Effectsof formocresolalone vs. formocresolwith eugenol onmacrophage adhesion to plastic surfaces JuanJos~ Segura, DDS, MD, PhD Alicia Jim~nez-Rubio, DDS,MD, PhD Juan Ram6n Calvo, MD, PhD Abstract However,the amountof formocresol that could be ab- Purpose:The purposeof this study wasto comparethe sorbed from pulpotomysites is minimal.13 In the light in-vitro effects of a European-basedformocresol formulation of the preceding discussion, the use of formaldehyde- that incorporateseugenol with formocresolalone on the ad- containingpastes on pulp tissues is controversial)’ hesionof macrophagesto plastic su~Caces. In Europe, therapeutic pulpotomiesof primary teeth Methods: MacrophagesTM were obtainedJ}om Wistar are performed using a formulation of formocresol rats. Theadherence capacity of macrophagesto a plastic sur- which incorporates eugenol (20% formaldehyde, 20% face~ wasdetermined. Assays were carried out in Eppendorf tricresol, and 20%eugenol). Eugenolis an ingredient tubes incubatedfor 15 rain at 37° C in a humidifiedatmo- in manyover-the-counter toothache drops, temporary sphere of 5%CO 2. The adherenceindex wascalculated. filling materials, restorative materials, bases, cements, Results:Results showedthat both formocresol/eugenoland intracanal medicaments,and in pastes used in endodon- |~--16 formocresolalone significantly decreasedthe adherencein- tics as a sealer for root canal fillings. It has been dex of macrophages.The formocresol formulation that claimedthat eugenoldoes not irritate the pulp if applied incorporatedeugenof wasmore potent in inhibiting mac- to dentin. In contrast, Glass and Zander]7 showedthat rophageadhesion than formocresolalone. eugenol induced chronic inflammation whenplaced on 18 Conclusions:Taking into accountthat adherenceto a an exposedpulp. Moreover,Br~innstr/Sm and Nyborg substrateis the first step in the phagocyticprocessof mac- and Webband Bussel119also demonstratedthat eugenol rophagesand in antigen presentation, both formocresol produces a markedinflammatory reaction in pulp. formulationscould inhibit macrophagefunction and modu- On the other hand, it has been shownthat inflamed pulp and periapical tissues contain a variety of immu- late immuneand inflammatory responses in dental pulp and nocompetent2° cells, with macrophagespredominating. periapical tissues (Pediatr Dent20:3 177-80,1998). Moreover, macrophagesare the most dominant immu- nocompetent cells through all stages of induced ormocresol (Buckley’s formula) consists of 19% periapical lesions.21’ 22 Macrophagesare implicated in formaldehydeand 35%cresol in a vehicle of 15% chronic inflammation and repair of pulpal and peri- F glycerin in water.1 This formocresolformulation, apical tissues. 23 They are known to have several as well as modificationsthat contain different propor- mediatorand regulatory functions, and are involved in tions of formaldehydeand cresol, are widely used by the entire spectrumof the defensereactions. 24 It is well Americandentists for pulpotomyprocedures in pri- 2’ documentedthat adherenceis the first step in the pha- maryteeth 3 and as an endodonticdrug to neutralize gocytic25 process of inflammatory macrophages. the septic and necrotic contents of root canal.< 5 < The in-vitro effects of formocresol alone and a Formocresolis highlytoxic to cells. 7 Tissuechanges formocresol formulation that incorporates eugenol on in dog livers and kidneys induced by the absorption of 8 the substrate adherence capacity of rat inflammatory formocresol from pulpotomysites have been shown. macrophagesare comparedin this study. Polyvinyl sponge implants containing full-strength formocresolcause fixation of fibroblasts in adjacent Methods cells.9 A 1:5 dilution of formocresolmarkedly sup ressed 1° Formocresol/eugenol(20% formaldehyde, 20°/0 tri- lactic dehydrogenaseactivity. At weakerconcentra- cresol, and 20% eugenol) was obtained from tions, formocresol does not fix the tissue, but might ~* LaboratonoBucca (Juan Alv~.rez Mendizabal,Madrid). create signs of cellular degeneration. Humanclinical Formocresol (42.1% cresol, 42.1% formaldehyde, studies have shownthat formocresol treatment causes 15.8% ethilic alcohol) was obtained from J. Bird severe inflammatoryreactions or necrosis of the pulp.12 Pediatric Dentistry -2&3, 1998 AmericanAcademy of Pediatric Dentistry 177 Moyer. RPMI-1640medium was obtained from Sigma ence characteristics to tissues as reported by Nogaet (St. Louis, MO). al.28 Formocresolalone or formocresol/eugenol(20 BL) The protocol was approved by our experimentation was dissolved directly in RPMI-1640medium to a fi- committee. Peritoneal macrophageswere elicited from nal dilution of 1:10, 1:100, or 1:1000in the incubation Wistarrats. 26 Briefly, each rat wasinjected intraperi- medium. RPMI-1640medium (20 BL) was added toneally with 5 mLof sterile 6%sodium caseinate. control samples. Adherenceassays were performedwith Animalswere killed after 4 days by decapitation and 15 min of incubation at 37°C in a humidified atmo- the peritoneal cavity was washed with 10 mLof cold sphere of 5% CO2 to provide a maximal adherence 0.9% NaCl. After a 2-min massage, the cell exudate index. 21 After gentle centrifugation to remove was removedwith a syringe and centrifuged for 10 min nonadherentcells (3 s in the vortex in position 5), 10- at 250 x g at 4°C. The contaminating red blood cells l.tL aliquots were taken from each sample and the were lysed with cold 0.2% NaC1.The remaining cells number of nonadherent macrophages/mL were were then washed with 0.9% NaC1by centrifugation, counted in Neubauer chambers. No agglutination of resuspended in RPMI-1640 medium, counted, ad- macrophageswas observed. The adherence index (AI) justed in the same mediumat 2-4 x 106 macrophages! was calculated according to the following equation: mLand immediately used for experiments. Meancells AI = 100 - Nonadherent macrophages/mL + per rat varied from 20-30 x 106, of which 85-95% Initial macrophages!mLx 100 were macrophagesby morphologicalcriteria in Giemsa All values were expressed as the mean+ standard and Papanicolaou staining techniques. Viability, as deviation of five separate experiments performed in determined by trypan-blue exclusion, was always triplicate, as indicated in the correspondingfigure. The greater than 94%. data were statistically evaluated by ANOVA.A value The quantification of substrate-adherence capacity of P < 0.05 wasconsidered statistically significant. was carried out according to the technique described previously by De la Fuente et al. 27 with minor modifi- Results cations. Aliquots of 180 IlL of cell suspension were dispensed in Eppendorftubes, which mimicthe adher- Both formocresol alone and formocresol/eugenol inhibited the substrate-adherence capacity of macroph- ages in a dose-dependent manner (Fig 1). Whenadded 100 to the incubationmedium at a final dilution of 1 : 1000, formocresol/eugenol decreased the AI by 12%(P 0.05). Moreover, the 1:100 dilution of formocresol/ eugenol significantly decreased the AI by 59%(P 8O 0.05). The 1:10 dilution of formocresol/eugenol strongly and very significantly decreasedthe AI by 94% (P< 0.01). Inhibition (half of the maximal)ofAI (ICs0) 60 was obtained at 1:327.3 formocresol/eugenoldilution. Formocresolalone at a final dilution of 1:1000 did not significantly decreased the AI of macrophages(P > 0.05). However,the 1:100 dilution of formocresol significantly decreased the AI by 26%(P < 0.05). The 1 : 10 dilution of formocresoldecreased the AI of mac- rophages by 78%(P < 0.05). Inhibition (half of 2O maximal) of AI (IC50) was obtained at 1:48.5 formocresol dilution. Discussion C 1:1000 1:100 1:10 In our study we demonstrate that both formocresol alone and formocresol/eugenol decrease the in-vitro Dilution substrate-adherencecapacity of rat peritoneal macroph- Fig1. Dilution-effectcurvefor the effects offormocresol aloneand ages to plastics surfaces. Althoughit has been shown formocresol/eugenolonAI of rat peritoneal macrophages. Macrophages that the adherence of macrophagesto plastic requires cell surface characteristics different fromadherence to (2-4x 106/inl)were incubated at 37°C in humidified,5 %2 inthe absence(C)or presence ofdecreasing dilutions (increasing collagen, fibronectin, or other cells, 29 the macrophage concentrations)offormocresol alone(C) and formocresol/eugenol (e). adhesion to a smoothplastic surface is comparableto After15 mm reaction wasstopped andthe AI calculated. Eachpoint is the that takingplace in animaltissues. 2c’-28’30.31 meanoffive experiments performed intriplicate. * P < 0.05 vs. control The sensitivity of cells to formocresol alone and (C):** P<0.01 vs. control(C). formocresol/eugenoldilutions as high as 1:100, which 178 American Academy of Pediatric Dentistry Pediatric Dentistry- 20:3, 1998 are similar to those found in periapical tissues, suggests 2. McDonaldRE, Avery DR: Tratamiento de las caries that their inhibitory effect on macrophage adhesion profundas, de la exposici6n pulpar y de los dientes despulpados.In OdontologlaPedi~itrica y del Adolescente, mayhave physiological signficance at the level of peri- 5th Ed. McDonaldRE, Avery DR,Eds. BuenosAires: Edi- apical tissues following formocresol/eugenol torial MddicaPanamericana SA, pp 408-435, 1990. pulpotomy if this substance leaks through the apical 3. MorawaAP, Stratton LH, Han SS, Copron RE: Clinical foramen and invades the periapical zone. evaluation of pulpotomiesusing dilute formocresol. ASDC Formocresol alone was less potent in inhibiting mac- J Dent Child 42:360-63, 1975. rophage