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VOLUME 14 , ISSUE 2 ( May-August, 2019 ) > List of Articles

Original Article

Titanium Kirschner Wires Resist Biofilms Better Than Stainless Steel and Hydroxyapatite-coated Wires: An In Vitro Study

James P McEvoy, Philip Martin, Arshad Khaleel, Shobana Dissanayeke

Keywords : Bacterial adhesion, Biofilms, External fixation, Infection, Orthopedics

Citation Information : McEvoy JP, Martin P, Khaleel A, Dissanayeke S. Titanium Kirschner Wires Resist Biofilms Better Than Stainless Steel and Hydroxyapatite-coated Wires: An In Vitro Study. 2019; 14 (2):57-64.

DOI: 10.5005/jp-journals-10080-1426

License: CC BY-NC-SA 4.0

Published Online: 25-12-2011

Copyright Statement:  Copyright © 2019; The Author(s).


Aim: External fixation surgery is frequently complicated by percutaneous pin site infection focused on the surface of the fixator pin. The primary aim of this study was to compare biofilm growth of clinically isolated pin site bacteria on Kirschner wires of different materials. Materials and methods: Two commonly infecting species, Staphylococcus epidermidis and Proteus mirabilis, were isolated from patients’ pin sites. A stirred batch bioreactor was used to grow these bacteria as single culture and co-cultured biofilms on Kirschner wires made of three different materials: stainless steel, hydroxyapatite-coated steel and titanium alloy. Results: We found that the surface density of viable cells within these biofilms was 3x higher on stainless steel and 4.5x higher on hydroxyapatitecoated wires than on the titanium wires. Conclusion: Our results suggest that the lower rates of clinical pin site infection seen with titanium Kirschner wires are due to, at least in part, titanium’s better bacterial biofilm resistance. Clinical significance: Our results are consistent with clinical studies which have found that pin site infection rates are reduced by the use of titanium relative to stainless steel or hydroxyapatite-coated pins.

  1. Bible JE, Mir HR. External fixation: principles and applications. J Am Acad Orthop Surg 2015;23(11):683–690. DOI: 10.5435/JAAOS-D-14-00281.
  2. Fragomen AT, Rozbruch SR. The mechanics of external fixation. HSS J 2007;3(1):13–29. DOI: 10.1007/s11420-006-9025-0.
  3. Mock C, Cherian MN. The global burden of musculoskeletal injuries: challenges and solutions. Clin Orthop Relat Res 2008;466(10):2306–2316. DOI: 10.1007/s11999-008-0416-z.
  4. Andruszkow H, Pfeifer R, Horst K, et al. External fixation in the elderly. Injury 2015;46:S7–S12. DOI: 10.1016/S0020-1383(15)30004-8.
  5. Kazmers NH, Fragomen AT, Rozbruch SR. Prevention of pin site infection in external fixation: a review of the literature. Strat Traum Limb Recon 2016;11(2):75–85. DOI: 10.1007/s11751-016-0256-4.
  6. DeJong ES, DeBerardino TM, Brooks DE, et al. Antimicrobial efficacy of external fixator pins coated with a lipid stabilized hydroxyapatite/chlorhexidine complex to prevent pin tract infection in a goat model. J Trauma 2001;50(6):1008–1014. DOI: 10.1097/00005373-200106000-00006.
  7. Antoci V, Ono CM, Antoci V, et al. Pin-tract infection during limb lengthening using external fixation. Am J Orthop 2008;37(9): E150–E154.
  8. Ferreira N, Marais LC. Prevention and management of external fixator pin track sepsis. Strat Traum Limb Recon 2012;7(2):67–72. DOI: 10.1007/s11751-012-0139-2.
  9. Bibbo C, Brueggeman J. Prevention and management of complications arising from external fixation pin sites. J Foot Ankle Surg 2010;49(1):87–92. DOI: 10.1053/j.jfas.2009.07.026.
  10. Loder RT. The influence of diabetes mellitus on the healing of closed fractures. Clin Orthop Relat Res 1988;232(232):210–216. DOI: 10.1097/00003086-198807000-00028.
  11. Brady RA, Calhoun JH, Leid JG, et al. Infections of orthopaedic implants and devices. In: Shirtliff ME, Leid JG. The Role of Biofilms in Device-Related Infections, Springer Series on Biofilms, vol. 3, Berlin: Springer; 2008. pp. 15–55.
  12. Ceroni D, Grumetz C, Desvachez O, et al. From prevention of pin-tract infection to treatment of osteomyelitis during paediatric external fixation. J Child Orthop 2016;10(6):605–612. DOI: 10.1007/s11832-016-0787-8.
  13. Davey ME, O’Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000;64(4):847–867. DOI: 10.1128/mmbr.64.4.847-867.2000.
  14. Gristina A. Biomaterial-centered infection: microbial adhesion versus tissue integration. Science 1987;237(4822):1588–1595. DOI: 10.1126/science.3629258.
  15. Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2004;2(2):95–108. DOI: 10.1038/nrmicro821.
  16. Ploux L, Ponche A, Anselme K. Bacteria/material interfaces: Role of the material and cell wall properties. J Adhes Sci Tech 2010;24(13-14): 2165–2201. DOI: 10.1163/016942410X511079.
  17. Campoccia D, Montanaro L, Arciola CR. The significance of infection related to orthopedic devices and issues of antibiotic resistance. Biomaterials 2006;27(11):2331–2339. DOI: 10.1016/j.biomaterials.2005.11.044.
  18. Jennison T, McNally M, Pandit H. Prevention of infection in external fixator pin sites. Acta Biomater 2014;10(2):595–603. DOI: 10.1016/j. actbio.2013.09.019.
  19. Clauss M, Graf S, Gersbach S, et al. Material and biofilm load of K wires in toe surgery: titanium versus stainless steel. Clin Orthop Relat Res 2013;471(7):2312–2317. DOI: 10.1007/s11999-013-2919-5.
  20. Pieske O, Geleng P, Zaspel J, et al. Titanium alloy pins versus stainless steel pins in external fixation at the wrist: a randomized prospective study. J Trauma Injury Infect Crit Care 2008;64(5):1275–1280. DOI: 10.1097/TA.0b013e31815e40e0.
  21. Silvestre MD, Bakaloudis G, Lolli F, et al. Late-developing infection following posterior fusion for adolescent idiopathic scoliosis. Eur Spine J 2011;20(S1):S121–S127. DOI: 10.1007/s00586-011-1754-1.
  22. Arens S, Schlegel U, Printzen G, et al. Influence of materials for fixation implants on local infection. J Bone Joint Surg Br 1996;78(4):647–651. DOI: 10.1302/0301-620X.78B4.0780647.
  23. Metsemakers WJ, Schmid T, Zeiter S, et al. Titanium and steel fracture fixation plates with different surface topographies: influence on infection rate in a rabbit fracture model. Injury 2016;47(3):633–639. DOI: 10.1016/j.injury.2016.01.011.
  24. Shida T, Koseki H, Yoda I, et al. Adherence ability of staphylococcus epidermidis on prosthetic biomaterials: an in vitro study. Int J Nanomed 2013;8:3955–3961. DOI: 10.2147/IJN.S51994.
  25. Moroni A, Cadossi M, Romagnoli M, et al. A biomechanical and histological analysis of standard versus hydroxyapatite-coated pins for external fixation. J Biomed Mater Res B Appl Biomater 2008;86(2):417–421. DOI: 10.1002/jbm.b.31036.
  26. Patel A, Ghai A, Anand A. Clinical benefit of hydroxyapatite-coated versus uncoated external fixation: a systematic review. Int J Orthop 2016;3(9):581–590. DOI: 10.17554/j.issn.2311-5106.2016.03.163.
  27. Arciola CR, Montanaro L, Moroni A, et al. Hydroxyapatite-coated orthopaedic screws as infection resistant materials: in vitro study. Biomaterials 1999;20(4):323–327. DOI: 10.1016/s0142-9612(98)00168-9.
  28. A 10 μL loop of each colony isolate was grown overnight at 37.5°C in 5 mL LB. Cells were harvested by centrifuging at 13,000 g for 3 minutes, washing in PBS, and a second centrifugation. The supernatant was discarded and bacteria were incubated first for 2 hours in 1.6 mL of 10 mM Tris-HCl (pH 8) with 4 μg of lysozyme (Sigma-Aldrich), followed by an hour with 25 μL proteinase-K (Sigma-Aldrich) and 200 μL lysis buffer (50 mM Tris, 100 mM EDTA, 1% SDS, pH 8.0). Extraction and purification was completed using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions. Extracted DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). If extracted isolates yielded a DNA concentration < 30 ng/μL then the extraction process was repeated with 5 × 15-second bursts of ultrasonic cell lysis (Microson XL-2000 ultrasonic cell disruptor, power setting 8) immediately after incubation with proteinase-K.
  29. Bacterial DNA was amplified using outer primers that targeted the 16S gene of all bacteria commonly associated with wound infections [28] and an inner primer that targeted the 16S hypervariable region unique to each species of bacteria. The primers used were: 16S Outer Forward (5′-GTG TAG CGG TGA AAT GCG-3′), 16S Outer Reverse (5′-ACG GGC GGT GTG TAC AA-3′), 16SInnerForward (5′-GGT GGA GCA TGT GGT TTA-3′), 16SInnerReverse (5′-CCA TTG TAG CAC GTG TGT-3′). A 50 μL master mix was prepared to the following final concentrations: 1 × Q5 reaction buffer (containing MgCl2), 200 μM dNTPs, 0.5 μM forward primer, 0.5 μM reverse primer, 0.02 U/μL Q5 polymerase, 10 ng template DNA. The nPCR reaction was completed on a Biometra TProfessional Thermocycler.
  30. Sauer P, Gallo J, Kesselová M, et al. Universal primers for detection of common bacterial pathogens causing prosthetic joint infection. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2005;149(2):285–288. DOI: 10.5507/bp.2005.043.
  31. Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol 1990;215(3):403–410. DOI: 10.1016/S0022-2836(05)80360-2.
  32. O’Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol Microbiol 1998;28(3):449–461. DOI: 10.1046/j.1365-2958.1998.00797.x.
  33. Donlan RM, Piede JA, Heyes CD, et al. Model system for growing and quantifying streptococcus pneumoniae biofilms in situ and in real time. Appl Environ Microbiol 2004;70(8):4980–4988. DOI: 10.1128/AEM.70.8.4980-4988.2004
  34. Bjerkan G, Witsø E, Bergh K. Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro. Acta Orthop 2009;80(2):245–250. DOI: 10.3109/17453670902947457.
  35. Herigstad B, Hamilton M, Heersink J. How to optimize the drop plate method for enumerating bacteria. J Microbiol Methods 2001;44(2):121–129. DOI: 10.1016/s0167-7012(00)00241-4.
  36. Hudetz D, Ursic Hudetz S, Harris LG, et al. Weak effect of metal type and Ica genes on staphylococcal infection of titanium and stainless steel implants. Clin Microbiol Infect 2008;14(12):1135–1145. DOI: 10.1111/j.1469-0691.2008.02096.x.
  37. Battin TJ, Sloan WT, Kjelleberg S, et al. Microbial landscapes: new paths to biofilm research. Nat Rev Microbiol 2007;5(1):76–81. DOI: 10.1038/nrmicro1556.
  38. Kiedrowski MR, Horswill AR. New approaches for treating staphylococcal biofilm infections. Ann N Y Acad Sci 2011;1241(1): 104–121. DOI: 10.1111/j.1749-6632.2011.06281.x.
  39. Lebeaux D, Chauhan A, Rendueles O, et al. From in vitro to in vivo models of bacterial biofilm-related infections. Pathogens 2013;2(2):288–356. DOI: 10.3390/pathogens2020288.
  40. Akens MK, Chien C, Katchky RN, et al. The impact of thermal cycling on staphylococcus aureus biofilm growth on stainless steel and titanium orthopaedic plates. BMC Musculoskel Disord 2018;19(1):1–6. DOI: 10.1186/s12891-018-2199-z.
  41. Raikar GN, Gregory JC, Ong JL, et al. Surface characterization of titanium implants. J Vac Sci Tech A: Vac Surf Films 1995;13(5): 2633–2637. DOI: 10.1116/1.579462.
  42. Chin MYH, Sandham A, de Vries J, et al. Biofilm formation on surface characterized micro-implants for skeletal anchorage in orthodontics. Biomaterials 2007;28(11):2032–2040. DOI: 10.1016/j. biomaterials.2006.12.014.
  43. Neoh KG, Hu X, Zheng D, et al. Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces. Biomaterials 2012;33(10):2813–2822. DOI: 10.1016/j.biomaterials.2012.01.018.
  44. Kerber SJ. Bioreactivity of titanium implant alloys. J Vac Sci Tech A: Vac Surf Films 1995;13(5):2619–2623. DOI: 10.1116/1.579460.
  45. Shah FA, Trobos M, Thomsen P, et al. Commercially pure titanium (cp-Ti) versus titanium alloy (Ti6Al4V) materials as bone anchored implants - is one truly better than the other? Mater Sci Eng C Mater Biol Appl 2016;62:960–966. DOI: 10.1016/j.msec.2016.01.032.
  46. Saithna A. The influence of hydroxyapatite coating of external fixator pins on pin loosening and pin track infection: a systematic review. Injury 2010;41(2):128–132. DOI: 10.1016/j.injury.2009.01.001.
  47. Oga M, Arizono T, Sugioka Y. Bacterial adherence to bioinert and bioactive materials studied in vitro. Acta Orthop Scand 1993;64(3):273–276. DOI: 10.3109/17453679308993623.
  48. Ravn C, Ferreira IS, Maiolo E, et al. Microcalorimetric detection of staphylococcal biofilm growth on various prosthetic biomaterials after exposure to daptomycin. J Orth Res 2018;36(10):2809–2816. DOI: 10.1002/jor.24040.
  49. Alam F, Balani K. Adhesion force of staphylococcus aureus on various biomaterial surfaces. J Mech Behav Biomed Mater 2017;65:872–880. DOI: 10.1016/j.jmbbm.2016.10.009.
  50. Pieske O, Pichlmaier L, Kaltenhauser F, et al. Hydroxyapatite-coated pins versus titanium alloy pins in external fixation at the wrist: a controlled cohort study. J Trauma 2011;70(4):845–851. DOI: 10.1097/TA.0b013e3181e97761.
  51. Teughels W, Van Assche N, Sliepen I, et al. Effect of material characteristics and/or surface topography on biofilm development. Clin Oral Implants Res 2006;17(Suppl 2):68–81. DOI: 10.1111/j.1600-0501.2006.01353.x.
  52. Stickler D, Hughes G. Ability of Proteus mirabilis to swarm over urethral catheters. Eur J Clin Microbiol Infect Dis 1999;18(3):206–208. DOI: 10.1007/s100960050260.
  53. Ica T, Caner V, Istanbullu O, et al. Characterization of mono- and mixed-culture Campylobacter jejuni biofilms. Appl Environ Microbiol 2012;78(4):1033–1038. DOI: 10.1128/AEM.07364-11.
  54. Cowan MM, Warren TM, Fletcher M. Mixed species colonization of solid surfaces in laboratory biofilms. Biofouling 1991;3(1):23–34. DOI: 10.1080/08927019109378159.
  55. Rao D, Webb JS, Kjelleberg S. Competitive interactions in mixedspecies biofilms containing the marine bacterium Pseudoalteromonas tunicata. Appl Environ Microbiol 2005;71(4):1729–1736. DOI: 10.1128/AEM.71.4.1729-1736.2005.
  56. Chang CC, Merritt K. Effect of Staphylococcus epidermidis on adherence of Pseudornonas aeruginosa and Proteus mirabilis to polymethyl methacrylate (PMMA) and gentamicin-containing PMMA. J Orth Res 1991;9(2):284–288. DOI: 10.1002/jor.1100090217.
  57. She P, Chen L, Qi Y, et al. Effects of human serum and apotransferrin on staphylococcus epidermidis RP62A biof ilm formation. Microbiologyopen 2016;5(6):957–966. DOI: 10.1002/mbo3.379.
  58. Roberts AEL, Kragh KN, Bjarnsholt T, et al. The limitations of in vitro experimentation in understanding biofilms and chronic infection. J Mol Biol 2015;427(23):3646–3661. DOI: 10.1016/j.jmb.2015.09.002.
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