Acquired pellicle and biofilm engineering with CaneCPI-5: Insights from proteomic and microbiomics analysis.

OBJECTIVE: In this in vivo proof-of-concept study, acquired pellicle engineering was implemented to promote alterations in the protein composition of the acquired enamel pellicle (AEP) and the bacterial composition of the dental biofilm after treatment with Sugarcane cystatin (CaneCPI-5). DESIGN: After prophylaxis, 10 volunteers rinsed (10 mL, 1 min) with the following solutions: 1) deionized water (H2O- negative control or 2) 0.1 mg/mL CaneCPI-5. The AEP and biofilm were formed along 2 or 3 h, respectively. The AEP was collected with electrode filter papers soaked in 3 % citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. The biofilm microbiome was collected with a dental curette. The DNA was extracted, amplified, and analyzed by 16S-rRNA Next Generation Sequencing (NGS). RESULTS: Treatment with CaneCPI-5 increased several proteins with antimicrobial, acid-resistance, affinity for hydroxyapatite, structural and calcium binding properties, such as Cysteine-rich-3 (6-fold-p = 0.03), Cystatin-B (5.5-fold-p < 0.01), Neutrophil-defensin 1 (4.7-fold-p < 0.01), Mucin (3.9-fold-p < 0.01), Immunoglobulin-heavy-constant (3.8-fold-p < 0.01) and Lactotransferrin (2.8-fold-p < 0.01). Microbiome revealed that several commensal bacteria had their abundance increased after rinsing with CaneCPI-5, such as Corynebacterium and Neisseria, while Streptococcus and Prevotella nigrescens were decreased. The results indicate the efficiency of CaneCPI-5 in promoting beneficial changes in the AEP and biofilm, making this phytocystatin a potential target for incorporation into dental products. CONCLUSION: Cane demonstrated the capability to alter the protein composition of the acquired enamel pellicle (AEP) and the initial colonizers of the biofilm, enhancing the presence of proteins and bacteria crucial for dental protection.

Acquired pellicle engineering with the association of cystatin and vitamin E against enamel erosion.

OBJECTIVE: Evaluate CaneCPI-5 associated with Vitamin E in acquired enamel pellicle (AEP) engineering to prevent dental erosion. METHODS: 180 human enamel specimens were divided into 12 groups and treated with the following solutions: Cane+VitT and Cane+VitS- CaneCPI-5 + Vit E; Vit+CaneT and Vit+CaneS- Vit E + CaneCPI-5; VitT and VitS- Vit E; CaneT and CaneS- CaneCPI-5; ControlT and ControlS - AmF/NaF/SnCl2; WaterT and WaterS- Deionized water. Groups' name followed by "T" were first treated (200 µl; 2 min) and then incubated in human saliva (200 µl; 1 h) to form the AEP. For groups followed by "S", the AEP was formed and then treatment was applied. The erosive challenge consisted of immersion in 1% citric acid (1 min, 1x/day, for 3 days). The percentage of superficial hardness loss (%SHL) and the relative surface reflection intensity (%SRI) were subjected to normality and homogeneity tests, Shapiro-Wilk and Levene tests, respectively. Subsequently, the data were analyzed using two-way ANOVA, Tukey's test and Pearson's correlation (p < 0.005). RESULTS: For%SHL and%SRI, water controls showed significantly lower protective capacity. Cane+VitT, Cane+VitS, and Vit+CaneS presented the lowest%SHL, and VitT and VitS did not differ from Vit+CaneT, but they were different from the other groups (p = 0.002). The greatest%SRI was found for the Cane+VitT, Vit+CaneT, VitT, Cane+VitS, Vit+CaneS, and VitS groups, which did not significantly differ. CaneT and ControlT, showed similar reflections compared to CaneS and ControlS. CONCLUSION: CaneCPI-5 and Vitamin E demonstrated a synergistic protective effect against initial erosion. CLINICAL SIGNIFICANCE: The results open up new possibilities for preventive approaches against erosion through the acquired pellicle engineering, with the combination of CaneCPI-5 and Vitamin E, which demonstrated to be more effective than commercial stannous mouthwash. Further research is warranted to explore the potential of this combination in diverse clinical settings.

Protein-based engineering of the initial acquired enamel pellicle in vivo: Proteomic evaluation.

OBJECTIVE: To study the proteomic alterations in the initial AEP after rinsing with CaneCPI-5, StN15 or Hb or their combination. MATERIALS AND METHODS: In five crossover phases, after prophylaxis, 10 volunteers in 5 consecutive days, rinsed (10 mL, 1 min) with the following solutions: deionized water (H2O- negative control- 1), 0.1 mg/mL CaneCPI-5 (2), 1.88×10-5 M StN15 (3), 1.0 mg/mL Hb (4) or their combination (5). The AEP formed after 3 min was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. RESULTS: Rinsing with the proteins/peptide increased the amounts of proteins in the AEP. The total numbers of proteins identified after rinsing with CaneCPI-5, StN15, Hb or their combination versus water, were 131, 167, 148 and 142, respectively. The treatment with the proteins/peptide or their combination increased proteins that bind calcium, phosphate and interact with distinct proteins, as well as proteins with antimicrobial and acid-resistant properties, such as, Cornifin-B (7.7, 12.6, and 4.3-fold for CaneCPI-5, StN15 and Hb, respectively), isoforms of Cystatin (2.2-2.4-fold for CaneCPI-5 and StN15), Proline-rich-protein 4 (4.3-fold; StN15), Histatin-1 (2.8-fold; StN15) and Hemoglobin (7.7-25-fold for Hb and Combination). Immunoglobulin, Keratin and Histone were exclusively identified upon treatment with the proteins/peptide, alone or combined. CONCLUSION: Rinsing with proteins/peptide, alone or combined, increased protective proteins in the initial AEP. CLINICAL RELEVANCE: Our results suggest that rinsing with the proteins/peptide or their combination increases the proteins capable of enhancing the protective function of the basal layer of AEP.