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In Vitro Antimicrobial and Cytotoxic Effects of Kri 1 Paste And SCIENTIFIC ARTICLE In vitro antimicrobialand cytotoxic effects of Kri 1 pasteand zinc oxide-eugenolused in primarytooth pulpectomies Kelly J. Wright, DMDSergio V. Barbosa, DDS, PhD Kouji Araki, DDS,PhD Larz S.W. Sp~ngberg,DDS, PhD Abstract The antimicrobial and cytotoxic effects of Kri I paste, an iodoform-basedprimary tooth filling material, were comparedwith zinc oxide-eugenol (ZOE), using in vitro techniques. Antimicrobial evaluation involved measuring inhibition zones Streptococcus faecalis on brain heart agar. Cytotoxicity evaluation involved direct cell-medicamentcontact experiments of 4-hr and 24-hr duration using fresh and set medicaments,and indirect cell-medicamentcontact experiments of 24-hr duration using fresh and set medicaments.ZOE produced a greater zone of bacterial inhibition than Kri 1 paste. Kri 1 paste cytotoxicity remainedhigh regardless of the amountof setting time in the 4-hr direct contact experiment, while ZOEcytotoxicity decreased with setting time. Both Kri I paste and ZOEhad high cytotoxicity regardless of setting time in the 24-hr direct cell-medicament contact test. ZOEcytotoxicity decreased to control levels after only 1 day of setting in the indirect contact experiments, comparedwith greater than 7 days for Kri I paste. The results suggest ZOEhas better antimicrobial activity than Kri I paste. ZOEalso has lower cytotoxicity, although prolongedcell-medicament contact mayresult in both medicamentshaving similarly high cytotoxicity. (Pediatr Dent 16:102-6, 1994) Introduction time of the material. No osteolytic changes were found To maintain function, esthetics, arch length, and arch surrounding ZOEimplants. Several studies have in- vestigated the antimicrobial action of Kri I in vivo 9’ 11-13 symmetry, primary teeth should be maintained in the 1~-16 dental arch until their proper exfoliation timeo1, 2 and in vitro. No studies have comparedthe antibac- Endodontic treatment of primary teeth has been a suc- terial action and attendant cytotoxicity of Kri 1 paste cessful method of maintaining nonvital primary teeth and ZOEin vitro. with and without periapical pathosis. However, suc- The purpose of this study was to examine the claim cess is related to strict tooth selection criteria and to of superior antimicrobial action for Kri I paste by com- thorough debridement of the canals followed by paring the antimicrobial and cytotoxic effects of Kri 1 obturation with a suitable filling material. 2, 3 paste and ZOEin vitro. Zinc oxide-eugenol (ZOE) has traditionally been used Methods and materials as a root canal filling material in the primary dentition. Recently, an iodoform paste (Kri I paste, Pharmachemie Antimicrobialevaluation AG, Zurich, Switzerland) has been recommended as an Kri 1 paste (iodoform 80.8%, camphor 4.86%, p- alternative medicament to ZOEin root canal therapy chlorophenol 2.025%, menthol 1.215%, was used as for nonvital primary teeth. ~ Walkhoff originally de- supplied. ZOE(zinc oxide U.S.P./eugenol U.S.P. -- scribed the formula for an iodoform paste in 1928 and Sultan Chemists, Inc., Englewood, NJ) was mixed to used7 it in permanentteeth. Later, Castagnola and Orlay, the same consistency as Kri I paste immediately before Juge, 8and Barker and Lockett 9reported using experimentation (2.5 g zinc-oxide to 1 ml eugenol). Walkhoff’s paste as a filling material in permanentteeth, Ninety-millimeter-diameter petri dishes were filled but long-term treatment success was compromised since with 12 ml of BBL Brain Heart Infusion with PABA the paste eventually resorbed, leaving a deficit in the (BBL Microbiology Systems, Becton Dickinson and Co., root canal. Cockeysville, MD)and BBLPurified Agaro Six-milli- The reported rationales for using Kri 1 paste over meter-diameter wells were created in the agar using ZOEin primary teeth are: ease of use, rapid resorption the end of a sterilized glass pipette. The wells were from periapical tissues, and superior antimicrobial ac- filled with 0.05 ml of either Kri 1 paste or ZOEdis- tion.4,SKri I paste is easier to use than ZOEbecause it is pensed from tuberculin syringes. supplied as a premixed paste that can be placed di- Aliquots of 0.1 ml of 106 CFU/mlStreptococcusfaecalis rectly into the root canal. Kri I resorbs rapidly from the (ATCC19433) in BBLthioglycollate medium were then periapical tissues, however, Woodhouse,et al. 1° have spread over the surface of the agar plates. The plates shown osteolytic changes in the bone surrounding Kri were inverted and incubated at 37°C for 24 hr. Two 1 paste implants in cats, persisting past the retention measurements of the diameter of bacterial growth inhi- 102 Pediatric Dentistry: March/April 1994 - Volume 16, Number 2 bition around each well were made using a millimeter where T = 51Cr released in test samples, b -- background ruler, and the values were averaged. These values were radiation and R = 5~Cr released in reference samples. then used to calculate area of bacterial inhibition (area For the indirect cytotoxicity evaluation, 0.1 ml of =n d2/4), after subtracting the area of the wells. Agar either Kri 1 paste or ZOEwas placed in the 6.5-ram- plates containing wells, but no medicaments, were diameter chamber insert of a cluster well cell culture spread with S. faecalis and incubated as positive con- plate. The porous bottoms of the cell culture inserts trols. Agar plates containing wells, but no medicaments (uniformly spread 0.4 gm holes) were 1 mmfrom the or S. faecalis, were also incubated to ensure mainte- bottom of the well. This allowed independent access nance of sterile conditions (negative controls). All ex- from the chamber insert into the well through the me- perimental procedures were carried out under sterile dium. One milliliter of 5~Cr-labeled L929cells was dis- conditions using a SterilGARD hood® (The Baker Com- pensed into each cell culture well. The chamber inserts pany, Sanford, ME). containing the test medicaments were then placed into the wells. Empty chamber inserts were placed in the Cytotoxicityevaluation cell culture wells for control. Randomly withdrawn Twoin vitro cytotoxicity test methods were used -- 0.5-ml cell samples were transferred to test tubes to be the direct cell-material contact test of Sp~ngberg17 and used as reference samples for calculating chromium theis indirect cell-material contact test of Safavi, et al. release. The plates containing the cells were incubated Five- to 7-day cultures of L929 mousefibroblast cells at 37°C for 4 hr immediately after mixing (fresh) and were used for the cytotoxicity tests. The culture me- after 1, 7, 14, and 28 days. dium used was minimal essential medium (MEM)with The chamber inserts were stored in PBS and incu- Eagle’s salts (Flow Laboratories, McLean, VA) supple- bated at 37°C and 100 % humidity between experiments. mented with 10% (v/v) fetal calf serum, 2 mM The percentage of 51Cr release in test and control samples glutamine, and 2.2 mg sodium bicarbonate per ml. was calculated by the same formula used in the direct Streptomycin (50 ~g/ml) and penicillin (100 IU/ml) toxicity test. were also added to the medium. The medium was The antimicrobial data were analyzed using changed every other day and on the day before the Student’s t-test while the cytotoxicity data were ana- experiments. lyzed using ANOVA. Radiochromium (SlCr) was supplied as sodium chro- mateTM in sterile isotonic saline (NewEngland Nuclear, Results Du Pont Company, Wilmington, DE). Cell monolayer On average, ZOE-filled wells produced a signifi- culturess were labeled by incubation with I I.tCi per 10 cantly greater area of inhibition (P < 0.01) than did the cells 20-24 hr before the experiments. The labeled cells Kri 1-filled wells (Fig 1). Positive controls showed were harvested using 0.125% trypsin and washed three confluent S. faecalis growth over the agar with no areas times in Ca~÷-free and Mg2÷-free phosphate buffered of inhibition around the wells. Negative controls con- saline (PBS) before use. The cells were resuspended firmed the maintenance of sterile conditions (absence MEMat a concentration of 4x104 cells/ml. of bacterial growth). Serial dilution and plating of the For direct cytotoxicity testing, 0.3 ml of ZOEor Kri 1 paste was placed in the bottom of 16-mm-diameter cell culture wells (Transwell; Costar, ® Cambridge, MA). 400. Materials were tested immediately after mixing (fresh), 350. or after 1-day or 7-day setting times. Twoml of 51Cr- labeled L929 cells were placed in the culture wells and EE 300. incubated in contact with the medicaments for 4 or 24 250. hr at 37°C. After incubation, 1 ml of the fluid in the test chamber 200.: was transferred to a test tube and centrifuged (500 x g) 150, for 10 min. One-half ml of the supernatant was trans- ferred to a test tube for counting in a gammaparticle 100, counter for 1 min. Negative controls of the L929 sus- 50" . pension were used. During cell dispensing, 0.5 ml aliquots of cell suspension were randomly distributed o into six test tubes and used as reference samples. The Kri1 ZOE percentage of SlCr released from the experimental and control samples was calculated according to the for- Medicament mula: Fig 1. Antibacterialaction of Kri 1 paste(N = 15)and ZOE (N 51Cr release (%) = (T-b) x 1 3) basedon Streptococcus faecalis inhibition on agar (mean + (R-b) SD).* = significantlygreater than Kri 1 paste(P < 0.01). Pediatric Dentistry: March/April 1994 -Volume 16, Number2 103 Both Kri I paste and ZOEwere highly cytotoxic on 4 hr exposure 24 hr exposure lOO--- contact of the fresh medicaments with L929 mouse fibroblast cells (Fig 3) and significantly different from the control (P < 0.01). There was no significant differ- ence between ZOEand control in the 1-, 7-, 14-, and 28- day experiment groups.
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