Longitudinal study of the three-dimensional development of subsurface enamel lesions during in vitro demineralisation

A longitudinal study was made of the 3D development of subsurface enamel lesions in whole human molars. X-ray microtomography (XMT) was used to measure the 3D distribution of linear attenuation coefficients in the tissue at 8-15 stages during cumulative times of 36-107 days demineralisation through approximately 1-mm-wide windows. Although lesion morphology was consistent with preferential anisotropic dissolution parallel to enamel prisms at the advancing front, detail (at a scale of approximately 100 microm) varied in relation to exposed surface sites separated by <1 mm. The distribution of mineral in the most superficial region varied across the exposed face of each lesion. Within lesions, localised foci of low mineral concentration (at a scale of approximately 200 microm) retained their general form through successive stages of demineralisation before coalescing. The most advanced regions within a lesion seemed to correspond with surface regions with lowest mineral concentration. These findings indicate that local variations in fractional pore volume of partially demineralised enamel influence the subsequent spatial development of lesions.     

X-Ray Microtomography Study to Validate the Efficacies of Caries Removal in Primary Molars by Hand Excavation and Chemo-Mechanical Technique

Background/Aims: Mechanical removal of carious dentine based on perceived hardness is subjective and tends to be excessively destructive; chemo-mechanical techniques have been proposed as being more objective and conservative. The aims of the present study are to use X-ray microtomography (XMT/micro-CT) to determine the three-dimensional mineral concentration distribution in sound, carious and excavated dentine using hand excavation (HE) and a chemo-mechanical, Carisolv (CS), removal technique for primary molars, and to compare the volume of sound dentine removed in order to validate the efficacies of these two techniques. Methods: Twenty-one primary molars with open carious cavities were hemisected. The carious tissue in one half was then removed by HE and the other by CS. XMT scans were taken before and after caries removal. After alignment, subtracted XMT images from the two scans revealing the tissues removed were generated, from which mineral distributions were determined, and volumes of sound dentine removed by each technique were calculated. Results: It was found that the sound dentine removed by HE and CS techniques accounted for 4.0 and 2.1% of total tissues removed, respectively. The mean cut-off linear attenuation coefficients at 40 keV to which HE and CS excavated to were 1.27 and 1.09 cm(-1), respectively. The corresponding Knoop hardness number for the cut-off for CS was 25 kg·mm(-2). Conclusion: It is concluded that using XMT, CS is validated to be more conservative than HE and preserves a layer of partially demineralised dentine with a mineral concentration >0.97 g·cm(-3).     

X-ray microtomographic study of mineral concentration distribution in deciduous enamel

OBJECTIVE: To determine the mineral concentration distribution in deciduous enamel by quantitative X-ray microtomography (XMT). DESIGN: Tooth rods ( approximately 2 mm x 2 mm) were removed from the mid-buccal region of 11 deciduous molars. Three XMT slices were taken at 1.5, 2.0 and 2.5 mm from the amelocemental junction. The distribution and variation in mineral concentration of enamel were studied from the XMT images. RESULTS: The mean mineral concentration for all the teeth was 2.81 (S.D. = 0.065) g cm(-3). There was no notable difference in the mean mineral concentration values between the three XMT slices of each tooth. However, there was up to 8% variation between different teeth (2.69-2.92 g cm(-3)). Gradients of increasing mineral concentration from the amelodentinal junction (ADJ) to the external surface were found, ranging from 0.08 to 0.60 mg cm(-3) microm(-1) with a mean of 0.366 mg cm(-3) microm(-1). The mineral concentration gradients in the occlusal slices were steeper than those in the cervical slices. The difference in mineral concentration between the inner and outer enamel ranged from 1.5 to 8.7%. CONCLUSION: In view of the large variation in both the means and the gradients of mineral concentration in deciduous molars, the mineral distribution of each experimental tooth should be measured as baseline data in studies of caries progression.     

Sequential 3D X-ray microtomographic measurement of enamel and dentine ablation by an Er:YAG laser

OBJECTIVE: To demonstrate the progression of crater growth during repeated sequential application of an Er:YAG laser to enamel and dentine, monitored using X-ray microtomography (XMT). DESIGN: A single centre study in which laser craters were created in blocks cut from human enamel and/or dentine under standardised and known conditions and then studied using XMT to obtain visualisation and quantification of the effects. SETTING: University setting, UK, 2001. MAIN OUTCOME MEASURES: Success was judged by an ability to obtain useful 3D XMT reconstructions of the blocks during crater development, and to make measurements from these data. These measurements were compared with data obtained from similar studies using different measurement techniques. RESULTS: Time sequences of 2D and 3D images were obtained which demonstrated the progression of laser craters in enamel and dentine. Quantitative measurements from these data enabled values to be derived for the rate of progression of crater depth per unit energy, and the volume of hard tissue removed per unit energy. These values were compared with data derived from other studies and shown to be broadly comparable. However, the present study is unique in that these values were obtained from a series of measurements of the same craters over time. CONCLUSIONS: 3D X-ray microtomography is shown to be a useful tool for quantitative measurements in dental research. For the Er:YAG laser, the relationship of laser crater depth and volume of mineral removed to applied energy was found to be linear.     

An X-ray microtomography study on the mineral concentration of carious dentine removed during cavity preparation in deciduous molars

Dentists use a number of criteria in order to assess when a cavity is caries free, amongst which hardness is probably the most widely used. However, the judgement is subjective. X-ray microtomography (XMT) is a non-destructive microscopic technique that allows in vitro specimens to be scanned, manipulated and then rescanned. In this study, a high-definition XMT scanner was used to determine the mineral distribution of carious dentine in 10 deciduous molars, and the extent of dentine removed by an experienced clinician was investigated. For each tooth, after an initial XMT scan, caries was removed using a steel bur in a slow hand-piece. The tooth was then repositioned and rescanned. Mineral concentrations were calculated from the linear attenuation coefficients assuming the mineral phase to be hydroxyapatite and the organic phase to be collagen. The volume of dentine tissues removed was calculated by subtracting data of the second scan from the first. The results showed that the mean modal mineral concentration for the 10 teeth was 1.42 g x cm(-3) for sound dentine. Because of uncertainty about collagen concentration in carious dentine, the mean modal mineral concentration for the carious dentine had a range of 0.37-0.5 g x cm(-3). It was found that the subjective criteria used by the operator could lead to inconsistency of cavity preparation. The cavities could be overprepared by 8.5-44.3% in volume. However, the overpreparation was not uniform throughout the cavity: residual demineralised dentine could still be detected in the postoperative scan in isolated regions.     

Mineral density of hypomineralised enamel

Objectives

To characterize molar–incisor hypomineralisation (MIH) defects of different severities quantitatively and qualitatively using X-ray microtomography (XMT) and to measure the range of reduction in mineral density (MD) of MIH enamel compared with the normal range.

Methods

Ten sound teeth and ten MIH teeth were scanned using a commercial XMT system. Four hydroxyapatite phantoms of different densities were used as calibration standards with each scan. A calibration equation derived from the phantoms with each tooth was used for MD calibration. MD was traced from the cementum–enamel junction (CEJ) to the cusp tip and from the dentine–enamel junction (DEJ) to the outer enamel surface.

Results

In sound teeth, MD increased from CEJ to cusp/incisal tip, while in MIH teeth MD dropped from the CEJ to the occlusal region, then increased again at the cusp tip. MD was highest midway between DEJ and outer enamel in sound teeth. In MIH, enamel showed normal thickness and MD was highest near the DEJ and then decreased towards the outer enamel. MD of MIH enamel was on average about 19% lower than sound enamel. The MIH defects seemed to follow the incremental lines of enamel formation.

Conclusions

MIH defects are hypomineralised defects of different severities that follow the natural incremental lines of enamel formation. Cuspal areas are usually only mildly affected and cervical enamel always appears to be sound.     

Crystallographic and structural alterations in the mineral phase of human enamel with carious attacks

The present study aims to obtain further information about the crystallographic and structural alterations in the mineral phase of enamel with the onset of caries. For this purpose, X-ray microbeam diffraction analysis was carried out on ground sections prepared from natural while spot lesions. Electron spin resonance (ESR) analysis was also performed on block samples cut from white spot lesions and from undamaged enamel of the same teeth. The results of crystallinity measurements showed that enamel apatite in demineralized lesions was lower in crystallinity than the apatite in the surface layer and surrounding sound enamel. This X-ray diffraction study also revealed the presence of two non-apatitic minerals in the de-mineralized lesions. One type of the mineral is whitlockite, giving spotty rings. The nature of the other mineral, giving a single ring-like reflection, remains to be elucidated. A comparison of ESR spectra taken from the caries-attacked and undamaged enamel provided evidence that the former samples have a lower degree of alignment of apatite microcrystals. The occurrence of non-apatitic mineral phases and the observed difference in microcrystalline alignment may be the results of a remineralization process.     

Crystallographic and structural alterations in the mineral phase of human enamel with carious attacks

The present study aims to obtain further information about the crystallographic and structural alterations in the mineral phase of enamel with the onset of caries. For this purpose, X-ray microbeam diffraction analysis was carried out on ground sections prepared from natural white spot lesions. Electron spin resonance (ESR) analysis was also performed on block samples cut from white spot lesions and from undamaged enamel of the same teeth. The results of crystallinity measurements showed that enamel apatite in demineralized lesions was lower in crystallinity than the apatite in the surface layer and surrounding sound enamel. This X-ray diffraction study also revealed the presence of two non-apatitic minerals in the demineralized lesions. One type of the mineral is whitlockite, giving spotty rings. The nature of the other mineral, giving a single ring-like reflection, remains to be elucidated. A comparison of ESR spectra taken from the caries-attacked and undamaged enamel provided evidence that the former samples have a lower degree of alignment of apatite microcrystals. The occurrence of non-apatitic mineral phases and the observed difference in microcrystalline alignment may be the results of a remineralization process.     

Dentinal carious lesion in three dimensions

AIM: The aim of this study was to show the morphology of the carious lesion in dentine in three dimensions (3D). DESIGN: A novel high-definition X-ray microtomography (XMT) scanner was used to scan 10 carious primary molars at a resolution of 15 x 15 x 15 microm3. A stack of approximately 640 XMT slices were recorded for each tooth. Using this data set and a volume rendering algorithm, each tooth was reconstructed in 3D. The VG Studio Max 1.0 visualization software package was used to make normal enamel and dentine transparent to show the carious lesions in 3D. A video film, comprised of the rendered images from 60 viewing angles rotating through 360 degrees , was produced to show the carious lesion and its relation to the pulp in a three-dimensional perspective (http://www.smd.qmul.ac.uk/dental/oralgrowdev/biophysics/xmt/images/carious.mpg). RESULTS: These images showed that carious lesions in dentine were bowl-shaped. The pulp adjacent to the carious lesion was also observed to mimic the base of the bowl-shaped lesion. CONCLUSIONS: It was concluded from the teeth studied that the shape at the base of the carious lesion in dentine is curved in 3D, rather than conical, as traditionally believed from two-dimensional image interpretation. Further 3D studies are needed to investigate whether the bowl-shaped carious lesions in dentine also apply to caries in other types of teeth.     

Wavelength-independent microradiography: a method for non-destructive quantification of enamel and dentin mineral concentrations using polychromatic x-rays

Wavelength-independent Microradiography (WIM), described in this paper, used polychromatic, high-energy (less than or equal to 60 kV) x-rays for determination of mineral concentrations in tooth material non-destructively. This was done with the aid of a reference step-wedge made of 94% aluminum, 6% zinc. The mass attenuation coefficient of this material has a wavelength-independent ratio to the mass attenuation coefficients of enamel and dentin. With this method, mineral concentrations of enamel and dentin samples, with a thickness up to 500 microns, were determined at 20- and at 60-kV tube voltage. The samples were demineralized for 72 and 144 h and measured again. Comparison of the data showed that mineral quantification was within 1.5%, independent of the x-rays used. Finally, these mineral concentrations--obtained from the Wavelength-independent Microradiography--were compared with measurements of the same samples by Longitudinal Microradiography. A correlation of 0.99 was found for enamel and one of 0.96 for dentin.     

3D X-ray microscopic study of the extent of variations in enamel density in first permanent molars with idiopathic enamel hypomineralisation

OBJECTIVE: To measure mineral concentration distributions within teeth with idiopathic enamel hypomineralisation, a condition in which developmental defects are seen in first permanent molars, and/or incisors. DESIGN: X-ray microtomographic and 3D x-ray microscopy. SETTING: UK University, 2001. MATERIALS AND METHODS: X-ray microtomographic measurements of the extent of hypomineralisation in two affected molars and two contralateral controls extracted from the same patient. RESULTS: The control molars were visibly normal. The affected molars showed hypomineralised yellow opaque enamel with regions of breakdown. X-ray microtomographic images showed; a 20% reduction in mineral concentration of affected enamel (most cases involved full enamel thickness); hypomineralised enamel had a mineral concentration gradient opposite to that of normal enamel; regions of hypomineralisation distributed randomly throughout affected teeth, (apart from cervical region which was less severely affected). CONCLUSIONS: The pattern of mineral concentration suggests a disturbance during the maturation process. Differences in susceptibility of the ameloblasts during different stages of dental development may explain the asymmetric distribution of the defects. Topical fluoride applications may help promote post eruption maturation of the surface layer in these teeth. The use of fissure sealants and adhesive materials appears to prevent further breakdown.     

Abrasion biopsy in studies of mineral density of experimental enamel lesions

The profile of mineral density of experimental enamel lesions was determined by means of sampling with a new abrasion biopsy technique. Three types of lesions were produced on bovine enamel slabs: (1) pre-softened in lactic acid buffer for 16 hr; (2) pre-softened and exposed to a test for acid resistance for seven days; and (3) pre-softened, exposed to the seven-day Intra-oral Cariogenicity Test with extra-oral immersions in 1000 ppm F solutions for one min twice daily (ICT/F), plus the seven-day test for acid resistance. Lesions were assessed with measurements of surface microhardness, microradiography of thin sections, and abrasion biopsy. For abrasion biopsy of the experimental lesions, 15 parallel layers of approximately 10 microns each were abraded simultaneously with reference slabs of sound enamel on strips of lapping film. The depth of abrasion for each sample was calculated from the phosphorus content of the reference sample. Exposure to the ICT/F formed an acid-resistant zone within the lesion which diminished the microhardness change, although it did not have an appreciable effect on the total lesion depth as assessed with microradiography; abrasion biopsy indicated the formation of a mineral-dense zone within the lesion. This recently-developed technique of abrasion biopsy of experimental lesions offers the opportunity to link the composition of the lesion to controlled experimental conditions which will improve our understanding of demineralizing and remineralizing reactions on a standard tooth substrate.     

Three-dimensional study of human dental fissure enamel by synchrotron X-ray microtomography

The three-dimensional morphology of human tooth fissures and the quantification of mineral distribution in fissure enamel are pertinent to the development and diagnosis of caries. Synchrotron X-ray microtomography was used to measure linear attenuation coefficients (at 25 keV) at high spatial resolution with a volume-imaging element (cubic voxel) of 4.9 × 4.9 × 4.9  µ m³ in a block from a human premolar that included part of a stained fissure. From the linear attenuation coefficient, the mineral concentration, expressed as g_HAp cm⁻³ (where HAp is stoichiometric hydroxyapatite), was calculated. The mean mineral concentration in bulk enamel was 2.84 g_HAp cm⁻³. Well-defined regions (1.5–2.6 g_HAp cm⁻³), extending up to ≈ 130  µ m from the base of some narrower lengths of the fissure and up to ≈ 50  µ m deep from the fissure surface, were attributed to hypomineralization. Other regions of low mineral concentration, some (1.4–2.3 g_HAp cm⁻³) lying within the expected course of the fissure base and some (2.2–2.7 g_HAp cm⁻³) deep to the pit, were also considered to be of developmental origin. However, a diffuse distribution of low mineral concentrations (2.2–2.7 g_HAp cm⁻³) in the pit walls was attributed primarily to demineralization from caries. The fissure contained heterogeneous material (≤ 0.5 g_HAp cm⁻³) exhibiting partial mineralization.     

Microradiography and electron-microprobe analysis of some caries-like lesions of enamel prepared in vitro in human teeth

Six lesions were made on the buccal surfaces of premolars. The volume percentage of mineral was determined as a function of depth by microradiography. Using the electron microprobe, the signals for Ca, Na, Mg, P and Cl were recorded as a function of depth both through the lesions and through the adjacent sound enamel. In the demineralized parts, there was a preferential loss of Na and a preferential retention of chloride. In the surface layers, the Na: Ca and Cl: Ca ratios were almost the same as in the adjacent sound enamel, indicating that the surface layers were not formed by gross dissolution of the original mineral followed by gross reprecipitation of another, less-soluble calcium phosphate, but remained probably because their microcrystals were protected by a thin layer of precipitated fluorapatite or fluoridated hydroxyapatite. The same had been found for surface layers in natural caries.     

Quantitative high contrast X-ray microtomography for dental research

ObjectivesX-ray microtomography (XMT or micro-CT) is a miniaturized version of medical CT and has been used extensively for in vitro dental research. The technique allows three-dimensional analyses of both structure and density (or concentration), the latter requiring some a priori knowledge of composition. The aim of this paper is to demonstrate the capabilities of an XMT scanner specifically designed for dental research, especially that requiring high contrast resolution or accurate mineral concentration quantification.MethodsThe MuCAT scanners designed at Queen Mary, University of London, use high dynamic range CCD cameras with time-delay integration readout to provide high quality tomographic images. Accuracy in mineral concentration quantification is achieved using a modelling approach to beam hardening correction with automatic calibration following every scan. Accuracy was tested by measuring the density of a hydroxyapatite disc, and the utility of the high contrast and high accuracy capabilities are demonstrated in three applications from ongoing dental research projects.ResultsQuantification accuracy in the hydroxyapatite disc density was better than 1%. In dental applications, slight differences in mineral concentration allowed features such as dead tracts to be visualized and ion transport from a glass-ionomer cement into demineralized dentine to be observed.ConclusionThe improved accuracy and contrast sensitivity, together with the non-destructive nature of XMT in general, facilitates precise studies of dynamic processes in teeth. The ability to differentiate subtle differences in mineral concentration allows dead tracts to be traced in three dimensions, linking external pathology in teeth to reactive processes from the pulp.Clinical significanceThe MuCAT scanners have been optimized for quantification of mineral concentration and are particularly useful for in vitro studies of de- and re-mineralization, excavation and other treatment methodologies, and for gaining further insight into tooth morphology and pathology. Results from such studies will inform development of clinical treatment and management.     

An X-ray microtomographic study of natural white-spot enamel lesions

White-spot enamel lesions are an early presentation of dental caries and are ideally managed by non-invasive procedures. The aim of this study was to characterize white-spot enamel lesions by x-ray microtomography. In particular, mineral content across the lesion from the surface to the base of the lesion was measured and surface layers defined. Molars with long buccal white-spot enamel lesions were collected, photographed, and each sectioned to produce 3 500-μm-thick sections. The sections were mounted and imaged by quantitative x-ray microtomography at a 15-μm voxel size. We analyzed line profiles through the middle of each 3D image to determine mineral content and depth. The surface layer thickness of the lesions ranged from 35 to 130 μm, with the maximum mineral content in this layer being 74% to 100% of that of sound enamel. The average mineral content across the lesions ranged from 1.73 to 2.48 g/cm(3). No significant differences could be found between lesions clinically categorized as active and those categorized as inactive. However, for depth-matched active and inactive lesions, the active lesions exhibited a more porous surface layer than the inactive lesions. White-spot enamel lesions are highly variable, with surface layers of considerable thickness.     

Diffusive transport within dentinal tubules: an X-ray microtomographic study

The hydrodynamic theory of dentine hypersensitivity proposes that external stimuli cause dentinal fluid movement within dentinal tubules thereby triggering mechanosensitive nerves and eliciting a pain response. The aim of this study was to employ X-ray microtomography (XMT) to monitor diffusion of caesium acetate through dentine to investigate the extent to which transport occurs within the primary tubules compared to that through branched microtubules believed to run perpendicular to the direction of the primary dentinal tubules. 2.0-mm thick coronal dentine disks masked to leave half of the upper surface exposed were imaged by XMT, initially in water, which was then replaced with an aqueous solution of 0.50 mol l(-1) caesium acetate. Further XMT images were acquired after 1 and 6 days immersion. The XMT images were used to measure the change in the X-ray linear attenuation coefficient resulting from caesium acetate ingress into dentine. There was clearly considerable ingress of caesium acetate into the dentine lying below the exposed surface, but considerably less beneath the sealed surface, suggesting that diffusive transport occurs predominantly in the direction of the primary dentinal tubules, with no significant lateral transport. Primary tubules are clearly the dominant transmission route for triggering the mechanosensitive nerves present at the dentine-pulp interface, and for delivery of nerve desensitising agents.     

Imaging artificial caries on the occlusal surfaces with polarization-sensitive optical coherence tomography

Polarization-sensitive optical coherence tomography (PS-OCT) is a nondestructive imaging system that can utilize near-infrared (IR) light to produce depth-resolved images of dental enamel and has the potential to monitor early enamel occlusal caries. The objective of this study was to investigate the relationship between the magnitude of backscattered light and depolarization recorded by PS-OCT with changes in the enamel mineral volume in an artificial caries model. Artificial lesions were created on a selected region on the occlusal surfaces of sound posterior teeth (n=10) using a well-characterized 14-day pH cycling model. An all-fiber-based PS-OCT system operating at 1,310 nm was used to collect serial images at day 0 and day 14 prior to tooth sectioning. The quantitative mineral content profile and relative mineral loss, DeltaZ (%volxmicrom), of the carious enamel samples were obtained from transverse sections using high-resolution digital microradiography (DM). Line profiles of PS-OCT and DM images were used to evaluate the artificial caries severity and depth. The integrated reflectivity of the perpendicular-axis PS-OCT image, quantifying lesion severity, was correlated to the DeltaZ of the caries lesions. There was also a strong correlation between the lesion depth calculated from both imaging modalities. PS-OCT can image and quantify artificial occlusal caries by measuring the increase in backscattering and depolarization of near-IR light. This optical method has promising applications for in vivo detection and monitoring of early enamel occlusal caries.     

In situ acid resistance of in vivo formed white spot lesions

Thin sections of natural enamel lesions, so-called white spots (WS), and areas of sound enamel (SEn) adjacent to the WS were exposed to an intraoral environment for 2 weeks. Thin sections of WS samples, clamped in a PMMA holder, were microradiographed before and after exposure to intraoral conditions. Acid resistance was evaluated by lesion depth and mineral changes during the cariogenic challenge. The results show that there were statistically significant differences in lesion depth, mineral loss and mineral volume percent at the surface before and after the intraoral cariogenic challenge at least at p<0.05, except for a change in mineral volume percent at the surface of WS samples. This exception indicates that no mineral change occurred in the surface layer of WS. The fact of 2.8 and 1.8 times higher ratios of SEn over WS of mean changes in lesion depth and mineral loss data, respectively, seems to indicate a quantitative difference in acid resistance level of WS lesions compared with the areas of SEn. Regarding the site of mineral changes, a distinctive feature of WS samples is that mineral loss occurs at the bottom of lesions. In contrast, areas of SEn produce a typical subsurface type of lesions. From this in situ study, it can be concluded that the surface of WS samples was apparently much more acid-resistant (at least approximately 2 times) than the areas of SEn that received a similar intraoral acid challenge.     

Chemical and structural challenges in remineralization of dental enamel lesions

Abstract – The aim of the present paper was to identify some chemical and structural factors which may prevent a full remineralization of caries lesions and to study whether it is possible to overcome such obstacles. Samples of powdered enamel apatite were equilibrated with solutions metastably supersaturated with respect to enamel hydroxyapatite and fluorapatite. After 10 min and 60 min of equilibration at 20°C the suspensions were centrifuged and the calcium and phosphate concentrations and the pH were determined in the supernatant. In parallel studies, 50–75-μm-thick sections of 27 fluorotic teeth of a severity of 5–7 according to Thylstrup and Fejerskov's classification were examined by microradiography and in polarized light using distilled water, and Thoulett's media or seen dry in air. Five obstacles inhibiting remineralization were identified: 1) Although remineralizing solutions or saliva are supersaturated with respect to enamel apatite the total amount of calcium and phosphate dissolved in it is small, so that after precipitation of the dissolved mineral only 1/20 000-1/30 000 of the volume of the mineralizing solution is occupied by mineral. 2) The concentration gradients from the mineralizing solution into the enamel is small, which indicates a slow diffusion into and out of the lesion. 3) The uptake of calcium and phosphate by the enamel apatite crystals is so rapid that the aqueous phase within the pores can be presumed to be only marginally supersaturated in the deeper parts of the lesion. 4) The surface layer of the enamel lesions was found to be a serious obstacle to remineralization so that a subsurface area remains hypomineralized after exposure to salivary remineralization even for a lifetime. 5) Nucleation of new apatite crystals to substitute lost crystals is an unsolved problem.