Introduction: Biomaterial associate infections (BAI) are still an issue in total joint replacement (TJR) surgery [1]. The initial bacterial adhesion to the biomaterial surface is a complex process related to physico-chemical interactions with the substratum [2,3]. Ultra High Molecular Weight Polyethylene (UHMWPE) is the most commonly used bearing material in modern TJRs. Many efforts have been made to improve the quality and the performance of UHMWPE, through reducing or eliminating the oxidation. In a previous study [4], the adhesion of Staphylococcus epidermidis, the leading etiologic agent of TJR infections, to different polyethylenes was quantified. The aim of this study was to quantify the adhesive strength of Staphylococcus aureus and Escherichia coli to standard UHMWPE, Vitamin E blended UHMWPE and oxidized UHMWPE. Methods: Biomaterials Cylindrical specimens (height 14 mm, diameter 5 mm) were punched out from compression molded sheets of standard GUR 1020 UHMWPE (PE) and of UHMWPE blended with 0.1% w/w vitamin E (VE-PE) (MediTECH/Quadrant, Fort Wayne, IN, USA). Half of the PE samples were exposed to accelerated ageing in a ventilated oven at 95°C for 210 h, to obtain oxidized UHMWPE (OX-PE). Sterilization was achieved through 70% ethanol immersion followed by washing in sterile demineralized water. Bacterial cultures Biofilm producing strains of S. aureus ATCC 29213 and E. coli ATCC 25922 were used for adhesion assays. Additionally biofilm producing S. aureus and E. coli recently isolated from orthopedic implant infections and previously characterized as in vitro biofilm producers were also tested. Each strain was cultured on Tryptic Soy Agar (TSA; Merck KGaA, Darmstadt, Germany); young colonies (18-24h) were picked to approximately 3-4 McFarland standard and inoculated into cryovials containing both cryopreservative fluid and porous beads to allow bacteria to adhere (Microbank, Biomérieux; Rome, Italy). After inoculation, cryovials were kept at -80°C for extended storage. Adhesion assays All strains stored at -80°C were cultured over night at 37°C in Tryptone Soya Broth (TSB; Basingstoke, UK). After incubation, bacteria were re-suspended in 100 ??l of TSB, harvested by 10 min centrifugation at 4,000 rpm and then diluted in TSB to 107 CFU/ml, as confirmed by colony counts on TSA. The sterile biomaterials were placed in 2 ml of bacterial suspension and incubated by shaking (3, 7, 24, 48h) at 37°C to allow in vitro bacterial adhesion, biofilm formation and its maturation. Controls were represented by bacteria incubated in TSB with no biomaterial. The number of strongly bound bacteria in the cylinders after incubation was quantified after sonication (40 kHZ) for 7 min at 22°C in 1.5 ml of sterile NaCl 0.9% (Bieffe Medital S.p.A., Grosotto, Italy). The number of CFU in each sonication product was quantified by serial plate counts into TSA. All the experiments were performed simultaneously for each biomaterial, assayed in triplicate and repeated a minimum of three times. Statistical analysis - The adhesion assay results, expressed as CFU/ml, were analyzed by descriptive statistics (mean values and standard deviations) and tested by unpaired T-Student test, to highlight significant differences (p<0.05) between the different biomaterials using the Graphpad Prism 6 software (San Diego, CA, USA). Results: Quantitative analysis of bacterial adhesion on various polyethylenes were performed by using ATCC biofilm producing S. aureus and E. coli. After 3h of incubation, the initial S. aureus adherence on the different biomaterials achieved adhesion rates similar on PE and VE-PE, ranging from 4.12x106 to 3.98x106 CFU/ml, but significantly higher on OX-PE (9.82x106 CFU/ml; p=0.0041). A similar trend of staphylococcal adhesion was detected even after 7 and 24h. In contrast, after 48h, a lower staphylococcal adherence on VE-PE (1.29 x107CFU/ml; p=0.0243) compared with that seen on both PE (3.18 x107 CFU/ml) and OX-PE (3.56 x107 CFU/ml ) was observed. No significant difference in the numbers of E. coli adhering to PE, VE-PE and PE-OX was noted after 3, 7, and 24h of incubation. On the other hand, at 48h of incubation the E. coli adherence to VE-PE was significantly lower (5.83x106 CFU/ml; p=0.0081) than to PE and OX-PE (1.15x107 and 1.46x107 respectively). The microbiological findings obtained with biofilm producing S. aureus and E. coli recently isolated from orthopedic implant infections were concordant with those registered with collection strains: again, after 48h of incubation, a significantly lower bacterial adhesion was detected on VE-PE for both S. aureus and E. coli compared with that registered on PE and OX-PE. Discussion: The VE stabilization has been recently proposed to improve oxidation resistance of UHMWPE while maintaining wear resistance and fatigue strength [5,6]. Previous studies [4] indicated that no significant difference of the surface roughness and static contact angle (CA) was found among the three biomaterial samples, while a lower CA was observed for OX-PE, if compared to the other two groups. With attenuated total reflectance (ATR)-fourier transform infrared (FTIR) spectroscopy, the only significant difference observed was in the spectrum of the aged group, OX-PE, indicating adsorption of protein-like substances on the polymer surface. The results of the adhesion assays of S. aureus and E. coli on PE, VE-PE and OX-PE samples led to the observation that the surface oxidation facilitates the microorganism adhesion, confirming what previously reported with S. epidermidis [4]. In contrast, a significant decreased bacterial adhesion was registered for both strains on VE-PE, in comparison with that observed on PE, within 48h of observation, underlying the role of VE, probably related to its well established antioxidant properties [6]. The mechanism by which VE may affect the bacterial adherence to UHMWPE it is currently unknown, even if potential effects of VE on infection are currently investigated [7]. From our previous results [4], the CA measurements showed no significant variations in hydrophilicity between PE and VE-PE suggesting that other, concurrent factors must be involved in the different bacterial adhesion. A direct effect of VE itself, even if present in very low concentration in the polyethylene matrix, on the bacterial adhesive ability cannot be ruled out. The results obtained by testing both collection and clinical strains of S. aureus and E. coli highlighted similar adhesion bacterial capacity although indicating some variability in the initial adherence to inert surfaces. These results are consistent to the trends previously reported with S. epidermidis [4], but in contrast with some literature data on collection S. aureus [7,8]. The hypothesis is that the very early stage of colonization process is strongly affected by intrinsic intra and inter-species variability among different bacterial genera [4,7,8,9]. To transfer into clinical practice experimentally obtained results, an ex vivo failed prosthetic component analysis will be necessary to correlate biomaterials surface with infection.

Surface properties of different UHMWPEs and bacterial adhesion.

BANCHE, Giuliana;ALLIZOND, VALERIA;BRACCO, Pierangiola;BRACH DEL PREVER, Elena Maria;CUFFINI, Annamaria
2014

Abstract

Introduction: Biomaterial associate infections (BAI) are still an issue in total joint replacement (TJR) surgery [1]. The initial bacterial adhesion to the biomaterial surface is a complex process related to physico-chemical interactions with the substratum [2,3]. Ultra High Molecular Weight Polyethylene (UHMWPE) is the most commonly used bearing material in modern TJRs. Many efforts have been made to improve the quality and the performance of UHMWPE, through reducing or eliminating the oxidation. In a previous study [4], the adhesion of Staphylococcus epidermidis, the leading etiologic agent of TJR infections, to different polyethylenes was quantified. The aim of this study was to quantify the adhesive strength of Staphylococcus aureus and Escherichia coli to standard UHMWPE, Vitamin E blended UHMWPE and oxidized UHMWPE. Methods: Biomaterials Cylindrical specimens (height 14 mm, diameter 5 mm) were punched out from compression molded sheets of standard GUR 1020 UHMWPE (PE) and of UHMWPE blended with 0.1% w/w vitamin E (VE-PE) (MediTECH/Quadrant, Fort Wayne, IN, USA). Half of the PE samples were exposed to accelerated ageing in a ventilated oven at 95°C for 210 h, to obtain oxidized UHMWPE (OX-PE). Sterilization was achieved through 70% ethanol immersion followed by washing in sterile demineralized water. Bacterial cultures Biofilm producing strains of S. aureus ATCC 29213 and E. coli ATCC 25922 were used for adhesion assays. Additionally biofilm producing S. aureus and E. coli recently isolated from orthopedic implant infections and previously characterized as in vitro biofilm producers were also tested. Each strain was cultured on Tryptic Soy Agar (TSA; Merck KGaA, Darmstadt, Germany); young colonies (18-24h) were picked to approximately 3-4 McFarland standard and inoculated into cryovials containing both cryopreservative fluid and porous beads to allow bacteria to adhere (Microbank, Biomérieux; Rome, Italy). After inoculation, cryovials were kept at -80°C for extended storage. Adhesion assays All strains stored at -80°C were cultured over night at 37°C in Tryptone Soya Broth (TSB; Basingstoke, UK). After incubation, bacteria were re-suspended in 100 ??l of TSB, harvested by 10 min centrifugation at 4,000 rpm and then diluted in TSB to 107 CFU/ml, as confirmed by colony counts on TSA. The sterile biomaterials were placed in 2 ml of bacterial suspension and incubated by shaking (3, 7, 24, 48h) at 37°C to allow in vitro bacterial adhesion, biofilm formation and its maturation. Controls were represented by bacteria incubated in TSB with no biomaterial. The number of strongly bound bacteria in the cylinders after incubation was quantified after sonication (40 kHZ) for 7 min at 22°C in 1.5 ml of sterile NaCl 0.9% (Bieffe Medital S.p.A., Grosotto, Italy). The number of CFU in each sonication product was quantified by serial plate counts into TSA. All the experiments were performed simultaneously for each biomaterial, assayed in triplicate and repeated a minimum of three times. Statistical analysis - The adhesion assay results, expressed as CFU/ml, were analyzed by descriptive statistics (mean values and standard deviations) and tested by unpaired T-Student test, to highlight significant differences (p<0.05) between the different biomaterials using the Graphpad Prism 6 software (San Diego, CA, USA). Results: Quantitative analysis of bacterial adhesion on various polyethylenes were performed by using ATCC biofilm producing S. aureus and E. coli. After 3h of incubation, the initial S. aureus adherence on the different biomaterials achieved adhesion rates similar on PE and VE-PE, ranging from 4.12x106 to 3.98x106 CFU/ml, but significantly higher on OX-PE (9.82x106 CFU/ml; p=0.0041). A similar trend of staphylococcal adhesion was detected even after 7 and 24h. In contrast, after 48h, a lower staphylococcal adherence on VE-PE (1.29 x107CFU/ml; p=0.0243) compared with that seen on both PE (3.18 x107 CFU/ml) and OX-PE (3.56 x107 CFU/ml ) was observed. No significant difference in the numbers of E. coli adhering to PE, VE-PE and PE-OX was noted after 3, 7, and 24h of incubation. On the other hand, at 48h of incubation the E. coli adherence to VE-PE was significantly lower (5.83x106 CFU/ml; p=0.0081) than to PE and OX-PE (1.15x107 and 1.46x107 respectively). The microbiological findings obtained with biofilm producing S. aureus and E. coli recently isolated from orthopedic implant infections were concordant with those registered with collection strains: again, after 48h of incubation, a significantly lower bacterial adhesion was detected on VE-PE for both S. aureus and E. coli compared with that registered on PE and OX-PE. Discussion: The VE stabilization has been recently proposed to improve oxidation resistance of UHMWPE while maintaining wear resistance and fatigue strength [5,6]. Previous studies [4] indicated that no significant difference of the surface roughness and static contact angle (CA) was found among the three biomaterial samples, while a lower CA was observed for OX-PE, if compared to the other two groups. With attenuated total reflectance (ATR)-fourier transform infrared (FTIR) spectroscopy, the only significant difference observed was in the spectrum of the aged group, OX-PE, indicating adsorption of protein-like substances on the polymer surface. The results of the adhesion assays of S. aureus and E. coli on PE, VE-PE and OX-PE samples led to the observation that the surface oxidation facilitates the microorganism adhesion, confirming what previously reported with S. epidermidis [4]. In contrast, a significant decreased bacterial adhesion was registered for both strains on VE-PE, in comparison with that observed on PE, within 48h of observation, underlying the role of VE, probably related to its well established antioxidant properties [6]. The mechanism by which VE may affect the bacterial adherence to UHMWPE it is currently unknown, even if potential effects of VE on infection are currently investigated [7]. From our previous results [4], the CA measurements showed no significant variations in hydrophilicity between PE and VE-PE suggesting that other, concurrent factors must be involved in the different bacterial adhesion. A direct effect of VE itself, even if present in very low concentration in the polyethylene matrix, on the bacterial adhesive ability cannot be ruled out. The results obtained by testing both collection and clinical strains of S. aureus and E. coli highlighted similar adhesion bacterial capacity although indicating some variability in the initial adherence to inert surfaces. These results are consistent to the trends previously reported with S. epidermidis [4], but in contrast with some literature data on collection S. aureus [7,8]. The hypothesis is that the very early stage of colonization process is strongly affected by intrinsic intra and inter-species variability among different bacterial genera [4,7,8,9]. To transfer into clinical practice experimentally obtained results, an ex vivo failed prosthetic component analysis will be necessary to correlate biomaterials surface with infection.
60th Annual Meeting of the ORS
New Orleans
March 15-18 2014
Annual Meeting Abstracts
Ortopaedic Research Society
1867
1867
http://www.ors.org/download-2014-abstracts/
standard UHMWPE; Vitamin E blended UHMWPE; oxidized UHMWPE; adhesive properties; Staphylococcus aureus; Escherichia coli
BISTOLFI A.; BOFFANO M.; BANCHE G.; ALLIZOND V.; BRACCO P.; CIMINO A.; BRACH DEL PREVER E.M.; CUFFINI A.M.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/153583
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