The composition and structure of bovine peritubular dentin: Mapping by time of flight secondary ion mass spectroscopy

Journal of

Structural Biology Journal of Structural Biology 156 (2006) 320–333 www.elsevier.com/locate/yjsbi

The composition and structure of bovine peritubular dentin: Mapping by time of flight secondary ion mass spectroscopy Bat-Ami Gotliv, Joshua S. Robach, Arthur Veis

*

Department of cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 303E Chicago Avenue, Chicago, IL 60611-3008, USA Received 13 December 2005; received in revised form 8 February 2006; accepted 9 February 2006 Available online 9 March 2006

Abstract The dentin layer of the tooth is a complex mineralized tissue traversed by a closely packed system of tubules. Each tubule is surrounded by highly mineralized tissue referred to as peritubular dentin (PTD). The remaining mineralized collagen network between the tubules is the intertubular dentin (ITD). A TOF-SIMS analysis of the PTD constituents has been used to compare the PTD to the ITD. The PTD differs from the ITD not only in the degree of mineralization but also in the amount and nature of the mineral elements and amino acids. The organic matrix of the PTD consists of a unique collagen free assembly of proteins rich in glutamic acid, where the ITD organic matrix is collagen-rich and Asp-rich. The apparent concentration of organic fragment ions observed in the PTD in the TOF-SIMS negative ion mode was much higher than expected. The PTD was found to be rich in Ca2+, Na+, Mg2+, and K+. The amount of Mg2+ and K+ in the PTD was significantly reduced after deproteination, while Ca2+ and Na+ were still accumulated in the PTD. This implies that Mg2+ and K+ are mainly associated with the organic matrix rather than with the mineral of the PTD.  2006 Elsevier Inc. All rights reserved. Keywords: Peritubular dentin; Intertubular dentin; TOF-SIMS; Tooth structure; Collagen; Non-collagenous proteins

1. Introduction Vertebrate tooth dentin is a complex tissue formed in an organized fashion by a layer of odontoblasts initially in opposition to the layer of ameloblasts that are involved in enamel formation. The secretory odontoblasts are elongated polarized cells that secrete collagen at the end facing the dentino–enamel junction (DEJ). The main body of each odontoblast retracts from the DEJ as the secreted dentin collagen thickens and mineralizes, forming a channel which becomes a dentinal tubule. The tubules penetrate the entire thickness of the dentin and predentin. The majority of the dentin collagen fibril network is deposited between the tubules, with each fibril axis approximately perpendicular to the direction of the tubule and nearly parallel to the DEJ. The network becomes mineralized forming the intertubular dentin (ITD). However, an elongated odontoblas*

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tic process remains within each tubule for a substantial portion of the total tubule length. The tubules become surrounded by annular collars of a differently structured mineralized material, the peritubular dentin (PTD) (Bradford, 1958; Frank, 1959; Fearnhead, 1957; Takuma, 1960). The tubule inner diameter is smallest near the DEJ but increases as the tubule extends towards the cell body at the odontoblast terminal web. In the coronal dentin the odontoblasts become increasingly crowded as they move into the restricted pulpal space. The cells move inward and downward, creating a sinuous path for each tubule. While the number of tubules is directly linked to the odontoblast number, the tubule curvature, and dimensions as well as the thickness of the PTD annulus, all vary from location to location. The relative matrix area occupied by the ITD ranges from about 12% at the mineralization front (MF) at the dentin–predentin junction to 96% near the DEJ where the tubules originate, while the inverse is true for the PTD which goes from a relative area of about 3% at the DEJ to over 60% at the MF (Pashley, 1989). The cellular

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odontoblastic process extends within the tubule, in a fully grown tooth, to about one-third the total length of the tubule (Gorraci et al., 1999). In the coronal dentin the PTD is more heavily mineralized than the ITD, with the difference in the mineral content ranging from less then 9% (Miller et al., 1971) to 40% (Frank, 1959) depending on location. In root dentin the width of the PTD annulus is generally small and the PTD may be missing in some instances (Takuma and Eda, 1966). The differences between the PTD and ITD mineral content control the mechanical properties of the dentin. Thus, the dentin microhardness varies in different parts of a given tooth (Pashley et al., 1985). Using AFM techniques Marshall et al. (1997), found that PTD hardness was not dependent upon location and had a uniform Young’s modulus, concluding that the changes in dentin microhardness with location could be attributed to changes in the hardness of ITD and not to a local increase in number of tubules. By comparing the microhardness of the tubular mantle dentin just below the DEJ with the bulk of the dentin, Wang and Weiner (1998) showed that the presence of PTD enhances dentin stiffness. Clearly, although the function of the PTD is not well established, the dentinal tubules and surrounding PTD have an important biomechanical function in dentin. The evident microheterogeneity in mechanical properties, mineral content, and matrix organization of dentin raises fundamental questions regarding the control over formation, growth, and composition relevant not only to the dentin layer but to mineralized tissues in general. Approximately 90% of the ITD organic matrix is type I collagen, with the remainder being a mixture of non-collagenous proteins (NCP) and proteoglycans. The major NCP is phosphophoryn (Dimuzio and Veis, 1978), a highly phosphorylated acidic protein rich in aspartic acid and phosphorylated serine residues, but several other acidic, phosphorylated proteins are present. The composition and the organization of the PTD is more problematic. The small scale of the PTD and close association between the PTD and the ITD, as well as the difficulties of studying heavily mineralized tissue, makes it very complicated to characterize intact PTD. In early studies, the PTD matrix was determined to be morphologically amorphous and, based on various staining techniques, to contain acid mucopolysaccharides (Takuma and Eda, 1966). More recently, transverse and longitudinal sections of mineralized human teeth, etched to reveal the underlying organic matrix and then stained with Stains-All, a metachromatic stain that emphasizes phosphoproteins and acidic glycosylated proteins, showed the PTD to be stained much more heavily than the surrounding ITD matrix. In transverse cross-section, however, the central tubule spaces were either essentially empty or contained unstained remnants of the odontoblastic processes (Weiner et al., 1999). The carbonated apatite of the PTD and ITD were crystallographically similar in spite of the apparent differences in matrix composition. Weiner et al. concluded that the

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PTD–ITD system showed that two distinct carbonated apatite based mineralized tissues could be organized and formed contiguously within the same organ utilizing different sets of matrix proteins. The isolated PTD proteins were phosphorylated but enriched in Glu residues while relatively low in Ser and Asp and devoid of Hyp, in contrast to the Hyp-rich, higher Asp and Ser content of the ITD NCP and collagen matrix. Thus, the PTD matrix did not appear to contain either collagen or phosphophoryn. Two of the major questions concerning the PTD structure relate to the presence of collagen and the continuity between ITD and PTD. Using electron microscopy, Takuma and Eda (1966) observed a small number of fibrils, identified as collagen, in the PTD of human teeth. Magne et al. (2002) used FTIR to analyze the collagen and mineral content of the PTD in horse dentin. They concluded that, within the limits of their resolution, collagen was the main protein component of the PTD. Weiner et al. (1999) on the other hand, concluded that the PTD did not contain collagen, and that the mineral-related proteins were distinctly different. Although it was possible to fractionate dentin by a density gradient method and obtain a PTD enriched preparation for analysis (Weiner et al., 1999) the methods used required an initial degradative treatment of the dentin, raising the possibility of losses and degradative changes in the isolated PTD proteins. In the work presented here, we have addressed the questions of PTD and ITD differences by examining the PTD and ITD constituents without the necessity for physical isolation of the structure, that is, in situ. The method adopted for this purpose is time of flight secondary ion mass spectroscopy (TOF-SIMS). In this technique, a beam of heavy metal ions is focused on the material in question, causing the spalling of ions and charged fragments from its surface. These ions are swept into a TOF-MS and identified. The beam can be rastered over the surface and the ions released at every point analyzed by mass to charge ratio. Thus, a map of the surface distribution can be determined (Van Vaeck et al., 1999). In this study, we have focused on mapping the distributions of the characteristic amino acid fragments and ionized element constituents of the PTD and ITD of bovine dentin. The remarkably distinct patterns show that the constituents of the PTD and ITD are different and that the PTD is not a collagen based tissue element. 2. Materials and methods 2.1. Bovine dentin preparation The freshly extracted lower jaws of 12–18 months old calves were collected (Aurora Packing Company, Aurora, IL) and immediately placed on ice. The unerupted molars, erupted molars and incisors were removed from the jaws the same day and washed five times for 20 min each wash in phosphate-buffered saline containing 5% penicillin– streptomycin–amphotericin solution (Invitrogen, Carlsbad,

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CL). The unerupted molars were immediately placed in phosphate-buffered saline containing 50% glycerol and stored at 80 C. The erupted molars and incisors were soaked overnight in 5% NaOCl at room temperature, washed extensively with distilled water, and then stored in phosphate-buffered saline containing 50% glycerol at 80 C. The NaOCl procedure was not applied to the unerupted molars. Immediately after thawing the stored molars incisors and unerupted molars were sectioned near the cervical line (crown–root junction) with a diamond saw (Isomet 1000, Buehler, Lake Bluff, IL). About three teeth of each type were used for analysis. The crown and root portions were collected and treated separately. Each part was then sectioned into three 3 mm sections parallel to the initial cut. The pulp tissue was removed and discarded. The 3 mm sections were fixed overnight in Karnovsky’s fixative (13% paraformaldehyde, 5% glutaraldehyde, and 0.1 M sodium phosphate buffer, 4 C). The fixed slices were washed with 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA), three times, 30 min each wash. Dehydration followed, using a graded series of ethanol solutions (30, 50, 70, 90, and 100%), with 15 min in each solution. The dehydrated sections were placed in propylene oxide (three times, 30 min each) and then incubated overnight in polypropylene oxide–Epon solution (1:1) (EMBED 813 kit, Electron Microscopy Services, Hatfield, PA). The sections were washed in fresh polypropylene oxide–Epon solution (1:2, 4 h) then incubated in 100% Epon (1 h) and polymerized for about 24 h at 60 C, until hardened.

liquid gallium producing a 25 keV ion probe (spot size 100–500 nm). The spectra were acquired over an area 30 lm · 30 lm under static conditions detecting either positive ion fragments or negative ion fragments and the masses of each ion fragment of interest were determined. At least four different scans were performed on different regions of each tooth section. The mass scales of the positive ion spectra were calibrated using: CH3 þ , C2 H3 þ , and C3 H5 þ . The mass scales of the negative ions spectra were calibrated using: CH, OH, and C2 H  . Sample surfaces were cleaned by sputtering for 10 min before collecting spectra for analysis. This procedure removes a few Angstroms from the surface, smoothing as well as cleaning it. The amino acid fragments were identified according to their mass as published in different studies of individual poly-amino acids absorbed to an artificial substrate (Dambach et al., 2004; Mantus et al., 1993; Samuel et al., 2001). The fragments used for imaging in the present work, those unique to specific amino acids and/or having a high intensity when examined as absorbed on a surface as poly-amino acids, are listed in Table 1. Since static SIMS is a qualitative rather than quantitative technique, the most important choice in deciding upon which fragment ion to select was the uniqueness of that fragment for identification purposes. Note that although the characteristic mass unit of Asp (88 m/z) is common also to Asn (Lhoest et al., 2001), this peak can be attributed mainly to Asp because of the high content of this amino acid in phosphophoryn

2.2. Preparation of SIMS samples

Table 1 Fragment ions followed by TOF-SIMS

The embedded 3 mm blocks were cut with the diamond saw, perpendicular to the tubule direction in the middle of the dentin layer. The dentin from the pulp side was removed and thin sections, 200–400 lm, were cut from the remaining dentin. These thin sections were polished on both sides using 4000 grit paper, followed by 0.3 lm aluminum paste (Buhler, Lake Bluff, IL). The polished sections were washed in double distilled water (DDW), with sonication for 1 min. The sections were immersed in ethanol–sodium ethoxide solution (1:1) then washed in 100% ethanol (10 min) to completely remove the Epon. The smear layer was removed by placing the sections in citric acid (0.01 M, pH 3) for 1 min, then washing in DDW for 5 min, and 100% ethanol (three times, 10 min each). In the final step before mounting, the samples in ethanol were sonicated for 1 min, and then dried. The dry samples were mounted on magnetic discs using adhesive copper tape (SPI, West Chester, PA).

Component

2.3. TOF-SIMS analysis

Fragment/ion detected

Mass units

Positive ions detected in dentin surface by TOF-SIMS Hydroxyproline Hyp C4H8NO+ Proline Pro C4H6N+ Methionine Met C2H5S+ Glycine Gly CH4N+ Alanine Ala C2H6N+ Valine Val C5H7O+ Aspartic acid Asp C3H6O2N+ Glutamic acid Glu C3H4O+ Serine Ser C3H3O2+ 2+ Ca2+ Ca Ca Mg Mg2+ Mg2+ + Na Na Na+ K+ K K+

86.06 68.05 61.01 30.03a 44.05b 83.09 88c 56 71 40.08 24.3 23 38.96

Negative ions detected in dentin surface by TOF-SIMS Protein OCN Protein CN Carboxylates COOH Phosphate P Phosphate PO3 Phosphate PO2 Phosphate PO

42 26 44.99 30.97 78.97 62.97 46.97

a

A PHI TRIFT III TOF-SIMS apparatus (Physical Electronics) was used. The primary ion source was a

Shortcut

b c

This fragment is common to many other amino acids. This is the most intense peak for both alanine and serine. This fragment is common to Asp and Asn.

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(Dimuzio and Veis, 1978), the major non-collagenous protein in the dentin. 2.4. Dentin demineralization Some samples were demineralized before SIMS analysis. After removing the Epon and washing the smear layer as described above the mineralized sections were rehydrated in a ‘‘reverse’’ ethanol gradient (100, 90, 70, 50, and 30% for 10 min at each step) and washed with DDW twice for 15 min. A solution of 0.5 M acetic acid containing 2% glutaraldehyde and 1% formaldehyde as cross-linking fixatives was added for 1 h or 5 min, for total demineralization or surface etching. After washing out the fixative, the samples were again dehydrated in the graded ethanol series, and then sonicated in 100% ethanol (5 min). The dry samples were mounted on the magnetic disc using adhesive copper tape and scanned in TOF-SIMS as described. 2.5. Fractured dentin Whole untreated coronal dentin was fractured mechanically in liquid nitrogen. The dentin fragments were washed in DDW as the only treatment, and stored at 80 C. After lyophilization, the dentin fragments were mounted as above on a magnetic disc so as to expose a surface to the spalling ion beam. 2.6. Exposing the mineral phase by digestion of the total organic matrix The macromolecules of the PTD and ITD were removed using ethylenediamine (Armstrong and Singer, 1965; Skinner et al., 1972). Coronal dentin, either polished sections or fractured surface particles, were fixed in Karnovsky’s fixative as described above and put in ethylenediamine solution (10 h, 60 C) (Sigma–Aldrich, St. Louis, MO). After washing in DDW three times, 20 min each, the samples were dehydrated in the graded ethanol protocol. The polished sections were mounted on magnetic discs with the adhesive copper tape for use in the TOF-SIMS. The fractured dentin particles were mounted on SEM pins using adhesive carbon tape. These were gold coated (6 nm layer of gold) and observed in a Hitachi 3500 SEM in the high vacuum mode. 2.7. Reference surface-bound amino acid SIMS spectra An amino acid mixture used for amino acid analysis standard (500 pmol each amino acid, Agilent Technologies, Waldbronn, Germany) was mixed with 500 pmol L-4-hydroxyproline (Fluka, Sigma–Aldrich, St. Louis, MO) and absorbed onto a fractured geological calcite (Wards Natural Science, Rochester, NY) and dried overnight. The fractured calcite was mounted on the magnetic discs with the cooper tape and observed in the TOF-SIMS. The amino acid fragments were identified according to the masses shown in Table 1.

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2.8. Magnesium staining Bovine molar sections embedded in Epon as described were cut into 200 lm sections with the diamond saw. These were polished from both sides, using the 4000 grit paper and 0.3 lm aluminum paste. Both mineralized and acetic acid demineralized sections were prepared, following the procedures described above. The sections were stained with magneson following the procedure of B.D.H. (1946) with some modifications. A 0.5% solution of magneson (p-nitrobenzene-azoresorcinol) in 2.0 M NaOH was mixed 1:1 with 30% NaOH. The mineralized and demineralized sections were soaked in this solution for 10 min in the dark. The sections were then washed in warm 30% NaOH and observed in the light microscope with transmitted light. Magnesium ions stain a deep blue. 2.9. Colocalization of components The colocalization Finder used in this study is part of the ImageJ 1.33u program (NIH). The basic ImageJ plugin highlights the colocalizated points of two eight-bits images. The two images will be affected to the two red and green channels of an RGB image. The colocalizated points will appear white by default (display value = 255). The plugin initially generates an eight-bit image with only the colocalizated points (image available by validating colocalizated points eight-bit), then it combines the three 8-bit images in an RGB image. Two points are considered as colocalizated if their respective intensities are strictly higher than the threshold of their channels (which are 50 gray levels by default: threshold channel 1 (0–255)), and if their ratio (of intensity) is strictly higher than the ratio setting value (which is 50% by default: ratio (0–100%)). Thus, two points are considered as colocalized if their respective intensities are strictly higher than the threshold of their channels, and if their ratio of intensity is strictly higher than the ratio setting value. 3. Results 3.1. Positive ion distribution in the coronal dentin surface The PTD was clearly observed in cross-section using the TOF-SIMS technique (Fig. 1) as a ring of mineralized tissue surrounding an essentially non-mineralized, empty central space. As expected, the PTD was distinguished from the ITD by a higher local calcium signal. The virtually empty tubule spaces or holes were observed clearly penetrating the dentin surface, which was cut perpendicular to the tubule direction. The distributions of Ca2+, Mg2+, K+, and Na+ as well as amino acid fragments corresponding to Glu, Ser, Ala, Met, Pro, Hyp, and Val were determined and are shown in Fig. 1. A distribution labeled Gly is also shown, but although the CH4N of 30.3 mass units was reported to be the highest intensity peak of glycine, this fragment is common to many other amino acids

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Fig. 1. Ion and amino acid fragment distributions in dentin surface of an erupted molar bovine tooth cut perpendicular to the tubule axis as observed by TOF-SIMS. The gradient in the intensity of the colors indicates variation in ion signal, thus a high ion signal shows as lighter color. For the specific fragments of each amino acid see Table 1. The scale bar is 10 lm. The graphs in (n) describe Ca2+ (a) and Na+ (b) distributions as gray color levels along the black line drawn across the tubule hole.

as well (Mantus et al., 1993). It is important to note that each Fig. 1 scan, a–m, represents the same surface at the same time and at the same magnification. Casual examination of the patterns in Fig. 1 suggests that the distributions of components are similar, but three factors must be considered: the intrinsic intensities of the particular component spalled from the surface; the surface concentration of that component; and the relative signal intensities of the individual sputtered ions. In Fig. 1, panel a, the Ca2+ ion signal around the tubule hole is, as expected, more intense in comparison to the ITD. However, the PTD image itself is not uniform across the PTD, a narrow ring of lower Ca2+ density surrounds the tubule space, as emphasized in the intensity trace through the tubule marked with a black line in panel a and shown in panel n of Fig. 1. Just as the ITD–PTD interface is not entirely abrupt, showing the merger of the two structures, the inner wall of the PTD annulus is not sharp, indicating that in these sections where the tubules do not contain a cell process, there is also a Ca2+ containing material of reduced Ca content along the PTD inner surface. The PTD also exhibited (in order of intensity) strong signals for Na+, K+, and

Mg2+. The Na+ (Fig. 1b) signal clearly showed a lower intensity ring around the PTD inner surface. It is evident in the intensity trace shown in panel n that in the marked tubule in panel b the tubule itself also contains Na+ containing components. The apparent tubule diameter thus seems smaller than the Ca2+ trace in panel a. Mg2+ and K+ had similar distributions, but by virtue of the apparently larger diameter of the tubule were at lower concentration within the tubule. Positive ion fragments from protein components also traced out the PTD (Fig. 1), with higher concentrations of Glu, Ser, and Met in the PTD than the ITD, and the total protein, represented by (Ser + Ala). Val and Asp are not concentrated in the PTD but are rather more homogenously distributed over the dentin surface. In fact, Asp much like Pro, Hyp, and Gly are most prominent in the ITD. A similar SIMS survey of the ion distributions was obtained by attempting to section the dentin so that the PTD and tubules were viewed somewhat more parallel to the tubule long axis than in cross-section (Fig. 2). In the section shown the PTD surfaces are seen at an oblique

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Fig. 2. Ions and amino acid fragment distributions in the dentin surface of erupted molar bovine teeth cut obliquely to the tubule long axis as observed by TOF-SIMS and SEM (SEM a and b). In SEM b, the PTD interior surface is observed at high magnification. Scale bar of the TOF-SIMS images is 10 lm.

angle, with tubules running at an angle to the polished surface. SEM of a similar polished oblique surface shows that the PTD interior surface is exposed (Fig. 2, SEM a and b). Surprisingly, these interior surfaces of the PTD are marked by an intense signal when observing the Ca2+ ion distribution. This signal is far greater than that in the ITD. However, the interior wall of the PTD (the PTD–tubule lumen interface) is also higher in the Mg2+, Na+, and K+, signals than in the ITD. Likewise, the Glu and total protein (Ser + Ala) signals are more intense along the PTD luminal surface than in the surrounding ITD while the Pro, Hyp, and Gly distributions indicative of the collagen of the ITD provide a negative image of the PTD inner surfaces, and hence are more restricted to the ITD. These data are in accord with the direct cross-sectional SIMS view of the PTD showing the high Ca2+, Na+, K+, and Mg2+ ion concentrations, and higher Glu and Ser-Ala contents of the PTD and its luminal contents. To show these amino acid distributions more clearly with respect to the Ca2+ ion distribution, the colocalization Finder plugin for ImageJ program was used, as explained in Section 2. In this protocol where the two species examined pixel by pixel colocalize above the threshold level they appear as a green dot. As shown in Fig. 3, there is extensive overlap between Ca2+ and Glu in the PTD inner tubule surface. However, the overlap between Ca2+/Pro, Ca2+/ Hyp, and Ca2+/Gly colocalization is observed only in the ITD region and not on the PTD walls. This is indicative of the calcified collagen in the ITD and a lack of significant collagen content in the PTD. Nevertheless, as emphasized

by the Ca2+/Glu overlay, the PTD does contain a Glu rich protein. The data shown in Figs. 1–3 were observed in erupted bovine molar dentin. Similar results were obtained in the unerupted molars and erupted incisors (data not shown). 3.2. Negative ion distributions in the coronal dentin surface As shown in Table 1, the negative ion spectra reflect the presence of phosphate and carboxylate ions and the fragments from the bombardment of the amide bonds. These carboxylate ions were observed in all the poly amino acids studied in TOF-SIMS negative mode. The intensity of individual carboxylate ions was common among the different poly-amino acids, meaning that the carboxylate ions are characteristic of total proteins rather than attributable to a specific amino acid. Fig. 4 shows the distribution of negative ions in a coronal dentin section cut perpendicular to the tubule long axis. In the negative ion mode the PTD region is highlighted by the high concentration of all the different derivatives of phosphate ions, which arise from the hydroxyapatite mineral and the phosphorylated acidic proteins. As observed in the positive ion mode distributions of the surface perpendicular to the tubules, the tubule centers are empty. The CN and OCN ions are clearly observed while the HCO2 signal was weak (data not shown). For each of these ions the intensities are higher over the PTD rather than in the ITD region implying either a higher protein concentration in the PTD surface, or a greater accessibility to the Ga+ ion beam. As seen in Fig. 4 the patterns of P, PO, and

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Fig. 3. Colocalization images of calcium ions and other fragments as indicated in each box title. The green color indicates the region of colocalization. In this region the ratio of intensity between two points of the images shown in Fig. 2 is higher than 50%. Scale bar is 10 lm.

Fig. 4. Negative ion distributions in bovine coronal dentin cut perpendicular to the dentin surface. Scale bar is 10 lm.

PO2 distribution are virtually identical, whereas the PO3 ions seem to be more prominent in the ITD matrix, coincident with the Asp rich proteins and collagen. Thus, the P, PO, and PO2 may be more prominent products of the hydroxyapatite.

3.3. Ethylenediamine deproteination Deproteination of the dentin matrix with ethylenediamine extracts virtually all of the organic components not occluded within the crystals, while mineral phase maintains

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its crystallinity and its major element chemical identity (Armstrong and Singer, 1965; Skinner et al., 1972). Fig. 5 shows positive element and fragment ion distributions in coronal dentin after ethylenediamine treatment. The Ca2+and the Na+ ions are still concentrated in the PTD. However, there is significant reduction in the amount of Mg2+ and K+ in the PTD in comparison to the untreated coronal dentin showed in Figs. 1 and 2. As expected, the amino acid fragments in the PTD are also significantly decreased; however, it is evident that amino acid derived

Fig. 5. Ion distributions in coronal bovine dentin after ethylenediamine deproteination. Scale bar is 10 lm.

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fragments are present, indicating the abundance of mineral-occluded protein in the PTD as well as in the ITD. After the ethylenediamine treatment the Glu and (Ala + Ser) fragments are more homogenously distributed in the dentin, there is no longer a distinct accumulation of the Glu-rich protein in the PTD, indicating that the Glu-rich protein surrounds but is not occluded in the PTD crystalline phase. Fig. 6 compares the Glu distribution in the mineralized dentin (Fig. 6A) versus deproteinated dentin (Fig. 6B). The Glu distributions are shown by the changes in gray level as observed across the selected tubule holes (white lines in Fig. 6). The minimum deproteinated dentin gray level intensity indicating low Glu concentration is more extended in comparison to the same region in the mineralized dentin intensity implying the removal of Glu from the PTD by deproteination. The maximum intensity regions of the deproteinated intensities are nearly constant signifying a more homogenous distribution of mineral-occluded Glu in the ITD. The Pro, Hyp, and Gly fragments, representing the collagen matrix are highlighted in the ITD regions. In the Pro and Gly scans, and less prominently in the Hyp scans, the tubule cross sections appear completely black (Fig. 5). The Pro, Hyp, and Gly very evident in the deproteinated ITD matrix suggest that non-mineral-occluded collagen fibrils also resist total removal during deproteination with ethylenediamine. Scanning electron microscopy (SEM) (Fig. 7) was used to compare the surfaces of molar coronal dentin before and after ethylenediamine deproteination treatment. The smooth surface of the PTD observed in untreated fractured coronal dentin (Fig. 7A) is considerably eroded after ethylenediamine deproteination treatment (Fig. 7B). Upon deproteination the plate-like PTD crystals remaining appear similar in size and arrangement to those in the ITD. The ITD region, however, maintains its appearance without significant change. The shift in PTD fracture surfaces from smooth to eroded upon deproteination is consistent with the interpretation of the SIMS data shown in

Fig. 6. Glu distribution in dentin untreated with ethylenediamine (A) and in deproteinated dentin (B). The insets are the gray level intensities along the white lines across the tubule holes in the pictures.

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Fig. 7. Scanning electron micrograph of coronal dentin fractured surfaces in cross-section and longitudinal section showing (A) untreated surfaces and (B) the dentin surfaces after ethylenediamine deproteination treatment. In the left panel the dentin was fractured perpendicular to the tubule direction and in the right side panel the view is along the tubule axes.

Fig. 6 is suggesting that the PTD has readily accessible protein outside of the crystal protected region. 3.4. Demineralization Fig. 8B shows the ion distributions in coronal molar dentin after demineralization with 0.5 M acetic acid for 1 h in the presence of cross-linking fixative. This acetic acid etching treatment was used for demineralization preparatory to immunohistochemical localization of the dentin noncollagenous protein–phosphophoryn (Rahima et al., 1988) and relied on the fact that phosphophoryn is not readily soluble in acetic acid (Butler et al., 1972; Takagi and Sasaki, 1986) as well as the cross-linking insolubilization of the

proteins by the glutaraldehyde and formaldehyde. The total positive ion image presented in Fig. 8 represents the combined distribution of all the ions and amino acid fragments that were followed in the study (Ca2+, Mg2+, Na+, K+, Glu, Asp, Ala + Ser, Ser, Gly, Pro, and Hyp). Fig. 8A shows the total positive ion distribution in a similar mineralized dentin surface. The two images are virtually the inverse of each other. The high concentration total signal of the PTD in the mineralized dentin (A), becomes the lower signal region in the demineralized section (B), while the demineralized ITD yields a more intense signal distribution in the ITD of B than A. Thus, the protein content of the total PTD after demineralization is lower than that in the ITD.

Fig. 8. Combined distribution of Ca2+, Mg2+, Na+, K+, Glu, Asp, Ala + Ser, Ser, Gly, Pro, and Hyp fragments in mineralized coronal molar dentin (A) and after 1 h demineralization (B) with 0.5 M acetic acid in the presence of fixative. Scale bar is 10 lm.

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3.5. Controls for TOF-SIMS validity In root dentin the PTD is generally small and missing in some instances (Takuma and Eda, 1966). Therefore, the root dentin provides a good control for the ion fragment distributions observed in the coronal dentin in Figs. 1 and 2. Fig. 9 presents the distribution of Ca2+, Na+, Mg2+, K+, Glu, Ala + Ser, and Pro fragments in the apical root dentin, perpendicular to the tubule direction. The Ca2+ rich ring observed as the PTD in the coronal dentin does not surround the tubule holes in the apical root dentin. In fact, all the detected ions and fragments appear to be homogeneously distributed in the apical root dentin. In other parts of the root, cut close to the cervical coronal– root junction, narrow PTD rings surrounding each tubule were observed (data not shown). The ions and amino acid fragments observed in those sections showed the same distribution as in the coronal dentin. Since the existence of Mg2+ in the dentin surface was very interesting, possibly relating to the mechanism of PTD formation, a chemical staining method was applied to localize the Mg2+. Thin molar coronal dentin sections were stained with magneson (p-nitrobenzene–azoresorcinol) before and after demineralization (Fig. 10). In this test the magneson interacts with Mg2+ to give a blue/purple color (Feigl, 1937). The ITD and the PTD in the mineralized dentin section stained in purple (Fig. 10A) indicating

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the high magnesium content. Demineralization was carried out as described above with 0.5 M acetic acid, but for only 5 min in contrast to 1 h. In the demineralized thin dentin section (100–50 lm) the ITD stained a lighter blue while the PTD was stained in dark blue, indicating the higher concentration of magnesium in the PTD (Figs. 10B and C). When a dentin section demineralized under the same conditions was examined with TOF-SIMS, it showed that the amount of calcium ions was significantly reduced but unlike the 1 h acetic acid demineralization, protein ion fragments still appeared in the PTD. The peak in the TOF-SIMS spectrum of 56 mass units, attributed to C3H4O+ from the Glu residues (Mantus et al., 1993; Samuel et al., 2001; Dambach et al., 2004), is intense and dominates in the fragment ions attributable to amino acids in the dentin surface (Fig. 1). The distribution of this peak showed it to be concentrated in the PTD. A mass peak of 56 could also arise from the presence of iron in the dentin, although there are no reports of the existence of iron in the dentin. Potassium ferricyanide interacts with iron, yielding a blue color (Evamy, 1963). Application of this qualitative test showed that there was no iron in the dentin and thus made us confident that the origin of the 56 mass unit peak was indeed from glutamic acid fragments. An amino acid mixture containing all of the amino acids that were followed in this study was adsorbed to geological

Fig. 9. Positive ion distributions in a molar root dentin section. This apical section was cut from the lower part of the root. Scale bar is 2.5 lm.

Fig. 10. Thin sections (50–100 lm) of molar coronal dentin stained with magneson. The blue and purple colors are the results of magneson reaction with magnesium. The dentin section in (A) is fully mineralized. The dentin sections in (B) (along the tubules) was demineralized with 0.5 M acetic acid for 5 min in the present of fixative. The inset (C) is higher magnification of section (B).

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calcite, to mimic the roughness of the dentin surface without the protein constituents. The fragmentation of the mixture was checked using the TOF-SIMS under the conditions of the dentin study. This control was important since in all the other TOF-SIMS studies used as reference for the amino acid fragmentation spectra, the poly-amino acids were adsorbed to a flat artificial surface such as mica or silicon substrate (Samuel et al., 2001). Since the roughness of the surface has a major impact on the SIMS results, a calcite surface seemed more appropriate. In addition, the ion beam source in the apparatus used was Ga+ ions, while other ion sources were used in the reference poly-amino acid studies. In this control we found that the same amino acid fragments which were reported in Table 1 were also spalled from the calcite surface, with the same relatively intensities as reported (Mantus et al., 1993; Samuel et al., 2001; Dambach et al., 2004). Finally, the dentin samples used in this study were fixed, dehydrated, embedded in EPON, and polished. The EPON was completely removed before TOF-SIMS analysis. To determine how the spectra obtained might be influenced by the polishing and other preparative steps, otherwise untreated fractured erupted molars and incisors were used. Although it was much harder to map the ionized fragments on the unpolished dentin surfaces, the fragment ions detected on fractured dentin surfaces show the same distribution patterns as observed for a polished surface. Since the fractured dentin was crushed in liquid nitrogen and lyophilized to complete dryness, solvent effects on the results were also eliminated. 4. Discussion The TOF-SIMS method has been used to in biochemical research to study proteins adsorbed on surfaces (Lhoest et al., 2001; Sanni et al., 2002), individual poly-amino acids (Mantus et al., 1993; Samuel et al., 2001), development of mineralization in cell cultures (Dambach et al., 2004), and most recently to track the distribution of magnesium in coral (Meibom et al., 2004). Lefe`vre et al. (1976) used secondary ion microscopy to study the dentin surface and follow the distribution of Ca2+, Mg2+, K+, and Na+. The TOF-SIMS microscope used in our study provided much more advanced features, not available at the time of the Lefe`ver study. Among these features are the restriction to ˚ , detection limits in the analysis of the surface to 10–20 A ppm-ppb sensitivity range, and spatial resolution less then 120 nm. Lodding (1997) used SIMS to follow the distribution of Ca2+, Na+, CN, O, and phosphate in the dentin. However, in their study the tubules and PTD were not examined in detail and the amino acid distributions were not followed. The data presented here is the first comprehensive map of the surface distribution of the main elements and amino acids in dentin. The PTD and the ITD were observed clearly and distinctly in both polished coronal dentin surfaces and fractured surfaces using TOF-SIMS. The tubule centers were

empty and appeared as holes or empty channels in all the sections examined. The ion distributions were consistent in erupted molars, incisors, and unerupted molars. Therefore we can conclude that the PTD with its specific attributes is formed at an early developmental stage and is not only present in the mature teeth. The distribution of the PTD, however, is not homogenous in the dentin. As we and others (Takuma and Eda, 1966) have observed, the PTD is missing from the apical root dentin. In the regions were the PTD exists it contains higher concentrations of Ca2+, Mg2+, K+, and Na+ in comparison to the ITD. Lefe`vre et al. (1976) came to the same conclusions in their early secondary ion microscopy study. The accumulation of Mg2+ in the PTD is particularly interesting. The presence of magnesium in dentin was reported in different studies to be between 0.9% (Jenkins, 1966) and 0.74% (Derise and Ritchey, 1974). The amounts of magnesium are lowest at the DEJ and increase toward the mineralization front at the predentin–dentin junction (Inoue et al., 1971; Johnson, 1972) and thus magnesium was suggested to play a specific and important role in tooth mineralization. There are three possible roles of magnesium in the dentin: 1 Magnesium can be incorporated in the apatite lattice; 2 Magnesium is present in a separate mineral phase; 3. Magnesium is associated with the organic macromolecules. Terpstra and Driessens (1986) considered the Mg2+ ion incorporation in the apatite lattice based on its ionic radii as compared with Ca2+ ion and concluded that the substitution of Ca2+ by Mg2+ could not be excluded. However, their data showed that Mg incorporation in the apatite lattice, if at all, was very limited. Selected area electron diffraction of dentin led Lefe`vre et al. (1976) to the conclusion that in addition to hydroxyapatite the PTD contained whitlockite, another calcium phosphate mineral. Magnesium whitlockite is a calcium orthophosphate crystal in which magnesium is partly substituted for calcium (Lagier and Baud, 2003). Magnesium whitlockite is present in the lumen of dentinal tubules of transparent carious dentin (Vahl et al., 1964), and in dental calculus (Jensen and Hansen, 1957). However, there are only minor differences in the diffraction patterns of hydroxyapatite and whitlockite, and these are very difficult to distinguish in healthy teeth. Schroeder and Frank (1985) studied human PTD by high-resolution transmission electron microscopy and identified only hydroxyapatite crystals. Trautz et al. (1964) stated that the Mg2+ concentration in teeth and bone is too low to form a detectable whitlockite phase. In the present study, we performed wide-angle X-ray diffraction on coronal dentin and compared it to the diffraction pattern of apical root dentin, which does not contain PTD. The only mineral observed in both dentin sections was hydroxyapatite and no other mineral phases could be identified (Data not shown). It might be that the content of whitlockite is so small that it cannot be identified when the whole dentin section is being examined and thus future X-ray studies should be cattied out on isolated PTD. However,

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our SIMS results imply a different role for the magnesium ions. The amount of Mg+ in the PTD was significantly reduced after deproteination with ethylenediamine, suggesting that major portion of the magnesium ions might be associated with organic matrix rather than the mineral phase. Magnesium staining with magneson after demineralization of a thin dentin section for 5 min with acetic acid in the present of fixative supported this conclusion. The magnesium ions were still concentrated on the tubule walls although most of the mineral had been removed. We can thus speculate that Mg2+ has some crucial role in the formation and stabilization of the PTD organic matrix, distinct from its role in the ITD. About 1 wt% of the tooth enamel layer is organic and Mg was suggested to be associated with the enamel proteins (Robinson et al., 1981). Marsh (1989) studied the interaction of dentin phosphophoryn with magnesium in vitro. She showed that at saturation magnesium interacts with phosphophoryn to form stable high molecular weight colloidal particles that are negatively charged. A similar mechanism could drive the accumulation of Mg with macromolecules unique to PTD. The non-collagenous proteins (NCP) constitute about 10% of the dentin organic matrix and the average percentage of magnesium in the dentin was reported to be 0.8% (Jenkins, 1966; Derise and Ritchey, 1974). Theoretically, the magnesium-associated macromolecules could comprise about 8% of the dentin NCP. However, it seems unlikely that the all the magnesium ions are associated with the organic macromolecules of the dentin. Mg2+ has inhibitory effects on the formation of apatite (Blumenthal et al., 1977) and hence its accumulation around the tubules may control the mineralization level of the PTD. In such a case, magnesium ions could be surface bound to the apatite mineral as a protein complex. Sodium and potassium ions were also found to be more concentrated in the PTD than in the ITD. The overall concentrations of these ions in the dentin was reported to be 0.75% for sodium (So¨remark and Samsahl, 1962) and 0.1% for potassium (Jenkins, 1966). Sodium and potassium were detected in the human dentin fluid by electron probe analysis (Haljama¨e and Ro¨ckert, 1970). Our ethylenediamine deproteination results suggest that since sodium ions remain in the PTD after this treatment (Fig. 5), then they are probably associated with the mineral phase of the PTD rather than the organic part. The potassium ions, however, disappeared after this treatment (Fig. 5). (Wiesmann et al., 1998) showed that K+ was localized at relatively high concentration in an ‘‘electron dense micro area’’ along the surface of the odontoblastic process in rat dentin near the dentin–predentin boundary, colocalized with a high phosphate content. They suggested that K+ could play an important role either directly by blocking mineral nucleation or indirectly as a consequence of its regulation of phosphotransferase reactions (Suelter, 1970). Potassium ion could thus prevent early apatite formation, or control the extent of

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PTD formation. It is noteworthy that rat teeth do not form PTD. The PTD and its tubule luminal interface is rich in Glu, Ser, and Met residues. The C2H6N+ fragment, which is a highly intense fragment ion contributed by both Ala and Ser is also concentrated in the PTD. Since direct amino acid analysis found Ser to be concentrated in a PTD-rich fraction of human dentin (Weiner et al., 1999), we can speculate that the C2H6N+ fragment was mainly derived from Ser rather then Ala. Weiner et al. also found that the proteins in the PTD are rich in Glu. Asp is the major amino acid in the main non-collagenous dentin protein–phosphophoryn. On the basis of these data Weiner et al. (1999) concluded that the proteins in the PTD-enriched fraction were not in the group of phosphophoryns. In fact the gel electrophoresis of the PTD-enriched fraction was much different from the whole dentin extract gel. We also can conclude that the macromolecules of the PTD are distinctly different from, and may have different properties or roles than the macromolecules in the ITD. The Glu and Ser rich protein(s) accumulated in the PTD dissolve easily in acetic acid, as demonstrated by the TOF-SIMS images of the demineralized dentin section. This is much different from the Asp-rich phosphophoryn, which does not dissolve in acetic acid (Butler et al., 1972; Takagi and Sasaki, 1986). The high concentration of Met in the PTD is consistent with the TOF-SIMS negative ion distribution. The negative ion images show that the PTD is rich in CN and OCN. These ions were found to appear in all the SIMS spectra of individual poly-amino acids. The intensity of these ions was also found to be identical among the different polyamino acids. Therefore we can regard the CN and OCN as protein matrix fragments in general and be considered as a measure of total protein as well. We can then conclude that, although the PTD is limited in total amount because of its restricted distribution, the concentration of non-collagenous proteins in the PTD is high relative to concentration of the non-collagenous protein concentration in the ITD. This conclusion is supported by the SEM observation of fractured dentin before and after deproteination with ethylenediamine showing the removal of NCP containing mineralized tissue. The smooth PTD surface is severely eroded after deproteination, indicative of its high protein content. Thus, in addition to being highly mineralized, the remaining organic matrix of the PTD is rich in a glutamic acid-rich protein(s). The unexpected high concentration of non-collagenous protein in the PTD and at the tubule luminal surface is also consistent with (and explains) the data of Weiner et al. (1999) showing that the PTD isolated from crushed and sonicated human dentin by gradient density centrifugation floated at a density of about 2.28 g/ml while pure apatite crystals were obtained in the gradient at >2.47 g/ml. Assuming that the protein component has an average density of 1.33 g/ml, this suggests that the PTD is about 13% non-collagenous protein. Rostrum bone is an example for another very highly mineralized tissue that contains significant amount of organic material.

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This hypermineralized tissue is rich in lipids (Zylberberg et al., 1998). The high concentrations of phosphate ions in the PTD can be derived from both the mineral part and the phosphorylated proteins. The presence of highly phosphorylated protein(s) in the PTD was observed in the histological staining of partially demineralized tooth sections (Takagi and Sasaki, 1986; Rahima et al., 1988; Weiner et al., 1999). The PTD stained intensely blue with Stains-All while the ITD stained purple with less intensity. We can now explain this different staining by either a high concentration of phosphorylated protein in the PTD as observed in the negative ion fragment distributions seen in the SIMS spectra (Fig. 4) or by the presence of Glu-rich unique PTD highly phosphorylated protein according to the SIMS positive ions distribution, or both. The ITD is composed primarily of mineralized collagen, with its unique Hyp, Pro, and Gly content. According to the positive ion SIMS images, these do concentrate in the ITD region and at the border with the PTD. But there is neither collagen nor collagen fragments in the PTD (Fig. 3). These results are consistent with Weiner et al. (1999), who found no collagen in the PTD-enriched fraction, but do differ from the FTIR identification of collagen by Magne et al. (2002). The FTIR identification of collagen depended, however, on the assignment of the amide I frequencies of the collagen peptide bonds. These may not be entirely unique to collagen, and the IR spectrum of the PTD protein has not been determined.

of water compresses the tubule radius, and therefore, raises its internal protein and mineral concentration. This may lead to the formation of the PTD mineral phase, modulated by the presence of the unique Glu-rich, Mg2+-rich PTD protein which coats the PTD crystal surfaces, possibly limiting excess PTD formation. It would appear that the PTD is deposited outside the odontoblastic process membrane since the crystal organization in the PTD and the ITD are similar, in agreement with our earlier observations that there is structural continuity at the border between the two regions (Weiner et al., 1999). The PTD mineral may be less stable than ITD hydroxyapatite because of the presence of small amounts of Mg whitlockite and K+. The next major efforts to understand dentin formation require answers to two very different questions. First, what is the nature of the unique Mg-binding PTD protein? Second, why is the PTD formation so limited in the apical root dentin? Answers to these questions will be the basis of our future studies. Acknowledgments This work has been supported by NIDCR Grant DE01374 (to A.V.). The SIMS and SEM study was carried out in the (EPIC) (NIFTI) (Keck-II) facility of NUANCE Center at Northwestern University. NUANCE Center is supported by NSF-NSEC, NSF-MRSEC, Keck Foundation, the State of Illinois, and Northwestern University. References

5. Possible mechanisms for PTD formation As the odontoblast retreats from the DEJ, the main odontoblastic process becomes surrounded by the extensive mineralized collagenous matrix of the ITD. The odontoblast body and its principal odontoblastic process retract apically in the direction of the pulp, leaving behind the space previously occupied by the process. This space becomes a continuous tubule spanning the length of the dentin. After the ITD has mineralized the PTD forms within the tubule lumen as a densely mineralized but essentially non-collagenous annular ring. The odontoblastic process is not uniform in cross-section, becoming wider as it approaches the odontoblast and much more narrow at the opposite end. As the tubular process exits the dentin into the non-mineralized predentin matrix the tubule diameter increases markedly (Marshall et al., 1997). Only the tubule surrounded by the mineralized dentin develops the PTD. Looking from the inverse perspective of formation of the mineralized matrix at the mineralization front, it is clear from the work of Weinstock and Leblond (1973, 1974) and Rabie and Veis (1995) that the non-collagenous components of the dentin are delivered directly at the mineralization front through exocytotic vesicles from the processes, concurrent with a major efflux of the mineral ions which, in the rat, build mineral at the rate of 10 nm/min. This efflux of exported proteins and mineral ions, along with the efflux

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