Advances in Dental Research

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The Application of in Vitro Models to Research on Demineralization and Remineralization of the Teeth D.J. White ADR 1995 9: 175 DOI: 10.1177/08959374950090030101 The online version of this article can be found at: http://adr.sagepub.com/content/9/3/175

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THE APPLICATION OF IN VITRO MODELS TO RESEARCH ON DEMORALIZATION AND REMINERALIZATION OF THE TEETH D.J. WHITE

The Procter & Gamble Company HCRC P.O. Box 8006 Mason, Ohio 45040-8006 Adv Dent Res 9(3): 175-193, November, 1995

Abstract—Progress in in vivo and in situ experimentation has led many researchers to speculate as to the relevance and importance of in vitro testing protocols in caries research. A Medline/Biosis search for the present review revealed well over 300 citations (since 1989) documenting in vitro tests associated with caries research on mineralization and fluoride reactivity. The present survey documents these recent applications of in vitro test methods in both mechanistic and 'profile'* caries research. In mechanistic studies, in vitro protocols over the past five years have made possible detailed studies of dynamics occurring in mineral loss and gain from dental tissues and the reaction dynamics associated with fluoride anticaries activity. Similarly, in profile applications, in vitro protocols make possible the inexpensive and rapid—yet sensitive—assessment of F anticaries efficacy within fluoride-active systems, and these tests represent a key component of product activity confirmation. The ability to carry out single variable experiments under highly controlled conditions remains a key advantage in in vitro experimentation, and will likely drive even further utilization, as advances continue in physical-chemical and analytical techniques for substrate analysis in these protocols. Despite their advantages, in vitro testing protocols have significant limitations, most particularly related to their inability to simulate the complex biological processes involved in caries. Key words: In vitro, caries, demoralization, remineralization. Presented at the Conference on Clinical Aspects of De/Remineralization of Teeth, June 11-14, 1994, Woodclijf Conference Center, Rochester, NY * "Profile ", for the purposes of this paper, refers to test protocols used by industrial firms in the screening of topical agents for efficacy in standardized tests aimed to meet American Dental Association or Food and Drug Administration requirements. ADA standardized tests include use of appropriate control agents/products as detailed in the proceedings of the 1990 Model Systems Conference.

I

t is safe to conclude that in vitro experiments are the most commonly applied methods in dental research. The scope and applicability of in vitro testing methods for demineralization, remineralization, and fluoridation were reviewed in detail at preceding conferences, including the 1990 Workshop on Technological Advances in Intra-oral Model Systems to Assess Cariogenicity [J Dent Res 71(4)(Spec Iss), 1992] and the 1989 Joint IADR/ORCA International Symposium on Fluorides [J Dent Res 69(2)(Spec Iss), 1990]. At the 1990 conference, this author undertook a review of various model systems used to evaluate the anticaries efficacy of topical fluoride agents. In that paper, the relative advantages and limitations of in vitro laboratory testing methods were compared with in situ and animal models (White, 1992). The literature search associated with this current review demonstrated that in vitro models/protocols remain at the forefront of caries research. A Medline/Biosis search of published and abstract literature revealed well over 300 stand-alone in vitro model studies carried out since 1989, encompassing demineralization, remineralization, and fluoridation of enamel and/or dentin. The purpose of the present review is to update the current state of the art in in vitro model testing of demineralization and remineralization evolving from the preceding conferences and summaries of recent literature. The review will be focused on two primary areas of laboratory research: (I) studies devoted to measurement of mineral changes in enamel and changes effected by topically applied agents, including remineralization, demineralization, and pH cycling protocols; and (II) studies devoted to measures of topical fluoridation efficiency, including fluoride uptake, retention, and release. IN VITRO STUDIES

OF REMINERALIZATION, DEMINERALIZATION, PH CYCLING (COMBINED) DE- AND REMINERALIZATION In vitro studies of remineralization— models and recent applications In vitro remineralization models include experiments directed toward determining fundamental factors important to caries lesion repair and the efficacy of treatment modalities in potentiating lesion consolidation. Remineralization protocols can be grouped into three general categories. In pH-lattice ion 'drift' methods, substrates are exposed to constant-volume supersaturated remineralization solutions, and crystallization is allowed to proceed non-

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WHITE

Calcium

ADV DENT RES NOVEMBER 1995

Calcium

1 5s

o§ c

Phosphate

o

I 3H 2 Levels in Saturated Solution

1 -

20

40

60

80

100

120

140

20

40

60

80

100

120

140

Remineralization Time

Remineralization Time

Fig. 1— Simulation of calcium and phosphate lattice ion concentration changes which typically occur in 'drift' experimental protocols.

Fig. 2—Simulation of calcium and phosphate lattice ion concentrations in constant-composition studies.

controlled, with associated decreases in lattice ion calcium and phosphate and, in unbuffered systems, pH. Fig. 1 illustrates concentration changes associated with mineralization processes in these experiments. These methods have the advantage of direct chemical measurement of remineralization within a given exposure period (ten Cate and Duijsters, 1982); however, they are limited in that the thermodynamic supersaturation driving remineralization decreases rapidly during treatment, thereby artificially restricting mineral redeposition. The relative thermodynamic supersaturation can be defined in terms of the activity products of ions contributing to the solubility product of the enamel phase in equilibrium—approximated as hydroxyapatite, as follows:

either flow-through (high volume) techniques or titrationcontrolled supersaturation. In constant-composition experiments, lattice ion concentrations and pH remain constant throughout remineralization, as illustrated in Fig. 2. In the flow-through techniques, supersaturation during remineralization is kept constant by means of a high volume of remineralization medium, thereby ensuring a constant thermodynamic driving force during remineralization (Buskes et al, 1985; Herkstroter et al, 1991). Flow-through designs permit multi-group studies to be carried out. The measurement of remineralization is confined to substrate changes. Fig. 3 shows the experimental design for the study of remineralization under flow-through constant-composition conditions (from Buskes et al, 1985). In titration-controlled constant-composition studies, the composition of the remineralization media is maintained by the controlled addition of calcium phosphate lattice ions and buffer titrants monitored potentiometrically by pH and/or calcium ionselective electrodes (Amjad et al, 1981; White et al, 1988b), as illustrated in Fig. 4. These techniques use the principles established for crystallization studies of apatites, as described originally by Nancollas and colleagues (Koutsoukos et al, 1980; Tomson and Nanccllas, 1978). The advantage of the titration-controlled constant-composition techniques is direct measurement of remineralization by solution changes (in this case titrant) without the limitations of changing supersaturation. Drawbacks of these techniques are the requirement for extra titrant assemblies for multi-group experiments and difficulty in predicting the phase composition of remineralizing minerals—a requirement to ensure titrant compensation of static supersaturation. Substrates used for in vitro remineralization studies typically include enamel or dentin slabs prepared with

K l/9

(1)

where the parentheses denote ion activities in solution, and K SP(HAP) represents the solubility product of hydroxyapatite mineral. In the presence of fluoride, Eq. (1) is modified in that the operative solubility product may be that of a fully or partially substituted fluoroapatite, and the ion product must include contributions from the fluoride ion activity in solution. (The calculation of the effective thermodynamic driving force in fact further depends upon ion association in plaque fluid and saliva and is the source of continued research.) The changes in supersaturation during remineralization can have an effect not only on the amount of mineral formed but also on both the type and location of mineral phases deposited (Nancollas, 1982; White etal, 1988b; LeGeros, 1991). The other two types of in vitro remineralization protocols include constant-composition methods which encompass


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177

IN VITRO MODELS FOR CARIES TESTING

I

00

00

,,H Combustion |lmpulso>

Double-Walled Reaction Vessel—* Thermostated at37C

Fig. 4—Constant-composition titration-controlled system for remineralization experiments. Here, titrants compensate for remineralization, with titrant amounts indicative of reaction.

Fig, 3—Experimental set-up of a typical constantcomposition 'high volume' system (from Buskes et al., 1985). Enamel specimens are held in H, as shown in panel 'a'. Mineralization titrant flows over specimens, as shown in '£>'. Electrodes E and sensor S ensure proper solution delivery to specimens. Solution flows through inlet (1) and outlet (2). The ability to mineralize/demineralize multiple treatment groups of specimens simultaneously is an advantage of this system. (Reproduced with permission o/Caries Research, S. Karger Publishers.) artificial caries lesions (e.g., White, 1987a) or in some cases directly include sections of natural lesions (Silverstone, 1983). More recently, researchers have applied the use of single sections of artificial caries lesions in in vitro remineralization models. The single-section substrates are highly valued in intra-oral models to reduce specimen variability associated with remineralization/demineralization (ten Cate, 1986; Mellberg et al., 1992; Stephen et al., 1992; Wefel and Jensen, 1992). In principle, single sections provide investigators with the ability to measure mineral changes accurately at repeated time periods of exposure, with re-

introduction of specimens into media to continue the process. Because of the limited surface area of the substrate, these techniques represent constant supersaturation without the need for flow-through or 'high volume' experimental logistics. Figs. 5 and 6 show the preparation and use of single sections in typical in vitro experiments. In some instances, single-section studies provide an ideal means to elucidate mechanistic factors important to caries prevention. For example, in a recent study, Exterkate et al. (1993) used single sections to examine rate and localization of remineralization of subsurface lesions at various Ca/P ratios and supersaturations. They observed that initial remineralization rates in subsurface lesions are extremely fast, followed by reduced rates with time. This result matches previous observations in the literature (ten Cate, 1990) and should be carefully considered by in situ researchers who place freshly prepared artificial caries lesions in intra-oral appliances and measure remineralization activity over extremely short periods. These researchers further observed that remineralization was easily localized to the surface zone and that Ca/P ratios strongly influenced the location of mineral deposition, with lesion consolidation encouraged under Ca/P ratios resembling apatite stoichiometry. Obviously, these types of insights are difficult to derive from in vivo or animal research experiments, thus highlighting the advantage of experimental control provided within in vitro models. Along similar lines, numerous in vitro investigations continue to examine in detail important features associated with remineralization which would be difficult if not impossible to study in vivo. These include mechanistic investigations of dentin remineralization (Arends et al., 1989b, 1990a; Clarkson et al. 1991; Klont and ten Cate, 1990, 1991), studies of the influence of lesion structure on remineralization/demineralization (Fusayama, 1991; Klont and ten Cate, 1991; Redelberger et al., 1991; Larsen and Pearce, 1992; Collys et al., 1993), studies of the influence of


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Solution in *

enamel slab from bovine incisor Demineralizing Solution

Vent

- » Solution out

Section

X-ray beam

Exposed surface

Fig. 6—Cell for flow-through mineralization of a single section, 'x' indicates surface against which microradiograms are measured. (Reproduced from Anderson and Elliott, 1992, with permission of the J Dent Res, American Association for Dental Research, publisher.)

embedding

trimming and removal of outer surface Fig. 5—Procedures involved in single-section preparation for in vitro or in situ research. Lab = labial surface. (Reproduced from Exterkate et al., 1993, with permission of the J Dent Res, American Association for Dental Research, publisher.)

solution chemical factors on remineralization and crystal growth (supersaturation, F, CO3, etc.) (Featherstone et al, 1990; Goldberg et al, 1990; Lammers et al. 1989, 1990, 1991a,b, 1992), and studies of the effects of surface-active agents and treatments on remineralization processes (Kiss et al, 1990; Borggreven et al, 1991; Johnsson et al, 1991; Arends et al, 1992a; Raj et al, 1992; Reddy and Indushekar, 1992; Richardson et al, 1993). In vitro studies of demineralization— models and recent applications Tooth decay is the result of progressive mineral loss from dental tissues. In vitro demineralization models enable

researchers selectively to examine fundamental processes associated with mineral loss from the teeth. Research on demineralization is extensive and has been reviewed recently (Featherstone et al, 1990; Larsen, 1990; ten Cate, 1990, 1992). As in remineralization, models devoted to demineralization include constant-composition methods (Buskes et al, 1987; Chow and Takagi, 1989; Gao et al, 1993) and methods allowing pH/lattice ion/undersaturation drift. In the latter methods, time-dependent decreases in undersaturation limit the extent of demineralization possible, depending upon the volume/surface area ratio within media. A major difference between demineralization and remineralization models involves substrates used in testing. While remineralization methods typically use lesions (treated or control) as substrate, demineralization models utilize a wider variety of substrates, including those pre-treated with chemical agents (fluoride), pre-treated and remineralized with chemical agents, or natural enamel or root surface lesions. Some recent applications of in vitro demineralization testing include mechanistic studies of solution/substrate factors affecting demineralization (Rentsch et al, 1990; Gao et al, 1991; Redelberger et al, 1991; Anderson and Elliott, 1992), direct comparisons of demineralization (and remineralization) in vitro and in vivo (Collys et al, 1991; Arends et al, 1992a,b), studies of factors contributing to the intrinsic resistance of mineralized tissue to acid demineralization (DeGroot et al, 1986; Creanor et al, 1989; Petzold et al, 1990; Faller et al, 1991; Arends et al, 1992a; Boonstra et al, 1993; Gangler et al, 1993), studies of demineralization kinetics of remineralized lesions (Iijma and Koulourides, 1989; Lammers et al, 1991a, 1992; Iijima et al, 1993; Tanaka et al, 1993), and investigations into the effects of solution- and substrate-bound fluoride on acid resistance (Feagin and Graves, 1988; Chu et al, 1989; Samarawickrama and Speirs, 1993). As would be expected,


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n

2 o


3 -

2 -

Mineral Gained

s Q

1

-

z Baseline Substrate Mineral Content

3 2 UJ

-1— Mineral Gained -2 -

I UJ

1

-3 -

0

O

1

2

3

4

5

6

7

TIME (ARBITRARY UNITS)

Fig. 7—Mineral flux associated with pH-cy cling progression and reversal models.

numerous in vitro demineralization studies continue to be published examining chemical treatments, agents, procedures, and natural substances which may inhibit or promote apatite enamel and dentin dissolution (e.g., Arends et al, 1986, 1989a, 1990a, 1992d; Clarkson et al, 1986; Gedalia et al., 1986, 1991; Okazaki et al, 1986; Crall and Bjerga, 1987; Nieuw-Amerongen et al, 1987; Stabholz et al, 1987; Collys et al, 1988, 1990; Ettinger et al, 1988; Purton and Rodda, 1988; Seppa, 1988; van Dorp et al, 1988; Borggreven et al, 1989; Derand et al, 1989; Ott, 1989; Tadmor et al, 1989; van der Linden, 1989; Boonstra et al, 1990, 1993; Grobler et al, 1990; Yue et al, 1990; Christoffersen et al, 1991; Larsen, 1991; Seppa and Forss, 1991; Donly and Ruiz, 1992; Featherstone et al, 1993; Hell wig et al, 1993; Kautsky and Featherstone, 1993; Lussi et al, 1993; Spets-Happonen et al, 1993; Grogono and Mayo, 1994; Hook et al, 1994; Sorvari et al, 1994a; Souto and Donly, 1994; Wikiel et al, 1994). In vitro demineralization protocols have also recently been applied as diagnostic tests for the modifying effects of laser treatments on enamel and dentin (Tagomori and Morioka, 1989; Oho and Morioka, 1990; Fox et al, 1992; Nammour et al, 1992). In vitro studies of pH cycling— models and recent applications 'pH cycling' refers to in vitro experimental protocols including exposure of substrates, enamel, or dentin, to combinations of remineralization and demineralization. These combination experiments are designed to simulate the dynamic variations in mineral saturation and pH associated

179

with the natural caries process. pH-cycling models, at their most basic, can involve a simple topical treatment of sound/carious enamel with an agent, followed by secondary acid exposures to measure effects on subsequent demineralization rates. Koulourides (1980), in pioneering studies of 'acquired acid resistance', in fact exposed artificial lesions to topical remineralization solutions and measured subsequent resistance of treated areas to secondary acid attacks, thus carrying out some of the first truly 'cycling experiments'. Today, pH-cycling models refer to more complex and detailed types of protocols. The genesis of modern pH-cycling protocols was produced by ten Cate and Duijsters (1982). This protocol involved numerous cycles of demineralization and remineralization, with mineral uptake and loss measured chemically with eventual matching of total mineral flux to distributions assessed by microradiography. In vitro pH-cycling models can generally be classified into progression (Featherstone et al, 1986; White and Featherstone, 1987; Page, 1991) or reversal models (White, 1987a, 1988), depending upon the net flux of mineral to/from the tooth substrate, as shown in Fig. 7. As reviewed by ten Cate (1990, 1992), modern pHcycling models have become methods of choice for many caries researchers applying in vitro techniques, because they provide better simulation of the caries process for both mechanistic studies and for profile evaluations of toothpastes and mouthrinses. The most significant development in the use of in vitro cycling models includes the recent applications of automated cycling techniques and comparison of treatment effects under various demineralization/remineralization ratios. Almqvist, in a series of studies (Almqvist et al, 1990; Almqvist and Lagerlof, 1993a,b), applied a continuous delivery system to provide control of multiple pH cycles and to provide fluoride clearance characteristics simulating intra-oral effects after topical treatments. The authors applied 125I absorptiometry for evaluation of lesion substrate mineral changes. Herkstroter et al (1991) applied automated pH cycling in a flow-through system to examine the effects of surface layers in sound enamel on lesion progress in the presence of fluoride. Robinson et al (1992) used chemical analysis in automated cycling to examine lesion progression under pHcycling conditions. Kirkham et al (1994) used automated cycling to compare the effects of various cycling ratios on lesion progression. Since their inception, pH-cycling protocols have served as powerful means for providing mechanistic insights into the caries process and preventive measures, and applications of these models for these purposes continue today. ADA profile applications of dentifrices in remineralization/demineralization models Following the conference on model systems in 1990, a major focus of researchers was on development of in vitro models meeting suggested ADA guidelines associated with topical evaluations of dentifrices (Chilton, 1992; Proskin, 1992). pH-


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TABLE 1 DOSE-RESPONSE EVALUATION OF DENTIFRICE EFFICACY IN pH-CYCLING LESION REVERSAL (Faller, 1992) AZ pH Cycling (Am x Vol%)

Dentifrice Formulation (ppm F as NaF)

+145 -86 -140 -190 -222

1 275 550 825 1100

cycling models have been the primary focus of efforts toward meeting these guidelines. The majority of this work has been carried out by industry, and unfortunately (for this review), most of this research is only in the form of abstracts. Nevertheless, the results of some of these efforts are cited here to illustrate progress in this regard. Several research groups have demonstrated the ability of pH-cycling protocols to meet dose-response (0, 250 ppm and 1100 ppm F as NaF or SMFP) activity in both enamel and dentin (Dunipace et al, 1992; Faller, 1992; Barrett-Vespone et al, 1993; Kim et al, 1994; Schemehorn et al, 1994). Results for a representative model are shown in Table 1, where the clear dose responses of NaF from 1 to 250 to 1100 ppm F are illustrated in the form of changes in the mineral content of lesions (AZ = area produced by lesion measured in volume % mineral x scanning depth) (Faller, 1992). Protocols successfully meeting ADA guidelines included measures of lesion reversal (Faller, 1992) and progression (Barrett-Vespone et al, 1993). Studies have also examined fluoride toothpaste efficacy in dose ranges above the conventional fluoride levels, in an effort to broaden model clinical relevance (Bowman et al, 1988; Featherstone et al,

1988a; Damato et al, 1990; Heilman et al, 1991). Several more recent studies applying pH-cycling models include those listed in Table 2. Featherstone et al (1992) used their pH-cycling lesion progression model to demonstrate that sodium tripolyphosphate, a tartar control crystallization inhibitor, had no effect on the activity of fluoridated dentifrice. This result matches well with previous results obtained with tartar control toothpastes incorporating pyrophosphate, examined in this same model (Featherstone et al, 1988b, 1989). From a methodological standpoint, Macpherson et al. (1991) used a pH-cycling protocol to observe that enamel collected from different sites exhibited different demineralization sensitivities. Schemehorn et al. (1992) demonstrated that pH-cycling models met validation standards using either bovine or human enamel substrates. In a practical utilization of in vitro techniques separate from F toothpaste evaluation, Joyston-Bechal and Kidd (1987) examined various artificial salivas for their ability to promote remineralization. With respect to utilization in profile applications and the emerging ADA guidelines, this author believes that the conclusions reached in the 1990 review of comparative

TABLE 2 SOME RECENT PROFILE APPLICATIONS OF pH-CYCLING MODELS Reference Barrett-Vespone et al, 1993 Kim era/., 1994 Schemehorn et al, 1994 Barrett-Vespone et al, 1994 Dunipace et al, 1992 Heilman^ a/., 1992 Faller, 1992 Heilman^ a/., 1991 Damato ef al, 1990 Heilman and Wefel, 1994

Remineralization Analysis

Observation

AZ*

NaF dose response SMFP dose response NaF dose response NaF and SMFP dose response NaF > SMFP > placebo NaF dose response NaF dose response NaF > placebo NaF rinse dose response above 1100 ppmF NaF in baking soda paste

Microhardness Microhardness

AZ AZ AZ, polarized light

AZ AZ, polarized light

AZ AZ, polarized light

*AZ = Mineral content (\xm x vol%) within enamel in cross-section.


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models systems still remain valid today (White, 1992). In vitro model systems remain easy to modify and validate in order to simulate clinical efficacy correlations under controlled experimental conditions, ideally permitting single variable experiments to be carried out. On the other hand, in vitro protocols provide only limited information predicting the efficacy of new chemical agents or comparing the clinical efficacy of different agents, due to their restricted capacity to account for the entire biological process involved in caries. Limitations of in vitro remineralization/demineralization models The limitations of in vitro test systems for both mechanistic and profile applications were summarized in a previous review (White, 1992). Overall, in vitro models are mechanistically limited in three key ways: inadequate simulation of biological aspects of caries, difficulty in matching solid/solution ratios occurring in vivo, and artifacts associated with substrate choice/reaction conditions. The inability to match the breadth of relevant biological conditions limits the clinical relevance of in vitro test results and profile applications of these models. From a biological perspective, it is virtually impossible for in vitro models adequately to simulate the complex and diverse intra-oral conditions contributing to caries development. Although researchers have previously investigated in vitro 'artificial mouth' systems, including bacterial plaque and artificial saliva (e.g., Viazis, 1989; Viazis et al, 1990; DeLong and Douglas, 1991; Simonsson et al, 1991; Sissons et al, 1991, 1992; Pearce et al, 1992; Sakaguchi et al, 1992; Douglas et al, 1993; Hellwig et al, 1993; Sorvari et ah, 1994b), the absolute merits of these model systems today must be critically assessed in light of advances in in situ and animal caries protocols. Most recent in vitro studies of de- and remineralization, including dental plaque/saliva, have been directed not toward simulation of 'artificial mouth' conditions, but rather toward examining the effects of selected salivary/plaque properties in a controlled environment in single variable experiments (e.g., Zameck and Tinanoff, 1987; van Loveren et al, 1991; Blake-Haskins et al, 1992; Firestone et al, 1993; Wahab et al, 1993). A second limitation of in vitro protocols is the difficulty in simulating the volume and composition of saliva and tooth surface area encountered in in vivo remineralization. The proper simulation of in vivo remineralization would require knowledge of fluid volume, coverage, and transport over substrate surfaces accounting for dynamic variations under dental plaque (Weatherell et al, 1986; Larsen and Fejerskov, 1989; Oliveby et al, 1990; Macpherson and Dawes, 1991, 1994; Pearce, 1991; Dawes and Macpherson, 1993). A further complication is that it is quite clear that different oral surfaces {e.g., maxillary/mandibular) are bathed in different volumes and source combinations of saliva (Dawes, 1993). Researchers typically attempt to account for saliva volume by varying volume/supersaturation (in 'drift assays') or by bathing media volume (in constant-composition flow-through

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systems). In the case of supersaturation adjustment (i.e., calcium/phosphate content of media), the increases in local concentrations of calcium and phosphate in vitro cannot possibly account for the large volumes of saliva secreted in vivo and in addition can create artifacts of surface-dominated mineralization. Within flow-through systems, larger volumes of remineralization solutions can be used, but these cannot simulate intra-oral surface area/volume precisely. In either case, artificial salivas cannot account for calcium binding and substrate-surface adsorption effects of saliva macromolecules and low-molecular-weight solutes which affect intra-oral mineralization (Arends et al, 1992d; Featherstone et al, 1993; Williams and Wu, 1993). In vitro systems using saliva as a remineralization medium (White 1987b, 1988) can experience artifacts due to extra-oral breakdown of saliva. Unfortunately, the simulation of intra-oral demineralization by in vitro protocols is equally poor. For demineralization, knowledge of acid anion profiles, pH changes on the lesion surface, diffusion rates of acids/salivary buffers, and kinetics of sugar/acid transport would be required for precise simulation of undersaturation variations associated with acid challenges to the tooth surface. Most test methods use constant-concentration acid challenges in solution volumes in large excess to substrate surface area, essentially mimicking constant-composition conditions, which are surely never present in in vivo demineralization. Thus, in vitro models cannot mimic solid surface area/solution ratios and saliva/plaque fluid composition encountered in vivo. These limitations should be kept in perspective in attempts to correlate in vitro changes directly with in vivo responses to agents and treatment modalities. A third mechanistic limitation encountered by in vitro models (and by in situ models as well) involves artifacts associated with choice of substrate and test conditions, particularly the time periods (rates) of de- and remineralization used in studies. With respect to time, it must be considered that in vitro test systems examining de- and remineralization necessarily accelerate the mineral dynamics associated with the caries process and reversal into much shorter time periods than occur in vivo. For example, it is not uncommon today to see the development of from 1 to 3 new carious surfaces, on average, within children in a three-year caries clinical trial. Conservatively assuming that an interproximal lesion must extend 1.5 mm from the enamel surface into dentin for x-ray detection, a newly developing lesion would have advanced 1500 urn within three years, or at a linear (see Arends et al, 1992d) rate of 1-2 urn a day. Artificial caries lesions are routinely placed into in vitro or in situ remineralization studies with lesion depths ranging from 50 to 150 urn. These lesion depths can be taken to represent the equivalent of some three months' clinical formation prior to topical exposure or further pH cycling. Such dynamics do not occur in vivo. In pH-cycling (progression) or demineralization studies, researchers typically see the development of 100-250-um lesions in periods as short as days or weeks, representing up to six to 12 months of clinical lesion progression. It is also important to emphasize that


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TABLE 3 PROFILE COMPARISON OF DENTIFRICES FOR FLUORIDE UPTAKE (Faller et al, 1994) Dentifrice Formulation Placebo 250 ppm NaF2 1100 ppm NaF + 5% KNO3 (marketed product) 1100 ppm NaF + 5% KNO3 (Crest Sensitiv. Protection) 1100 ppm NaF (Crest® Control)

F Uptake in pH Cycling (|Lig/cm2)

Rat Caries Inhibition %

2.5 a ' ! 8.2b

25b

8.4b

50c

26.0c

86d

nm3

80d

]

ppm NaF = as F. Means with different superscript letters significantly different at p < 0.05. 3 nm = not measured.

2

substrate selection/preparation can have a strong impact upon experimental results in vitro, with the choice of material affecting secondary rates of reactivity as well as sensitivity to various agents (Strang et al, 1987, 1988; Damato et al., 1988; Wucherpfenning et aly 1990; Mellberg, 1991, 1992; Schafer et al., 1992). Investigators should keep these effects in mind in mechanistic and profile applications of in vitro models.

FLUORIDE UPTAKE—'BIOAVAILABILITY' Fluoride uptake is the primary 'bioavailability' measurement available for validating the activity of fluoridated dental products. In The Pharmacological Basis of Therapeutics, Gilman et al (1990) observed: "'Bioavailability' is the term used to indicate...the extent to which a drug reaches its site of action or reaches a biological fluid from which the drug has access to its site of action". By this definition, the measurement of fluoride uptake into enamel and dentin satisfies the measurement of a drug reaching its site of action, while the measurement of fluoride levels within in vivo plaque or saliva satisfies the measurement of drug levels in biological fluids with access to the site of action (Raven et al, 1991; Duckworth et al, 1992a,b). Measurements of fluoride uptake (within a fluoride-active class—e.g., NaF vs. NaF; SMFP vs. SMFP) into carious enamel are required by ADA and FDA for confirmation of the generic efficacy of fluoridated dentifrices and rinses. As profile tests, F uptake measures are more important evidence of bioequivalence than analytical measures of topical fluoride content, because bioavailability measures account for overall F reactivity, which can be affected by solution complexation, diffusion, local pH, and surface-active interfering agents. The latter are not expressed in simple availability measures. Today, mechanistic studies of fluoride reactivity have expanded beyond simple uptake measurements to include assessment of the type, location,

and subsequent secondary reactivity of reaction products formed within and on substrates. In vitro models are especially suitable for careful study of the mechanistic aspects of fluoridation, since experiments can be performed free of risks of contamination due to other dietary and topical sources of fluoride. Models used in in vitro fluoride uptake evaluations and current trends in mechanism-based research are briefly highlighted below. In vitro fluoride uptake in carious enamel It is well-known (and continuously reconfirmed—e.g., Chan et al, 1991) that fluoride reactivity from virtually all topical systems is increased dramatically in carious vs. sound enamel. As a result of this, the majority of required in vitro (and in situ) screening evaluations of toothpastes and mouthrinses include studies of fluoride uptake into artificial caries lesions. The measurements of uptake, remineralization, and secondary acid resistance make possible the combined measures of efficiency of fluoride delivery and impact of delivered fluoride on both lesion repair and resistance to caries progression. In typical in vitro models, artificial lesions are treated with the formulation/agent of interest, and the amount of fluoride acquired under standardized conditions is analytically determined (White, 1992). Analytical methods essentially involve isolation of the lesion material (by either biopsy, chemical etching, or abrasion), solubilization of the lesion material, and measurement by fluoride electrode. Today, fluoride uptake measures are often included as a subset analysis within pH cycling de- and remineralization protocols, thereby facilitating the unique measurement of bioavailability (uptake) and pharmacological activity (remineralization/demineralization) within the same model. Fluoride uptake assessments provide important bioavailability assays for toothpastes and mouthrinses, since they can quickly identify deficiencies in formulations in the transport of fluoride to enamel (White, 1992). For example,



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as shown in Table 3, relatively simple experiments have revealed substantial deficiencies in fluoride delivery from marketed products, which translated into reduced anticaries activity in animal models as well (Faller et al, 1994; Shaffer et al.f 1994). The methods, benefits, and limitations of fluoride uptake measurements for both profile and mechanistic applications have been reviewed in detail (Arends and Christoffersen, 1990; Chow, 1990; Margolis and Moreno, 1990; White and Nancollas, 1990; Ingram etal, 1992; White, 1992). In vitro fluoride uptake in sound enamel The fluoride levels in most over-the-counter (OTC) dentifrices and mouthrinses are not sufficient to produce significant amounts of reactivity with sound enamel. As a result of this, fluoride uptake from dentifrices and mouthwashes is typically assessed in artificial caries lesions rather than in sound enamel. However, sound enamel measurements still remain a focus of researchers evaluating professionally applied fluorides, where the concentration and pH of application would drive significant reactivity on non-decalcified surfaces. In a typical protocol, either powdered or sound enamel and dentin are treated with professional gel or varnish, and fluoridation is analytically measured as described earlier (e.g., Barbakow et al, 1988; White, 1992). Today, these measures are often accompanied by measurements of the surface properties of treated substrates and the F release from the treated surfaces. In the case of professionally applied products, bioavailability may be defined in a number of ways: the amount of fluoride taken up by enamel/dentin, the retention of fluoride within enamel and/or dentin, or the ability of the treated surfaces to provide low levels of fluoride in saliva. Measurements of fluoride 'release' as a primary bioavailability measure in in vitro evaluations include recent studies of topically applied solutions (Saxegaard and Rolla, 1988; Skartveit et al, 1991), F-releasing resins and restorative materials (Temin et al, 1989; Arends et al, 1990b; Arends and van der Zee, 1990; Chan et al, 1990; Dionysopoulos et al, 1990; Donly and Ruiz, 1992; Dijkman et al, 1993), bonding systems (Tsanidis and Koulourides, 1992), professionally applied gels (Mok et al, 1990; Takagi et al, 1992), occlusal sealants (van Dorp and ten Cate, 1992), and varnishes (Koch etal, 1988; Seppa, 1988; Bruun and Givskov, 1991; Eronat et al, 1993). Recent studies have also examined the fluoridation of cementum (Brewer et al, 1987). In vitro fluoride uptake—mechanism studies—reaction products, location of deposition, and release in solution Despite the vast number of studies relating enamel/lesion fluoride uptake to anticaries effects, research suggests that our understanding of the relation between fluoridation and caries prevention is incomplete. In particular, it is now recognized that fluoride agents can provide similar levels of fluoride uptake on sound enamel or in carious enamel while differing dramatically in three critical elements affecting efficacy: (1) nature and composition of the fluoridation reaction products, (2) the location of deposited fluoride,

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and/or (3) the secondary reactivity of the acquired fluoride (e.g., mobility). At the Callaway Gardens Joint IADR/ORCA symposium [J Dent Res 69(2)(Spec Iss), 1990], a series of reviews documented the literature relating enamel/dentin fluoridation to caries prevention and provided these general conclusions: • Fluoride uptake onto enamel and into lesions (that is, incorporated into the mineral component of the tooth) is necessary but not sufficient in describing caries protection from fluoride—caries protection must be defined in terms of mineral lost and mineral gained; • Fluoride in the enamel/dentin mineral phase provides only modest protection against acid demineralization and provides little benefit to remineralization; • Fluoride in solution provides significant benefits in reducing tooth demineralization rates and in enhancing remineralization rates. Consistent with these observations, considerable in vitro research has been devoted to detailed analysis of reaction products formed by fluoride, the location of fluoride deposition, and the secondary reactivity of fluoride taken up by enamel and dentin—particularly the provision of fluoride in the liquid phase to enhance remineralization and diminish demineralization. These research efforts have been assisted substantially by improvements in physical-chemical and spectroscopic methods for the assessment of the location and composition of fluoride on surfaces and in solution. With respect to reaction products, researchers continue to speculate on the potential anticaries effects of calcium fluoride reaction products in and on enamel (Dijkman and Arends, 1988; Rolla, 1988; Saxegaard and Rolla, 1988, 1989; Ogaard et al, 1990; Cruz et al, 1991, 1992, 1993; Bruun and Givskov, 1993). Recently, Rolla and colleagues have used in vitro techniques systematically to document factors affecting calcium fluoride formation and equilibria in plaque, enamel, and dentin (Cruz et al, 1991, 1993). Experiments confirmed that the formation of calcium fluoride from topical application of NaF is both concentration- and pH-dependent, with apatite dissolution accompanying redeposition of fluoride (Duschner and Uchtmann, 1988a,b; Saxegaard and Rolla, 1988). Both NaF and SMFP dentifrices have been suggested to form calcium fluoride within plaque and teeth (Arends et al, 1988). Once formed, calcium fluoride equilibria were shown to be strongly influenced by saliva inorganic and organic components (Christofferson et al, 1988; Lagerlof et al, 1988; Saxegaard, 1988; Saxegaard et al, 1988; Rykke et al, 1989; Matsuo et al, 1990). Newly developed microraman techniques (Tsuda and Arends, 1993a,b) and NMR (White et al, 1988a, 1992) can precisely identify CaF2 as a reaction product in enamel and dentin. Using NMR, Nelson and colleagues (Arends et al, 1984; White et al, 1994) identified a new F reaction product, Non Specifically Adsorbed Fluoride (NSAF), as an important product of enamel fluoridation. As shown in Fig. 8, NSAF represents fluoride ions hydrogen-bonded to apatitic surfaces


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through surface acid phosphate species. Initially identified as a reaction product of low-level fluoridation of enamel, NSAF has been observed as an initial reaction product within carious enamel from topical dentifrice use as well and may be indicative of the solution F L species described by Arends (1995). In some practical applications, Barkvoll et al. (1988) used an in vitro protocol to demonstrate that calcium fluoride formation was strongly affected by product components like surfactant-sodium lauryl sulfate. Similarly, Franco and Cury (1994) demonstrated that F uptake from aqueous solution was inhibited by surfactant available in commercial 'plaque removal' mouthwash. In another in vitro study, Yu et al. (1993) observed CaF2-like deposits on enamel following treatments with tannin-fluoride mixtures. Chow and coworkers (Chow and Takagi, 1991; Chow et al, 1992; Takagi et al., 1992) have used in vitro studies in their efforts to develop new cariostatic reaction systems containing fluorosilicate/monocalcium phosphate combinations providing elevated levels of CaF2 formation. Studies of the precise anticaries actions of fluoride are complicated by the fact that dental caries is a subsurface phenomenon rather than simple erosion. Controlled singlevariable in vitro protocols are ideal for the study of factors affecting the location of deposition, equilibration, and migration of fluoride within enamel and dentin (Clarkson et al, 1988; Iijima and Koulourides, 1989). This research has been facilitated by improved physical analytical techniques such as SIMS (Secondary Ion Mass Spectrometry) (Petersson et al, 1989; Corpron et al, 1992; Iijima et al, 1993) and microprobe methods (Tveit et al., 1988) which can complement cross-sectioned mineral evaluations from transversal microradiography (Nelson et al, 1992). From these and other studies, it is becoming increasingly clear that topical fluoride reactivity from most sources occurs (at least initially) in enamel and dentin (lesion) surface layers, with redistribution occurring under secondary acid attack. F redistribution is often associated with mineral laminations within lesions. Chu et al (1989) have developed computer models to describe F migration and uptake within developing lesions.

0.1-=

0.0110 DAYS 15 Fig. 9—Measurement of fluoride loss (every 24 h) after topical treatment as an example of traditional measurement ofF lability. (Reproduced from Skartveit et al., 1991, with permission of Ada. Odontol Scand, Scandinavian University Press, publisher.)

As suggested earlier, researchers today are interested not only in the location and composition of mineral reaction products of fluoride treatment but also in the solution equilibrium resulting from these products under both de- and remineralizing conditions. Of particular interest are investigations of the labile (solution) fluoride levels in pseudo-equilibrium within lesions, plaque fluid, and saliva (Shern et al, 1987; Saxegaard and Rolla, 1989; Margolis, 1990; Sidi and Wilson, 1991; Duckworth et al, 1992a,b, 1994; Featherstone and Zero, 1992; Margolis and Moreno, 1992; ten Cate, 1992). A focus of in vitro research in recent years has thus been to characterize the effects of topical treatments in providing 'solution' levels of fluoride. At its simplest, the delivery of 'labile' fluoride can be assessed by measures of the release of fluoride from enamel/dentin substrates into aqueous solution following treatments. This is commonly applied to studies of fluoridating properties of varnishes and resins (e.g., Skartveit et al, 1991), as illustrated in Fig. 9. Takagi et al (1992) have developed a novel titration technique to monitor directly the uptake and release of labile fluoride in lesions following topical treatment. Their method involves potentiostat-controlled (F ion selective electrode) back-titration of fluoride from treated caries lesions into a low-level baseline equilibrium solution. The technique provides an excellent measure of solution fluoride equilibria which complements conventional fluoride uptake measurements. Results of their studies are illustrated


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different fluoride sources.) There would appear to be no difficulty in designing in vitro models meeting validation requirements of regulatory bodies for fluoride uptake, as evidenced by recent research, and this aspect will not be discussed further here. Such excellent empirical correlations may seem surprising in light of the preceding discussion on the relative importance of bound and labile fluoride for caries prevention, the impact of fluoride location on potential reactivity, and the critical importance of reaction products on both. Most likely, the predictive capability of fluoride uptake for anticaries activity within active systems is related to two factors: (1) Uptake models measure efficiency of fluoride delivery—this should, in principle, be related to topical efficacy (where uptake is time-dependent); and Fig. 10—Labile fluoride as measured in titration system of Takagi et al., 1992. F loss can be directly measured in g, accounting for titrant dilution. Loosely bound fluoride measured by titration is here plotted against time following treatment with various fluoride preparations. (Reproduced with permission o/Caries Res, S. Karger Publishers.)

in Fig. 10. Sieck et al. (1990) similarly described a constantcomposition method for assessing loosely bound vs. fixed fluoride during F washing from enamel. On a different scale, Vogel et al. (1988) have pioneered the measurement of F within the lesion at various locations within single sections undergoing in vitro demineralization and remineralization using ultra-micro-analytical techniques in their laboratory. As an alternative approach, Larsen et al. (1993) have recently examined fluoridation and remineralization of apatite powders in vitro at solid solution ratios mimicking in vivo microscopic solution chemical conditions. It is noteworthy that researchers interested in evaluating potential anticaries properties of fluoridated dental materials (such as bonding agents, restorative materials, etc.) use F release measurements essentially as a bioavailability assay, directing correlations to expected clinical caries protection of these materials in adjacent locations. F uptake assessments in profile applications As mentioned earlier, current profile assessments of fluoride bioavailability in vitro involve relatively simple measures of fluoride uptake (Naleway, 1985). In fact, F uptake is the best in vitro bioavailability marker for fluoride reactivity within proven clinical fluoride systems. In vitro studies of fluoridation have been shown to correlate well to both animal caries and in situ fluoridation/remineralization results within active systems and also to clinical effectiveness of different fluoride agents (White, 1992). (As for remineralization and demineralization protocols, the clinical relevance of in vitro F uptake measurments is limited in comparisons of new fluoridating agents or in comparing

(2) Greater F uptake in tissue likely manifests itself as increased local solution F as well as mineral F. Under acid challenges, such treated mineral likely provides solution fluoride within the lesion. Limitations to fluoride uptake Fluoride uptake protocols are experimentally affected by parameters similar to those of model systems designed to assess de- and remineralization. Thus, the choice of substrate (sound enamel, etched enamel, softened enamel, subsurface decalcified enamel), treatment conditions (type of treatment, time of treatment, washing, equilibration), and substrate equilibration media (volume, composition, pH) all greatly affect measured fluoridation results {e.g., Mellberg et al., 1991). Studies examining fluoridation of enamel and dentin also show a rather unique difference from remineralization/demineralization assessments in that they exhibit variability in the measured 'parameter' defining efficacy. For remineralization and demineralization models, mineral lost/gained is direct evidence of efficacy in experimental protocols. However, for F uptake, efficacy measures can include not only the amount of fluoride taken up but also the form of deposit. For example, published studies have reached entirely different conclusions regarding topical activity, depending upon whether the measured 'fluoridation' parameter involved fluoride uptake (total), KOH extractable fluoride, or firmly bound F (Klimek et al, 1982; Hellwig et al., 1987). In addition, as described earlier, there remains no general agreement as to whether F uptake protocols should direct assessments to simple evaluations of uptake, or include location and secondary reactivity of reacted fluorides. This remains a significant limitation in the evaluation of new fluoridation systems, since clinical standards are not readily available by which one can assess either the 'site' or the 'action' associated with 'bioavailability'. As for demineralization and remineralization, in vitro fluoridation assays suffer from limitations related to lack of simulation of biological conditions, including the actions of salivary phosphatases (of importance in SMFP/NaF comparisons). Last, as for all test methods, fluoridation results obtained from in


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vitro protocols must be carefully examined for potential artifacts emanating from choices of treatment concentrations, periods, washing, etc. (e.g., Faller et al., 1990; Mellberg and Fletcher, 1990). Overall, F bioavailability assessments of oral products require the assessment of fluoridation efficiency in addition to de- and remineralization effects. The evaluation of new fluoridation agents or comparisons of different fluoride sources require more than simple uptake assessments, including more detailed measures of reaction products, location of reacted fluoride, and solution equilibria of reacted fluoride under conditions relevant to clinical caries formation and prevention (White, 1992).

SUMMARY The breadth and scope of research briefly described in this review ably demonstrate that in vitro demineralization, remineralization, and fluoridation studies remain important tools of dental researchers for both fundamental and applied (profile) testing purposes. The main advantage of in vitro testing which drives continued utilization (despite limitations) is that it provides investigators with the capability to perform single-variable experiments under controlled conditions. Such experimentation permits mechanistic studies to be performed which are difficult if not impossible to carry out in vivo (for example, see Carey et al., 1991). Furthermore, the controlled and easily modified conditions permitted by in vitro profile tests readily permit researchers using these models to produce data meeting regulatory (viz. ADA) guidelines. The translation of in vitro findings to clinical expectations for new active systems (whose mechanism of action may be unclear) or in comparing different topical agents is quite tenuous, owing to the limited capability of in vitro methods in simulating biological conditions (White, 1992). Nevertheless, well-designed in vitro experimentation into demineralization, remineralization, and fluoridation reactions represents a major focus of mechanism-based dental research into caries prevention, despite advances in in situ and animal caries models.

REFERENCES Almqvist H, Lagerlof F (1993a). Effect of intermittent delivery of fluoride to solution on root hard-tissue de- and remineralization measured by 125I absorptiometry. / Dent Res 72:1593-1598. Almqvist H, Lagerlof F (1993b). Influence of constant fluoride levels in solution on root hard tissue de- and remineralization measured by 125I absorptiometry. Caries /tes 27:100-105. Almqvist H, Lagerlof F, Angmar-Mansson B (1990). Automatic pH-cycling caries model applied on root hard tissue. Caries Res 24:1-5. Amjad Z, Koutsoukos PG, Nancollas GH (1981). The mineralization of enamel surfaces. A constant composition

kinetics study. J Dent Res 60:1783-1792. Anderson P, Elliott JC (1992). Subsurface demineralization in dental enamel and other permeable solids during acid dissolution. / Dent Res 71:1473-1481. Arends J (1995). The application of in vitro models to research on demineralization and remineralization of the teeth. Reaction paper. Adv Dent Res 9: 194-197 . Arends J, Christoffersen J (1990). Nature and role of loosely bound fluoride in dental caries. J Dent Res 69(Spec Iss):601-605. Arends J, van der Zee Y (1990). Fluoride uptake in bovine enamel and dentin from a fluoride-releasing composite resin. Quintessence Int 21:541-544. Arends J, Nelson DGA, Dijkman AG, Jongebloed WL (1984). Effects of various fluorides on enamel structure and chemistry. In: Cariology today. Guggenheim B, editor. Basel: Karger, pp. 245-258. Arends J, Schuthof J, Christoffersen J (1986). Inhibition of enamel demineralization by albumin in vitro. Caries Res 20:337-340. Arends J, Reintsema H, Dijkman AG (1988). 'Calciumfluoride-like' material formed in partially demineralized human enamel in vivo owing to the action of fluoridated toothpastes. Ada Odontol Scand 46:347-353. Arends J, Ogaard B, Ruben J, Wemes J, Rolla G (1989a). Influence of glutardialdehyde on dentine demineralization in vitro and in vivo. Scand J Dent Res 97:297-300. Arends J, Christoffersen J, Ruben J, Jongebloed WL (1989b). Remineralization of bovine dentine in vitro. The influence of the F content in solution on mineral distribution. Caries Res 23:309-314. Arends J, Ruben JL, Christoffersen J, Jongebloed WL, Zuidgeest TGM (1990a). Remineralization of human dentine in vitro. Caries Res 24:432-435. Arends J, Ruben J, Dijkman AG (1990b). Effect of fluoride release from a fluoride-containing composite resin on secondary caries: an in vitro study. Quintessence Int 21:671-674. Arends J, Smits M, Ruben JL, Christoffersen J (1990c). Combined effects of xylitol and fluoride on enamel demineralization in vitro. Caries Res 24:256-257. Arends J, Christoffersen J, Buskes JAKM, Ruben J (1992a). Effects of fluoride and methanehydroxydiphosphonate on enamel and on dentine demineralization. Caries Res 26:409-417. Arends J, Christoffersen J, Christoffersen MR, Ogaard B, Dijkman AG, Jongebloed WL (1992b). Rate and mechanism of enamel demineralization in situ. Caries Res 26:18-21. Arends J, Dijkman AG, Huizinga E, van der Kuyl M, Boerma A, Ruben J, et al. (1992c). The influence of saliva constituents and properties on enamel remineralization in situ. In: Clinical and biological aspects of dentifrices. Embery G, Rolla G, editors. Oxford: Oxford Medical Publications, pp. 145-155. Barbakow F, Sener B, Imfeld T, Saltini C (1988). In vitro enamel fluoride retention after brushing with dentifrices


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containing 0.025% fluoride. Schweiz Montsschr Zahnmed 98:126-130. Barkvoll P, Rolla G, Lagerlof F (1988). Effect of sodium lauryl sulfate on the deposition of alkali soluble fluoride on enamel in vitro. Caries Res 22:139-144. Barrett-Vespone NA, Featherstone JDB, Shields CP, Faller RV (1993). Fluoride dose response during pH-cycling of deciduous and permanent enamel (abstract). / Dent Res 72:234. Barrett-Vespone NA, Featherstone JDB, Shields CS, Faller RV (1994). In vitro efficacy of a stabilized stannous fluoride toothpaste (abstract). J Dent Res 73:241. Blake-Haskins JC, Mellberg JR, Snyder C (1992). Effect of calcium in model plaque on the anticaries activity of fluoride in vitro. J Dent Res 71:1482-1486. Boonstra WD, ten Bosch JJ, Arends J (1990). Protein and mineral release during in vitro demineralization of bovine dentine. J Biol Buccale 18:43-48. Boonstra WD, de Vries J, ten Bosch JJ, Ogaard B, Arends J (1993). Inhibition of bovine dentine demineralization by a glutardialdehyde pretreatment: an in vitro caries study. Scand J Dent Res 101:72-77. Borggreven JMPM, Driessens FCM, Hoeks TL, Zwanenburg B (1989). Effect of surface-active compounds on demineralization of dental enamel. Caries Res 23:238-242. Borggreven JMPM, Driessens FCM, Hoeks TL, Zwanenburg B (1991a). Effect of 2-0 stearoylglycerol-1,3 bisphosphate on in vitro demineralization of dental root surfaces. Caries Res 25:264-267. Borggreven JMPM, Lammers PC, Hoeks T, Zwanenburg B, Driessens FCM (1991b). In vitro remineralization of caries lesions treated with surface active phosphates. Caries Res 25:34-38. Bowman WD, Wietfeldt JR, Agricola FO, Warner R, Morgan NE, Faller RV (1988). The effect of soluble strontium on the remineralization and fluoride uptake by carious enamel from sodium fluoride (abstract). / Dent Res 67:257. Brewer KP, Retief DH, Wallace MC, Bradley EL (1987). Cementum fluoride uptake from topical fluoride agents. Gerodontics 3:212-214. Bruun C, Givskov H (1991). Formation of CaF2 on sound enamel and in caries like enamel lesions under different forms of fluoride applications in vitro. Caries Res 25:96-100. Bruun C, Givskov H (1993). Calcium fluoride formation in enamel from semi- or low-concentrated F agents in vitro. Caries Res 27:96-99. Buskes JAKM, Christoffersen J, Arends J (1985). Lesion formation and lesion remineralization under constant composition conditions. A new technique. Caries Res 19:490-496. Buskes JAKM, de Josselin de Jong E, Christoffersen J, Arends J (1987). Microradiographic study on the demineralization of thick enamel sections: A constant composition study. Caries Res 21:15-21. Carey CM, Vogel GL, Chow LC (1991). Permselectivity of sound and carious human dental enamel as measured by membrane potential. J Dent Res 70:1479-1485.

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Chan DCN, Swift EJ Jr, Bishara SE (1990). In vitro evaluation of a fluoride-releasing orthodontic resin. J Dent Res 69:1576-1579. Chan JC, Hill FJ, Newman HN (1991). Uptake of fluoride by sound and artificially carious enamel in vitro following application of topical sodium and amine fluorides. J Dent 19:110-115. Chilton N (1992). Consensus conference on intra-oral models: experimental design and analysis. J Dent Res 71:953. Chow LC (1990). Tooth bound fluoride and dental caries. J Dent Res 69(Spec Iss):595-600. Chow LC, Takagi S (1989). A quasi-constant composition method for studying the formation of artificial caries-like lesions. Caries Res 23:129-134. Chow LC, Takagi S (1991). Deposition of fluoride on tooth surfaces by a two solution mouthrinse in vitro. Caries Res 25:397-401. Chow LC, Takagi S, Shih S (1992). Effect of a two solution fluoride mouthrinse on remineralization of enamel lesions in vitro. J Dent Res 71:437-443. Christoffersen J, Christoffersen MR, Kibalczyc W, Perdok WG (1988). Kinetics of dissolution and growth of calcium fluoride and effects of phosphate. Acta Odontol Scand 46:325-336. Christoffersen J, Christoffersen MR, Ruben J, Arends J (1991). The effect of EHDP concentration on enamel demineralization in vitro. J Dent Res 70:123-126. Chu JS, Fox JL, Higuchi WI (1989). Quantitative study of fluoride transport during subsurface dissolution of dental enamel. J Dent Res 68:32-41. Clarkson BH, Hall DL, Heilman JR, Wefel JS (1986). Effect of proteolytic enzymes on caries lesion formation in vitro. J Oral Pathol 15:423-429. Clarkson BH, Hansen SE, Wefel JS (1988). Effect of topical fluoride treatments on fluoride distribution during in vitro caries-like lesion formation. Caries Res 22:263-268. Clarkson BH, Feagin FF, McCurdy SP, Sheetz JH, Speirs R (1991). Effects of phosphoprotein moieties on the remineralization of human root caries. Caries Res 25:166-173. Collys K, Slop D, Coomans D (1988). The influence of trace elements on demineralization and remineralization properties of tooth enamel in vitro. Trace Elements Med 5:161-169. Collys K, Slop D, De Langhe L, Coomans D (1990). A comparison of the influence of lanthanum and fluoride on de- and remineralization of bovine enamel in vitro. J Dent Res 69:458-462. Collys K, Cleymaet R, Coomans D, Slop D (1991). Acidetched enamel surfaces after 24 hour exposure to calcifying media in vitro and in vivo. J Dent 19:230-235. Collys K, Cleymaet R, Coormans D, Michotte Y, Slop D (1993). Rehardening of surface softened and surface etched enamel in vitro and by intraoral exposure. Caries Res 27:15-20. Corpron RE, More FG, Mount G (1992). Comparison of fluoride profiles by SIMS with mineral density of


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