Biofilm development of <i>Porphyromonas gingivalis</i> on titanium surfaces in response to 1,4-dihydroxy-2-naphthoic acid—a hybrid <i>in vitro</i> – <i>in silico</i> approach

Colonization of titanium dental implants by the oral pathogen <i>Porphyromonas gingivalis</i> can lead to peri-implant diseases and, ultimately, implant failure. <i>P. gingivalis</i> growth can be stimulated by 1,4-dihydroxy-2-naphthoic acid (DHNA), a menaquinone precursor from various oral bacteria; however, its impact on biofilm formation remains unclear. The aim of the study was to evaluate <i>P. gingivalis</i> growth and metabolic activity over 6 days in response to DHNA on two titanium grade IV surfaces with different roughness using a hybrid <i>in vitro-in silico</i> approach. <i>P. gingivalis</i> growth was modestly stimulated by DHNA and exhibited an inverse correlation with ammonia concentration in culture medium. Notably, this growth pattern transitioned from an initial linear phase to a later exponential phase, with DHNA-treated biofilms reaching this exponential shift at an earlier stage than untreated controls. Confocal microscopy revealed that DHNA-treated biofilms exhibited surface-dependent growth patterns, with larger biofilm volumes observed on rougher surfaces in later biofilm stages, compared with smoother surfaces. Regardless of surface characteristics, the area occupied by biofilms and the size of the aggregates exhibited a consistent and progressive increase over time and were larger in late DHNA-treated biofilms. The experimental data were used to calibrate a coupled finite element method (FEM)-based model that simulated <i>P. gingivalis</i> biofilm dynamics and nutrient utilization. Summarizing, DHNA moderately stimulated <i>P. gingivalis</i> growth, accelerated its transition to ammonia-independent growth, and promoted an increase in biofilm area and aggregate size. Our coupled approach offers significant potential for advancing <i>in vitro</i> biofilm research.IMPORTANCEThe results of our hybrid <i>in vitro-in silico</i> experiments advance the research on <i>P. gingivalis</i> physiology and its DHNA-dependent colonization of implant surfaces. Our findings reveal that DHNA accelerates <i>P. gingivalis</i> growth, induces aggregation, and promotes colonization of titanium surfaces. For the first time, DHNA-induced <i>P. gingivalis</i> growth acceleration and an earlier shift away from ammonia dependency were observed fluorometrically, highlighting ammonia assimilation as a promising marker of <i>P. gingivalis</i> physiology during early biofilm expansion. Understanding how growth factors together with surface properties influence <i>P. gingivalis</i> colonization offers a basis for future preventive strategies. Our study's stringent characterization of 3D surface texture parameters is expected to improve the reproducibility of biofilm-surface interactions experiments. The findings were validated using a continuum-based <i>in silico</i> model, initiating a hybrid approach where computational models complement <i>in vitro</i> research. Our interdisciplinary approach offers a versatile framework for investigating additional aspects of oral biofilms on titanium.