Biofilm Growth on Nanostructured Block Copolymer Surfaces

Objectives: To determine the effect of block copolymer nanomorphology, known to affect adhesion and morphology of proteins, on the growth of 24h Streptococcus mutans biofilms.

Methods: Random, diblock and triblock copolymers of methyl methacrylate-2-hydroxyethyl methacrylate (MMA-HEMA) and MMA-acrylic acid (AA) in a 1:1 mole ratio were synthesized and cast on a silicon wafer.  The nanomorphology was verified by atomic force microscopy.  Streptococcus mutans (strain UA159) biofilms were grown microaerophilically on UV-sterilized specimens (batch culture, 0.5X Tryptone-yeast-extract medium with 10mM sucrose, 37°C, 100rpm). After 24h bacterial growth, specimens were stained (BacLightª LIVE/DEAD, Invitrogen) and imaged (Leica TCS SP2-MP; n=3 locations/specimen). Biofilm structural parameters calculated using ISA3D software were: biovolume (μm³), maximum biofilm thickness (μm), and biofilm roughness coefficient. Parameters were compared using General-Linear-Models and post hoc Student-Newman-Keuls tests (SNK; α=0.05) to determine differences in biofilm structures for live- (Syto-9 stain) and dead/damaged-cells (Propidium Iodide stain).

Results: Statistically significant differences existed among the MMA-HEMA and MMA-AA copolymer groups for each parameter (p<0.01), and between the live and dead/damaged cells (p<0.01).  MMA-AA copolymers in general had larger mean biovolumes of both living and dead/damaged cells than MMA-HEMA copolymers, but MMA-HEMA random copolymer had higher mean maximum biofilm thickness and biofilm roughness coefficient values of living and dead/damaged cells than the other copolymers.  In addition, significant differences were found in maximum biofilm thickness as a function of nanomorphology for the live cells (Table 1).

Table 1.  Mean (standard deviation) values; superscript letters indicate SNK groupings within columns

Conclusions:  MMA-HEMA copolymers were more effective at killing the 24h S. mutans biofilms than MMA-AA copolymers.  This preliminary study also indicates that block copolymer nanomorphology supports less biofilm growth.