A Biofilm Pocket Model to Evaluate Different Non-Surgical Periodontal Treatment Modalities in Terms of Biofilm Removal and Reformation, Surface Alterations and Attachment of Periodontal Ligament Fibroblasts

Abstract

Background and aim: There is a lack of suitable in vitro models to evaluate various treatment modalities intending to remove subgingival bacterial biofilm. Consequently, the aims of this in vitro-study were: a) to establish a pocket model enabling mechanical removal of biofilm and b) to evaluate repeated non-surgical periodontal treatment with respect to biofilm removal and reformation, surface alterations, tooth hard-substance-loss, and attachment of periodontal ligament (PDL) fibroblasts.

Material and methods: Standardized human dentin specimens were colonized by multi-species biofilms for 3.5 days and subsequently placed into artificially created pockets. Non-surgical periodontal treatment was performed as follows: a) hand-instrumentation with curettes (CUR), b) ultrasonication (US), c) subgingival air-polishing using erythritol (EAP) and d) subgingival air-polishing using erythritol combined with chlorhexidine digluconate (EAP-CHX). The reduction and recolonization of bacterial counts, surface roughness (Ra and Rz), the caused tooth substance-loss (thickness) as well as the attachment of PDL fibroblasts were evaluated and statistically analyzed by means of ANOVA with Post-Hoc LSD.

Results: After 5 treatments, bacterial reduction in biofilms was highest when applying EAP-CHX (4 log10). The lowest reduction was found after CUR (2 log10). Additionally, substance-loss was the highest when using CUR (128±40 µm) in comparison with US (14±12 µm), EAP (6±7 µm) and EAP-CHX (11±10) µm). Surface was roughened when using CUR and US. Surfaces exposed to US and to EAP attracted the highest numbers of PDL fibroblasts.

Conclusion: The established biofilm model simulating a periodontal pocket combined with interchangeable placements of test specimens with multi-species biofilms enables the evaluation of different non-surgical treatment modalities on biofilm removal and surface alterations. Compared to hand instrumentation the application of ultrasonication and of air-polishing with erythritol prevents from substance-loss and results in a smooth surface with nearly no residual biofilm that promotes the reattachment of PDL fibroblasts.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. A-D. Dentin Specimen, Biofilm Formation, and Pocket Model.

Schematic illustration and corresponding photographs of the biofilm formation on dentin specimen (A+B) and the insertion of the dentin specimen into the artificial pocket model (C+D).

Fig 2. A-D. Dentin Specimen, Biofilm Formation, and Pocket Model.

Schematic illustration and corresponding photographs of the four treatment modalities hand instrumentation (A), ultrasonication (B), erythritol air-polishing (EAP; C), and EAP with chlorhexidine digluconate (D).

Fig 3. A-C. Biofilm removal.

Graph depicting the mean colony forming units (log10 CFU, ±SD, n = 10; A, B) and SEM photographs (C) after one (A, C) and 5 times (B) biofilm formation (CON) and followed by instrumentation of the four different treatment methods (hand instrumentation (CUR), ultrasonication (US), erythritol air-polishing (EAP), and EAP with chlorhexidine digluconate (EAP-CHX). CFU were pronouncedly reduced by US and EAP-CHX (* p

Fig 4. A-C. Reformation of biofilm.

Graph presenting the mean colony forming units (log10 CFU, ±SD, n = 10; A, B) and SEM photographs (C) after one (A, C) and 5 times (B) biofilm formation (CON) followed by instrumentation of the four different treatment methods (hand instrumentation (CUR), ultrasonication (US), erythritol air-polishing (EAP), and EAP with chlorhexidine digluconate (EAP-CHX). All treatment modalities were followed by an additional biofilm formation cycle). Reformation of biofilm was delayed by US and EAP and EAP-CHX (* p

Fig 5. A-D. Biofilm removal and recolonization of selected bacterial species.

Counts of selected bacterial species after biofilm removal and recolonization determined by real-time PCR. Graph presenting the mean log10 counts (±SD, n = 10) after one (A, C) and 5 times (B, D) biofilm formation and followed by instrumentation of the four different treatment methods (hand instrumentation (CUR), ultrasonication (US), erythritol air-polishing (EAP), EAP with chlorhexidine digluconate (EAP-CHX), and an untreated control (con) (A, B) as well as after an additional biofilm formation (C, D) (* p¶p<0.05; ¶¶ p<0.01, ¶¶¶ p<0.001 compared to CUR, † p<0.05; †† p<0.01 compared to US, § p<0.05; §§ p<0.01 compared to EAP).

Fig 6. A+B. Tooth hard-substance-loss by different treatment methods.

Graph presenting the thickness difference before and after one (A) and 5 times (B) instrumentation of the dentin specimens using four treatment methods (hand instrumentation (CUR), ultrasonication (US), (erythritol air-polishing (EAP), EAP with chlorhexidine digluconate (EAP-CHX)), and an untreated control (con). Instrumentation using CUR was related to significantly higher hard-substance loss compared to the other groups (** p

Fig 7. A-D. Tooth surface roughness.

Graph depicting the average surface roughness Ra (A, B) and the arithmetic mean height of the surface profile Rz (C, D) after one (A, C) and 5 times (B, D) instrumentation of the dentin specimens using four treatment methods (hand instrumentation (CUR), ultrasonication (US), (erythritol air-polishing (EAP), EAP with chlorhexidine digluconate (EAP-CHX)), and an untreated control (con). Instrumentation using CUR and US resulted in an increased average surface roughness after 5 times instrumentation (* p

Fig 8. A-C. Attachment of periodontal ligament (PDL) fibroblasts after one treatment.

Graphs presenting the mean attached PDL fibroblasts (A), release of IL-8 (B) and microscopic photographs (C) after one biofilm formation and one instrumentation of the dentin specimens using four treatment methods (hand instrumentation (CUR), ultrasonication (US), (erythritol air-polishing (EAP), EAP with chlorhexidine digluconate (EAP-CHX)), and an untreated control (con). IL-8 was measured in media after 40 h incubation. (* p

Fig 9. A-C. Attachment of periodontal ligament (PDL) fibroblasts after five treatments.

Graphs presenting the mean attached PDL fibroblasts (A), release of IL-8 (B) and microscopic photographs (C) after five biofilm formations, five instrumentations of the dentin specimens using four treatment methods (hand instrumentation (CUR), ultrasonication (US), (erythritol air-polishing (EAP), EAP with chlorhexidine digluconate (EAP-CHX)), and an untreated control (con) and a complete biofilm removal. IL-8 was measured in media after 40 h incubation. (* p