Proteolysis by MMP20 Prevents Aberrant Mineralization in Secretory Enamel.

The present study was conducted to investigate the role of proteolysis by matrix metalloproteinase 20 (MMP20) in regulating the initial formation of the enamel mineral structure during the secretory stage of amelogenesis, utilizing Mmp20-null mice that lack this essential protease. Ultrathin sagittal sections of maxillary incisors from 8-wk-old wild-type (WT), Mmp20-null (KO), and heterozygous (HET) littermates were prepared. Secretory-stage enamel ultrastructures from each genotype as a function of development were compared using transmission electron microscopy, selected area electron diffraction, and Raman microspectroscopy. Characteristic rod structures observed in WT enamel exhibited amorphous features in newly deposited enamel, which subsequently transformed into apatite-like crystals in older enamel. Surprisingly, initial mineral formation in KO enamel was found to proceed in the same manner as in the WT. However, soon after a rod structure began to form, large plate-like crystals appeared randomly within the developing KO enamel layer. As development continued, observed plate-like crystals became dominant and obscured the appearance of the enamel rod structure. Upon formation of these plate-like crystals, the KO enamel layer stopped growing in thickness, unlike WT and HET enamel layers that continued to grow at the same rate. Raman results indicated that Mmp20-KO enamel contains a significant portion of octacalcium phosphate, unlike WT enamel. Although normal in all other respects, large, randomly dispersed mineral crystals were observed in secretory HET enamel, although to a lesser extent than that seen in KO enamel, indicating that the level of MMP20 expression has a proportional effect on suppressing aberrant mineral formation. In conclusion, we found that proteolysis of extracellular enamel matrix proteins by MMP20 is not required for the initial development of the enamel rod structure during the early secretory stage of amelogenesis. Proteolysis by MMP20, however, is essential for the prevention of abnormal crystal formation during amelogenesis.

Decellularized Swine Dental Pulp Tissue for Regenerative Root Canal Therapy.

In the current theme of dental pulp regeneration, biological and synthetic scaffolds are becoming a potential therapy for pulp revitalization. The goal is to provide a suitable environment for cellular infiltration, proliferation, and differentiation. The extracellular matrix (ECM) represents a natural scaffold material resembling the native tissue chemical and mechanical properties. In the past few years, ECM-based scaffolds have shown promising results in terms of progenitor cells recruitment, promotion of constructive remodeling, and modulation of host response. These properties make ECM-derived scaffolds an ideal candidate for pulp regenerative therapy. Development of strategies for clinically relevant tissue engineering using dental pulp extracellular matrix (DP-ECM) can provide an alternative to conventional root canal treatment. In this work, we successfully decellularized ECM derived from porcine dental pulp. The resulting scaffold was characterized using immunostaining (collagen type I, dentin matrix protein 1, dentin sialoprotein, and Von Willebrand factor) and enzyme-linked immunosorbent assay (transforming growth factor ß, vascular endothelial growth factor, and basic fibroblast growth factor) for extracellular proteins where the ECM retained its proteins and significant amount of growth factors. Furthermore, a pilot in vivo study was conducted where the matrix was implanted for 8 wk in a dog root canal model. Our in vitro and preliminary in vivo data show that the decellularized ECM supports cellular infiltration together with the expression of pulp-dentin and vascular markers (DSP and CD31) compared to the controls. Herein, we show the feasibility to produce a decellularized ECM scaffold and validate the concept of using ECM-based scaffolds for pulp regeneration.

Biomimetic coating of magnesium alloy for enhanced corrosion resistance and calcium phosphate deposition.

Degradable metals have been suggested as biomaterials with revolutionary potential for bone-related therapies. Of these candidate metals, magnesium alloys appear to be particularly attractive candidates because of their non-toxicity and outstanding mechanical properties. Despite their having been widely studied as orthopedic implants for bone replacement/regeneration, their undesirably rapid corrosion rate under physiological conditions has limited their actual clinical application. This study reports the use of a novel biomimetic peptide coating for Mg alloys to improve the alloy corrosion resistance. A 3DSS biomimetic peptide is designed based on the highly acidic, bioactive bone and dentin extracellular matrix protein, phosphophoryn. Surface characterization techniques (scanning electron microscopy, energy dispersive X-ray spectroscopy and diffuse-reflectance infrared spectroscopy) confirmed the feasibility of coating the biomimetic 3DSS peptide onto Mg alloy AZ31B. The 3DSS peptide was also used as a template for calcium phosphate deposition on the surface of the alloy. The 3DSS biomimetic peptide coating presented a protective role of AZ31B in both hydrogen evolution and electrochemical corrosion tests.

Structural changes in amelogenin upon self-assembly and mineral interactions.

Amelogenin, the major protein of forming dental enamel, plays a crucial role in the biomineralization of this tissue. Amelogenin is soluble at low pH and self-assembles to form higher order structures at physiological pH. To understand the mechanisms of its assembly and interactions with calcium phosphate mineral, we conducted FTIR spectroscopy (FTIRS) studies of pH-triggered assembly of recombinant porcine amelogenin rP172 and its interactions with mature hydroxyapatite and apatitic mineral formed in situ. Analysis of our data indicated that rP172 at pH 3.0 exists in an unfolded disordered state, while increases in pH led to structural ordering, manifested by increases in intra- and intermolecular ß-sheet structures and a decrease in random coil and ß-turns. Amelogenin assembled at pH 7.2 was also found to contain large portions of extended intramolecular ß-sheet and PPII. These FTIRS findings are consistent with those previously obtained with other techniques, thus verifying the validity of our experimental approach. Interestingly, interactions with mineral led to a reduction in protein structural organization. The findings obtained show that amelogenin has intrinsic structural flexibility to accommodate interactions with both forming and mature calcium phosphate mineral phases, providing new insights into the potential importance of amelogenin-mineral interactions in enamel biomineralization.

Leucine-rich amelogenin peptides regulate mineralization in vitro.

Amelogenin's capacity to regulate enamel formation is related to its conserved N- and C-terminal domains, its ability to self-assemble, and its ability to stabilize amorphous calcium phosphate (ACP) - a capacity enhanced by amelogenin phosphorylation. This in vitro study provides further insight into amelogenin function, using variations of the Leucine-Rich Amelogenin Peptide (LRAP), an alternative splice product comprised solely of amelogenin's N- and C-terminal domains. Peptide self-assembly was studied by dynamic light-scattering and transmission electron microscopy (TEM). TEM, selected area electron diffraction, and Fourier transform-infrared spectroscopy were also used to determine the effect of phosphorylated and non-phosphorylated LRAP on calcium phosphate formation. Results show that phosphorylated and non-phosphorylated LRAP can self-assemble into chain-like structures in a fashion dependent on the C-terminal domain. Notably, this capacity was enhanced by added calcium and to a much greater degree for phosphorylated LRAP. Furthermore, phosphorylated LRAP was found to stabilize ACP and prevent its transformation to hydroxyapatite (HA), while aligned HA crystals formed in the presence of non-phosphorylated LRAP. The N- and C-terminal amelogenin domains in non-phosphorylated LRAP are, therefore, sufficient to guide ACP transformation into ordered bundles of apatite crystals, making LRAP an excellent candidate for biomimetic approaches for enamel regeneration.

Potential role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro.

N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (-P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(-P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

Compositional determinants of mechanical properties of enamel.

Dental enamel is comprised primarily of carbonated apatite, with less than 1% w/w organic matter and 4-5% w/w water. To determine the influence of each component on the microhardness and fracture toughness of rat incisor enamel, we mechanically tested specimens in which water and organic matrix were selectively removed. Tests were performed in mid-sagittal and transverse orientations to assess the effect of the structural organization on enamel micromechanical properties. While removal of organic matrix resulted in up to a 23% increase in microhardness, and as much as a 46% decrease in fracture toughness, water had a significantly lesser effect on these properties. Moreover, removal of organic matrix dramatically weakened the dentino-enamel junction (DEJ). Analysis of our data also showed that the structural organization of enamel affects its micromechanical properties. We anticipate that these findings will help guide the development of bio-inspired nanostructured materials for mineralized tissue repair and regeneration.

Role of macromolecular assembly of enamel matrix proteins in enamel formation.

Unlike other mineralized tissues, mature dental enamel is primarily (> 95% by weight) composed of apatitic crystals and has a unique hierarchical structure. Due to its high mineral content and organized structure, enamel has exceptional functional properties and is the hardest substance in the human body. Enamel formation (amelogenesis) is the result of highly orchestrated extracellular processes that regulate the nucleation, growth, and organization of forming mineral crystals. However, major aspects of the mechanism of enamel formation are not well-understood, although substantial evidence suggests that protein-protein and protein-mineral interactions play crucial roles in this process. The purpose of this review is a critical evaluation of the present state of knowledge regarding the potential role of the assembly of enamel matrix proteins in the regulation of crystal growth and the structural organization of the resulting enamel tissue. This review primarily focuses on the structure and function of amelogenin, the predominant enamel matrix protein. This review also provides a brief description of novel in vitro approaches that have used synthetic macromolecules (i.e., surfactants and polymers) to regulate the formation of hierarchical inorganic (composite) structures in a fashion analogous to that believed to take place in biological systems, such as enamel. Accordingly, this review illustrates the potential for developing bio-inspired approaches to mineralized tissue repair and regeneration. In conclusion, the authors present a hypothesis, based on the evidence presented, that the full-length amelogenin uniquely regulates proper enamel formation through a process of cooperative mineralization, and not as a pre-formed matrix.

Fluorosis: a new model and new insights.

Fluoride is an effective agent for the prevention of dental caries. However, the mechanism of how excessive fluoride exposure causes fluorosis remains uncertain. Zebrafish (Danio rerio) exhibit periodic tooth replacement throughout their lives, thereby providing continuous access to teeth at developmental stages susceptible to fluoride exposure. Zebrafish teeth do not contain true enamel, but consist of a hard enameloid surface. Therefore, we asked whether zebrafish could be used as a model organism for the study of dental fluorosis. Scanning electron microscopy of fluoride-treated teeth demonstrated that the enameloid was pitted and rough, and FTIR analysis demonstrated that the teeth also contained a significantly higher organic content when compared with untreated controls. Furthermore, we demonstrate for the first time that decreased expression of an important signaling molecule (Alk8) in tooth development may contribute to the observed fluorotic phenotype, and that increased cell apoptosis may also play a role in the mechanism of fluorosis.

Decreased mineral content in MMP-20 null mouse enamel is prominent during the maturation stage.

During enamel development, matrix metalloproteinase-20 (MMP-20, enamelysin) is expressed early during the secretory stage as the enamel thickens, and kallikrein-4 (KLK-4, EMSP1) is expressed later during the maturation stage as the enamel hardens. Thus, we investigated whether the physical properties of the secretory-/maturation-stage MMP-20 null enamel were significantly different from those of controls. We demonstrated that although, in relative terms, the weight percent of mature mineral in the MMP-20 null mouse enamel was only 7-16% less than that in controls, overall the enamel mineral was reduced by about 50%, and its hardness was decreased by 37%. Percent mineral content by weight was assessed at 3 different developmental stages. Remarkably, the biggest difference in mineral content between MMP-20 null and controls occurred in the nearly mature enamel, when MMP-20 is normally no longer expressed. This suggests that MMP-20 acts either directly or indirectly to facilitate the removal of maturation-stage enamel proteins.

Self-assembly and mineralization of peptide-amphiphile nanofibers.

We have used the pH-induced self-assembly of a peptide-amphiphile to make a nanostructured fibrous scaffold reminiscent of extracellular matrix. The design of this peptide-amphiphile allows the nanofibers to be reversibly cross-linked to enhance or decrease their structural integrity. After cross-linking, the fibers are able to direct mineralization of hydroxyapatite to form a composite material in which the crystallographic c axes of hydroxyapatite are aligned with the long axes of the fibers. This alignment is the same as that observed between collagen fibrils and hydroxyapatite crystals in bone.

A transmission electron microscope study using vitrified ice sections of predentin: structural changes in the dentin collagenous matrix prior to mineralization.

The assembly of the collagenous organic matrix prior to mineralization is a key step in the formation of bones and teeth. This process was studied in the predentin of continuously forming rat incisors, using unstained vitrified ice sections examined in the transmission electron microscope. Progressing from the odontoblast surface to the mineralization front, the collagen fibrils thicken to ultimately form a dense network, and their repeat D-spacings and banding patterns vary. Using immunolocalization, the most abundant noncollagenous protein in dentin, phosphophoryn, was mapped to the boundaries between the gap and overlap zones along the fibrils nearest the mineralization front. It thus appears that the premineralized collagen matrix undergoes dynamic changes in its structure. These may be mediated by the addition and interaction with the highly anionic noncollagenous proteins associated with collagen. These changes presumably create a collagenous framework that is able to mineralize.

Peritubular dentin formation: crystal organization and the macromolecular constituents in human teeth.

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite-based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.

Cellular control over spicule formation in sea urchin embryos: A structural approach.

The spicules of the sea urchin embryo form in intracellular membrane-delineated compartments. Each spicule is composed of a single crystal of calcite and amorphous calcium carbonate. The latter transforms with time into calcite by overgrowth of the preexisting crystal. Relationships between the membrane surrounding the spiculogenic compartment and the spicule mineral phase were studied in the transmission electron microscope (TEM) using freeze-fracture. In all the replicas observed the spicules were tightly surrounded by the membrane. Furthermore, a variety of structures that are related to the material exchange process across the membrane were observed. The spiculogenic cells were separated from other cell types of the embryo, frozen, and freeze-dried on the TEM grids. The contents of electron-dense granules in the spiculogenic cells were shown by electron diffraction to be composed of amorphous calcium carbonate. These observations are consistent with the notion that the amorphous calcium carbonate-containing granules contain the precursor mineral phase for spicule formation and that the membrane surrounding the forming spicule is involved both in transport of material and in controlling spicule mineralization.