Therapeutic Potential of Chlorhexidine-Loaded Calcium Hydroxide-Based Intracanal Medications in Endo-Periodontal Lesions: An Ex Vivo and In Vitro Study

Abstract

Endo-periodontal lesions are challenging clinical situations where both the supporting tissues and the root canal of the same tooth are infected. In the present study, chlorhexidine (CHX)-loaded calcium hydroxide (CH) pastes were used as intracanal medications (ICMs). They were prepared and tested on pathogens found in both the root canal and the periodontal pocket. Exposure to 0.5% and 1% CHX-loaded ICMs decreased the growth of Porphyromonas gingivalis and was effective in eradicating or inhibiting an Enterococcus faecalis biofilm. CH was injected into the root canal of extracted human teeth immersed in deionized water. CHX-loaded ICMs resulted in the transradicular diffusion of active components outside the tooth through the apex and the lateral dentinal tubules, as shown by the release of CHX (from 3.99 µg/mL to 51.28 µg/mL) and changes in pH (from 6.63 to 8.18) and calcium concentrations (from 2.42 ppm to 14.67 ppm) after 7 days. The 0.5% CHX-loaded ICM was non-toxic and reduced the release of IL-6 by periodontal cells stimulated by P. gingivalis lipopolysaccharides. Results indicate that the root canal may serve as a reservoir for periodontal drug delivery and that CHX-based ICMs can be an adjuvant for the control of infections and inflammation in endo-periodontal lesions.

Keywords: endo-periodontal lesions; intracanal medication; ion release; local drug delivery; periodontal cells.

Figure 1

Time–kill kinetics curves and areas under the curves (AUC) of the time–kill kinetics for the CH + 0.5%, 1%, 2%, and 4% CHX solutions (*): significant difference (p < 0.05) between the CH formulation and the test formulations (ANOVA test).

Figure 2

pH changes over 28 days in the diffusion medium of teeth not containing (control) or containing an intracanal medication (calcium hydroxide alone [CH] or CH + 1% chlorhexidine [CH + CHX]) through the apex (apical) or the dental tubuli (lateral). There was a significant difference (*) (p < 0.05) at all times between the control and the test formulations (CH and CH + CHX) (ANOVA test).

Figure 3

Changes in calcium ion concentrations in the diffusion medium of teeth not containing (control) or containing an intracanal medication: calcium hydroxide alone (CH) or CH + 1% chlorhexidine (CH + CHX), through the apex (apical) or the dental tubuli (lateral). There was a significant difference with the control group (*) (p < 0.05) and the CH group (#) (p < 0.05) (ANOVA test).

Figure 4

The evolution of chlorhexidine concentrations in the diffusion medium of teeth containing an intracanal medication: calcium hydroxide alone (CH) or CH + 1% chlorhexidine (CH + CHX) through the apex (apical) or the dentinal tubuli (lateral). Standard deviation: 95% confidence interval. (*): significant difference (p < 0.05) between the CH + CHX (apical) group and the other groups (ANOVA test).

Figure 5

Proliferation of (a) periodontal ligament fibroblasts (PDLs), (b) osteoblasts, and (c) cementoblasts after a 1-day contact with 1, 1/2 or 1/10 diluted calcium hydroxide extracts with (CH + 0.5% CHX and CH + 1% CHX) or without chlorhexidine (CH) (n = 3). The measurements were determined using an Alamar Blue^® assay, and the data are expressed as means and standard deviations. Difference between metabolically inactive and metabolically active cells. Standard deviation: 95% confidence interval; (*) significant difference with control group (p < 0.05); (#) significant difference with CH (p < 0.05), (α) significant difference with 0.5% CHX group (p < 0.05) (ANOVA test).

Figure 6

Intracellular alkaline phosphatase (ALP) activity of (a) osteoblasts and (b) cementoblasts stimulated with calcium hydroxide extracts with 0.5% chlorhexidine (CH + 0.5% CHX), or 1% chlorhexidine (CH + 1% CHX), or without chlorhexidine (CH) for 1 and 10 days (n = 3); no statistical difference (ANOVA test).

Figure 7

Semi-quantification of calcium deposits in (a) osteoblasts and (b) cementoblasts using Alizarin Red Staining after stimulation with calcium hydroxide alone (CH) or calcium hydroxide + chlorhexidine (CH + 0.5% CHX and CH + 1% CHX) for 14 days (n = 3) and corresponding images obtained using an optical microscope. Significant difference with control group (*) (p < 0.05) (ANOVA test).

Figure 8

Expression of (a) tumor necrosis factor-alpha (TNF-α) and (b) interleukin 6 (IL-6) by periodontal ligament fibroblasts after a 24 h exposure to LPS followed by treatments with calcium hydroxide extracts with 0.5% chlorhexidine (CH + 0.5% CHX) or 1% chlorhexidine (CH + 1% CHX) or without chlorhexidine (CH), (n = 3); control+: culture control or positive control (cells not stimulated by LPS). Significant difference with control group (*) (p < 0.05); significant difference with CH (#) (p < 0.05) (ANOVA test).

Figure 9

Diffusion model protocol with an incisor (IC) following chemo-mechanical preparation: (a1,b1) labio-lingual views, (a1) for apical diffusion and (b1) for lateral diffusion following the cavity preparation, (a2,b2), following the varnish application, (a3,b3) root attachment to the vial cap, and (a4,b4) immersion in 10 mL of deionized water and incubation at 37 °C in a humidified atmosphere.