Introduction: Infection due to bacterial adhesion and biofilm formation represetns a major drawback to the use of blood-contacting devices. Previously we have developed textured polyurethane biomaterials for reducing bacterial adhesion and inhibiting biofilm formation. In this work, we applied the texturing pattern to functionalized polymers bearing antimicrobial properties, and show a combination of antimicrobial functionalization and physical approaches can produce additive effects on biological responses and enhance the hemocompatibility of biomaterials.
Material and Methods: Carbosil polyurethane (PU, 2080A) and phosphazene polymers (trifluoroethoxy (TFE), crosslinakble trifluoroethoxy (XLTFE), and octafluoropentoxy (OFP)) were textured with ordered arrays of pillars using a soft lithography two-stage replication molding technique[1]. The Carbosil polyurethane material was doped with S-nitroso-N-acetylpenicillamine (SNAP) with different contents of SNAP in PU (5, 10, and 15 wt%) for nitric oxide (NO) release[2]. Antimicrobial properties of materials were evaluated by the optical density (OD600) of microbial cultures incubated with materials and strain S. epidermidis RP62A. Bacterial adhesion and biofilm formation on material surfaces were carried out in a multiwell plate or rotating disk system (RDS)[1] with same strain. Bacterial adhesion was counted under a fluorescent optical microscope. To observe biofilm formation, samples were in incubated in multiwell plates under static condition for 2 days or RDS system under shear for 8 days. Samples were stained with FTIC conjugated wheat germ agglutinin, and biofilm observed by fluorescence.
Results and Discussion: Characterization of biomaterial surfaces: AFM images showed material surfaces textured with ordered arrays of pillars, as expected, except for TFE and XLTFE materials that were difficult to texture.Water contact angle measurements show surface hydrophobicity increased after texturing due to entrapped air.
Antimicrobial properties of materials:The optical densities of culture media indicate the growth of bacteria in the presence of a variety of polymers (Fig. 1). Carbosil PU 0% SNAP and PUU films show the similar optical density as that of control (no polymers). Significant inhibition of bacterial growth was observed in the cultures for Carbosil 10% and 15% SNAP polymers, indicating NO release inhibits the growth of bacteria. A slight decrease of OD600 was observed for all phosphazene polymers.
Fig. 1 Optical density of culture media indicating antimicrobial properties of polymers.
Bacterial adhesion on functionalized textured polymer surfaces: Higher bacterial adhesion was observed on smooth surfaces without functionalization (Fig. 2). Surface texturing with pattern decreased bacterial adhesion on all surfaces, except for TFE and XLTFE polymers, indicating the importance of surface texturing in inhibiting adhesion. Furthermore, a significant decrease in adhesion was observed on textured PU surfaces containing 10% or 15% SNAP as well as on OFP textured surfaces, suggesting antimicrobial functionalization and texturing produce a synergistic effect on inhibiting bacterial adhesion, as the OFP polymer alone inhibited bacterial growth only slightly.
Fig. 2 Bacterial adhesion on polymer surface in PBS for 1 hr at 37°C under static condition.
Biofilm formation: Under static condition, biofilms were observed on Carbosil PU surfaces containing 0% SNAP and 5% SNAP after 2 days, while no biofilms were observed on surfaces containing 10% and 15% SNAP. Under shear stresses, no biofilms were observed on smooth or textured PU surfaces containing 15% SNAP after 8 days. Long term exposure experiments for biofilm formation on functionalized textured surfaces are on-going.
Fig. 3 Biofilm on textured 500/500 nm patterned surfaces containing (a) 0% SNAP, (b) 15% SNAP under static condition for 2 days, and (c) 15% SNAP under shear for 8 days.
Conclusion: A combination of antimicrobial functionalization of polymers and surface texturing provided an improved approach to inhibiting bacterial adhesion and biofilm formation, and thus to potentially preventing biomaterial related infections.
References:
[1] Xu, L.C., Siedlecki, C.A. Acta Biomaterialia. (2012) 8, 72-81
[2] Brisbois E.J., Handa H., Major T.C., Bartlett R.H., Meyerhoff M.E., Biomaterials (2013), 34, 6957-6966