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.

However, the epitaxial growth of enamel with a foreign ACP phase has not been achieved in vitro. Our previous study demonstrated that ~20-nm ACP particles can adsorb and even assemble onto enamel HAP crystals, but these particles failed to induce the epitaxial growth of enamel crystals ( 8 ). In general, coalescence and fusion between particles occur more readily at smaller sizes ( 26 ). Accordingly, a question arises: What is the minimum size of the ACP particles that can be used in HAP growth studies? Recent studies have suggested that both Posner’s clusters, which are 0.95 nm in size ( 27 ), and calcium phosphate ion clusters (CPICs), with sizes of a few nanometers ( 28 ), can serve as the basic building blocks of ACP and HAP ( 29 , 30 ). However, these ultrasmall ion clusters themselves are extremely unstable and can spontaneously aggregate and even nucleate within a few seconds ( 28 ). Although several additive-stabilized CPICs ( 31 , 32 ), especially polymer-induced liquid precursor, have been proposed and synthesized, they cannot be applied to the repair of enamel, which is almost purely inorganic, because the irremovable organics would destroy the integrity of the enamel mineral phase, weakening the exceptional mechanical strength of this hard tissue.

During the biomineralization of hard tissue, the microstructures of natural materials are precisely controlled and duplicated ( 21 ). There is growing evidence that biomineralization at the growth frontier occurs in an integrated crystalline-amorphous interface: The crystalline mineral phase is coated by its amorphous phase (precursor) to ensure continuous epitaxial construction (e.g., the crystal growth frontiers of the zebrafish fin bone and nacre) ( 22 – 25 ). Inspired by these biological processes, we suggest that a rationally designed structure between HAP and amorphous calcium phosphate [ACP; Ca 3 (PO 4 ) 2 ·nH 2 O] may mimic the biomineralization frontier to induce the epitaxial regeneration of enamel.

Biomineralization produces numerous biological composites with excellent mechanical performance, of which tooth enamel is the hardest ( 1 , 2 ). The primary mineral phase [~96 weight % (wt %)] of enamel consists of nonstoichiometric fluoridated carbonate apatite crystals ( 3 , 4 ) that are tightly packed with well-defined orientations to ensure a high striking strength ( 5 , 6 ). In general, hydroxyapatite [HAP; Ca 10 (PO 4 ) 6 (OH) 2 ] is used as a simplified mineral model to investigate enamel formation and reconstruction ( 7 – 10 ). Although enamel formation (amelogenesis) is a part of the overall process of biological development, mature enamel is acellular and scarcely self-repaired after damage ( 11 ). Therefore, caries or dental decay is one of the most prevalent chronic diseases in humans worldwide ( 12 ). Despite the great attempts made at enamel remineralization by using various strategies, such as direct solution mineralization ( 13 , 14 ), protein/peptide-induced mineralization ( 15 – 17 ), hydrogel-driven mineralization ( 7 , 18 , 19 ), and precursor assembly ( 8 , 9 , 20 ), no applicable repair for clinical development has been achieved because the complicated hierarchical structure of natural enamel cannot be replicated at large scale in laboratories.

RESULTS

Characterization of CPICs Different from other irremovable organic additives, especially polymers, triethylamine (TEA) is a small molecule that easily volatilizes in ambient environments. Our study revealed that TEA is an effective stabilizer of CPICs, and its controllable removal can result in pure HAP formation. We generated our CPICs on a large scale by mixing two ethanol solutions: one containing phosphoric acid (H 3 PO 4 ; 9.8 mM) and another containing calcium chloride dihydrate (CaCl 2 ·2H 2 O; 13.1 mM) and TEA (263.0 mM). Transmission electron microscopy (TEM; Fig. 1A) images show clusters with an average diameter of 1.5 ± 0.3 nm, and dynamic light scattering (DLS) measurements confirmed their size of 1.6 ± 0.6 nm, in line with previously reported cluster sizes (1.0 to 1.6 nm) (28). In contrast to other ultrasmall clusters, the resulting CPICs were stable in ethanol for at least 2 days without any aggregation or size increase (fig. S1). We confirmed the stabilizing effect of TEA on the CPICs by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The stretching vibrations of C─N in the FTIR spectrum indicated the presence of TEA in the CPICs. Typically, this peak arose at 1200 cm−1 in ethanol solution (fig. S2A) but shifted to 1203 cm−1 in the CPICs (Fig. 1B), indicating the interaction between the TEA and the CPICs. In the NMR spectra, the chemical shift (δ) of the TEA methylene protons was 2.56 parts per million (ppm) for free TEA but 3.01 ppm for TEA in the CPICs (Fig. 1C and fig. S2B); meanwhile, the δ of the 31P of PO 4 3− in the CPICs shifted from 0 (free H 3 PO 4 ) to −3.03 ppm because of the presence of TEA (fig. S2C), reflecting the interaction with TEA. The binding between TEA and phosphate results in the stabilization effect of TEA on CPICs. 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).

Construction of a mimetic biomineralization frontier However, TEA is volatile and can be readily removed from the CPIC solution with ethanol evaporation. With ethanol volatilization at room temperature, the content of TEA in the CPICs continued to decrease to initiate ACP formation, which could be explained by the reduced stability (Fig. 1, D and F). Because no ethanol or TEA could be detected in the resulting bulk ACP by FTIR spectroscopy (Fig. 1E) or gas chromatography–mass spectrometry (GC-MS; fig. S2D), we completely removed the organics including TEA during the material evolution, ensuring pure inorganic calcium phosphate production. In contrast to the other bulk ACP (fig. S3, B to D) consolidated from nanoparticles (fig. S3A), no particle, grain, or material boundaries could be observed within the CPIC-induced ACP by scanning electron microscopy (SEM; Fig. 1D) and atomic force microscopy (AFM; fig. S4), implying a structural continuity. This structural continuity can be extended to HAP-ACP interface to establish the mimetic biomineralization frontier. By loading onto a TEM grid, the synthetic single-crystalline HAP rods (fig. S5) were dipped into a CPIC ethanol solution and then withdrawn (Fig. 2A). After an air dry treatment, the observation under high-resolution TEM (HRTEM) showed that a continuous ACP layer first formed onto the HAP. Subsequently, the epitaxial growth of the HAP crystal along the c-axial direction occurred (Fig. 2, A to C). The amorphous character of the ACP layer was proven by selected area electron diffraction (SAED; Fig. 2B, inset), and the elements were analyzed by using energy-dispersive x-ray spectroscopy (EDXS) (fig. S6C). It should be emphasized that the resulting ACP layer from the CPICs was well integrated onto the HAP without any gap. Because the crystalline phase is directly and tightly covered with a continuous layer of the disordered amorphous phase, this established HAP-ACP for epitaxial growth is exactly the same as the previously found biomineralization frontier in nature (22–25, 33). In contrast, no conventional ACP (with a typical size of ~20 nm) could construct such a structurally continuous interface from the crystalline to amorphous phase because the substrate-particle boundary cannot be avoided (fig. S6, A and D) (34). Although these ACP particles adsorbed onto the HAP rod with the subsequent crystallization, the existing crystal-particle boundary blocked the epitaxial growth from HAP to ACP, resulting in polycrystals (fig. S6B). Therefore, the observed directional growth is attributed to the continuous integration of HAP and ACP rather than the particle attachment. Analogously, the directional crystallization in biomineralization is suggested as an epitaxial solid-state transition at the well-established crystalline-amorphous interface (22, 24, 25, 35). 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). The application of a biomimetic frontier can be extended to enamel repair for epitaxial construction. Figure 2D shows that the CPICs exhibited excellent biocompatibility with the native enamel, which was reflected by their perfect wettability. In this experiment, we dropped 100 μl of a CPIC ethanol solution (2 mg/ml) on an enamel window (4 mm by 5 mm). After air-drying at room temperature for 15 min, an ~3-μm ACP layer was found as a continuous coating on the enamel surface (Fig. 2E). The details of the enamel-ACP interface revealed by HRTEM observation demonstrate the structural integration and continuity of the boundary between the enamel and the resulting ACP (Fig. 2F).

CPIC-repaired tooth enamel Human enamel is characterized by a well-organized structure with hierarchical complexity: Well-aligned enamel rods are interwoven with inter-rods, which produces the characteristic “fish scale–shaped” structure (36, 37). In general, enamel rods run in a perpendicular direction to the enamel surface, and inter-rod enamel is at an angle of approximately 60° to the enamel rods (37). Although there have been many attempts at enamel remineralization, none could reproduce the characterized fish scale–shaped structure. However, this unique feature of natural enamel could be precisely replicated within 48 hours by using the CPIC material. The repaired enamel had the same morphological texture as native enamel because they were indistinguishable by SEM (Fig. 3, A and C). The boundary between the repaired and native enamel (protected by nail varnish during the repair) demonstrates successful epitaxial growth, and we also confirmed the formation of a new HAP layer by AFM (Fig. 3B). Notably, the resulting HAP and the assembled structures in the repaired layer were exactly the same as the native materials. Although both enamel rods and inter-rods are HAP, they have different orientations in the enamel. In our work, both the enamel rods and inter-rods could be epitaxially grown simultaneously in the repair process (Fig. 3D). This coinstantaneous duplication of the HAP with differential orientations during the enamel reconstruction means that each individual epitaxial growth process is specific and controllable at the nanoscale, affording a high structural resolution to benefit the construction of materials with complicated architectures. A cross-sectional SEM image shows that the thickness of the enamel-identical repair layer was approximately 2.0 to 2.8 μm (Fig. 3, E and F), with well-organized and uniform characteristics. In the control experiment, this fish scale–shaped structure that is characteristic of the enamel could not be reproduced by using conventional ACP nanoparticles (fig. S7). The epitaxial growth of the enamel rods could be confirmed by HRTEM observation along a single rod from the native area to the regrowth (Fig. 3G). Longitudinal examinations showed that the newly formed HAP phase developed on a natural HAP crystal with an identical orientation, which was in the crystallographic c-axial direction (Fig. 3, H to J). We monitored the evolution of the mineral phase in the repaired layer of enamel by using x-ray diffraction (XRD; Fig. 3K): The introduction of CPICs (line b) produced an ACP precursor layer on the enamel (line c; the HAP signals were contributed by the enamel substrate). Subsequently, the ACP gradually evolved to HAP on the enamel (line d; the decreasing ACP signals with the increasing HAP signals in comparison with those in line c), and eventually, a well-crystallized HAP layer was observed (line e). All of the diffraction peaks in the repaired layer and their relative intensities were identical to those of the original enamel window (line a), implying an identically organized crystallographic structure at the macroscopic level. The precise reconstruction of the enamel structure from the nanoscale to the macroscale was achieved. 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). The repair of whole tooth enamel could be achieved by this biomimetic tactic. For comparison, we symmetrically divided an etched whole tooth enamel into two parts (Fig. 4, A to C): The left side was protected by nail varnish as the control, and the right side was used for the repair treatment with CPICs. Calcein, a molecule with green fluorescence under ultraviolet (UV) irradiation, was used to label the newly grown HAP. We validated the large-scale enamel repair by confocal laser scanning microscopy (CLSM) and SEM at multiple scales on a cross section of the tooth enamel (Fig. 4, D and F), which showed that the thickness of the repaired layer could reach ~2.7 μm (Fig. 4E). Moreover, SEM at higher magnification confirmed that the HAP crystals grown in the repaired layer shared the notable structure and orientation of the natural enamel. 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).