Remineralization of Caries-Like Lesions of Enamel With

Scientific Article

Remineralizationof caries-like lesions of enamelwith acidulatedcalcifying fluids: a polarized light microscopicstudy

Catherine M. Flaitz DDS, MS M. John Hicks DDS, MS, PhD, MD

Abstract 45%of 10-year-old children and 72% of 14-year-old adolescents have experiencedcaries. 1, 2 By age 17 years, The purposeof this in vitro study was to evaluate the dental caries has occurred in almost 85%of these late effects of acidulated calcifying fluids (CF) adolescents.i, 2 The caries prevalence data from the remineralization of caries-like lesions of enamel. Lesions 1 were created in sound enamelusing an acidified gel. Cen- NIDRsurvey represent only clinically detectable caries and do not include caries that would be diagnosed tral longitudinal sections weretaken from each tooth (N 20)following lesion formation to serve as control lesions by radiographic examination. Furthermore, it is well (CL). Each tooth was sectioned into quarters and each knownthat enamel caries may exist for a considerable quarter was assigned to one of four groups: 1) 1 mMcal- length of time prior to either radiographic or clinical detection. 4 The prevalence of such undetectable white cium CFat pH 7.0 (CF1); 2) 1 mMcalcium CF at pH (ACF1); 3) 3 mMcalcium CF at pH 7.0 (CF3); 4) spot lesions within the pediatric, adolescent or adult calcium CFat pH5.0 (ACF3).Tooth quarters were treated populations is not known. This information reinforces the need to develop innovative techniques and alter with assigned CFs(HAP [Ca/P = 1.63], pH7.0 or 5.0, 0.05 mMFl) for ten 60-sec periods. Longitudinal sections were existing regimens for prevention and remineralization of both clinically detectable enamellesions with intact preparedand imbibedin water for polarized light study. Mean lesion depths were determined and compared surfaces and clinically undetectable white spot lesions. A variety of calcifying fluids, synthetic salivas, and (ANOVAand DMR). With I mMcalcium CFs, mean le- oral fluids have been developed in the past to sion depths were 187 l~m for CL, 154 l~m for CF1and 133 ~tm for ACF1. With 3 mMcalcium CFs, mean lesion remineralize enamel caries, s-~7 Unfortunately, remin- depths were 192 t~m for CL, 172for CF3and 149 for ACF3. eralization of naturally occurring white spot lesions Acidulated (22%, 29%) and nonacidulated (10%, 18%) and caries-like lesions of enamel requires numerous CFsresulted in significant reductions(P < 0.05) in lesion exposures to the treatment fluid for considerable time depths when comparedwith control lesions. Acidulated periods. Recently, acid-etching of caries-like lesions of CFs(13%, 14%)resulted in significant lesion depth re- enamel prior to treatment with calcifying fluids has ductions (P < 0.05) when comparedwith nonacidulated been shownto enhance remineralization, using a regi- CFs. The acidulated I mMcalcium CF producedthe great- men that would appear to be clinically applicable/ est degree of remineralization. Acidulation of CFs en- With this encouraging information, it maybe possible hancedthe degree of remineralization over that attained to facilitate remineralization by acidulating calcifying fluids, thereby eliminating the need for the acid-etch by nonacidulated CFs. This improvement in remineralizingability of acidulated calcifying fluids may step prior to calcifying fluid treatment. be due to creation of a more reactive enamel surface. The purpose of this in vitro study was to evaluate the effects of acidulation of synthetic calcifying fluids (Pediatr Dent 18:205-9, 1996) containing 1 mMand 3 mMcalcium on remineralization of caries-like lesions of enamelusing polar- ental caries within the pediatric and adolescent ized light microscopic techniques. ~population continues to be a disease of consid- DLerable clinical importance. - Althougha recent Methods and materials NIDRcaries prevalence surveyI indicated that slightly Twenty macroscopically caries-free human molar less than 50%of children and adolescents were caries- teeth were chosen for this laboratory study. Following free, these results are somewhatmisleading. 2 In fact, a fluoride-free prophylaxis, the specimens were coated

PediatricDentistry - 18:3,1996 AmericanAcademy of PediatricDentistry 205 with an acid-resistant varnish except for two windows ing polarized light microscopic techniques. Photomi- of sound enamel on both buccal and lingual surfaces. crographs of the lesions were projected onto a digi- The specimens were then exposed to a dialyzed-recon- tized tablet and five measurements were made along stituted acidified gel18 (1.0 mMcalcium, 0.6 mMphos- the advancing front of the body of the lesion to de- phate, 0.05 mMfluoride at pH 4.75 _+ 0.02) to create termine mean body of the lesion depth. In addition, cariesqike lesions in the exposed enamel windows. mean surface zone depth was obtained by taking five After 10 weeks, central longitudinal sections were pre- measurements along the inner aspect of the surface pared from each specimen to serve as control lesions zone. Data from 40 paired lesions from each of the prior to experimental treatment. After obtaining cen- experimental and control groups were available for tral longitudinal sections, the teeth were sectioned into statistical analysis. Comparisons were made among quarters and specific quarters from each tooth were the mean depths for each group using ANOVAand assigned to a treatment group: Duncan’smultiple range test for a paired design with 1.1 mMcalcium calcifying fluid (CF) at pH 7.0 an alpha level of P 0.05. (mesiolingual) Results 2. 1 mMcalcium CF at pH 5.0 (mesiobuccal) 3.3 mMcalcium CF at pH 7.0 (distolingual) With I mMcalcifying fluids, lesion depths for both 4. 3 mMcalcium CF at pH 5.0 (distobuccal). nonacidulated (pH 7.0) and acidulated (pH 5.0) After tooth quarter preparation, a fluoride-free (Table, Fig 1) were significantly reduced (P < 0.05) toothbrush prophylaxis was performed to remove re- whencompared with that of the paired control lesions. sidual acidified gel from the specimens. Acid-resistant The body of the lesion depth was reduced by 18%with varnish was applied to the cut faces of the specimens the nonacidulated CF and by 29%with the acidulated CF. Acidulation of the 1 mMcalcium CF resulted TABLE. REMINERALIZATIONOF CARIES-LIKE LESIONS OF ENAMEL: in a further 14%reduc- EFFECTOF ACIDULATEDCALCIFYING FLUIDS tion in body of the lesion Surface Zone DepthBody of Lesion Reduction depth when compared (Mean + SD) Depth (Mean + SD) in Lesion Depth with paired lesions treated with the non- 1 mMcalcium calcifying~fluids acidulated CF (P < 0.05). The mean surface zone Control lesions 22-+ 5 l~m 187-+g’h 19 ~m depth with the acidu- 18% e r~ ’1 lated CF was increased Calcifying fluid at pH7.0 19_+4 Bma, 154_+15 ~mg,i, I 2.9%o by 18% (P > 0.05) and a’ f h’ 37% (P < 0.05) when Calcifying fluid at pH5.0 26_+ 4 lum 133+ 12 ~rn i, n compared with paired 10%I 3 rnMcalcium calciJidingyquids control lesions and 11% nonacidulated CF- treat- Control lesions 18b,c + 6 ~u-n 192k _+ 171.tmJ, ed lesions, respectively. 10%’1 With 3 mMcalcifying Calcifying fluid at pH7.0 29 _ 5 ~Ffl b’ d, e 172_+ 13 ~mi,t, m I 22%I fluids, acidulation re- 13% suited in a 22%reduc- Calcifying fluid at pH5.0 37 + 7 Brnc’ a, f 149-+ 15 l~nk’ 1, n I tion (P < 0.05) in mean body of the lesion depth when compared with ANOVA& DMR-paired sample results: meanswith same letters significantly different (P < 0.05). that for paired control and adjacent to the enamel windows, leaving two win- lesions (Table, Fig 2). In contrast, remineralization dowswith caries-like enamel lesions per tooth quarter the lesions with the nonacidulated CF produced a exposed. The 1 rnM and 3 mMcalcium calcifying flu- 10% decrease in lesion depth when compared with ids at pH7.00 _+ 0.02 and pH5.00 _+ 0.02 were prepared control lesions (P < 0.05). The body of the lesion from hydroxyapatite (Ca:P ratio = 1.63), with addition depth was reduced by 13%with acidulation of the 3 of sodiumchloride to adjust ionic strength and 0.05 mM mMcalcium CF (P < 0.05) when compared with the (1 ppm) fluoride. 12,13,17 The specimens underwent 10 nonacidulated CF. Surface zones from lesions treated separate 60-sec exposures to the assigned calcifying with either nonacidulated (61%) or acidulated (105%) fluid (2 ml/specimen). After each exposure period, the CFs had significant (P < 0.05) depth increases when specimens were rinsed with deionized distilled water compared with paired control lesions. Acidulation of for 30 min. After the final treatment and following the the 3 mMcalcium CF resulted in a further 28%in- water rinse, longitudinal sections from each tooth quar- crease in surface zone depth when compared with ter were taken, imbibed in water, and examined us- nonacidulated CF (P < 0.05).

206 American Academy of Pediatric Dentistry Pediatric Dentistry - 18:3, 1996 more reactive, somewhat porous enamel surface.19" -' It is well known that the acid-etch technique allows for creation of char- acteristic etching patterns and microporosities that Fig 1: Remineralization of caries-like lesions of enamel with 1 mm calcium calcifying fluid extend into the enamel to (arrow = surface zone; B = body of lesion; water imbibition, polarized light microscopy; depths of 50-100 pm; original magnification 250x). thereby allowing resin a. Representative paired lesion from control group. material to penetrate into b. Representative paired lesion remineralized with nonacidulated (pH 7.0) calcifying fluid. the etched enamel.23-25 c. Representative paired lesion remineralized with acidulated (pH 5.0) calcifying fluid. In contrast, the acidulation of topical fluoride agents provides a means for enhanced and rapid substitution of hydroxyl groups for fluorine ions.20"22'26"28 The incorpo- ration of fluoride into hydroxyapatite produces a fluoridated hydroxyapa- Fig 2: Remineralization of caries-like lesions of enamel with 3 mm calcium calcifying fluid (arrow = surface zone; B = body of lesion; water imbibition, polarized light microscopy; tite, which has a signifi- original magnification 250x). cantly increased solubil- a. Representative paired lesion from control group. ity coefficient and is able b. Representative paired lesion remineralized with nonacidulated (pH 7.0) calcifying fluid. to resist demineralization c. Representative paired lesion remineralized with acidulated (pH 5.0) calcifying fluid. by organic acids produced by dental plaque When comparisons were made between the 1 mM to a greater extent than hydroxyapatite. calcium CFs and the 3 mM calcium CFs, certain differ- The current in vitro investigation took advantage of ences were noted (Table 1). The nonacidulated and the beneficial effects of acidulation to enhance acidulated 1 mM calcium CFs possessed significant remineralization of caries-like lesions of enamel by syn- reductions (10 and 11%, P < 0.05) in body of the lesion thetic calcifying fluids. Although significant depths when compared with their corresponding 3 mM remineralization may be achieved with calcifying flu- calcium CFs. For all calcifying fluids, the greatest re- ids at neutral pH,7'8; 10"17 our study demonstrated the duction in body of the lesion depth was in with those advantage of acidulation of calcifying fluids. While the lesions treated with the acidulated 1 mM calcium CF. nonacidulated calcifying fluids produced reductions in In contrast, surface zone depth was increased signifi- lesion depths of 18% for the 1 mM calcium CF and 10% cantly (42 and 53%, P < 0.05) with the 3 mM calcium for the 3 mM calcium CF, acidulation of these CFs. In addition, the greatest increase in surface zone remineralizing agents resulted in significantly greater depth was seen with the acidulated 3 mM calcium CF. lesion depth reductions (29% for 1 mM calcium CF and 22% for 3 mM calcium CF). This degree of Discussion remineralization was realized while maintaining intact Dental scientists initially explored the effects of di- negatively birefringent (pore volume < 5%) surface lute acid on prevention and restoration of dental car- zones. In fact, the surface zone depths were substan- ies during the mid 1950s and early 1960s.19-22 Currently, tially increased following treatment with acidulated the acid-etch technique is a well-established clinical calcifying fluids when compared with nonacidulated procedure used to retain preventive and restorative calcifying fluids. The findings in our study compare resin materials; while acidulated phosphate fluoride favorably with those previously reported7 when acid gels and rinses are the preferred professionally applied etching of caries-like lesions of enamel preceded topical fluoride agent for caries prevention in most remineralization with calcifying fluids. In that particu- dental practices. Both the acid-etch technique and lar in vitro study,7 the combination of acid etching with acidulation of sodium fluoride with phosphoric acid calcifying fluid treatment resulted in a lesion depth depend upon creating a certain degree of enamel dis- reductions of 34% for 1 mM calcium CF and 24% for 3 solution to produce the desired effects.19'28 The action mM calcium CF. Surface zones from the etched, of the acidic environment of etching solutions and remineralized lesions were increased by more than acidulated phosphate fluoride results in removal of su- 80% with both 1 mM calcium and 3 mM calcium CFs. perficial organic pellicle and debris, and exposure of a Both this and previous in vitro studies7 utilized the

Pediatric Dentistry - 18:3,1996 American Academy of Pediatric Dentistry 207 same experimental design with ten separate, 60-sec adaptation of calcifying fluids for prophylaxis against exposures to the calcifying fluids. The substitution the formation of clinically detectable lesions, and the of acidulation of the calcifying fluids for acid etch- reversal of clinically undetectable white spot lesions ing the lesions prior to calcifying fluid treatment and hypocalcified enamel. appears to have a similar effect on remineralization of caries-like enamel lesions. Conclusions The pattern of enamel caries remineralization and The conclusions from this in vitro polarized light mechanism of mineral deposition depend on the satu- microscopic study that evaluated the effects of acidu- ration levels of the mineral phases composing the cal- lated calcifying fluids on remineralization of cariesqike cifying fluid. 1°,13,14,17 Prior analysis of the 3 mMcalcium lesions of enamel are: CF has indicated certain mineral phases are supersatu- 1. Remineralization of caries-like lesions of enamel rated and include hydroxyapatite, fluorapatite, may be accomplished with both acidulated and octacalcium phosphate, and tricalcium phosphate. Be- nonacidulated calcifying fluids using a series of cause of these supersaturated mineral phases in the 3 relatively short treatment periods. mMcalcium CF, rapid precipitation of newly formed 2. The greatest lesion depth reduction was realized crystals -- termed crystal nucleation-- within the pore with the acidulated calcifying fluid containing structure of enamel caries is the mechanism involved I mMcalcium; however the greatest increase in in remineralization. Remineralization by nucleation surface zone depth was found with the acidu- results in the formation and deposition of small diam- lated calcifying fluid containing 3 mMcalcium. eter (50 to 75 nm) crystals within the surface zone and 3. Acidulation of calcifying fluids enhanced the superficial aspect of the body of the lesion. Due to the degree of remineralization over that attained by rapid deposition of crystals, the more superficial aspect nonacidulated calcifying fluids. This improve- of the lesion is remineralized, and access to the under- ment in remineralizing ability of acidulated cal- lying pore structure of the enamel lesion is obstructed. cifying fluids may be due to the removal of su- With the I mMcalcium CF, remineralization of enamel perficial organic pellicle and debris, and the lesions involves a more prolonged deposition of min- creation of a more reactive enamel surface. eral phases on existing crystals within the lesion, Dr. Flaitz is associate professor, divisions of oral pathologyand termed crystal growth. This results in a more gradual pediatric dentistry, University of Texas-HoustonHealth Science "plugging" of porosities within the lesion and allows Center, Houston,Texas. Dr. Hicks is assistant professor, depart- access to the more deeply situated regions of the lesion. ment of pathology, Baylor College of Medicine,Texas Children’s Remineralization of enamel lesions with the I mMcal- Hospital, and adjunct professor, division of pediatric dentistry, University of Texas-HoustonHealth Science Center, Houston, cium CF by crystal growth produces crystal diameters Texas. of 50-150 nm within the lesion. This phenomenon oc- curs because only hydroxyapatite and fluorapatite are 1. National Caries Program, NIDR.Epidemiology and oral disease prevention program: oral health of United States supersaturated in this remineralizing fluid. The mecha- school children: the national survey of dental caries in US nisms of caries remineralization for the I mMcalcium school children: 1986-1987. NIHPublication No. 89-2247, CF by crystal growth and 3 mMcalcium CF by crystal Bethesda, MD:National Institutes for Health, 1989. nucleation provide an explanation for the differences 2. Hicks MJ, Flaitz CM:Epidemiology of dental caries in the pediatric and adolescent population: a review of past and in remineralization pattern between these calcifying current trends. J Clin Pediatr Dent 18:43-49, 1993. fluids. With the 3 mMcalcium CF, the more superfi- 3. Hicks MJ, Flaitz CM,Silverstone LM:The current status of cial aspect of the lesion tends to be affected; whereas, dental caries in the pediatric population. J Pedod10:57-62, with the i mMcalcium CF, the entire lesion depth may 1985. be affected, This difference results in a greater reduc- 4. Silverstone LM:Relationship of the macroscopic,histologi- cal and radiographic appearanceof interproximal lesions in tion in lesion depth for 1 mMcalcium CF than for 3 mM humanteeth: in vitro study using artificial caries technique. calcium CF, but a greater increase in surface zone depth Pediatr Dent 3:414-22, 1981. for 3 mMcalcium CF than for I mMcalcium CFo Both 5. Redelberger T, Klinger HG,Wiedemann W: Effect of pore remineralizing fluids may provide significant protec- topology on remineralization rate of surface-softened tion against caries progression. enamel and synthetic hydroxyapatite: an experimental com- parison and mathematicalmodel. Caries Res 25:88-95, 1991. Remineralization may be enhanced significantly by 6. Hicks MJ, Flaitz CM,Silverstone LM:Initiation and progres- acidulation of synthetic calcifying fluids while main- sion of caries-like lesions of enamel:effect of periodic treat- taining an intact surface over the enamel lesion. Al- ment with synthetic saliva and sodiumfluoride. Caries Res though our study was carried out in a laboratory set- 19:481-89,1985. 7. Flaitz CM,Hicks MJ: Role of the acid-etch technique in ting, the experimental regimen and treatment times remineralizationof caries-like lesions of enamel:a polarized may be transferred easily to the clinical setting. Perhaps light and scoanningelectron microscopicstudy. ASDCJ Dent in the near future, clinical investigations into Child 61:21-28, 1994. remineralization of clinically detectable white spot le- 8. LammersPC, Borggreven JMPM,Driessens FCM:Influence sions may be realized. Just as important is the clinical of fluoride on in vitro remineralizationof artificial subsur-

208 AmericanAcademy of Pediatric Dentistry PediatricDentistry - 18:3, 1996 face lesions determinedby a sandwichtechnique. Caries Res 19. Buonocore MG:A simple method of increasing the adhe- 24:81-85, 1990. sion of acrylic filling materials to enamelsurfaces. J DRes 9. Collys K, Cleymaet R, CoomansD, Slop D: Acid-etched 34:849-53,1955. enamelsurfaces after 24 hr exposureto calcifying mediain 20. BrudevoldF, SavoryA, GardnerDE, Spinnelli, M, Speirs R: vitro and in vivo. J Dent19:230-35, 1991. Studyof acidulated phosphatefluoride solutions I: in vitro 10. Silverstone LM,Hicks MJ, Featherstone MJ: Dynamicfac- effects on enamel. ArchOral Biol 8:167-77, 1963. tors affecting lesion initiation and progression in human 21. AasendenR, Brudevold F, McCannHG: The response of in- dental enamel. Part I. The dynamicnature of enamel car- tact and experimentally altered humanenamel to topical ies. QuintessenceInt 19:683-711,1988. fluoride. Arch Oral Biol 13:543-52,1968. 11. G~inglerP, HoyerI: In vivo remineralization of etched hu- 22. DePaolaPF, AasendenR, BrudevoldF: The use of topically manand rat enamel. Caries Res 18:336-43, 1984. applied acidulated phosphate fluoride preceded by mild 12. Silverstone LM:Remineralization phenomena.Caries Res etchingof the enamel:a one year clinical trial. ArchOral Biol 11(suppl 1):59-84, 1977. 16:1155-63,1971. 13. Silverstone LM, Wefel JS, ZimmermanBF, Clarkson BH, 23. HicksMJ, Silverstone LM:Acid-etching of caries-like lesions Featherstone MJ:Remineralization of natural and artificial of enamel: a polarized light microscopicstudy. Caries Res lesions in humandental enamel in vivo. Effect of calcium 18:315-26,1984. concentration of the calcifying fluid. Caries Res 15:138-57, 24. Hicks MJ,Silverstone LM:Acid-etching of caries-like lesions 1981. of enamel:a scanningelectron microscopicstudy. Caries Res 14. Silverstone LM:Remineralization and enamel caries: new 18:327-35,1984. concepts. Dent Update10:261-73, 1983. 25. Silverstone LM:Fissure sealants: laboratory studies. Caries 15. ten Cate JM, ArendsJ: Remineralizationof artificial enamel Res 8:2-26, 1974. lesions in vitro. Caries Res 11:277-86,1977. 26. Joyston-Bechal S, DuckworthR, Braden M: The mechanism 16. Gelhard RBFM,ten Cate JM, Arends J: Rehardeningof arti- of uptake of F18 by enamel from NaF and APFsolutions ficial enamellesions in vivo. Caries Res 13:80-83,1979. labeled with F18. Arch Oral Biol 18:1077-89,1973. 17. Silverstone LM,Wefel JS: The effect of remineralization on 27. Nancollas GH:Physicochemistry of demineralization and artificial caries-like lesions and their crystal content. J Crys- remineralization. J Dent Res 53:297-302,1974. tal Growth53:148-59, 1981. 28. Hicks MJ, Flaitz CM,Silverstone LM:Fluoride uptake in 18. Feagin FF, Clarkson BH, Wefel JS: Chemicaland physical vitro of soundenamel and caries-like lesions of enamelfrom evaluation of dialyzed-reconstituted acidified gelatin sur- fluoride solutions of relatively low concentration. J Pedod face lesions of humanenamel. Caries Res 19:219-27, 1985. 11:47-61, 1986.

Mild illness no reason to delay childhood immunizations

RESEARCHERSSAY MISSED VACCINATIONS CONTRIBUTE TO has not been universally followed, delaying vaccina- DISEASEOUTBREAKS tions for some children," according to the research- There is no reason to delay vaccinating mildly ill ers, who point to concerns among many physicians children for measles, mumps and other ailments, of vaccine failure and increased side effects when according to an article in a recent issue of The Jour- vaccines are administered during an acute illness. hal of the American Medical Association. In the current study, 157 of the 386 children had Gall E. King, MD, MPH, Centers for Disease one or more mild illnesses including upper respira- Control and Prevention, and colleagues studied 386 tory tract infection, ear infection, and diarrhea. children, aged 15-23 months, at six county health The researchers found seroconversion (develop- department immunization clinics in the Atlanta area ment of antibodies) rates to measles, mumps and from February 1992 to April 1993. They examined rubella antigens slightly, but not significantly, higher the response to the measles-mumps-rubella vaccine for children with mild illness compared to children among children with and without illness. who were well at the time of immunization. For The researchers write: "Vaccinations that are instance, the seroconversion rate for measles was missed because of mild illness are an important cause 99% for children with a mild illness compared to of under-vaccination among preschool and children 97% for well children. And, multiple symptoms in in high-risk populations and have contributed to the mildly ill children were not found to increase the occurrence of disease outbreaks. Reducing all missed likelihood of vaccine failure. opportunities to vaccinate is one of the standards for Overall seroconversion rates were 98% for pediatric immunization practices ... (and) is also measles, 83% for mumps and 98% for rubella. important component in the strategy for reaching The researchers conclude: "Mild illness or a his- the U.S. disease reduction and immunization goals tory of recent mild illness is not associated with for the year 1996." reduced seroconversion to MMRor with increased The American Academy of Pediatrics has long adverse events, and such illnesses are not associated advised physicians not to view mild illness as a rea- with a reduced level of antibody response to MMR son to delay vaccinations, but the "recommendation vaccine."

Pediatric Dentistry- 18:3, 1996 AmericanAcademy of Pediatric Dentistry 209