Chang Du

Analysis of Secondary Structure and Self-Assembly of Amelogenin by Variable Temperature Circular Dichroism and Isothermal Titration Calorimetry

Amelogenin is a proline‐rich enamel matrix protein known to play an important role in the oriented growth of enamel crystals. Amelogenin self‐assembles to form nanospheres and higher order structures mediated by hydrophobic interactions. This study aims to obtain a better insight into the relationship between primary–secondary structure and self‐assembly of amelogenin by applying computational and biophysical methods. Variable temperature circular dichroism studies indicated that under physiological pH recombinant full‐length porcine amelogenin contains unordered structures in equilibrium with polyproline type II (PPII) structure, the latter being more populated at lower temperatures. Increasing the concentration of rP172 resulted in the promotion of folding to an ordered β‐structured assembly. Isothermal titration calorimetry dilution studies revealed that at all temperatures, self‐assembly is entropically driven due to the hydrophobic effect and the molar heat of assembly (ΔHA) decreases with temperature. Using a computational approach, a profile of domains in the amino acid sequence that have a high propensity to assemble and to have PPII structures has been identified. We conclude that the assembly properties of amelogenin are due to complementarity between the hydrophobic and PPII helix prone regions. Proteins 2009.

Apatite/Amelogenin Coating on Titanium Promotes Osteogenic Gene Expression

Osteoblast differentiation and extracellular matrix production are pivotal processes for implant osseointegration or bone tissue engineering. We hypothesized that a biomimetic coating on titanium surfaces, consisting of apatite and amelogenin, would promote such processes. Human Embryonic Palatal Mesenchymal pre-osteoblasts were used as a model for the evaluation of cell adhesion and spreading patterns, as well as mRNA expression of certain osteoblastic gene products. Real-time PCR showed significant (p < 0.05) increase in expression of type I collagen, alkaline phosphatase, and osteocalcin from cells grown on titanium with an apatite/amelogenin composite, as compared with that from cells grown on a pure titanium or apatite coating only. Osteocalcin expression was specifically stimulated by amelogenin added to the culture media. Enhanced attachment and cell spreading were also observed. The biomimetic coating promoting cell adhesion and osteoblast differentiation may have great potential for future dental and biomedical applications.

Enamel Proteases Reduce Amelogenin-Apatite Binding

Organic matrix degradation and crystal maturation are extracellular events that occur simultaneously during enamel biomineralization. We hypothesized that enamel proteases control amelogenin-mineral interaction, which, in turn can affect crystal nucleation, organization, and growth. We used a recombinant amelogenin (rP172), a homolog of its major cleavage product (rP148), and a native amelogenin lacking both N- and C-termini (13k). We compared apatite binding affinity between amelogenins and their digest products during proteolysis. We further compared binding affinity among the 3 amelogenins using a Langmuir model for protein adsorption. Amelogenin-apatite binding affinity was progressively reduced with the proteolysis at the C- and N- termini by recombinant pig MMP-20 (rpMMP20) and recombinant human kallikrein-4 (rhKLK4), respectively. The binding affinity of amelogenin to apatite was found to be in the descending order of rP172, rP148, and 13k. Analysis of our data suggests that, before its complete degradation during enamel maturation, stepwise processing of amelogenin by MMP-20 and then KLK4 reduces amelogenin-apatite interaction.

Tooth regeneration: challenges and opportunities for biomedical material research

Tooth regeneration presents many challenges to researchers in the fields of biology, medicine and material science. This review considers the opportunities for biomedical material research to contribute to this multidisciplinary endeavor. We present short summaries and an overview on the collective knowledge of tooth developmental biology, advances in stem-cell research, and progress in the understanding of the tooth biomineralization principles as they provide the foundation for developing strategies for reparative and regenerative medicine. We emphasize that various biomaterials developed via biomimetic strategies have great potential for tooth tissue engineering and regeneration applications. The current practices in tooth tissue engineering approaches and applications of biomimetic carriers or scaffolds are also discussed.

Immunogold Labeling of Amelogenin in Developing Porcine Enamel Revealed by Field Emission Scanning Electron Microscopy

The present study describes a method using immunohistochemical labeling in combination with high-resolution imaging (field emission scanning electron microscopy) to investigate the spatial localization of amelogenins on apatite crystallites in developing porcine enamel. Cross-sections of developing enamel tissue from freeze-fractured pig third molar were treated with antiserum against recombinant mouse amelogenin and immunoreactivity confirmed by Western blot analysis. The samples were then treated with the goat anti-rabbit IgG conjugated with 10-nm gold particles. The control samples were treated with the secondary antibody only. The in-lens secondary electrons detector and quadrant back-scattering detector were employed to reveal the high-resolution morphology of enamel structures and gold particle distribution. The immunolabeling showed a preference of the gold particle localization along the side faces of the ribbon-like apatite crystals. The preferential localization of amelogenin in vivo on enamel crystals strongly supports its direct function in controlling crystal morphology.