the success of root canal treatment depends on the eradication of microorganisms from the root canal system and prevention of re - infection . due to its complex anatomy , with multiple fins , isthmuses , ramifications and accessory canals , it is virtually impossible for mechanical instrumentation to shape and clean the entire root canal system . according to a study conducted by bystrm and sundqvist , the number of intracanal microorganisms reduced 1001000 fold following hand instrumentation with stainless steel files and using sterile saline as a root canal irrigant . thus , using an appropriate irrigation solution is essential to remove residual tissue and to kill microorganisms . the antimicrobial effectiveness of naocl is based on its high ph ( hydroxyl ions action ) . the high ph of naocl disturbes the completeness of the cytoplasmic membrane via inhibiting enzymes irreversibly , biosynthetic changes in cellular metabolism and depriving phospholipids observed in peroxidation of lipids . furthermore , naocl possesses excellent tissue dissolving ability . in other words , tetracyclines are absorbed to dentin immediately and are deliver thereafter without missing their antibacterial effectiveness . in the field of endodontics , tetracyclines have been used to irrigate root - end cavities , to remove the smear layer from the instrumented canals   and as an intra - canal medicament . currently , there are two antibiotic - based root canal irrigation solutions in the field of endodontics . in addition , its antibacterial substantivity has been proved in some studies [ 10 , 11 ] . the other antibiotic - based irrigant , tetraclean , ( ogna laboratori farmaceutici , muggi , italy ) , similar to mtad , is a mixture of an antibiotic , an acid and two detergents ( propylene glycol and cetrimide ) . however , the concentration of the antibiotic , doxycycline ( 50 mg ml ) and the type of detergent ( polypropylene glycol and cetrimide ) differ from those of mtad . recently , mohammadi et al . showed that the substantivity of tetraclean was significantly greater than mtad and 5.25% naocl . mtad as well as tetraclean do not dissolve organic tissues . therefore , naocl should be used to dissolve organic tissues . .   showed that the antimicrobial substantivity of mtad was reduced when mtad was applied to 1.3% naocl - irrigated dentin . recently , mohammadi et al .   showed that substantivity of tetraclean was reduced in naocl - irrigated dentin . a study demonstrated that pretreatment of naocl - irrigated dentin with ascorbic acid prevented the decrease in mtad substantivity . considering the closed similarity between the composition of mtad and tetraclean , the aim of this study was to assess the effect of pretreatment of naocl - irrigated dentin with ascorbic acid on the substantivity of tetraclean . the method of the present study was modified from that described by haapasalo and rstavik . intact bovine central incisor teeth were used . in order to clean and disinfect the surface , the teeth were kept in 0.5% naocl solution ( merck , germany ) for up to 7 days . the clinical crown and apical third were removed from each tooth using a rotary diamond saw at 1000 rpm ( isomet plus precision saw , buehler , il , usa ) under water - cooling . the cementum was removed using polish paper ( ecomet 3 , variable - speed grinder - polisher , buehler , il , usa ) , which resulted in a center - holed piece of root dentin with a 6-mm outer diameter ( fig 1 ) . using a diamond saw , the remained piece of each tooth was cut into 4-mm - thick slices . using an iso 023 slow speed round bur , the canals of the 4-mm blocks were enlarged ( standardized ) . during the procedures , all teeth and dentin slices were preserved in vials containing tap water to prevent dehydration . in order to remove the smear layer , each dentin block was placed inside 3 ml of 17% edta with a ph of 7.2 placed in an ultrasonic bath for 10 min and irrigated under tap water for 1 hour . thereafter , the mentioned process was conducted with 5.25% naocl ( merck , darmstadt , germany ) instead of edta . the specimens were then placed in bhi broth ( oxoid , basingstoke , uk ) and autoclaved at 121 c with 1 atm pressure for 15 minutes . the specimens were kept in an incubator at 37c for 24 h to monitor the efficacy of the sterilization . a total of 80 specimens were randomly divided into five groups as follows : 
 group 1 ( 20 specimens ) : tetraclean ( ogna laboratori farmaceutici , muggi , italy ) irrigationgroup 2 ( 20 specimens ) : 5.25% naocl and tetraclean irrigationgroup 3 ( 20 specimens ) : 5.25% naocl , ascorbic acid and tetraclean irrigation group 1 ( 20 specimens ) : tetraclean ( ogna laboratori farmaceutici , muggi , italy ) irrigation group 2 ( 20 specimens ) : 5.25% naocl and tetraclean irrigation group 3 ( 20 specimens ) : 5.25% naocl , ascorbic acid and tetraclean irrigation furthermore , 10 sterile and 10 infected dentin tubes were used as negative and positive control groups , respectively . in order to contaminate the dentin tubes , isolated 24-h colonies of pure cultures of e. faecalis ( atcc 29212 ) suspended in 5 ml of bhi the bottles containing each specimen in groups 1 , 2 , 3 and 4 were opened under laminar flow . two milliliters of sterile bhi was removed with sterile pipettes and replaced with 2 ml of bacterial inoculum . the bottles were closed and kept at 37c for 14 days . to prevent medium saturation , 1 ml of freshly prepared bhi was replaced by 1 ml of contaminated bhi every 2 days . each specimen was removed from its bottle under aseptic conditions after 14 days and the canal was irrigated with 5 ml of sterile saline and dried with sterile paper points . the outer surface of the specimens was covered with two layers of nail varnish to prevent contact of the medicament with the external surface . afterwards , specimens were fixed at the bottom of wells of 24-well cell culture plates using decontaminated sticky wax , which also obliterated the apical surface of the root canal . finally , the irrigating solutions were inserted into the canal lumen with sterile 3-ml plastic syringes and 27-gauge needles until the dentin tubes were totally filled . solutions were removed using sterile paper points ten minutes after placement into the lumen . in group 2 the naocl solution was then removed with sterile paper points and dentin tubes were filled with tetraclean for 10 minutes . however , in group 3 samples , dentin tubes were filled with 10% ascorbic acid solution for 10 minutes before using tetraclean and after removing 5.25% naocl solution . the specimens were then incubated at 37c for 28 days to maintain humidity . at experimental times of 0 , 7 , 14 , 21 and 28 days , dentin chips were removed from the canals with sequential sterile low - speed round burs with increasing diameters of 025 , 027 , 029 , 031 and 033 iso sizes , respectively . the powder dentin samples obtained with each bur were immediately collected in separate test tubes containing 3 ml of freshly prepared bhi . the positive control group showed viable bacteria at all experimental times , which indicated the efficiency of the method . in contrast , the negative control group showed no viable bacteria at all experimental times . the number of cfu was minimum in the first cultures ( day 0 ) in all experimental groups and there were statistically significant differences between the obtained results at any time period ( p = 0.03 ) . the tetraclean group showed the most effective antibacterial action at all five experimental periods ( p < 0.001 ) . the naocl / ascorbic acid / tetraclean group showed similar antibacterial substantivity to the tetraclean group at all time periods ( p= 0.002 ) . enterococcus faecalis was chosen for the following reasons : ( i ) its prevalence in root - filled teeth with periradicular lesions using culturing and polymerase chain reaction ( pcr ) methods is 2470% and 6777% , respectively . ( ii ) its capacity to endure prolonged periods of starvation until an adequate nutritional supply becomes available has been demonstrated . the method of the present study was a modification of that proposed by haapasalo and orstavik . using the mentioned method , each consecutive bur size removes only 0.1 mm of the dentin thickness around the root canal . according to the microcomputed tomography ( ct ) studies , comparatively huge areas of the root canal surfaces leave untouched by files . therefore , using an irrigation solution is necessary to improve the debridement of the canals . for improvement of the efficacy of root canal irrigants the closeness of this approximation is related to the wettability of the irrigant on dentin surface and wettability of irrigants has a strict correlation with their surface tension . the force between molecules that produces a tendency for the surface area of a liquid to decrease  . surface tension is the force between molecules that decreases the surface area of the irrigant . this force reduces the penetration depth of the liquid into a capillary tube . enhancing the wettability of an antimicrobial irrigant increase its protein solubility and improves its efficacy in the uninstrumented parts of the root canal . despite the strong antimicrobial activity of naocl against e. faecalis , even in the form of biofilm , its high surface tension   does not allow it to reach bacterial stocks in the depth of dentinal tubules and canal irregularities . to overcome this problem , some antibiotic - based irrigation solutions ( i.e. mtad and tetraclean ) with low surface tension have been introduced . new generation of endodontic irrigants combine a powerful detergent effect with a strong antimicrobial efficacy . in fact , in the present study , the effect of ascorbic acid ( vitamin c ) was assessed on the reversing the oxidative potential of naocl as a reducing agent . findings demonstrated that treatment of naocl - irrigated dentin with 10% ascorbic acid for 10 minutes prevents reduction of the substantivity of tetraclean by prohibiting oxidation of doxycycline by naocl . reducing the activity of ascorbic acid ascorbic acid also prevents photodegradation of tetracycline isomers ( doxycycline and minocycline ) [ 27 , 28 ] . using a human dentin tube model , mohammadi and shahriari   demonstrated that residual antibacterial activity of mtad was significantly greater than chx . in another study using bovine dentin tube model , mohammadi et al .   demonstrated that the residual antibacterial activity of tetraclean was significantly greater than mtad and 5.25% naocl . another study demonstrated that the residual antibacterial activity of tetraclean was significantly greater than hypoclean ( a naocl - based irrigation solution with reduced surface tension ) and 5.25% naocl . .   demonstrated that when mtad was used as initial rinse followed by the use of various concentrations of naocl as final rinse , a chemical reaction happened between naocl and the residual mtad , which caused formation of a brown solution in the root canal . they proposed that such a phenomenon might be caused by the dentinal absorption and release of doxycycline present in mtad and the final rinse of naocl . the reaction of naocl with either doxycycline or citric acid in mtad is not an acid - base reaction . rather , it is a redox reaction that involves the oxidation of doxycycline by naocl . examined the effect of naocl - mtad interaction on the antimicrobial substantivity of mtad in dentin . findings demonstrated that the use of 1.3% naocl as an initial rinse resulted in a 30% reduction in the substantivity of mtad . this study   also showed that treating the naocl - irrigated dentin with ascorbic acid prevented the interaction between naocl and mtad . in another study , mohammadi et al .   found that substantivity of tetraclean in naocl - treated dentin reduced significantly . the reduction of the substantivity of tetraclean and mtad can be attributed to the partial loss in the substantivity from the doxycycline component . within the limitations of the present study , in naocl - irrigated dentin , ascorbic acid prevented the decrease in the substantivity of tetraclean .