Inhibition of Staphylococcus epidermidis biofilms using polymerizable vancomycin derivatives

Abstract

Background: Biofilm formation on indwelling medical devices is a ubiquitous problem causing considerable patient morbidity and mortality. In orthopaedic surgery, this problem is exacerbated by the large number and variety of material types that are implanted. Metallic hardware in conjunction with polymethylmethacrylate (PMMA) bone cement is commonly used.

Questions/purposes: We asked whether polymerizable derivatives of vancomycin might be useful to (1) surface modify Ti-6Al-4V alloy and to surface/bulk modify PMMA bone cement to prevent Staphylococcus epidermidis biofilm formation and (2) whether the process altered the compressive modulus, yield strength, resilience, and/or fracture strength of cement copolymers.

Methods: A Ti-6Al-4V alloy was silanized with methacryloxypropyltrimethoxysilane in preparation for subsequent polymer attachment. Surfaces were then coated with polymers formed from PEG(375)-acrylate or a vancomycin-PEG(3400)-PEG(375)-acrylate copolymer. PMMA was loaded with various species, including vancomycin and several polymerizable vancomycin derivatives. To assess antibiofilm properties of these materials, initial bacterial adherence to coated Ti-6Al-4V was determined by scanning electron microscopy (SEM). Biofilm dry mass was determined on PMMA coupons; the compressive mechanical properties were also determined.

Results: SEM showed the vancomycin-PEG(3400)-acrylate-type surface reduced adherent bacteria numbers by approximately fourfold when compared with PEG(375)-acrylate alone. Vancomycin-loading reduced all mechanical properties tested; in contrast, loading a vancomycin-acrylamide derivative restored these deficits but demonstrated no antibiofilm properties. A polymerizable, PEGylated vancomycin derivative reduced biofilm attachment but resulted in inferior cement mechanical properties.

Clinical relevance: The approaches presented here may offer new strategies for developing biofilm-resistant orthopaedic materials. Specifically, polymerizable derivatives of traditional antibiotics may allow for direct polymerization into existing materials such as PMMA bone cement while minimizing mechanical property compromise. Questions remain regarding ideal monomer structure(s) that confer biologic and mechanical benefits.

Fig. 1

The flow chart illustrates a basic research design having three separate arms of inquiry following chemical synthesis of polymerizable vancomycin derivatives. The number of samples of each material type examined are shown by “n”.

Fig. 2

Vancomycin was modified at the primary amines illustrated. Both PEG-acrylate and acrylamide polymerizable derivatives of vancomycin were synthesized. The PEG-acrylate derivative had a 3400 g/mol PEG spacer. For VA-1 and VA-2, the numeral refers to the site of modification, primary or secondary amine, respectively. R₁ is classically referred to as the vancomycin V₃ position, and R₂ is termed the X₁ position.

Fig. 3A–D

A schematic of the Ti surface modification procedure is shown. (A) The native oxide surface is represented. (B) The native oxide surface is thickened and the number of surface hydroxyl groups are presumably increased. (C) Surface hydroxyls are reacted with methacryloxypropyltrimethoxysilane in anhydrous toluene. This reaction effectively places a layer of methacrylate groups on the surface that can participate in free radical polymerization and anchor polymer chains to the surface. (D) Covering the methacrylated surface with a monomer solution containing a polymerizable antibiotic (VPA[3400] in this case), another polymerizable species (PEG[375]-acrylate), and a photoinitiator followed by exposure to ultraviolet radiation results in polymerization. A fraction of the polymer chains are covalently attached to the surface, albeit in a geometrically more complex structure than that shown here. In this reaction scheme, a polyacrylate backbone is formed with pendant VPA(3400) and PEG(375). Here, assuming ideal polymerization, n = 9 and n = 80.

Fig. 4A–E

Scanning electron microscopy (×1000) provides a qualitative means of visualizing changes in Ti-6Al-4V surface structure (A) before and (B) after oxidation with H₂SO₄:H₂O₂ solution. This oxidation procedure serves to increase the number of surface hydroxyl groups available for subsequent functionalization with a silane reagent. XPS allows for elemental characterization of Ti-6Al-4V surfaces having undergone modification as described in Fig. 3. The number of electrons ejected per second is plotted against electron binding energy. (C) The native oxide is shown. Oxygen and titanium are the primary elements present, but some level of N, C, Na, and Cl contamination exists as well. Residual Ar is present from the XPS surface preparation procedure. (D) The oxidation process increased the oxide content of the surface and possibly changed the structure of the Ti 2p peak. These findings are consistent with oxide replacement of Ti-6Al-4V in the surface layer. (E) Silanization leads to a further attenuated Ti 2p peak, an increased C 1 s peak, and the appearance of Si 2 s and Si 2p peaks. This collection of observations is consistent with the formation of a siloxane layer over the Ti-6Al-4V oxidized surface.

Fig. 5A–E

(A) Three Ti alloy discs exposed to biofilm-forming Staphylococcus epidermidis culture for 66 hrs are shown. The composition of the surface coatings (top surface only) are illustrated (arrows). (B) The same three discs are shown from a second perspective. Bare oxide surface is completely covered with biofilm. The coated portion of the PEG(375)-acrylate disc is visually clean. The coated portion of the VPA(3400)-PEG(375)-acrylate disc is visually clean as well. These figures clearly demonstrate the antibiofilm properties of PEG-type coatings. (C) A scanning electron microscopy (SEM) image of S. epidermidis biofilm at ×5000 magnification is shown (66 hours in culture). Cocci are apparent as is glycocalyx. (D) An SEM image at ×750 magnification shows attachment of S. epidermidis on PEG(375)-acrylate-coated Ti alloy (24 hours in culture). Images of this sort (without obscuring glycocalyx) were used to quantify attached organisms on both PEG and VPA(3400) surfaces. The white spots are bacteria. (E) Both surfaces reduced the number of attached organisms. However, data suggest that having VPA(3400) in the copolymer reduces bacterial attachment more effectively than PEG(375)-acrylate alone.

Fig. 6A–B

(A) Staphylococcus epidermidis biofilms readily proliferate on PMMA bone cement and vancomycin-loaded PMMA bone cement. Even high-dose vancomycin loading does not prevent biofilm formation after initial burst release, although it tends to increase experimental variability. This outcome may be related to inconsistent elution of small amounts of entrapped antibiotic. (B) Various species were added to PMMA bone cement to evaluate antibiofilm activity. Eighty-eight-hour time points are shown. Neither of the vancomycin-acrylamides were effective. Both VPA(3400) and PEG(3000)-acrylate reduced adherent organisms with respect to PMMA. These observations suggest ethylene glycol functionalities are largely responsible for the observed antibiofilm properties in this assay.

Fig. 7A–B

(A) Representative stress-strain curves are shown for PMMA bone cement with various additives (compressive tests). Loading vancomycin-acrylamide-2 (VA-2) at 10 wt% results in properties similar to those of PMMA, suggesting that polymerizable antibiotic derivatives may avoid some of the problems classically encountered with high-dose vancomycin loading. However, adding VPA(3400) at 10 wt% severely compromises mechanical properties and may lead to microcracking. This behavior is likely the result of the bulky PEG linker or its hydrophilicity. It is expected that an optimum number of ethylene glycol repeats would result in properties between those of VA-2 and VPA(3400) while at the same time offer antibiofilm properties. (B) Key mechanical properties are illustrated.