A mathematical algorithm to identify toxicity and prioritize pollutants in field sediments.

In order to evaluate sediment toxicity, a mathematical algorithm was developed to compute the toxicity of multiple component mixtures acting in an additive manner. A statistical approach was devised to determine the presence of potential interactive effects among mixture components. The algorithm uses three kinds of data to obtain an integrative approach to sediment toxicity assessment: Microtox toxicity data (EC50 values), sediment pollutant concentration measurements, and sequential extraction (SEQ) data to investigate metal partitioning. To simplify the analysis of complex mixtures using a prioritization scheme based on intrinsic toxicity and relative abundance, a toxicity index (TI) was employed as an indicator of adverse ecological impact. In general, the ranking of contaminants using the TI approach was found to be most efficient in reducing computational time, and concentrations using bioavailability data from SEQ was found to be the best theoretical predictor of the experimental mixture toxicity value. Only a few pollutants that were present at the greatest abundance were needed to provide a good approximation of the calculated EC50 found when all components were included. Not only does this substantially reduce the computational time needed to determine the EC50, it could in some cases dramatically reduce the pollutant monitoring effort required to track toxicity effectively. This approach would have substantial implications for both risk assessment and for remediation strategies, making them more efficient by focusing on the priority pollutants identified.

Bioaccumulation of lead in Xenopus laevis tadpoles from water and sediment.

The overall objective of this research was to monitor the uptake kinetics of lead in an amphibian model and correlate metal content with embryo development. Based upon the concentration of lead found in the water and sediment of a Louisiana swamp adjacent to a Superfund site, a controlled laboratory experiment exploring lead uptake from water and sediment by Xenopus laevis tadpoles was conducted. For 5 weeks, tadpoles were exposed to water and a simulated sediment, kaolin, spiked with 1, 5, or 10 times the concentration of lead found in field water and sediment samples. Additionally, organisms were exposed to the 5 x condition for 3 and 6 weeks. The experimental controls consisted of unexposed tadpoles and ones exposed to lead originating from water or sediment exclusively. At the end of the exposure periods, developmental data, i.e., body weight and developmental stage, were recorded, and the tadpoles were analyzed for whole body lead concentration. Lead extraction was accomplished by dry ashing, and its amount was quantified polarographically. Results showed that lead inhibited the normal development of these amphibians, in a manner that generally was more severe as exposure level increased. The hindrance of tadpole development also coincided with an increase in whole body lead concentration at higher exposures. Temporally, at the 5 x exposure concentration, the mean lead level increased with time, but this difference was not statistically significant (P<.05). Additionally, control animals exposed to lead (either in water or in sediment) showed no statistical difference with regard to weight and lead uptake, indicating that lead originating from both water and sediment is incorporated into the tadpole. The controlled laboratory experimental protocol used here is thus capable of investigating the uptake of a single metal (Pb in this case) and determining its effect on the development of tadpoles while differentiating the significance of multiple sources of exposure.

Examination of in vivo influences on bioluminescent microbial assessment of corrosion product toxicity.

The composition of ionically dissolved and precipitated corrosion products from both free corrosion of ASTM F75 Co-Cr-Mo and galvanostatic polarization of Co-Cr-Mo and F138 316L stainless steel was determined using differential pulse polarography and inductively coupled plasma atomic emission spectroscopy. A bacterial bioluminescence assay, Microtox, was used to assess the toxicity of the solid and dissolved corrosion products produced by galvanostatic polarization and the individual ions within them. The role of in vivo salinity, temperature, and protein content as modulators of corrosion product toxicity assessment was investigated empirically and mechanistically. Co-Cr-Mo products were found to be more toxic than those of 316L, and the most toxic ions were Cr6+, Ni2+, and Co2+. Ringer's solution potentiated the toxicity of the more toxic metal ions and reduced the toxicity of the less toxic ions. Using theoretical analysis in conjunction with experimental measurements, the ions in both alloys were found to interact in an antagonistic fashion. The presence of albumin was found to decrease metal toxicity, presumably by chelation.

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement.

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.

Evaluation of metallic and polymeric biomaterial surface energy and surface roughness characteristics for directed cell adhesion.

Directed cell adhesion remains an important goal of implant and tissue engineering technology. In this study, surface energy and surface roughness were investigated to ascertain which of these properties show more overall influence on biomaterial-cell adhesion and colonization. Jet impingement was used to quantify cellular adhesion strength. Cellular proliferation and extracellular matrix secretion were used to characterize colonization of 3T3MC fibroblasts on: HS25 (a cobalt based implant alloy, ASTM F75), 316L stainless steel, Ti-6Al4V (a titanium implant alloy), commercially pure tantalum (Ta), polytetrafluoroethylene (PTFE), silicone rubber (SR), and high-density polyethylene (HDPE). The metals exhibited a nearly five-fold greater adhesion strength than the polymeric materials tested. Generally, surface energy was proportional to cellular adhesion strength. Only polymeric materials demonstrated significant increased adhesion strength associated with increased surface roughness. Cellular adhesion on metals demonstrated a linear correlation with surface energy. Less than half as much cellular proliferation was detected on polymeric materials compared to the metals. However the polymers tested demonstrated greater than twice the amount of secreted extracellular matrix (ECM) proteins on a per cell basis than the metallic materials. Thus, surface energy may be a more important determinant of cell adhesion and proliferation, and may be more useful than surface roughness for directing cell adhesion and cell colonization onto engineered tissue scaffoldings.

Correlation of field-measured toxicity with chemical concentration and pollutant availability.

Direct field toxicity tests were performed in two Louisiana waterways, Bayous Trepagnier and St. John, on sediments containing organic/heavy metal mixtures. Our approach involved bioluminescent bacterial toxicity assays (using DeltaTox, which qualitatively identifies polluted areas, and Microtox, which quantifies toxicity). Samples were more completely analyzed in our laboratory using inductively coupled plasma atomic emission spectroscopy (ICP-AES) and gas chromatography/mass spectrometry (GC/MS). Results indicate that lead is the primary toxic metal at the sites examined, though concentrations of metals fluctuate due to spatial variation and the dynamic nature of the waterways. Polycyclic aromatic hydrocarbons (PAHs) are the most abundant group of organics measured and appear to contribute to the overall toxic response. DeltaTox located toxic hotspots where there was an average light loss of 53-100%. Toxicity results from both assays agree but are well correlated with concentration measurements only for certain sediment fractions. Overall, the DeltaTox/Microtox approach appears to be rapid and cost effective for on-site hotspot identification, and may increase understanding of hazards associated with heavy metal and organic contaminants in these waterways.

Toxicity measurement of orthopedic implant alloy degradation products using a bioluminescent bacterial assay.

The toxicity of aqueous metal solutions representative of ionic degradation products from orthopedic implant alloys was determined using a bacterial bioluminescence assay, Microtox. The toxicity of forms of the individual elements released from ASTM F75 Co-Cr-Mo (Co-Cr-Mo), F138 316L stainless steel (316L), and F136 Ti-6Al-4V (Ti-6Al-4V) was first determined, and a mathematical model was developed to predict the toxicity of mixtures of these ions. Aqueous metal solutions were then mixed according to the proportions of the ions found in these alloys, and their toxicity was measured with Microtox. Mixture behavior was classified as synergistic, antagonistic, or additive by comparing measured toxicity to predicted toxicity. Since relating these tests to actual implant corrosion processes can be confounded by selective leaching, the predicted and measured toxicity of aqueous metal solutions mixed according to proportions representative of selective leaching were next determined, and the mixture behaviors were classified as before. The most toxic individual alloying elements were found to be hexavalent Cr, Ni, and Co, in that order: a finding in accord with prior biocompatibility research. Co-Cr-Mo was found to be the most toxic alloy mixture of both those combined according to alloy composition and those combined to reflect selective leaching. The Ti-6Al-4V mixtures were found to behave synergistically, while the Co-Cr-Mo and 316L mixtures behaved antagonistically. By providing insight into degradation product toxicity and elemental interaction, these experiments demonstrate the utility of employing bioluminescent bacterial assays to investigate biocompatibility of implant materials. Further studies to more closely simulate in vivo conditions, though, are required to fully gauge their potential in this regard.

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials.

Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.

Titanium metal release after posterior cervical spine plate placement in a canine model.

The local tissue concentration of released titanium from Ti-6Al-4V posterior cervical spine plates in canines was determined using inductively coupled plasma atomic emission spectroscopy. The plates were also evaluated for percentage of surface area damage. The highest titanium levels (> 100 ppm dry weight) and most severe surface damage were associated with screw-plate interfaces. A model to explain the metal release mechanisms was proposed, consisting of a combination of fretting wear, diffusion through the passive oxide layer, and dissolution of this layer.

Cell adhesion to biomaterials: correlations between surface charge, surface roughness, adsorbed protein, and cell morphology.

Adhesion of cells to a biomaterial surface can be a major factor mediating its biocompatibility. In this investigation, jet impingement techniques were used to quantify strength of cellular adhesion to various material surfaces. The metals tested: HS25 (a cobalt-based alloy similar to F75), 316L stainless steel, Ti-6Al-4V, and commercially pure tantalum, exhibited nearly a fivefold increase in adhesion strength above that characteristic of the polymeric materials tested (PTFE, silicone rubber, and HDPE). The present study examines physical and biological factors that might influence fibroblast adhesion to the biomaterial surface. The relation between surface charge and cellular adhesion was investigated in a controlled manner by measuring adhesion strength over a range of charge densities. The cells showed charge and electrical potential-dependent adhesion maxima, suggesting that surface alloying for optimum adherence may be possible. In a preliminary series of experiments adsorbed serum protein layers on a series of materials of differing adherence were investigated using gel electrophoresis to assess protein composition. Analysis of adsorbed proteins revealed little difference in relative abundance or total adsorption quantity. SEM micrographs of cells on Ti-6Al-4V and silicone rubber (high and low adhesion materials, respectively) demonstrated differences in cell morphology and cell density.

Corrosion and other electrochemical aspects of biomaterials.

Metallic materials are used extensively as orthopedic implants, dental materials, sensing elements of bioelectrodes, and other applications. The electrochemical behavior of these biomaterials is of interest for a variety of reasons. The corrosion resistance of an implant material influences its functional performance and durability and is a primary factor governing biocompatibility. Among the aspects affecting biocompatibility are the amounts and forms of released corrosion products and their disposition in the body after release. Electrochemical principles are very useful for understanding the factors affecting corrosion resistance and also form the foundation for many biosensors that measure the concentration of various chemical entities (including released corrosion products and naturally occurring substances). Many electrochemical measurement techniques have been used to study biomaterials for years (e.g., polarization curve measurement), while others (such as polarography and AC impedance methods) have been applied more recently. This work focuses on four main topics. The first is the nature of the body's environment as it affects in vivo electrochemical phenomena, that is, the chemical, mechanical, biological, and bioelectrical phenomena that affect the behavior and performance of biomaterials. The second deals with methodology--the techniques used for corrosion measurement and concentration determination, the appropriate environment (laboratory, cell culture, in vivo, etc.), and experimental problems encountered. The third topic treated is the knowledge accumulated regarding the performance of implant alloys in various applications, for example, the forms of corrosion to which they are susceptible, etc. Finally, improvements that may come about in the future regarding both materials and testing methodology are considered.

Use of a.c. impedance methods to study the corrosion behaviour of implant alloys.

The a.c. impedance method is a very useful technique for studying the corrosion behaviour of surgical implant alloys. In this article information is presented on how to efficiently and reliably acquire and analyse experimental data through the use of Nyquist, Bode and Rre versus omega Rim plots. Also, experiments were conducted in which 316L stainless steel specimens were subjected to polarization conditions which caused pitting corrosion. The increased surface area caused by the pitting, inferred from a.c. impedance measurements of specimen capacitance, was found to be well correlated with ASTM standard methods for assessment of pitting.

Stress-enhanced ion release--the effect of static loading.

Static stresses affect the corrosion behaviour of 316L stainless steel, Ti-6AI-4V, and a Co-Cr-Mo alloy. Several corrosion parameters are modified by stress, although the changes most relevant to the clinical situation are lowering of breakdown potentials and increases in corrosion currents. AC impedance techniques to measure capacitance allowed the latter effect to be partitioned into components of true current density and true area changes. Although loading past the yield point can definitely cause stress-enhanced ion release (SEIR), it is not required. SEIR can also be caused by elastic loading. The basic mechanism for this phenomenon appears to be passive film disruption followed by slow repassivation kinetics. Polished, grit-blasted, and porous-coated surfaces were examined. The porous-coated materials seemed to be most susceptible to SEIR. If effects similar to those observed here apply to in vivo conditions, then tests on unstressed alloys in vitro could grossly underestimate ion release rates of stressed implant devices in vivo.

Factors which influence the accuracy of corrosion rate determination of implant materials.

This study has revealed several factors which influence the accuracy and interpretation of electrochemical test results with surgical implant materials. These include variation between corrosion rate determination methods, IR drop, diffusional effects, and inherent statistical variations. The AC impedance method (ACI) is particularly useful for studying electrochemical mechanisms, measuring IR drop, and separating stress-enhanced ion release effects into components related to current density and surface area changes. This method can also measure true surface area even of irregular objects and can detect changes in area caused by cracking or surface plastic deformation. The corrosion rate method which is most accurate, in an absolute sense, must be determined by chemical analysis of the electrolyte.

The effect of surface preparation on metal/bone cement interfacial strength.

This study is concerned with finding practical ways for strengthening metal/bone cement (M/BC) interfaces via surface alterations and identifying fundamental mechanisms underlying M/BC adherence. Shear strengths have been inferred from torsion tests using shear-lag analysis. The variables examined with regard to their effects on interfacial strength are substrate material, surface roughness, interface porosity, passivation and sterilization, surface cleaning procedures, and use of bone cement precoated metals. M/BC interfaces can be substantially strengthened by applying the bone cement to the metal with high pressure. This would be a practical way to strengthen interfaces for precoated implants. The acrylic polymerized in vivo would employ the usual low pressure method. Otherwise, the main method for improving M/BC interfaces is through changing surface topography. Cleaning or chemical treatments have relatively minor effects. Roughened surfaces, as expected, produce stronger interfaces. Dramatic strength improvements occurred with a porous arc plasma sprayed layer on the substrate. Surprisingly, highly polished surfaces also improve interface strength (compared to less polished surfaces). The hypothesis is advanced that M/BC adherence depends upon superposition of mechanical interlocking and atomic interaction effects, with the latter predominating for finer finishes and vice versa. Differences exist between materials which are independent of roughness.

The influence of static stress on the corrosion behavior of 316L stainless steel in Ringer's solution.

The decrease in corrosion resistance of 316L stainless steel due to static stress was studied in vitro using a 37 degree C Ringer's solution electrolyte. Both potentiodynamic polarization and coulometric techniques were used. Cyclic anodic polarization tests with highly loaded fracture mechanics samples revealed a lowering of breakdown potential and disruption of passive films compared to unstressed controls. Measurements of the time-averaged current density due to a 100 mV anodic overpotential showed that a stress level causing plastic deformation increases the current density by more than an order of magnitude compared to samples stressed to the yield stress or nonloaded controls. The significance of these findings for surgical implant devices in service is discussed.

pH shifts and precipitation associated with metal ions in tissue culture.

Recently there has been interest in tests to assess the physiological response to surgical implant corrosion products. Both cell culturing in metal-bearing solutions and intramuscular injection of such solutions have been carried out. This paper examines the effects of the constraints of multicomponent equilibrium conditions on the characteristics of the solutions used in these biocompatibility tests. It is demonstrated that, unbuffered, they will have pH values shifted from neutral and that, in the buffered state, these solutions may contain both dissolved metal ions and insoluble hydroxides. The implications of these characteristics for the interpretation of the results of biocompatibility tests are discussed.

Determination of mineral-organic bonding effectiveness in bone--theoretical considerations.

It is postulated that the effectiveness of bonding between the mineral and organic phases could be an important influence on the behavior of bone with respect to its mechanical properties, metabolic activity, and aging effects associated with these factors. Changes in bonding effectiveness might also be related to the etiology of osteoporosis. If this hypothesis is correct, it would be of interest to determine the amount of debonding present in bone. An analysis that employs both macromechanical and micromechanical composite theory is performed to show how this quantity could be calculated. The approach taken is first to determine the elastic moduli of a characteristic volume from bulk elastic properties of bone and the mineral crystallite orientation distribution. Voigt and Reuss type averages are used to obtain upper and lower bounds. Modifications of the Halpin-Tsai equations that apply to chopped fiber composites are then used to calculate the amount of debonding between the phases in the characteristic volume. All of the parameters employed in the theory are measurable using established techniques. To apply the theory quantitatively the following information must be known: 1) the density and elastic moduli of the bone (and its phases), and 2) the mineral orientation distribution.

Unilateral posterior arch fractures of the atlas.

Unilateral posterior arch fractures of the atlas are discussed with two clinical examples and an experimental study of their mechanism. Laboratory fracturing of posterior arches of atlas specimens with a specially adapted universal testing machine produced nonsimultaneous fractures of the two sides in four of six specimens. In three of these specimens, a complete fracture on one side was temporarily displaced because the orientation of the leverage acting on the other side changed from sagittal to oblique. The consequent increase in the effective length of the lever arm reduced the angular deformation and strain on the second side. The second fracture occurred only after additional deflection of the posterior tubercle by up to 3 mm reproduced on the second side about the same angle of deformation that had caused the first fracture. A posterior arch fracture occurring by this mechanism will remain unilateral if the deflection is arrested before failure of the second side.

In vivo and in vitro studies of the stress-corrosion cracking behavior of surgical implant alloys.

Behavior of implant alloys exposed simultaneously to tensile stresses and corrosion environments has been examined. In the in vivo studies, a stainless steel and a titanium alloy exhibited cracklike features when loaded to the yield stress sigma y and implanted for 16 weeks. A cobalt-chromium alloy stressed beyond sigma y exhibited them in plastically deformed areas. A cobalt-chromium-nickel-molybdenum alloy appeared to be immune. In vitro samples loaded to various stress levels were immersed in Ringer's solution at 37 degrees C. Half of them were subjected to applied anodic potentials; the remaining control group was not. The applied potentials were dc potentials of magnitude similar to those generated by bioelectric effects. No attempt was made to duplicate time dependence or wave forms. Cracklike features were observed in the stainless steel and in the titanium alloy loaded to or beyond sigma y and polarized for 38 weeks. None were observed below sigma y. For the controls, no cracklike features were observed at any stress level after 53 1/2 weeks. Polarization measurements and potential versus time measurements were performed to study possible mechanisms for crack propagation. These investigations suggest that the in vivo corrosion environment is more severe than a 37 degrees C Ringer's solution because of the influence of both bioelectric effects and organic constituents. The implications of these studies for the performance of prosthetic devices is discussed.