Biofilm-forming organisms continue to be a central concern in orthopaedic procedures which involve the placement of metallic hardware in conjunction with PMMA bone cement. We present initial work to evaluate the usefulness of vancomycin-PEG-acrylate and vancomycin-acrylamide species for the inhibition of S. epidermidis biofilm formation on both Ti-6Al-4V orthopaedic alloy and on PMMA bone cement.. We evaluated: (1) whether VPA(3400) polymerized to Ti-6Al-4V alloy reduces bacterial attachment; (2) the antibiofilm effects (reduced biofilm mass) of VPA(3400) and two vancomycin-acrylamide derivatives when individually copolymerized with PMMA bone cement; and (3) the compressive mechanical effects of adding polymerizable antibiotics to PMMA bone cement.
We acknowledge limitations to our experiment. First is that the biofilm growth conditions presented may not reflect those encountered in vivo. A biofilm bioreactor might offer a more sophisticated approach to simulating growth conditions [3, 10, 34]. However, it is inherently difficult to define the complex milieu of an infection site, and the assays used here were sufficient to define differences between material types. Second, the traditional method of quantifying adherent biofilm is by measurement of viability by prior desorption (ultrasonication) and subsequent agar plating , though there is often considerable variability in the absolute bacterial count obtained by different methods . Quantification by SEM is often limited  secondary to problems of bacterial enumeration arising from bacterial agglomerates and matrix-embedded bacteria [25, 55, 60] and from the possibility that some of the biofilm structure will be lost during preparation [23, 63]. For this reason, our SEM quantification methods were limited to measurement of initial bacterial attachment where extensive glycocalyx was not present and where relative bacterial surface density was low. We expect the reported bacterial surface densities (Fig. 5) to be technique dependent as with other methods . Dry mass has also been used to quantify biofilm proliferation [51, 59]; however, the gravimetric assay used here to evaluate adherent biofilm mass on PMMA surfaces does not provide adequate precision for discriminating between the effects of VPA(3400) and PEG(3000)-acrylate. Third, although we demonstrated various statistically significant effects attributable to polymerizable antibiotic species, whether these effects will have clinical importance is a question for future work.
In experiments with polymer-coated Ti alloy, VPA(3400) appears to add an additional level of protection to PEG-type coatings (Fig. 5). Surface-contact killing may contribute to the decreased number of adherent bacteria during the initial stages of biomaterial colonization. It is likely this period is critical in true physiological infections in which the bacterial load in the surrounding fluids is generally expected to be low. In comparison, a recently described antimicrobial technique involves the covalent attachment of an antibiotic (presumably a monolayer) to titanium [5-7, 21, 32, 47]. Building on surface modification methods described by Nanci et al. , the authors were able to attach vancomycin to titanium via two aminoethoxyethoxyacetate linkers. One study suggested Staphylococcus aureus attachment may be blocked using this surface-modification platform . However, because aminoethoxyethoxyacetate linkers (sold commercially as “Mini-PEG”) effectively put a thin PEG layer on a surface (four ethylene glycol units), some of the observed antibiofilm properties may be related to the linker chemistry. Another study reported as few as four ethylene glycol repeats can provide antibiofouling properties for 3 weeks or longer with some cell types .
The data show incorporation of PEG moieties into the PMMA architecture retards but ultimately does not prevent biofilm adherence. This effect may be related to blockage of initial bacterial attachment, as suggested in other studies with PMMA [16, 53], but may be overcome as the surface is saturated with bacterial glycocalyx and cellular debris. Neither of the vancomycin-acrylamide derivatives showed an antibiofilm effect. This behavior is consistent with previous observations  suggesting the PEG linker is critical for antibacterial activity once vancomycin derivatives are polymerized from solid substrates. In an alternative approach, a quaternary amine dimethacrylate (QADMA) species was copolymerized with PMMA bone cement, and antibiofilm properties against Escherichia coli were examined [16, 53], but no quantification of adherent organisms was undertaken. Data suggested QADMA blocked the attachment of Escherichia coli; whether this was related to the killing of microorganisms is less clear.
Data with coated Ti alloy surfaces suggest copolymerizing a PEGylated vancomycin species, VPA(3400), with PEG(375)-acrylate is more effective than PEG alone at blocking S. epidermidis biofilms. The PEG spacer itself likely contributes to the antibiofilm effect, but SEM data indicate the pendant vancomycin molecule improves the antimicrobial effect under some growth conditions. Loading PMMA bone cement with certain polymerizable vancomycin derivatives may eventually be useful for retarding biofilm adherence without compromising mechanical properties, although the current formulations did one or the other, but not both. A vancomycin-acrylamide additive resulted in compressive mechanical properties almost identical to PMMA controls but did not inhibit biofilm growth. VPA(3400) reduced biofilm growth but compromised mechanical properties. Experiments described here should facilitate the development of new antibiofilm biomaterial surfaces.
We thank J. McCormick for assistance with XPS surface characterization.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
1. Adams, K., Couch, L., Cierny, G., Calhoun, J. and Mader, JT. In vitro and in vivo evaluation of antibiotic diffusion from antibiotic-impregnated polymethylmethacrylate beads. Clin Orthop Relat Res.
1992; 278: 244-252.
2. Alcantar, NA., Aydil, ES. and Israelachvili, JN. Polyethylene glycol-coated biocompatible surfaces. J Biomed Mater Res.
2000; 51: 343-351. 10.1002/1097-4636(20000905)51:3<343::AID-JBM7>3.0.CO;2-D
3. An, UH., Mcglohorn, JB., Bednarski, BK., Martin, KL. and Friedman, RJ. An Open Channel Flow Chamber for Characterizing Biofilm Formation on Biomaterial Surfaces. Methods Enzymol.
2001; 337: 79-88. 10.1016/S0076-6879(01)37008-8
4. Anguita-Alonso, P., Rouse, MS., Piper, KE., Jacofsky, DJ., Osmon, DR. and Patel, R. Comparative study of antimicrobial release kinetics from polymethylmethacrylate. Clin Orthop Relat Res.
2006; 445: 239-244.
5. Antoci, V Jr. Adams, CS., Hickok, NJ., Shapiro, IM. and Parvizi, J. Vancomycin bound to Ti rods reduces periprosthetic infection: preliminary study. Clin Orthop Relat Res.
2007; 461: 88-95.
6. Antoci, V Jr. Adams, CS., Parvizi, J., Ducheyne, P., Shapiro, IM. and Hickok, NJ. Covalently attached vancomycin provides a nanoscale antibacterial surface. Clin Orthop Relat Res.
2007; 461: 81-87.
7. Antoci, V Jr. King, SB., Jose, B., Parvizi, J., Zeiger, AR., Wickstrom, E., Freeman, TA., Composto, RJ., Ducheyne, P., Shapiro, IM., Hickok, NJ. and Adams, CS. Vancomycin covalently bonded to titanium alloy prevents bacterial colonization. J Orthop Res.
2007; 25: 858-866. 10.1002/jor.20348
8. Antonios, V., Berbari, E. and Osmon, D. In: Pace, JL., Rupp, ME. and Finch, RG. (eds.), Treatment protocol of infections of orthopedic devices. Biofilms, Infection, and Antimicrobial Therapy.
2006: Boca Raton, FL: CRC Press; 449-478.
9. Askew, MJ., Kufel, MF., Fleissner, PR Jr. Gradisar, IA Jr. Salstrom, SJ. and Tan, JS. Effect of vacuum mixing on the mechanical properties of antibiotic-impregnated polymethylmethacrylate bone cement. J Biomed Mater Res.
1990; 24: 573-580. 10.1002/jbm.820240504
10. Bott, T. and Grant, DM. Biofilms in Flowing Systems. Methods Enzymol.
2001; 337: 88-103. 10.1016/S0076-6879(01)37009-X
11. Boulmedais, F., Frisch, B., Etienne, O., Lavalle, P., Picart, C., Ogier, J., Voegel, JC., Schaaf, P. and Egles, C. Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials. Biomaterials.
2004; 25: 2003-2011. 10.1016/j.biomaterials.2003.08.039
12. Bourne, RB. Prophylactic use of antibiotic bone cement: an emerging standard-in the affirmative. J Arthroplasty.
2004; 19: (4 Suppl 1):69-72. 10.1016/j.arth.2004.03.005
13. Brien, WW., Salvati, EA., Klein, R., Brause, B. and Stern, S. Antibiotic impregnated bone cement in total hip arthroplasty. An in vivo comparison of the elution properties of tobramycin and vancomycin. Clin Orthop Relat Res.
1993; 296: 242-248.
14. Costerton, JW., Stewart, PS. and Greenberg, EP. Bacterial biofilms: a common cause of persistent infections. Science.
1999; 284: 1318-1322. 10.1126/science.284.5418.1318
15. Dalsin, JL., Lin, L., Tosatti, S., Voros, J., Textor, M. and Messersmith, PB. Protein resistance of titanium oxide surfaces modified by biologically inspired mPEG-DOPA. Langmuir.
2005; 21: 640-646. 10.1021/la048626g
16. Deb, S., Doiron, R., DiSilvio, L., Punyani, S. and Singh, H. PMMA bone cement containing a quaternary amine comonomer with potential antibacterial properties. J Biomed Mater Res B Appl Biomater.
2008; 85: 130-139.
17. DiMaio, FR., O'Halloran, JJ. and Quale, JM. In vitro elution of ciprofloxacin from polymethylmethacrylate cement beads. J Orthop Res.
1994; 12: 79-82. 10.1002/jor.1100120110
18. Donati, D. and Biscaglia, R. The use of antibiotic-impregnated cement in infected reconstructions after resection for bone tumours. J Bone Joint Surg Br.
1998; 80: 1045-1050. 10.1302/0301-620X.80B6.8570
19. Dong, B., Jiang, H., Manolache, S., Wong, AC. and Denes, FS. Plasma-mediated grafting of poly(ethylene glycol) on polyamide and polyester surfaces and evaluation of antifouling ability of modified substrates. Langmuir.
2007; 23: 7306-7313. 10.1021/la0633280
20. Dunne, WM Jr. Bacterial adhesion: seen any good biofilms lately? Clin Microbiol Rev.
2002; 15: 155-166. 10.1128/CMR.15.2.155-166.2002
21. Edupuganti, OP., Antoci, V Jr. King, SB., Jose, B., Adams, CS., Parvizi, J., Shapiro, IM., Zeiger, AR., Hickok, NJ. and Wickstrom, E. Covalent bonding of vancomycin to Ti6Al4V alloy pins provides long-term inhibition of Staphylococcus aureus colonization. Bioorg Med Chem Lett.
2007; 17: 2692-2696. 10.1016/j.bmcl.2007.03.005
22. Fan, X., Lin, L. and Messersmith, PB. Cell fouling resistance of polymer brushes grafted from Ti substrates by surface-initiated polymerization: effect of ethylene glycol side chain length. Biomacromolecules.
2006; 7: 2443-2448. 10.1021/bm060276k
23. Fassel, TA. and Edmiston, CE. Bacterial biofilms: strategies for preparing glycocalyx for electron microscopy. Methods Enzymol.
1999; 310: 194-203. 10.1016/S0076-6879(99)10017-X
24. Gerhart, TN., Roux, RD., Hanff, PA., Horowitz, GL., Renshaw, AA. and Hayes, WC. Antibiotic-loaded biodegradable bone cement for prophylaxis and treatment of experimental osteomyelitis in rats. J Orthop Res.
1993; 11: 250-255. 10.1002/jor.1100110212
25. Hannig, C., Follo, M., Hellwig, E. and Al-Ahmad, A. Visualization of adherent micro-organisms using different techniques. J Med Microbiol.
2010; 59: 1-7. 10.1099/jmm.0.015420-0
26. Hanssen, AD. Prophylactic use of antibiotic bone cement: an emerging standard-in opposition. J Arthroplasty.
2004; 19: (4 Suppl 1):73-77. 10.1016/j.arth.2004.04.006
27. Hanssen, AD., Rand, JA. and Osmon, DR. Treatment of the infected total knee arthroplasty with insertion of another prosthesis. The effect of antibiotic-impregnated bone cement. Clin Orthop Relat Res.
1994; 309: 44-55.
28. Harris, LG., Tosatti, S., Wieland, M., Textor, M. and Richards, RG. Staphylococcus aureus adhesion to titanium oxide surfaces coated with non-functionalized and peptide-functionalized poly(L-lysine)-grafted-poly(ethylene glycol) copolymers. Biomaterials.
2004; 25: 4135-4148. 10.1016/j.biomaterials.2003.11.033
29. Heck, D., Rosenberg, A., Schink-Ascani, M., Garbus, S. and Kiewitt, T. Use of antibiotic-impregnated cement during hip and knee arthroplasty in the United States. J Arthroplasty.
1995; 10: 470-475. 10.1016/S0883-5403(05)80148-2
30. Hoff, SF., Fitzgerald, RH Jr. and Kelly, PJ. The depot administration of penicillin G and gentamicin in acrylic bone cement. J Bone Joint Surg Am.
1981; 63: 798-804.
31. Hutchison, JB., Haraldsson, KT., Good, BT., Sebra, RP., Luo, N., Anseth, KS. and Bowman, CN. Robust polymer microfluidic device fabrication via contact liquid photolithographic polymerization (CLiPP). Lab Chip.
2004; 4: 658-662. 10.1039/b405985a
32. Jose, B., Antoci, V Jr. Zeiger, AR., Wickstrom, E. and Hickok, NJ. Vancomycin covalently bonded to titanium beads kills Staphylococcus aureus. Chem Biol.
2005; 12: 1041-1048. 10.1016/j.chembiol.2005.06.013
33. Kendall, RW., Duncan, CP. and Beauchamp, CP. Bacterial growth on antibiotic-loaded acrylic cement. A prospective in vivo retrieval study. J Arthroplasty.
1995; 10: 817-822. 10.1016/S0883-5403(05)80081-6
34. Kharazmi, A., Giwercman, B. and Hoiby, N. Robbins Device in Biofilm Research. Methods Enzymol.
1999; 310: 207-215. 10.1016/S0076-6879(99)10018-1
35. Klekamp, J., Dawson, JM., Haas, DW., DeBoer, D. and Christie, M. The use of vancomycin and tobramycin in acrylic bone cement: biomechanical effects and elution kinetics for use in joint arthroplasty. J Arthroplasty.
1999; 14: 339-346. 10.1016/S0883-5403(99)90061-X
36. Ko, YG., Kim, YH., Park, KD., Lee, HJ., Lee, WK., Park, HD., Kim, SH., Lee, GS. and Ahn, DJ. Immobilization of poly(ethylene glycol) or its sulfonate onto polymer surfaces by ozone oxidation. Biomaterials.
2001; 22: 2115-2123. 10.1016/S0142-9612(00)00400-2
37. Kuechle, DK., Landon, GC., Musher, DM. and Noble, PC. Elution of vancomycin, daptomycin, and amikacin from acrylic bone cement. Clin Orthop Relat Res.
1991; 264: 302-308.
38. Lawson, MC., Bowman, CN. and Anseth, KS. Vancomycin derivative photopolymerized to titanium kills S. epidermidis. Clin Orthop Relat Res.
2007; 461: 96-105.
39. Lawson, MC., Hoth, KB., Shoemaker, R., Bowman, CN. and Anseth, KS. Polymerizable vancomycin derivatives or bactericidal biomaterial surface modification: structure-function evaluation. Biomacromolecules.
2009; 10: 2221-2234. 10.1021/bm900410a
40. Maddikeri, RR., Tosatti, S., Schuler, M., Chessari, S., Textor, M., Richards, RG. and Harris, LG. Reduced medical infection related bacterial strains adhesion on bioactive RGD modified titanium surfaces: a first step toward cell selective surfaces. J Biomed Mater Res A.
2008; 84: 425-435.
41. Masri, BA., Duncan, CP. and Beauchamp, CP. Long-term elution of antibiotics from bone-cement: an in vivo study using the prosthesis of antibiotic-loaded acrylic cement (PROSTALAC) system. J Arthroplasty.
1998; 13: 331-338. 10.1016/S0883-5403(98)90179-6
42. Masri, BA., Duncan, CP., Beauchamp, CP., Paris, NJ. and Arntorp, J. Effect of varying surface patterns on antibiotic elution from antibiotic-loaded bone cement. J Arthroplasty.
1995; 10: 453-459. 10.1016/S0883-5403(05)80145-7
43. McLaren, AC., Nelson, CL., McLaren, SG. and DeCLerk, GR. The effect of glycine filler on the elution rate of gentamicin from acrylic bone cement: a pilot study. Clin Orthop Relat Res.
2004; 427: 25-27. 10.1097/01.blo.0000143556.41472.2a
44. Nanci, A., Wuest, JD., Peru, L., Brunet, P., Sharma, V., Zalzal, S. and McKee, MD. Chemical modification of titanium surfaces for covalent attachment of biological molecules. J Biomed Mater Res.
1998; 40: 324-335. 10.1002/(SICI)1097-4636(199805)40:2<324::AID-JBM18>3.0.CO;2-L
45. Nelson, CL., McLaren, AC., McLaren, SG., Johnson, JW. and Smeltzer, MS. Is aseptic loosening truly aseptic. Clin Orthop Relat Res.
2005; 437: 25-30. 10.1097/01.blo.0000175715.68624.3d
46. Neut, D., Belt, H., Horn, JR., Mei, HC. and Busscher, HJ. The effect of mixing on gentamicin release from polymethylmethacrylate bone cements. Acta Orthop Scand.
2003; 74: 670-676. 10.1080/00016470310018180
47. Parvizi, J., Wickstrom, E., Zeiger, AR., Adams, CS., Shapiro, IM., Purtill, JJ., Sharkey, PF., Hozack, WJ., Rothman, RH. and Hickok, NJ. Frank Stinchfield award. Titanium surface with biologic activity against infection. Clin Orthop Relat Res.
2004; 429: 33-38. 10.1097/01.blo.0000150116.65231.45
48. Peeters, E., Nells, HJ. and Coenye, T. Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods.
2008; 72: 157-165. 10.1016/j.mimet.2007.11.010
49. Penner, MJ., Duncan, CP. and Masri, BA. The in vitro elution characteristics of antibiotic-loaded CMW and Palacos-R bone cements. J Arthroplasty.
1999; 14: 209-214. 10.1016/S0883-5403(99)90128-6
50. Penner, MJ., Masri, BA. and Duncan, CP. Elution characteristics of vancomycin and tobramycin combined in acrylic bone-cement. J Arthroplasty.
1996; 11: 939-944. 10.1016/S0883-5403(96)80135-5
51. Percival, S. and Walker, JT. Methods used to assess biofouling of material used in distribution and domestic water systems. Methods Enzymol.
2001; 337: 187-200. 10.1016/S0076-6879(01)37014-3
52. Popat, KC., Mor, G., Grimes, CA. and Desai, TA. Surface modification of nanoporous alumina surfaces with poly(ethylene glycol). Langmuir.
2004; 20: 8035-8041. 10.1021/la049075x
53. Punyani, S., Deb, S. and Singh, H. Contact killing antimicrobial acrylic bone cements: preparation and characterization. J Biomat Sci-Polym E.
2007; 18: 131-145. 10.1163/156856207779116748
54. Ruiz-Taylor, LA., Martin, TL., Zaugg, FG., Witte, K., Indermuhle, P., Nock, S. and Wagner, P. Monolayers of derivatized poly(L-lysine)-grafted poly(ethylene glycol) on metal oxides as a class of biomolecular interfaces. Proc Natl Acad Sci USA.
2001; 98: 852-857. 10.1073/pnas.98.3.852
55. Schaudinn, C., Carr, G., Gorur, A., Jaramillo, D., Costerton, JW. and Webster, P. Imaging of endodontic biofilms by combined microscopy (FISH/cLSM-SEM). J Microscopy
2009; 235: (Pt. 2):124-127. 10.1111/j.1365-2818.2009.03201.x
56. Sebra, RP., Masters, KS., Bowman, CN. and Anseth, KS. Surface grafted antibodies: controlled architecture permits enhanced antigen detection. Langmuir.
2005; 21: 10907-10911. 10.1021/la052101m
57. Sebra, RP., Masters, KS., Cheung, CY., Bowman, CN. and Anseth, KS. Detection of antigens in biologically complex fluids with photografted whole antibodies. Anal Chem.
2006; 78: 3144-3151. 10.1021/ac052246y
58. Sebra, RP., Reddy, SK., Masters, KS., Bowman, CN. and Anseth, KS. Controlled polymerization chemistry to graft architectures that influence cell-material interactions. Acta Biomater.
2007; 3: 151-161. 10.1016/j.actbio.2006.07.010
59. Staudt, C., Horn, H., Hempel, DC. and Neu, TR. Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms. Biotechnol Bioeng.
2004; 88: 585-592. 10.1002/bit.20241
60. Surman, SB., Walker, JT., Goddard, DT., Morton, LHG., Keevil, CW., Weaver, W., Skinner, A., Hanson, K., Caldwell, D. and Kurtz, J. Comparison of microscope techniques for the examination of biofilms. J Microbiol Methods.
1996; 25: 57-70. 10.1016/0167-7012(95)00085-2
61. Belt, H., Neut, D., Schenk, W., Horn, JR., Mei, HC. and Busscher, HJ. Staphylococcus aureus biofilm formation on different gentamicin-loaded polymethylmethacrylate bone cements. Biomaterials.
2001; 22: 1607-1611. 10.1016/S0142-9612(00)00313-6
62. Wagner, VE., Koberstein, JT. and Bryers, JD. Protein and bacterial fouling characteristics of peptide and antibody decorated surfaces of PEG-poly(acrylic acid) co-polymers. Biomaterials.
2004; 25: 2247-2263. 10.1016/j.biomaterials.2003.09.020
63. Walker, JT., Verran, J., Boyd, RD. and Percival, S. Microscopy methods to investigate structure of potable water biofilms. Methods Enzymol.
2001; 337: 243-255. 10.1016/S0076-6879(01)37018-0
64. Webster, R., Didier, E., Harris, P., Siegel, N., Stadler, J., Tilbury, L. and Smith, D. PEGylated proteins: evaluation of their safety in the absence of definitive metabolism studies. Drug Metab Dispos.
2007; 35: 9-16. 10.1124/dmd.106.012419
65. Winblade, ND., Schmokel, H., Baumann, M., Hoffman, AS. and Hubbell, JA. Sterically blocking adhesion of cells to biological surfaces with a surface-active copolymer containing poly(ethylene glycol) and phenylboronic acid. J Biomed Mater Res.
2002; 59: 618-631. 10.1002/jbm.1273
66. Yaniv, M., Dabbi, D., Amir, H., Cohen, S., Mozes, M., Tsuberi, H., Frietkin, M., Dekel, S. and Ofek, I. Prolonged leaching time of peptide antibiotics from acrylic bone cement. Clin Orthop Relat Res.
1999; 363: 232-239. 10.1097/00003086-199906000-00030