Enamel is unique. It is the only epithelial-derived mineralized tissue in mammals and has a distinct micro- and nanostructure with nanofibrous apatite crystals as building blocks. It is synthesized by a highly specialized cell, the ameloblast, which secretes matrix proteins with little homology to any other known amino acid sequence, but which is composed of a primary structure that makes it competent to self-assemble and control apatite crystal growth at the nanometer scale. The end-product of ameloblast activity is a marvel of structural engineering: a material optimized to provide the tooth with maximum biting force, withstanding millions of cycles of loads without catastrophic failure, while also protecting the dental pulp from bacterial attack. This review attempts to bring into context the mechanical behavior of enamel with the developmental process of amelogenesis and structural development, since they are linked to tissue function, and the importance of controlling calcium phosphate mineralization at the nanometer scale. The origins of apatite nanofibers, the development of a stiffness gradient, and the biological processes responsible for the synthesis of a hard and fracture-resistant dental tissue are discussed with reference to the evolution of enamel from a fibrous composite to a complex, tough, and damage-tolerant coating on dentin.

Objective

To study the mechanisms which promote the interactions of amelogenin proteins with the forming mineral to establish suitable conditions for the biomimetic synthesis of enamel in vitro.

Design

Saturated calcium phosphate solutions were used in conjunction with recombinant amelogenin proteins to induce mineral formation on glass-ceramics substrates containing oriented fluoroapatite crystals (FAP). The height of mineral layers formed on these substrates within 24 h was measured by atomic force microscopy (AFM).

Experimental variables

The effect of protein concentration, pH and degree of saturation (DS) on the growth of apatite mineral was evaluated. Mineralization experiments were performed at 0, 0.4 and 1.6 mg/ml amelogenin concentrations. Mineralization solutions were used at pH values of 6.5, 7.4, 8.0 and 8.8 and DS of calcium and phosphate between 9 and 13.

Outcome measure

Height and morphology of mineralized layer formed on glass-ceramic substrates as determined from AFM measurements.

Results

Homogeneous nucleation and crystal growth of thin layers on the FAP were observed, when calcium and phosphate ions were added. The height of these layers grown on (001) planes of FAP was strongly dependent on the protein concentration and pH. At concentrations of 0 and 0.4 mg/ml crystal grew 5-15 nm on the FAP, while they grew approximately to 200 nm at 1.6 mg/ml. The enhanced crystal growth was observed only at pH 6.5, 7.4 and 8.0, while layers only 20 nm thick were obtained at pH 8.8. An increase in DS resulted in uncontrolled growth of calcium phosphate mineral covering large areas of the substrate.

Conclusions

Protein concentration, pH and the saturation of the mineralizing solution need to be considered carefully to provide suitable conditions for amelogenin-guided growth of apatite crystals.

Dental enamel forms through a protein-controlled mineralization and enzymatic degradation with a nanoscale precision that new engineering technologies may be able to mimic. Recombinant full-length human amelogenin (rH174) and a matrix-metalloprotease (MMP-20) were employed in a pH-stat titration system that enabled a continuous supply of calcium and phosphate ions over several days, mimicking the initial stages of matrix processing and crystallization in enamel in-vitro. Effects on the self-assembly and crystal growth from a saturated aqueous solution containing 0.4 mg/ml rH174 and MMP-20 with the weight ratio of 1:1000 with respect to rH174 were investigated. A transition from nanospheres to fibrous amelogenin assemblies was facilitated under conditions that involved an interaction between rH174 and the proteolytic cleavage products. Despite continuous titration, the levels of calcium exhibited a consistent trend of decreasing, thereby indicating its possible role in the protein self-assembly. This study suggests that mimicking enamel formation in-vitro requires the synergy between the aspects of matrix self-assembly, proteolysis and crystallization.

The formation of aligned fibrous apatite crystals in enamel is predominantly attributed to the involvement of amelogenin proteins. We developed a model to study interactions of matrix proteins with apatite mineral in vitro and tested the hypothesis that amelogenin solubility affects the ability to induce protein-guided mineralization. Crystal growth experiments were performed on fluoroapatite (FAP) glass-ceramics in mineralizing solutions containing recombinant full-length amelogenin (rH174) at different concentrations. Using atomic force microscopy, we observed that mineral precipitated randomly on the substrate, but also formed thin layers (height, 10 nm) on FAP within 24 hrs. This growth pattern was unaffected when 0.4 mg/mL of rH174 was added. In contrast, crystals grew on FAP at a rate up to 20 times higher, at 1.6 mg/mL protein. Furthermore, nanospheres and mineral bound specifically to FAP and aligned in strings approximately parallel to the c-axis of FAP, leading us to the conclusion that amelogenin proteins indeed control direction and rate of growth of apatite in enamel.

The developing enamel matrix is a highly dynamic system mainly composed of the full-length amelogenin and its proteolytic cleavage products. In this study, size, zeta-potential, and the isoelectric points of nanoparticles of the recombinant full-length human amelogenin (rH174) and two proteolytic products (rH163 and rH146) were analyzed by dynamic light-scattering and electrokinetic measurements. We tested the hypothesis that zeta-potential may be used as a control parameter in directing the self-assembly of amelogenins. Extensive aggregation of amelogenin molecules with the particle size reaching about one micron occurred at a mildly acidic to neutral pH, and coincided with the red shift of the internal fluorescence. Zeta-potential was between +/- 15 mV in the same pH range, indicating that amelogenin aggregation occurred when surface potentials were minimal. This suggests that electrostatic interactions may be another crucial factor, aside from hydrophobic interaction, in the aggregation and hierarchical assembly of spherical particles of amelogenins into supramolecular structures of a higher order.

The recent discovery of conditions that induce nanoribbon structures of amelogenin protein in vitro raises questions about their role in enamel formation. Nanoribbons of recombinant human full-length amelogenin (rH174) are about 17 nm wide and self-align into parallel bundles; thus, they could act as templates for crystallization of nanofibrous apatite comprising dental enamel. Here we analyzed the secondary structures of nanoribbon amelogenin by x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) and tested if the structural motif matches previous data on the organic matrix of enamel. XRD analysis showed that a peak corresponding to 4.7 Å is present in nanoribbons of amelogenin. In addition, FTIR analysis showed that amelogenin in the form of nanoribbons was comprised of β-sheets by up to 75%, while amelogenin nanospheres had predominantly random-coil structure. The observation of a 4.7-Å XRD spacing confirms the presence of β-sheets and illustrates structural parallels between the in vitro assemblies and structural motifs in developing enamel.

Objectives

Acetate and lactate are important cariogenic acids produced by oral bacteria. They produced different residual dentin structures in artificial lesions of similar depth. We evaluated if such lesions responded in the same way to a polymer-induced-liquid-precursor (PILP) remineralization.

Design

Dentin blocks obtained from human third molars, divided into 6 groups (n=3). Blocks were demineralized with acetate (66h) or lactate (168h) buffer at pH 5.0 to create 140μm target lesion depths. A-DEM and L-DEM groups received no remineralization. Other groups were remineralized for 14days. 100μg/mL polyaspartate was added into the remineralizing buffer for A-PIL and L-PIL, whereas A-CAP and L-CAP were treated with the same solution but without polyaspartate. Cross-sectioned blocks were examined for shrinkage and AFM-topography. Line profiles of reduced elastic modulus (E_r) were obtained by AFM-based nanoindentation across the lesion. Ultrastructures were examined with TEM.

Results

A-PIL and L-PIL recovered in shrinkage to the original height of the dentin and it appeared normal with tubules, with increases in E_r at both outer flat and inner sloped zones. At the sloped zone, acetate lesions lost more E_r but recovery rate after PILP was not statistically different from lactate lesions. A-CAP and L-CAP showed surface precipitates, significantly less recovery in shrinkage or E_r as compared to PILP groups. TEM-ultrastructure of PILP groups showed similar structural and mineral components in the sloped zone for lesions produced by either acid.

Conclusions

The PILP process provided significant recovery of both structure and mechanical properties for artificial lesions produced with acetate or lactate.

Objectives

We studied artificial dentin lesions in human teeth generated by lactate and acetate buffers (pH 5.0), the two most abundant acids in caries. The objective of this study was to determine differences in mechanical properties, mineral density profiles and ultrastructural variations of two different artificial lesions with the same approximate depth.

Methods

0.05M (pH 5.0) acetate or lactate buffer was used to create 1) 180μm-deep lesions in non-carious human dentin blocks (acetate 130h; lactate 14days); (2) demineralized, ∼180μm-thick non-carious dentin discs (3 weeks). We performed nanoindentation to determine mechanical properties across the hydrated lesions, and micro X-ray computed tomography (MicroXCT) to determine mineral profiles. Ultrastructure in lesions was analyzed by TEM/selected area electron diffraction (SAED). Demineralized dentin discs were analyzed by small angle X-ray scattering (SAXS).

Results

Diffusion-dominated demineralization was shown based on the linearity between lesion depths versus the square root of exposure time in either solution, with faster kinetics in acetate buffer. Nanoindentation revealed lactate induced a significantly sharper transition in reduced elastic modulus across the lesions. MicroXCT showed lactate demineralized lesions had swelling and more disorganized matrix structure, whereas acetate lesions had abrupt X-ray absorption near the margin. At the ultrastructural level, TEM showed lactate was more effective in removing minerals from the collagenous matrix, which was confirmed by SAXS analysis.

Conclusions

These findings indicated the different acids yielded lesions with different characteristics that could influence lesion formation resulting in their distinct predominance in different caries activities, and these differences may impact strategies for dentin caries remineralization.

Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp^-/-) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the "polymer-induced liquid-precursor" (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp^-/- and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp^-/- dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair.