Engineering Mechanisms Causing Adverse Biofilm Responses to 3D Printed Titanium Surfaces

Biofilm accumulation in the oral cavity is in constant flux and often opposes mucosal surface cells in an allegory of war (Ebersole 2017). Bacterial biofilms compete for strong surface attachment on surfaces including implants, which can lead to diseases such as peri-implantitis. Peri-implantitis occurs in approximately one third of patients receiving dental implants (Elemek 2020). Treatments such as regular debridement and/or episodic antibiotic therapy, are required to avoid bone loss around the implant and potential bone-implant failure. Also, there are challenges of oral microbes developing resistance against antibiotics in part because the antibiotic has diffused through only part of the microbial biofilm causing the remaining biofilm to adapt. With issues such as implant infection, antimicrobial resistance and integration failure, it is critical for dental professionals and engineers to work together to continuously improve biomaterials used for dental implants to minimize peri-implantitis. Many dental materials are customized to each patient, with the exception of dental implants. The emergence of additively manufactured (AM) biocompatible metals (e.g., pure Ti, Ti-6Al-4V alloy), however, has made this possible. AM parts are built layer by layer as opposed to subtractive methods as is the case for traditional machining. This process controls porosity of the built material, enables density gradient designs, and improves freedom of design. Porosity control is particularly significant for implant materials to match the mechanical properties of bone, which contains 45-60% minerals, 20-30% matrix, and 10-20% water. The porous nature of bone can be matched using Laser Powder Bed Fusion (LPBF), one of the most common AM methods for printing metal parts. Porosity control and freedom of design in AM titanium offers an opportunity to improve biocompatibility. Because bacterial biofilms are competing in this environment, attention is needed to understand how biofilm properties (e.g., adhesion, diffusivity) are affected by the surfaces produced by the AM process. Our overall goal is to advance experimental mechanics-based techniques for measuring biofilm response, offering insights into adverse biofilm structure and adhesion on 3D printed metal implants. Adverse biofilm structures are those biofilm configurations that resist deformation and have large adhesion strength (difficult removal opposing debridement) and resist small particle diffusion (difficult to kill by diffusing antimicrobials). In addition to biofilm response, a numerical factor that makes the interpretation of the measurements easy to implement is needed. For example, if biofilm adhesion is decreased but host cell adhesion is also decreased then the surface conditioning is not a viable solution. Our Adhesion Index is a non-dimensional parameter that indicates when favorable adhesion is taking place, i.e., when cell adhesion is much greater than biofilm adhesion. In addition to providing insight into biofilm response, this work will also improve biocompatibility screening. The PI acquired significant preliminary data exhibiting the ability of the laser-induced spallation technique to measure adhesion of bacterial biofilms on modified and unmodified titanium through NIDCR R03 funding. Here we will adapt the technique to measure biofilm adhesion on 3D printed metal additive surfaces to gain important biofilm response information to enable fabrication of customized implants. Aim 1: Determine the structural and deformation properties of single and multispecies oral biofilms on relevant and futuristic titanium implant materials. We will enumerate and determine structural characteristics (i.e., 3D morphology and stratification) of single species biofilms and two multispecies biofilms: healthy model of S. gordonii, S. oralis, and S. sanguinis and disease model of S. gordonii, P. gingivalis, and F. nucleatum on titanium. Titanium substrates used throughout all aims are: Additively manufactured (AM) commercially pure Ti, AM Ti-6Al-4V, and traditionally machined Ti and Ti-6Al-4V. We will perform confocal laser scanning microscopy on biofilms formed on all titanium surfaces. We will measure viscoelastic properties of biofilms formed on titanium and test the null hypothesis that the deformation mechanics of single species biofilms are equal across healthy and disease model biofilms independent of titanium surface. Aim 2: Quantify spatial and diffusive properties of single and multispecies oral biofilms. We will test the null hypothesis that diffusion coefficients of nanoparticles within single species biofilms are equal to both healthy and disease model biofilms. We will develop a protocol capable of single particle tracking within biofilms formed on titanium surfaces and obtain mesh size measurements. We will test the null hypothesis that the mesh size of healthy biofilms is equal to disease biofilms and invariant to proximity to titanium substrate. We anticipate tools developed to describe and predict diffusion, especially when diffusion is confined, in reduced flow conditions, would significantly aid pharmaceutical industries designing nanoparticles for the treatment of oral biofilms. Aim 3: Measure the Adhesion Index of additively manufactured titanium using our laser-induced spallation technique. We will test the null hypothesis that the adhesion of single species biofilms is equal to the adhesion of both healthy multispecies biofilms and disease multispecies biofilms. We will also test the null hypothesis that the adhesion of biofilms is insensitive to titanium substrate type. We will test the hypothesis that Adhesion Index is equal across all titanium substrates. Adhesion Index quantification through laser spallation measurements will provide a relevant, needed, metric for improved biocompatibility screening.