Kirk J. Bundy

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.

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.

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.

Bioaccumulation of chromium in red swamp crayfish (Procambarus clarkii)

Abstract Crayfish were exposed to a range of potassium dichromate concentrations (0.15, 0.30, 3.0 and 30 mg l−1) for periods up to 7 weeks. Chromium bioaccumulation in all tissues over the 7 week exposure period was not consistently time- and dose-dependent. The order of distribution of chromium into the various tissues was dependent upon the exposure concentration of the metal. Chromium clearance studies conducted 1 and 3 weeks following exposure demonstrated a concentration reduction in most tissues only at the highest exposure concentration of chromium (30 mg l−1). Histological studies demonstrated damage to both the gills and hepatopancreas at the lowest exposure concentration. The results suggest that the red swamp crayfish, Procambarus clarkii, is a useful biomarker for chromium exposure.

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.

Assessment of dental material degradation product toxicity using a bioluminescent bacterial assay

OBJECTIVES This study examined dental material degradation product toxicity using the Microtox bacterial bioluminescence assay as well as the effects on toxicity of selective leaching, chelation with protein, the physical form of the products, and synergistic/antagonistic interactions among released ions. METHODS Polarization was used to produce ionically dissolved (ID) and precipitated corrosion products from Litecast B alloy specimens, which were then chemically analyzed to determine their composition and to identify metal valence states. Corrosion product toxicity, as well as that of the individual alloying elements, alone and in the presence of mucin, was analyzed using Microtox. A mathematical approach identified synergistic/antagonistic interactions and determined element contribution to product toxicity. The mechanism by which the Microtox test bacterium interacts with solid products was explored. The toxicity of methyl methacrylate (MMA) monomer was also examined. RESULTS Precipitated corrosion products were found to be more toxic than ID products. The metals in the precipitate have been shown to be available to the test bacterium. Be and Ni were the most toxic elements in the products and contributed significantly to their toxicity. Synergistic and slightly antagonistic interactions were observed in the ID and precipitated products, respectively. Mucin decreased toxicity of all elements except Be. MMA monomer toxicity was found to be low compared to metal toxicity. SIGNIFICANCE Microtox is useful for evaluating dental degradation product biocompatibility and has significant promise for use in other types of studies, such as determining the effectiveness of antimicrobial treatments.

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.

Characterization of the Corrosion Behavior of Porous Biomaterials by A-C Impedance Techniques

In this research, the corrosion behavior of Ti-6Al-4V extralow interstitial (ELI) and Co-Cr-Mo alloys with polished, grit-blasted, and porous-coated surfaces has been investigated. The A-C impedance technique and a variety of D-C methods were used for this purpose. Both highly stressed and nonstressed specimens were tested. Stress-enhanced release of corrosion products was demonstrated to occur in some cases. The A-C impedance method, which is capable of measuring both corrosion current and true surface area, showed that this effect was mainly due to an area increase caused by plastic deformation. The advantages and characteristics of the A-C impedance method for corrosion measurements of implant materials are also described.

Surface charge, biofilm composition and cellular morphology as related to cellular adhesion to biomaterials

Our previous studies have used jet impingement techniques to quantify strength of cell adhesion to various material surfaces. Surface charge and energy parameters were found to mediate the strength of cellular adhesion. The present investigation examines biological factors which might influence fibroblast adhesion to biomaterials-biofilm composition and cell morphology. Also, the relationship of surface charge to cellular adhesion has been investigated in a controlled way by measuring adhesion strength over a range of surface charge densities. The cells showed potential dependent adhesion maxima which suggests that surface alloying for optimum adherence may be possible. The adsorbed serum protein biofilms on a series of materials of differing adherence were investigated using gel electrophoresis. The profiles of adsorbed proteins revealed little difference in relative abundance or total adsorption quantity. SEM micrographs of the cells on a titanium alloy and silicone rubber (high and low adhesion materials, respectively) showed morphologic and cell density differences.

Differential Pulse Polarographic Analysis of Lead and Chromium Content in Louisiana Waters

In this investigation, polarographic analyses of water, sediment, and animal samples from Devils Swamp near Baton Rouge, Louisiana, have been conducted. The focus of this work has been on detection of lead and chromium levels. Lead has been found to be relatively uniformly distributed among the various size fractions of the sediment and is present at a mean level of 18.7 µg g-1. In the water the concentration is about 15 µg L-1 of which 75% is bound in suspended particulates and the remainder is dissolved. Preliminary results indicate that more chromium than lead is present in bone and muscle of bullfrogs, and, for each metal, there is a higher concentration in muscle than bone. The mean lead muscle tissue concentration is 550 µg kg-1, which suggests that bioaccumulation of this metal is occurring, assuming that water contact or ingestion are the main routes of exposure. An important aspect of this research has been optimizing polarographic methodology for performing chromium speciation studies. Methods for determining the amounts of hexavalent and trivalent chromium in mixtures containing the two have been developed.