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.

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 field of dental materials has undergone more of a revolution than an evolution over the past 100 y. The development of new products, especially in the past half century, has occurred at a staggering pace, and their introduction to the market has been equally impressive. The movement has mostly come in the area of improved esthetics, marked by the gradual replacement of dental amalgam with dental composite and all-metal and porcelain-fused-to-metal indirect restorations with reinforced dental ceramics, all made possible by the rapid improvements in dental adhesive materials. This article covers the time course of dental materials development over the past century in which the Journal of Dental Research has been published. While there have been advances in nearly all materials used in the field, this article focuses on several areas, including dental amalgam, dental composites and light curing, dental adhesives and dental cements, ceramics, and new functional repair materials. A few short statements on future advances will be included at the end.

Direct placement restorative materials must interface with tooth structures that are often compromised by caries or trauma. The material must seal the interface while providing sufficient strength and wear resistance to assure function of the tooth for, ideally, the lifetime of the patient. Needed are direct restorative materials that are less technique-sensitive than current resin-based composite systems while having improved properties. The ideal material could be successfully used in areas of the world with limited infrastructure. Advances in our understanding of the interface between the restoration adhesive system and the stages of carious dentin can be used to promote remineralization. Application of fracture mechanics to adhesion at the tooth-restoration interface can provide insights for improvement. Research in polymer systems suggests alternatives to current composite resin matrix systems to overcome technique sensitivity, while advances in nano- and mesoparticle reinforcement and alignment in composite systems can increase material strength, toughness, and wear resistance, foreshadowing dental application.

The dentino-enamel junction (DEJ) connects enamel, that covers the outer surface of a tooth, to a thicker underlying dentin. The DEJ is a critical interface that permits joining these materials that have widely dissimilar mechanical properties. AFM-based nanoindentation and Raman microspectroscopy were used to define the width and composition of human molar DEJ. Indentation elastic modulus and hardness of enamel, dentin, and DEJ were determined along lines of indents made at 2 μm intervals across the DEJ. Indents made at maximum loads at each end of the indent lines were used to make visible markers allowing Raman microspectroscopy at 1 μm intervals across the DEJ, while using the nanoindent markers for orientation and location. Functional DEJ width estimates were made based on results from nanoindentation and Raman microspectroscopy. DEJ width estimates ranged from 4.7 (±1.2) μm to 6.1 (±1.9) μm based on hardness and 4.9 (±1.1) μm to 6.9 (±1.9) μm based on modulus. DEJ width based on Raman peak intensity variations were 8.0 (±3.2) μm to 8.5 (±3.1) μm based on the phosphate peak, and 7.6 (±3.2) μm to 8.0 (±2.6) μm for C-H stretching mode. These estimates are in the range of DEJ width estimates reported using nanoindentation.

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.