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

Abstract

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 and separate PTD from the less dense ITD by the use of aqueous sodium phosphotungstate step density gradients, after degrading the ITD collagen with NaOCl. Here, PTD-containing fragments were characterized by SEM and TOF-SIMS surface structure analysis. The TOF-SIMS data show that the isolated PTD does not contain collagen, but its surface is rich in glutamic acid-containing protein(s). The TOF-SIMS spectra also indicated that the intact PTD fragments contain phospholipids, and chemical analyses showed phosphatidylserine, phosphatidylinositol and phosphatidylcholine as the principal lipid components. In SEM sections, untreated PTD shows as a smooth collar around the tubule, but after digestion with ethylenediamine to remove all organic components, the porous nature of the mineral phase of small, thin platy apatite crystals becomes evident. Thus, the organic matrix of PTD appears to be a proteolipid-phospholipid complex.

Animal and human dentin microstructure and elemental composition

“…Similar results were obtained in some other studies [15,18,24]. The overall Mg concentrations reported for canine, equine, and human dentin are 1.545% [41], 0.63% in PTD and 0.56% in ITD [25], and 0.858% [42], respectively.…”

“…Atoms with higher atomic number appear brighter and those with lower atomic number darker. In dentin brighter PTD indicates mainly higher concentration of Ca and P ions and thus a higher degree of mineralization comparing to darker ITD [14,18,25]. As expected, EDXS measurements of PTD and ITD confirmed that the main dentin constitutive elements were Ca and P. Interestingly, in all species investigated higher amounts of P were assigned in PTD compared to ITD.…”

“…In human, PTD mineral content by volume is approximately 90% [3], but if PTD contains any collagen remains controversial [11][12][13][14][15][16][17][18]. The bulk of the dentin comprises intertubular dentin (ITD), whose organic content is approximately three times higher than in PTD [14].…”

Scanning Electron Microscopic Study on the Fibrillar Structures within Dentinal Tubules of Human Dentin

“…For some time, dentin tissue has been extensively analyzed by means of scanning electron microscopy, and in the published studies details of new dentinal structures were described (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). In 1964, Scott (24) described that OPs in the inner third of the dentin were associated with thin fibrils and thought they were OP ramifications.…”

Molecular Insights into Covalently Stained Carious Dentine Using Solid-State NMR and ToF-SIMS

“…In comparison to enamel, dentine consists of a larger variety of proteins [Linde, 1989;Fujisawa and Kuboki, 1990;Linde and Goldberg, 1993], where linkages to the mineral appear to arise from acidic non-collagenous proteins [Boskey, 1989;Butler and Ritchie, 1995;Duer et al, 2009;Iline et al, 2015]. Linkages have also been reported to involve glutamic acid-rich protein of high-molecular-mass calcium-proteolipid-phospholipid-phosphate complexes elucidated by time-of-flight secondary ion mass spectrometry (ToF-SIMS) [Gotliv and Veis, 2009].…”