BackgroundSodium hypochlorite (NaOCl) is essential to endodontic irrigation because it uniquely dissolves organic matter and disinfects. Chelators remove the smear layer. Sequences of NaOCl and the chelator ethylenediaminetetraacetic acid (EDTA) are in common use because alone neither fulfils all the core functions required of an irrigant. In 2005, the concept of continuous chelation was introduced, whereby a chelator is combined with NaOCl for simultaneous antimicrobial and proteolytic actions and with the capacity to remove smear layer. Not only is this simpler compared to standard irrigation, but some authors report improved antimicrobial effectiveness, enhanced dentinal debris removal, and improved bonding of endodontic materials to dentine. Combinations of alkaline EDTA with NaOCl have been investigated, although the only commercial application of this technique employs the chelator etidronate. The major shortcoming of etidronate-NaOCl mixtures is that a chemical reaction occurs between etidronate and NaOCl, causing reduced NaOCl concentrations over time. Given that continuous chelation may hold advantages, finding a chelator more compatible with NaOCl than etidronate would improve the lifespan of continuous chelation mixtures.ObjectivesThe initial objective was to examine the effect of heating to intracanal temperature on known continuous chelation mixtures. A number of chelators were then screened, aiming to identify one, which when combined with NaOCl had better stability with NaOCl than etidronate and was capable of removing smear layer. Once identified, the NaOCl stability of such a mixture was examined at root canal temperature and in refrigerated storage. Subsequent objectives focused on comparing the identified mixture to etidronate-hypochlorite mixtures, standard sequences and NaOCl with respect to organic tissue dissolution, antimicrobial activity and tooth fracture resistance.MethodsFree available chlorine (FAC), pH and temperature were used to assess NaOCl stability in known continuous chelation mixtures at root canal temperature versus room temperature. Iodometric titration was used to determine the FAC. This methodology was then employed to identify a stable chelator-NaOCl solution from several novel chelator mixtures at room temperature. The chelator inclusion criteria were the calcium ion ligand stability constant and chemical structure. Once a stable mixture was identified, scanning electron microscopy (SEM) in conjunction with image analysis software was employed to assess smear layer removal. The NaOCl stability was then tested for the mixture, as previously described, at root canal temperature and additionally in cold storage. The novel mixture and other irrigant regimens were then compared with respect to important irrigant functions. Tissue dissolution was assessed by measuring the weight loss of porcine mucosa samples at 32oC. The antimicrobial capacity against 7-day Enterococcus faecalis biofilms on hydroxyapatite discs was examined using microbial counts, SEM imaging and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) metabolic activity assay. Compressive strength testing, using an Instron universal testing machine, evaluated the mechanical properties of teeth treated with continuous chelation mixtures versus standard sequences.Results1. At 20 minutes, heating from 23oC to 35oC accelerated FAC falls in etidronate and EDTA mixtures with NaOCl. This reduced the therapeutic window of etidronate mixtures to 20 minutes and rendered EDTA mixtures unsuitable for use. Declines in FAC were accompanied by falls in pH.2. The chelators EDTA, egtazic acid (EGTA), pentetic acid (DTPA), cyclohexanediaminetetraacetic acid (CDTA), aminotris(methylenephosphonic acid) (ATMP), hydroxyphosphonic acetic acid, clodronate and etidronate were chosen for assessment of their stability with NaOCl. Clodronate was the only chelator that when combined with NaOCl maintained its FAC and was associated with neither a temperature spike nor a pH fall.3. Clodronate-NaOCl mixtures removed smear layer and did not erode the peritubular dentine.4. FAC in clodronate mixtures was unaffected by heating to root canal temperature over 3 hours. Clodronate-hypochlorite mixtures lost minimal FAC in cold storage over 3 months.5. At a clodronate concentration able to remove smear layer, clodronate-NaOCl mixtures dissolved porcine mucosa better than etidronate mixtures and equally as well as the control NaOCl.6. Colony counting methodology on E. faecalis biofilms on hydroxyapatite discs was invalidated.7. The resistance to compressive force was the same in teeth irrigated with the sequence NaOCl/EDTA/NaOCl or with etidronate or clodronate mixtures with NaOCl.8. SEM imaging showed that clodronate-NaOCl, etidronate-NaOCl and NaOCl all removed biofilm on hydroxyapatite discs. The XTT assay showed no differences between continuous chelation mixtures and NaOCl.ConclusionsClodronate, compared with etidronate, had superior NaOCl stability in continuous chelation mixtures. Clodronate-NaOCl mixtures removed smear layer, dissolved organic material and disinfected. The addition of clodronate to NaOCl neither impaired antimicrobial activity nor the dissolution of organic tissue by NaOCl. The use of clodronate in continuous chelation did not negatively impact the mechanical properties of teeth compared with a standard sequence. ​​​​​​​