Katharine Merritt

Detection of bacterial adherence on biomedical polymers

The ability to adhere to materials and promote formation of a biofilm is an important feature of the pathogenicity of some organisms, most notably the coagulase negative staphylococci. Various methods to detect bacterial adherence are available, but detection of adherence to nontransparent materials can be difficult. It was the purpose of this study to establish the suitability of a dye elution technique to detect adherence. The technique involves fixing the bacterial film with formalin, staining with crystal violet, eluting the dye with ethanol, and determining the optical density of the solution using 96-well plates and an enzyme immunosorbent assay reader with a 540-nm filter. This technique distinguished a known adherent from a known nonadherent organism and demonstrated that the presence of protein can inhibit adherence, and that adherence of different organisms to different biomedically important polymers can be measured. The dye elution technique was used to evaluate the adherence of known slime-producing polysaccharide antigen-positive (PSA+) and known nonslime-producing polysaccharide antigen-negative (PSA-) Staphylococcus epidermidis organisms to implant grade materials (polyethylene, expanded polytetrafluoroethylene, Dacron, silicone, and collagen) as well as to polystyrene.

Production of monoclonal antibodies to study corrosion products of CO-CR biomaterials

Sensitivity to nickel, cobalt, and chromium is common among the general population. The identification of these sensitivities is generally by the detection of cell-mediated immunity. We have reported previously the use of an indirect enzyme-linked immunosorbent assay method to quantitate metal-specific antibodies in patients with total joint replacements. To study the haptenic potential of these metal ions, rabbit albumin-glutathione-metal complexes with chromium, cobalt, or nickel were injected into mice. The splenocytes from one mouse in each group which developed a strong antibody against GSH-metal complexes were isolated and fused with myeloma cells to produce monoclonal antibodies. Chromium, cobalt, and nickel antibodies had similar affinity and bound with the specific GSH-metal complex. There was very little cross-reactivity between these antibodies. An inhibition assay using these monoclonal antibodies was demonstrated to be a simple technique, suitable for quantitation of free metal in solution.

Distribution of nickel and cobalt following dermal and systemic administration with in vitro and in vivo studies.

Contact dermatitis following skin contact with ionic metals occurs in about 15% of the human population, but systemic responses are not as common. It is generally believed that skin contact with metal ions leads to different biological processing compared with systemic contact. The purpose of the experiments presented here was to examine elimination of nickel and cobalt salts following skin application and following injection into the deep tissue of hamsters. In addition uptake of the metal by fibroblasts and by keratinocytes was examined in vitro. The data indicate that elimination of nickel and cobalt in the urine following systemic injection was rapid, as expected, but that elimination following skin application was delayed. Much of the metal was retained in the skin. Uptake of the metal by keratinocytes at low doses was greater than the uptake by the fibroblasts. At high doses the differences were not as great. Thus the conclusion is drawn that metal salts are retained in the skin for an extended period of time and could lead to prolonged antigen processing and consequent immune responses in the dermal tissue. Skin application leads to some systemic distribution but the systemic application did not lead to skin accumulation.

Effects of Ca and H2O2 added to RPMI on the fretting corrosion of Ti6Al4V

Titanium and its alloys have demonstrated considerable success in various surgical procedures including orthopedic, dental, and cardiovascular surgery. However, particulate debris from corrosion and wear is present in a considerable quantity in tissue local to the implant. This study evaluated the effect of Ca, since it is present in both serum and bone, and H2O2, since it is produced through local inflammation, on the amount of titanium release. Four sets of Ti6Al4V plates and Ti6Al4V screws were used. Each set was designated to one of four solutions: RPMI (cell culture growth media), RPMI with CaCl2, RPMI with CaCO3, and RPMI with H2O2. A fretter was used to cause corrosion by creating micromotion between two screws and a two-hole plate of Ti6Al4V. After fretting for 72 h, weight loss of the plate and screws and the amount of Ti and vanadium (V) in solution was used to assess the amount of fretting corrosion which had occurred. Results of weight loss and Ti in solution indicated that the presence of H2O2 increased the amount of particulate debris produced in RPMI as compared with RPMI alone. The addition of CaCl2 to RPMI also increased both weight loss and Ti in solution compared with RPMI alone. The addition of CaCO2, however, did not give values significantly different from RPMI alone. Comparison of weight loss and Ti in solution indicated that the increase in fretting corrosion was not different between RPMI with CaCl2 and RPMI with H2O2. The particulate wear debris from the four solutions was black in color and the size of the particulate produced was compared using a Coulter Multisizer. The results indicated that particles produced in the four solutions were not different, with mean values between 1.324 and 1.100 microns, and they were similar in size to the particulate found in tissues surrounding failed total hip replacements. In order to better understand the role of Ca in the fretting corrosion of Ti6Al4V, energy dispersive x-ray analysis (EDXA) using SEM was used to determine elemental composition of one countersink surface of a plate which had been run four times in RPMI with CaCl2. The presence of Ca in the bulk was not significant (% composition < 0.5%). However, Ca was present in two surface particles which were examined at a magnification of 55,000, with a Ca% composition of 63.2% and 19.2%. While results from this study indicate that both soluble Ca(CaCl2) and H2O2 increase the fretting corrosion of Ti6Al4V, the insoluble form of Ca, which would be found in bone and hydroxyapatite, has no effect. These data indicate that it is important to specify the media used in corrosion, dissolution, and elution experiments.

Characterization of calcium phosphate powders by ESCA and EDXA

Calcium phosphate (CaP) materials can be well characterized by traditional methods such as wet chemistry and X-ray diffraction (XRD). These methods, however, offer limitations when non-destructive evaluation of CaP coatings on curved surfaces is required. Since the source powders for these coatings are generally commercially available CaP powders, careful characterization of the source powders may allow inferences to be made regarding the effects of plasma spraying on coating composition. Nine commercially available CaP powders were characterized by scanning electron microscopy, wet chemistry and XRD. These techniques showed that major differences exist between individual powders claiming to be hydroxyapatite. Analysis of these nine powders by electron spectroscopy for chemical analysis (ESCA) and energy dispersive X-ray analysis (EDXA) suggest that these techniques can provide the chemical composition of CaP in a non-destructive manner and thus may be of use in determining the composition of CaP in configurations (such as coatings on metal surfaces) not readily amenable to traditional methods. A calibration curve is required, however, to relate this surface chemical composition result to the materials bulk composition as determined by wet chemistry analysis. Errors of less than 10% can be obtained using ESCA and EDXA. These studies suggest that non-destructive chemical composition evaluation by EDXA and ESCA may also be applicable to CaP coatings.

Use of Fibrinolytic Agents to Coat Wire Implants to Decrease Infection: An Animal Model

RATIONALE AND OBJECTIVES. Infection is a serious complication of metallic prosthesis implantation and may necessitate removal of the prosthesis. This study uses an animal model to evaluate the effects of coating stainless steel wire implants with fibrinolytic agents to prevent infection after bacterial contamination. METHODS. Three types of steel wire implants were used: plain stainless steel, heparin-coated steel, and urokinase-heparin- coated steel. Wire implants were incubated in a known concentration of Staphylococcus epidermidis and placed into the subcutaneous tissues of three groups of anesthetized hamsters. The implants and surrounding tissues were excised after 1 week and submitted for quantitative cultures. RESULTS. Using 100 organisms as the upper allowable limit to categorize abscesses as noninfected, the following rates of noninfectivity were observed: group 1 (control), 0% noninfected; group 2 (heparin-coated wire), 40% noninfected; and group 3 (urokinasc-heparin-coated wire), 50% noninfected. The noninfectivity rates of groups 2 and 3 were significantly higher than the rate of group 1 (P < 0.001). There was no significant difference between groups 2 and 3 (P=0.19). CONCLUSIONS.Both the heparin-coated and urokinase-heparin-coated wire exhibited significantly decreased infection rates compared with uncoated wire; the heparin coating may inhibit bacterial adherence. The urokinase coating of the heparin-coated to further decrease the infection rate, but not to a statistically significant degree.

Young Investigator Award. In vivo treatment of infected prosthetic graft material with urokinase: an animal model.

PURPOSE Pyogenic infection of vascular grafts represents a serious complication that may necessitate graft removal. If better treatment methods could be developed, perhaps some infected grafts could be salvaged and not removed. This study reports an animal model that evaluates the sterilization of contaminated vascular graft material implants with urokinase and antibiotics. MATERIALS AND METHODS Polytetrafluoroethylene (PTFE) implants were incubated overnight in a known concentration of bacteria (Staphylococcus epidermidis) and were then implanted subcutaneously into four groups of anesthetized hamsters. The first group (control) received no treatment. The second group received urokinase injections twice daily into each abscess. The third group received intraabscess urokinase and systemic gentamicin twice daily. The fourth group received only systemic gentamicin. The hamsters were killed after 1 week. The graft implants and surrounding tissues were excised and submitted for quantitative cultures. RESULTS With use of a cutoff value of 100 organisms per milliliter, below which the abscesses were considered noninfected, the following rates of noninfectivity were observed: group 1 (control), 5% noninfected; group 2 (urokinase only), 19.4%; group 3 (urokinase and gentamicin), 63.2%; and group 4 (gentamicin only), 32.5%. The noninfectivity rate of group 3 was significantly higher than that of all other groups combined (P < .001) and was significantly better than that of group 4 alone (P = .013). CONCLUSION The combination of intraabscess urokinase and systemic gentamicin is very synergistic in graft sterilization. Urokinase may assist in the degradation of both fibrin and the biofilm produced by S epidermidis, thus improving penetration of antibiotics and local host defense mechanisms.

Accelerated testing of a composite spine plate

Abstract A polymer composite was evaluated for use as an orthopaedic, variable screw placement (VSP) spine plate. Fretting fatigue or cyclic loading that causes micromotion at interfaces in the spine plate could cause significant wear of the composite plate. Therefore, a test method was developed to assess a continuous carbon fibre polysulphone composite plate in in vitro fretting fatigue. A relevant failure criterion was established and employed to determine the effect of torque on the fatigue life of the plate. Two failure mechanisms were observed and the plate displacement behaviour was indicative of these mechanisms. Furthermore, an optimum implant torque was defined. The results demonstrate the importance of tailoring the material to the implant application. Therefore several material improvements have been implemented.