Hydroxyapatite Crystal Production by Epithelial Keratinocytes in vitro

Objectives: In vitro utilization of oral epithelial keratinocytes to produce Hydroxyapatite crystals that resemble human tooth enamel
Methods: Cell culture: Immortalized Murine Oral Keratinocytes (IMOK) are chronically treated with rmTGFb1 for 10 days. Cells were harvested and resuspended in Matrigel and seeded into 24-well culture plates and treated with serotonin for 10 days. The cells will then stay for 10 days in a mineralization solution. After fixation, the biomaterial (natural enamel) is then harvested and molded into a sphere.
IHC: Cells were processed in culture plates or 20mM frozen sections of biomaterial. Samples were incubated with primary antibody and then with secondary antibody followed by treatment with avidin-biotin solution. Diamino-bensidine solution was used as a chromagen.
Engineering methods: Sample characterization was performed using a dual-beam scanning electron microscope (SEM) with focused ion beam (FIB) Both secondary and backscattered electron images were recorded for all samples. Using the FIB, a thin lamella of the sanitized biomaterial was lifted out from the sample for scanning transmission electron microscope (STEM) imaging. In order to determine the elemental composition of samples, x-ray energy dispersive spectroscopy (EDS) was performed on both the bulk specimen and lift-out lamella.
Results: 10 days treatment with rmTGFb1on (IMOK) induced a phenotypic changes including cellular hypertrophy, separation of the cells and acquisition of a cuboid shape. After that, organoids were formed by culturing the cells in Matrigel and treatment with serotonin. immunohistochemistry showing positive amelogenin and alizarin red staining demonstrating mineralization of the organoids.
Material was harvested and collected into an aggregate block. SEM image of showed the crystalline structure and x-ray energy dispersive spectroscopy, showed the composition of the synthesized sample
Conclusions: Characterization of the structure using scanning electron microscopy and related techniques of the grown materials (natural enamel) reveals a strong similarity to the hydroxyapatite crystals that occur in tooth enamel