The Composition of Bovine Peritubular Dentin: Matching TOF-SIMS, Scanning Electron Microscopy and Biochemical Component Distributions

Peritubular dentin (PTD) is a hypermineralized phase within the dentinal tubules in some vertebrate teeth as an interface between the intertubular dentin (ITD) and the cell processes. Our aim has been to understand the composition, structure and role of PTD as a mineralized tissue. We have utilized the technique of time of flight secondary ion mass spectrometry (TOF-SIMS) to map the distribution of positive and negative inorganic ions as well as organic components in the fully mineralized, intact PTD structure in bovine tooth cross-sections, and correlated these with scanning electron microscopy (SEM) in standard and backscatter modes. In recent work, we developed a procedure to freeze fracture the teeth Published online: August 26, 2008 Dr. Arthur Veis Department of Cell and Molecular Biology, Feinberg School of Medicine Northwestern University, 303 E. Chicago Avenue Chicago, IL 60611 (USA) Tel. +1 312 503 1355, Fax +1 312 503 2544, E-Mail [email protected] © 2008 S. Karger AG, Basel Accessible online at: www.karger.com/cto Abbreviations used in this paper ITD intertubular dentin PTD peritubular dentin SEM scanning electron microscopy TOF-SIMS time of flight secondary ion mass spectrometry D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /5 /2 01 7 1: 57 :2 1 P M Composition and Localization of Peritubular Dentin Constituents Cells Tissues Organs 2009;189:12–19 13 provided information on the major molecules and their characteristics, while physical techniques, such as X-ray crystallography, have described the mineral crystals and how they are packed and organized. However, since the biochemical approaches require the disruption and dissolution of the tissue in order to collect the matrix components, their correlation with each other in position and function has generally had to be deduced by indirect means. For example, in mineralized tissues, component localization by means of immunocytochemistry has uncertainties, since the mineral phase must be removed to expose the epitopes of the relevant matrix macromolecules, and the procedures used for this may introduce artifacts. Thus, proteins occluded in the mineral phase, but not accessible during tissue fixation could be missed as important modulators of the physical properties of the mineral phase. Techniques of surface analysis in metallurgy and surface chemistry have been developed which are applicable to the in situ analysis of mineralized tissues without prior degradative treatments. In the present study, one of these methods, time of flight secondary ion mass spectrometry (TOF-SIMS), has been applied in concert with biochemical analyses and other physical methods to examine the composition of the organic components of bovine peritubular dentin (PTD) compared with the composition of intertubular dentin (ITD). Materials and Methods TOF-SIMS Features In TOF-SIMS, a focused high-energy pulsed beam (nanosecond pulses) of metal ions, such as gallium (Ga + ) or gold (Au + ), is rastered across the surface of a sample. These ions penetrate the surface and transfer their energy to the molecules in the surface, resulting in the formation of charged ions originating within and depending upon the composition of the material. These secondary ions are ejected from the surface and drawn into the mass spectrometer, where their mass is analyzed by their time of flight from the surface to the detector. The ejected secondary ions may be simple, monoatomic ions, such as Ca + or Mg + , or more complex structures from the breakdown of organic molecules. Each pixel on the surface releases a spectrum describing the masses of all ions originating within that area, and the composition of the released ions depends on the depth of penetration of the primary beam. In order to map the surface, the density of the beam is kept low, in the order of 10 12 ions/cm 2 , much lower than the approximately 10 15 atoms/cm 2 residing in a monolayer. Thus, differential sampling of the spectrum from adjacent pixels by the pulsed beam is possible. The sampling depth of static SIMS is only a few atom diameters, 10–20 Å. Thus, a protein molecule with a dimension of greater than 40 Å would be only partially interacting to give rise to charged fragments, ranging from single amino acid fragments to larger polypeptides. In commercial instruments available, the upper limit to organic fragment size is about 2,000 Da. The mass resolution of TOF-SIMS is superb, at 10,000 (m/ m), so that ion fragments differing by only a few millimass units can be reliably distinguished from each other. In practice, the spectrum will contain many mass fragments of different intensity. The investigator must select the most pertinent characteristic fragment masses to follow for determining the distribution of sample surface constituents. Data Acquisition A PHI THRIFT III (Physical Electronics, Chanhassen, Minn., USA) TOF-SIMS apparatus was used with a liquid Ga primary ion source, producing a 25-keV ion beam. After the sample was mounted, the surface was cleaned of absorbed substances, and smoothed by sputtering with the Ga+ beam for 1 min. Spectra were then acquired over a 30 ! 30 m surface area (256 ! 256 pixels) in both positive and negative ion modes. The masses were calibrated with CH3 + , C2H + , C3H5 + , CH – , OH – and C2H – ions. Amino acid fragment ions were identified from the SIMS spectra of polyamino acids absorbed on silica wafers [Mantus et al., 1993; Samuel et al., 2001; Dambach et al., 2004], phospholipid fragment ion identification was based on the data of Ostrowski et al. [2005]. Since the polypeptides and phospholipids yield many fragments, we selected those fragments shown in earlier studies to have the highest signal intensities, at masses minimally overlapping with ion fragments originating from other components. For example, the C2H6N + fragment is the most intense peak (M = 44.05) from both Ala and Ser. A less intense, but not overlapping peak at M = 71 from Ser was used to distinguish between these amino acids. Bovine Tooth and PTD Preparation Unerupted and erupted bovine molar teeth were collected, placed on ice and processed as described [Gotliv and Veis, 2007] as rapidly as possible. The intact teeth were divided into coronal and root portions using an Isomet 1000 diamond saw. The coronal sections were embedded in epon and cut into thin sections to expose surfaces for hand polishing with 4,000-grit paper followed by 0.3m aluminum paste [Gotliv et al., 2006]. After washing and sonication to remove any debris and particulate matter [Gotliv et al., 2006], the sections were examined by either TOF-SIMS or scanning electron microscopy (SEM). The planes of the observed surfaces for sectioning and polishing were carefully selected to be as perpendicular as possible to the direction of the dentinal tubules, or at an angle of about 30° to the tubule axis to expose the inside of the tubule surface. Some of the initial coronal sections were also taken whole, frozen at –80°, crushed to a powder and then exposed to 5% NaOCl at room temperature for 2 weeks to digest the ITD collagenous matrix and expose the mineralized PTD. The treated crude PTD was then fractionated on a stepwise aqueous sodium polytungstate density gradient [Weiner et al., 1999]. The particles collected at each step boundary were subjected to SEM, using a Hitachi S-3500 SEM in the high-vacuum mode, and SIMS analyses. For SEM, the samples were mounted on SEM pins using adhesive carbon tape and coated with a 6-nm layer of gold. Biochemical Analysis The PTD powder and various density fractions were demineralized in 0.8 M HCl at 4 ° C and the soluble portion dialyzed against D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /5 /2 01 7 1: 57 :2 1 P M Gotliv /Veis Cells Tissues Organs 2009;189:12–19 14 deionized water using a 3500 MWCO Spectra/Por membrane. The retained material in the fractions shown to contain PTD by the SEM analyses were then subjected to a variety of procedures: SDS-PAGE; agarose/polyacrylamide gel electrophoresis; amino acid analysis; phosphate, sulfate sialic acid, neutral and amino sugar, uronic acid analyses; lectin binding; lipid extraction and analysis; all described in detail in Gotliv and Veis [2007].