Gradient Magnesium Content Affects Nanomechanics via Decreasing the Size and Crystallinity of Nanoparticles of Pseudoosteodentine of the Pacific Cutlassfish, Trichiurus lepturus Teeth

Abstract

The formation of biomaterials such as enamel, dentin, and bone is important for many organisms, and the mechanical properties of biomaterials are affected by a wide range of structural and chemical factors. Special dentins exist in extant aquatic gnathostomes, and many more are present in fossils. When a layer of compact orthodentine surrounds the porous osteodentine core in the crown, the composite dentin is called pseudoosteodentine. Using various high-resolution analytical techniques, including micro-computed tomography (micro-CT), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), Raman spectroscopy, and nanoindentation, we analyzed the micro- and nanostructures, chemical composition, and mechanical properties of pseudoosteodentine in the Pacific cutlassfish, Trichiurus lepturus teeth. Nanoscale oval-shaped hydroxyapatite (HA) crystals were distributed in a disordered manner in the pseudoosteodentine, and a cross-sectional analysis showed that the mineral crystallinity and crystalline particle size of the outer orthodentine were greater than those of middle and inner osteodentine. Moreover, the outer orthodentine comprised a mixture of smaller crystals and larger, more mature crystals. The nano-hardness and nano-stiffness of outer orthodentine were significantly higher than those of middle and inner osteodentine along a radical direction. The hardness and stiffness of pseudoosteodentine were inversely proportional to its magnesium (Mg) content. These data are consistent with the concept that Mg delays crystal maturation. The crystal size, crystallinity, nano-hardness, and nano-stiffness of pseudoosteodentine all decreased commensurately with the increase of its Mg concentration. The pseudoosteodentine of T. lepturus also can be regarded as a functional gradient material (FGM) because its mechanical properties are closely related to its chemical composition and nanostructure. Special pseudoosteodentine may therefore serve as a design standard for biomimetic synthetic mineral composites.

Figure 1

Tissue structure of dentin (pseudoosteodentine) of Trichiurus lepturus. (A) 3D reconstruction image of an upper jaw of T. lepturus. Large teeth are examined in this study indicated by a rectangular box. (B) 3D reconstruction image of a functional premaxillary tooth. Dentin makes up most of the tooth and overlain little by a hypermineralized enameloid. The white dotted line indicates the detection location. (C) SEM image of the polished transverse section of a premaxillary tooth. Instead of a pulp cavity, a tooth is replaced by a porous osteodentine (os). (D) Larger view of the boxed area in C. Osteodentine is covered by a thin layer of compact hard tissue. (E) Outer orthodentine layer of a transversely fractured tooth contains parallel dentinal tubules, which is a typical characteristic of orthodentine. (F) Inner osteodentine layer of a transversely fractured tooth resembles bone. Dentinal tubules radiate from the dentinal osteons. en, enameloid; de, dentin; or, orthodentine; os, osteodentine; do, dentinal osteons; dt, dentinal tubule; and it, interosteonal tissue.

Figure 2

Crystalline particle size, crystallinity, and composition of pseudoosteodentine of Trichiurus lepturus. (A) Nanoparticles of outer orthodentine layer from a transversely fractured tooth (diameter: 33.84 ± 6.15 nm). (B) Nanoparticles of middle osteodentine layer from a transversely fractured tooth (diameter: 21.87 ± 4.03 nm). (C) Nanoparticles of inner osteodentine layer from a transversely fractured tooth (diameter: 16.55 ± 3.54 nm). (D) Raman spectra of pseudoosteodentine assigned to hydroxyapatite (HA). (E) The full width at half maximum (FWHM) of the ν₁ PO₄^3– band showing the crystallinity of pseudoosteodentine gradually decreases from outside to inside. Avoid overlap by shifting the spectrum. or, orthodentine; and os, osteodentine.

Figure 3

Nano-hardness and nano-stiffness of pseudoosteodentine of Trichiurus lepturus. (A) Indentation hardness (HIT). (B) Vickers indentation hardness. (C) Elastic modulus. (D) Indentation elastic modulus. (E) Relationship between indentation hardness and crystal size. (F) Correlation between elastic modulus and crystal size. T. lepturus-O, outer layer of orthodentine; T. lepturus-M, middle layer of osteodentine; and T. lepturus-I, inner layer of osteodentine. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Distribution and concentration of chemical elements obtained by EDS microanalysis of the pseudoosteodentine of Trichiurus lepturus. (A) SEM image of the pseudoosteodentine of a transverse section from outer orthodentine to inner osteodentine. (B–E) Distribution of carbon (C), magnesium (Mg), phosphorus (P), and calcium (Ca). (F) The locations of a cross section marked with straight lines are used for point elemental analysis. (G) Comparison of Mg content. (H) The negative relationship between Mg content (wt %) and crystalline particle size of pseudoosteodentine. Fifty-nine indents were conducted on the sample. (I and L) Comparison of C, P content, Ca content, and the Ca/P ratio. T. lepturus-O, outer layer of orthodentine; T. lepturus-M, middle layer of osteodentine; and T. lepturus-I, inner layer of osteodentine. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Gradient change of psuedoosteodentine as derived from teeth of Trichiurus lepturus, based on Mg content, crystal size, crystallinity, nano-hardness, and nano-stiffness. Scanning electron micrograph at low magnification of embedded and etched T. lepturus tooth showed the SEM at high magnification were acquired on the three square regions (none etched) of outer, middle, and inner psuedoosteodentine, respectively. (A–A′) Scanning electron micrograph and schematic model of outer psuedoosteodentine (orthodentine). (B–B’) Scanning electron micrograph and schematic model of middle psuedoosteodentine (osteodentine). (C–C’) Scanning electron micrograph and schematic model of inner psuedoosteodentine (osteodentine).