Repair of tooth enamel by a biomimetic mineralization frontier ensuring epitaxial growth

Abstract

The regeneration of tooth enamel, the hardest biological tissue, remains a considerable challenge because its complicated and well-aligned apatite structure has not been duplicated artificially. We herein reveal that a rationally designed material composed of calcium phosphate ion clusters can be used to produce a precursor layer to induce the epitaxial crystal growth of enamel apatite, which mimics the biomineralization crystalline-amorphous frontier of hard tissue development in nature. After repair, the damaged enamel can be recovered completely because its hierarchical structure and mechanical properties are identical to those of natural enamel. The suggested phase transformation-based epitaxial growth follows a promising strategy for enamel regeneration and, more generally, for biomimetic reproduction of materials with complicated structure.

Fig. 1. Synthesis and characterization of CPICs and the manufacture of bulk ACP.

(A) TEM image of CPICs. Inset: DLS size distributions of the CPICs in ethanol solution. (B) FTIR spectra of the gel-like CPICs. a.u., arbitrary units. (C) ¹H NMR spectra of TEA and CPICs. (D) SEM image of bulk ACP formed on glass, which was fabricated by the aggregation and fusion of CPICs with solvent volatilization. Inset: X-ray diffraction (XRD) of bulk ACP. (E) FTIR spectra of bulk ACP materials. (F) Schematic of ACP formation as the stabilizer (TEA) was removed by using CPICs. Scale bars, 20 nm (A) and 5 μm (D).

Fig. 2. Construction of a mimetic biomineralization frontier for epitaxial crystal growth by using CPICs.

(A) Schematic of the epitaxial growth of crystalline HAP from the construction of the amorphous frontier on its surface. (B) The HAP crystal (marked with a yellow dotted line) coated with a continuous ACP layer (marked with a blue dotted line). Inset: SAED pattern of the amorphous layer. (C) The epitaxial growth of HAP was detected in the exact same region as in (B). The new crystal regrowth is marked with a red dotted line. Inset: SAED pattern of HAP and regrown crystal, which indicates that these crystals are aligned parallel to the crystallographic c axis of the HAP. (D) A CPIC ethanol solution dropped on the surface of enamel and air-dried for 15 min. (E) Cross-sectional SEM image of enamel coated with an ACP layer. (F) HRTEM image of a focused ion beam (FIB)–prepared ultrathin section of enamel coated with a continuous ACP layer (green region). Inset: SAED patterns of the original enamel rod and repaired layer. Scale bars, 10 nm (B and C), 1 mm (D), 1 μm (E), 50 nm (F), 2 nm⁻¹ (C, inset), and 5 nm⁻¹ (F, inset).

Fig. 3. Replication of the complicated structure of enamel.

(A) SEM image showing both acid-etched enamel and repaired enamel. (B) A three-dimensional AFM image of repaired enamel. (C) High-magnification SEM image of the red circle in (A). (D) Cross-sectional view of final repaired enamel, where both enamel rods and inter-rods were repaired. R and IR represent for enamel rod and inter-rod, respectively. (E and F) Enamel rods with different orientations can be repaired. (G) TEM image of a longitudinal section of the reconstructed layer on natural enamel, including the native and repaired zones. Inset: SAED of the native enamel (selected area, white cycle) and repaired enamel (selected area, yellow cycle) demonstrated that their long axes correspond to the crystallographic c axis of the HAP. A Pt layer was sputtered to protect the enamel surface from ion beam damage during the milling processes. Lattice fringes of a single enamel rod developed from the native area to the repaired area: The regenerated region (J) had the same characteristics as the natural region (H), and there was no boundary between them (I), demonstrating structural continuity. (K) The XRD spectra indicate the evolution process from CPICs to HAP on the enamel window: the etched enamel (line a), the initial gel-like CPICs coated on the enamel (line b; air-dried for 5 min), the ACP layer resulting from the CPICs (line c; air-dried for 15 min), an intermediate state of the evolution from ACP to HAP (line d; remineralization for 24 hours), and the final crystalline HAP layer on the enamel (line e; remineralization for 48 hours). Scale bars, 20 μm (A), 2 μm (C to F), 500 nm (G), 5 nm (H to J), and 2 nm⁻¹ (G, insets).

Fig. 4. Repair of whole tooth enamel and its mechanical and microtribological properties.

(A) SEM image of native acid-etched enamel. Inset: High-magnification SEM image of acid-etched enamel. (B) Digital image of a whole tooth, in which the left area was covered with acid-resistant varnish (displayed as dark) and the right area was repaired with CPICs containing calcein (displayed as yellow). (C) SEM image of repaired enamel. Inset: High-magnification SEM image of repaired enamel. (D and E) CLSM images of cross sections of the whole tooth. The repaired layer was labeled with calcein, which emitted green fluorescence. The thickness of the repaired layer was approximately 2.7 μm. (F) Cross-sectional view of the SEM image of the repaired enamel on a large scale. Inset: The transition zone from the native to repaired enamel. (G) Calculated hardness and elastic modulus of the enamel samples. (H) Coefficient of friction of the enamel samples measured at a constant normal force of 500 mN. Scale bars, 20 μm (A and C), 5 mm (B), 1 mm (D), 10 μm (E and F), 1 μm (A, inset, and C, inset), and 2 μm (F, inset).