Biofilm and cell adhesion strength on dental implant surfaces via the laser spallation technique.

OBJECTIVE: The aims of this study are to quantify the adhesion strength differential between an oral bacterial biofilm and an osteoblast-like cell monolayer to a dental implant-simulant surface and develop a metric that quantifies the biocompatible effect of implant surfaces on bacterial and cell adhesion. METHODS: High-amplitude short-duration stress waves generated by laser pulse absorption are used to spall bacteria and cells from titanium substrates. By carefully controlling laser fluence and calibration of laser fluence with applied stress, the adhesion difference between Streptococcus mutans biofilms and MG 63 osteoblast-like cell monolayers on smooth and rough titanium substrates is obtained. The ratio of cell adhesion strength to biofilm adhesion strength (i.e., Adhesion Index) is determined as a nondimensionalized parameter for biocompatibility assessment. RESULTS: Adhesion strength of 143 MPa, with a 95% C.I. (114, 176), is measured for MG 63 cells on smooth titanium and 292 MPa, with a 95% C.I. (267, 306), on roughened titanium. Adhesion strength for S. mutans on smooth titanium is 320 MPa, with a 95% C.I. (304, 333), and remained relatively constant at 332 MPa, with a 95% C.I. (324, 343), on roughened titanium. The calculated Adhesion Index for smooth titanium is 0.451, with a 95% C.I. (0.267, 0.622), which increased to 0.876, with a 95% C.I. (0.780, 0.932), on roughened titanium. SIGNIFICANCE: The laser spallation technique provides a platform to examine the tradeoffs of adhesion modulators on both biofilm and cell adhesion. This tradeoff is characterized by the Adhesion Index, which is proposed to aid biocompatibility screening and could help improve implantation outcomes. The Adhesion Index is implemented to determine surface factors that promote favorable adhesion of cells greater than biofilms. Here, an Adhesion Index ≫ 1 suggests favorable biocompatibility.

Adhesion of biofilms on titanium measured by laser-induced spallation.

Eradication of established implant-associated and bacterial biofilm-forming infections remains difficult in part because these biofilms remain well-adhered to the implant surface. Few experimental techniques are available to measure macro-scale strength of bacterial biofilm-implant adhesion. We have adapted the laser spallation technique to compare the macro-scale adhesion strength of biofilms formed on titanium. By using a rapid pressure wave (35 ns) to load the interface, we prevent disturbance of the biofilm surface prior to measurement, and preclude the time necessary for the biofilm to respond to and adapt under loading. Biofilms of Streptococcus mutans, a Gram-positive bacterium associated with human dental caries (cavities) were cultured directly on commercially pure titanium within our custom substrate assembly. Growth conditions were varied by adding sucrose to the Todd Hewitt Yeast (THY) broth: THY control, 37.5 mM, 75 mM, 375 mM, and 750 mM sucrose. Multiple locations on each biofilm were loaded using the laser spallation technique. Loading pressure wave amplitude was controlled by adjusting laser fluence, energy per area. Initially, addition of sucrose to the media increased biofilm adhesion to titanium. However, once a saturation concentration of 75 mM sucrose was reached, increasing the sucrose concentration further resulted in a decrease in biofilm adhesion. This study is the first demonstration of the adaptation of the laser spallation technique to measure bacterial biofilm adhesion. Establishment of this macro-scale biofilm adhesion measurement technique opens the door for many biofilm-surface adhesion studies. We anticipate further work in this area towards understanding the complex relationships among bacteria species, environmental factors, surface characteristics, and biofilm adhesion strength.