Supplementary Material for: Acquired Pellicle and Biofilm engineering by rinsing with hemoglobin solution

Introduction: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the "acquired pellicle engineering" concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization.
Objective: Combining in vivo and in vitro proof-of-concept protocols we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization.
Methods: In the in vivo studies, 10-volunteers, in 2 independent experiments, rinsed (10mL,1min) with: deionized water-negative control or 1.0mg/mL Hb. The AEP and biofilm formed along 2 or 3h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA Next-Generation-Sequencing (NGS). In vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with: 1)Phosphate-Buffered Solution-PBS, 2)0.12% Chlorhexidine, 3)500ppm NaF; 4)1.0mg/mL Hb; 5)2.0mg/mL Hb, and 6)4.0mg/mL Hb. The biofilm was cultivated for 5-days. Resazurin, colony-forming-units(CFU) and Transversal Microradiography(TMR) were performed.
Results: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU and enamel demineralization compared to PBS.
Conclusion: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering.