Remineralization of deep enamel dentine caries lesions INTRODUCTION The past decades have led to major changes in restorative

and preventive dentistry. Various investigators reported that early caries lesions can be remineralized from saliva. This process was studied in detail in laboratory experiments and in clinical trials.1,2 Adhesive dentistry has resulted in new non-metallic filling materials, such as composites based on acrylate resin and glass ionomer cements (GIC) based on an acid base precipitation mechanism. Restorations made with adhesive materials no longer require a large cavity preparation, but merely the removal of the tissue affected by caries. Moreover, the notion became generally accepted that restorative intervention is generally the beginning of a long sequence of re-restorations, often leading to crowns and implants, irrespective of how well the first filling was prepared. The concept of minimal intervention or minimal invasive dentistry (MID) has combined these three major (paradigm) shifts in operative dentistry and this philosophy is currently accepted worldwide. MID now has its own materials, congresses and research organization.3 In caries prevention, most protocols are still built around fluoride, although improving oral hygiene, sugar substitutes and antimicrobials are also part of the more comprehensive packages of non-invasive care. New methodologies of caries diagnosis have been developed, using the early changes of fluorescence induced by caries in the tissue detected by Quantitative Light Fluorescence (QLF),4 or with chromophores produced by bacteria (in the Diagnodent device)5 as indicators. Remineralization of enamel and dentine is studied from two perspectives. First of all, it is the process of mineral deposition from saliva or plaque fluid filling up small enamel or dentine defects formed during the demineralization episodes resulting from acid attack on the tooth. The relative magnitudes of demineralization and remineralization determine whether a tooth surface remains sound or a caries lesion develops. This lesion may then increase in severity eventually resulting in a (deep) cavity. (For more details about this caries equilibrium, see Featherstone.2) Alternatively, remineralization is studied and described as the repair of established lesions. Such lesions have developed over a long period but may be filled in with calcium phosphates when external conditions favour mineral deposition. This type of remineralization may either be complete and partial; when the mineral precipitating in the lesion is less soluble than the original tissue, this remineralization will help in preventing or limiting future tissue loss. Remineralization of superficial enamel lesions is well documented in hundreds of studies completed at numerous laboratories in the last century. Studies on the basic mechanism of remineralization and on methods to stimulate this process have led to the conclusion that the caries preventive effect of fluoride is beyond any doubt. This is partly attributed to the

enhancing effect of fluoride on calcium phosphate precipitation, hence remineralization.6 A topic that has received limited attention is whether there is a point of no return beyond which remineralization can or does no longer occur. Koulourides (one of the pioneers in remineralization research), stated that if caries has weakened the tooth structure to below a hardness of 150 Knoop Hardness Numbers (KHN), remineralization could no longer be achieved.7,8 Conceptually this was seen as the point where the mineral structure is destroyed to the extent that reprecipitation of mineral on remaining hydroxyapatite crystallites was no longer possible. In caries diagnosis using X-rays, the extent of caries is graded at various depth levels in the enamel and dentine, realizing that loss of tissue has occurred also considerably beyond the depth identified on the X-ray picture. The current consensus is that caries beyond the dentino-enamel junction (DEJ) should be treated with restorations, and lesions up to that point should receive extra preventive care. However, it was never studied whether deep lesions, extending into dentine, can be remineralized if such lesions are subjected to a continuous remineralization scheme. Obviously, such lesions should be protected from mechanical damage. Ideally, a study with this objective should be carried out in vivo or using an in situ model. However, given the fact that remineralization is a very slow process, it seemed technically impossible to complete such a study with volunteer subjects or with patients, within an acceptable time period. This article reiterates previous results from in vitro remineralization experiments of deep lesions, extending into dentine.9 The aim was to explore whether such lesions can still be remineralized and how this could be affected by treatments that would stimulate or inhibit calcium phosphate precipitation. These findings are then discussed from a theoretical and clinical perspective. Experimental design and results The experiment was performed in groups of 100 lm thick sections cut from ground bovine incisors. Sections were fully embedded in Araldite, and after setting of the resin the outer 200 lm of the enamel was cut with a diamond coated wire sectioning machine. This was done to remove surface enamel and to reduce the enamel thickness to the DEJ. Lesions through enamel and into dentine were formed during 1015 days in individual solutions containing 1.5 mM CaCl2, 0.9 mM KH2PO4 and 50 mM acetate buffer (pH 4.8), with the addition of 0.1 ppm KF to prevent surface loss.10,11 Next the lesions were immersed in solutions supersaturated and stochiometric to hydroxyxapatite (HAP), so as to achieve remineralization (1.5 mM CaCl2, 0.9 mM KH2PO4 20 mM HEPES buffer pH 7.0 and 130 mM KCl). All steps in this process were monitored and recorded by taking microradiographs, as was the remineralization phase by taking microradiographs weekly during 200 days. Profiles were scanned at fixed positions of the specimens. Five independent specimens were run at each condition.

Treatments tested were control (no additional treatments), weekly five-minute fluoride rinses with 1000 ppmF, addition of 1 ppmF to the remineralizing solution, and a five-minute single treatment with the calcium phosphate precipitation inhibitor bisphosphonate (as 2 mMethaneHydroxy-BisPhosphonate). The primary findings of these experiments are given in Fig 1. To average out variation between specimens in enamel thickness and overall lesion depth, data are expressed as percentage repair in four zones: outer enamel, inner enamel, outer dentine, inner dentine. The relative remineralization parameter shows that remineralization in dentine (panels C and D) may be up to 80 per cent after 200 days, while remineralization in inner enamel (panel B) plateaus at around 40 per cent. Only in the outer enamel (panel A) is the remineralization significantly affected by the various treatments, thus resulting in relative remineralization values between 40 and 100 per cent. (For full experimental details and results, see ten Cate.9) Theoretical considerations Whether remineralization occurs in enamel-dentine lesions extending beyond the DEJ depends on several factors. First, the concentration of mineral ions at the site of precipitation should exceed supersaturation to hydroxyapatite. This requires that not all calcium and phosphate ions entering through the lesion pores have already been precipitated in the layers closer to the surface. Secondly, the site of precipitation requires nuclei for precipitation, considering that substantially larger degrees of supersaturation are needed for de novo precipitation of apatite or precipitation onto remaining organic matrix than on fragments of enamel or dentine apatite crystals. Detailed electron microscopic analysis of crystallites in various zones of the lesion confirmed that remineralization occurs by growth of existing crystals to dimensions larger than the original crystallites.12 Rate control Considering the theory of crystallization kinetics, a precipitation at greater depth requires that the precipitation reaction is slow compared to mass transfer of ions, thus allowing diffusion to supply ions throughout the lesion. If such a condition is met, the mineral ion concentrations are uniform throughout the lesion. Little data are available to confirm this assumption. With Arrhenius plots (temperature dependence of reactions), we previously determined that the activation energies of remineralization of subsurface lesions versus surface softened enamel were significantly different. We then concluded that diffusion processes were rate limiting in the lesion remineralization (judging from the lower activation energies), while surface softened lesion remineralization was controlled by surface reactions.13 The current findings seem to contradict these early observations. Demineralization

More potentially relevant data are available on enamel demineralization, showing that its rate is determined by diffusion processes, although differences in mechanism were noted between demineralization in vitro and in vivo.14,15 Recently, we developed an artificial groove model, in thin sections, to simulate demineralization in fissures. In this model we periodically monitoredmineral loss with microradiography and pH and calcium concentrations throughout the dept h of the grooves with microelectrodes. The observed gradients in pH and calcium activities pointed to diffusion inhibition for demineralization even in the 250 lm wide grooves (ten Cate and Buijs, unpublished data). Making this comparison it should be noted that precipitation rates are probably 10 times slower than dissolution rates at relevant super- and undersaturation conditions. Enamel-dentine continuum Even if enamel and dentine form a continuum within the tooth, their respective apatite crystallites differ in size and composition, reflected in differences in solubility. In an aqueous environment this would eventually lead to the complete dissolution of the dentine crystallites in favour of the enamel crystallites, a process known as Ostwald ripening in crystal chemistry. Findings in accordance with this principle were observed when enamel and dentine (lesions) were de- and remineralized, respectively, when placed in juxtaposition.16 Matrix For in vivo remineralization, additional mechanisms could play a role as indicated by in situ studies completed by van Strijp and colleagues.17 In a comparison of various fluoride toothpastes they observed full remineralization of dentinal lesions in many of the participating subjects, and small changes in the contralaterally placed enamel lesions. This observation hints that remineralization conditions for dentine lesions may be favourable compared to enamel lesions, although the full explanation of this finding is yet lacking. One may formulate the hypothesis that the demineralized organic matrix of dentine may constitute a scaffold to enhance remineralization. Furthermore, non-collagenous components of this matrix (SIBLINGs, osteocalcin, proteoglycans).18,19 may interact directly with crystal formation and crystal growth during dentine remineralization. In the context of the current experiment, this all could suggest that remineralization of dentine proceeds faster than for enamel and would create a concentration `sink' beyond the DEJ. Modelling Many of the abovementioned findings need further study. However, given the duration of remineralization studies of deep lesions, it seems worthwhile to consider in silico experiments; computer simulations of remineralization using model parameters that can

be determined individually or are available in the literature (rates of apatite precipitation, dissolution, diffusion constants, etc.). Obviously, as with any simulation model, this approach helps to identify or illustrate the relative importance of the individual steps in a complex process, in this case diffusion, precipitation, tortuosity, etc. It is beyond the scope of this presentation to describe the numerical approach in detail or give all the equations for the separate steps in the process (for details see ten Cate).20 Examples of such computational exercises are included as illustration in Fig 2. Enhancing remineralization of deep lesions Fluoride Traditionally, the focus in the development of agents aimed to enhance remineralization has been on fluoride. The presence of low levels of fluoride increases the degree of supersaturation with respect to fluoridated hydroxyapatite. This thermodynamic property is the rationale for the enhancement of remineralization by fluoride. This, however, could lead to excessive remineralization of the surface layer of lesions and consequently lesion arrestment.21 In the described experiments (Fig 1) the tested agents (fluoride and bisphosphonates) were found to give rise to diverging results in the outer enamel, but these treatments did not significantly affect precipitation of mineral in the inner enamel and dentine. Fluoride treatments, whether as 1000 ppm topical treatments or continuously present at 1 ppm, were both beneficial for repair of deep lesions, at least in the outer enamel. Calcium After analysing comprehensive literature data on in situ remineralization, saliva and plaque mineral ion compositions and saliva flow dynamics, we have proposed that, in addition to fluoride, calcium may be rate limiting in remineralization.22 Since then many new products, including toothpastes and chewing gums, have been formulated with the aim to supply calcium ions to the oral cavity.23,24 Recently, clinical studies and in situ trials have confirmed the potential of this remineralization approach.25,26 There seems scope to also study such products with the aim to enhance deep rather than superficial lesions. Clinical aspects Obviously, conditions in the mouth are very different from the ideal remineralization conditions used in the experiments described in this article. An important in vivo challenge to the remineralization of deep lesions are the periods of low pH in the plaque which worsen the degree of mineralization rather than improving it. Moreover, the teeth are subject to mechanical forces which could break the surface of the lesion and create a retention site for bacteria. Once the surface is irreparably damaged, clearly the chance for a non-invasive repair of the lesion is lost. CONCLUSIONS The experiments described in this study show that remineralization of lesions extending into the dentine is possible. This process takes a considerably long time,

which clinically is unacceptable. Nevertheless, studies to further our knowledge in this field would contribute to the area of minimal intervention dentistry