanofiber-coated substrates, and correlated with fiber density and diameter. Directed migration of LECs was observed in the presence of aligned nanofibers, whereas random fiber alignment slowed down migration and growth of LECs. Cell guidance was significantly enhanced in the presence of more hydrophobic PLDL polymer nanofibers compared to PLGA (10:90). Subsequent experiments with tube-forming assays reveal the ability of resorbable hydrophobic nanofibers >300 nm in diameter to promote cell guidance in collagen gels without direct cell-fiber contact, in contrast to the previously reported contact-guidance phenomena. Our results show that endothelial cell guidance is possible within nanofiber/collagen-gel constructs that mimic the native extracellular matrix in terms of size and orientation of fibrillar components.

Confined compression of collagen hydrogels.

Reconstituted collagen hydrogels are often used for in vitro studies of cell-matrix interaction and as scaffolds for tissue engineering. Understanding the mechanical and transport behaviours of collagen hydrogels is therefore extremely important, albeit difficult due to their very high water content (typically >99.5%). In the present study the mechanical behaviour of collagen hydrogels in confined compression was investigated using biphasic theory (J Biomechemical Engineering 102 (1980) 73), to ascertain whether the technique is sufficiently sensitive to determine differences in the characteristics of hydrogels of between 0.2% and 0.4% collagen. Peak stress, equilibrium stress, aggregate modulus and hydraulic permeability of the hydrogels exhibited sensitivity to collagen content, demonstrating that the technique is clearly able to discriminate between hydrogels with small differences in collagen content and may also be sensitive to factors that affect matrix remodelling. The results also offer additional insight into the deformation-dependent permeability of collagen hydrogels. This study suggests that confined compression, together with biphasic theory, is a suitable technique for assessing the mechanical properties of collagen hydrogels.

Acute inflammatory response to cobalt chromium orthopaedic wear debris in a rodent air-pouch model.

This study used a rodent air-pouch model to assess the acute inflammatory response to cobalt-chromium (CoCr) alloy wear debris from a metal-on-metal hip resurfacing implant that may contribute to joint failure. Air-pouches were injected with either sterile phosphate-buffered saline, 1 µg lipopolysaccharide (LPS) or 2.5 mg CoCr wear debris. The in situ inflammatory response was monitored 4, 24, 48 and 72 h and 7 days later. A flow cytometric analysis of the inflammatory exudates showed that CoCr wear debris induced a different inflammatory pattern compared with LPS. LPS induced a strong early (4 h) neutrophil influx, with monocyte/macrophage influx peaking at 24 h, whereas CoCr wear debris initiated almost equal numbers of early monocyte/macrophage and neutrophil recruitment. Histological analyses also showed CoCr debris accumulated in the pouch wall and this was accompanied by vast cellular infiltration and fibrosis around the debris throughout the duration of the experiment. Assessment of inflammatory gene transcripts from air-pouch tissue showed that CoCr wear debris increased the expression of cytokines involved in promoting inflammation and fibrosis (IL-1ß, TGF-ß) and chemokines that promote the recruitment of neutrophils and monocytes/macrophages (CXCL2 and CCL2). The data suggest that inflammatory responses to CoCr debris induce a specific acute process in which the recruitment of monocytes/macrophages is key.

Distribution of metal released from cobalt-chromium alloy orthopaedic wear particles implanted into air pouches in mice.

Metal-on-metal hip replacement implants generate wear debris and release ions both locally and systemically in patients. To investigate dissemination of metal, we determined blood and organ levels of cobalt (Co), chromium (Cr), and molybdenum (Mo) following the implantation of Co-Cr alloy wear debris in mice using skin pouches as a model system. We observed increased metal levels in blood for up to 72 h; the levels of Co were highest and remained elevated for 7 days. Co levels were elevated in all organs studied (liver, kidney, spleen, lung, heart, brain, and testes), with the peak at 48 h; highest levels were measured in liver and kidney (838.9 ± 223.7 ng/g in liver, and 938.8 ± 131.6 ng/g in kidney). Organ Cr levels were considerably lower than Co levels, for example, Cr in kidney was 117.2 ± 12.6 ng/g tissue at 48 h. Co is more mobile than Cr, reaching higher levels at earlier time points. This could be due to local tissue binding of Cr. Exposure to Co-Cr particles in vivo altered antioxidant enzyme expression and activities. We observed induction of catalase protein in the liver and glutathione reductase (GR) and peroxidase (GPx) proteins in the spleen. Activities of catalase and GPx in the liver were significantly increased while that of GR was decreased in the kidney. Organs of mice with Co-Cr particle implantation were exposed to increased metal levels capable of inducing reactive oxygen species scavenging enzymes, suggesting the tissue may be subjected to oxidative stress; however, the overall antioxidant defence system was not markedly disturbed.

Inactivation of microorganisms within collagen gel biomatrices using pulsed electric field treatment.

Pulsed electric field (PEF) treatment was examined as a potential decontamination method for tissue engineering biomatrices by determining the susceptibility of a range of microorganisms whilst within a collagen gel. High intensity pulsed electric fields were applied to collagen gel biomatrices containing either Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida albicans, Saccharomyces cerevisiae or the spores of Aspergillus niger. The results established varying degrees of microbial PEF susceptibility. When high initial cell densities (10(6)-10(7) CFU ml(-1)) were PEF treated with 100 pulses at 45 kV cm(-1), the greatest log reduction was achieved with S. cerevisiae (~6.5 log(10) CFU ml(-1)) and the lowest reduction achieved with S. epidermidis (~0.5 log(10) CFU ml(-1)). The results demonstrate that inactivation is influenced by the intrinsic properties of the microorganism treated. Further investigations are required to optimise the microbial inactivation kinetics associated with PEF treatment of collagen gel biomatrices.