High-pressure replica technique for in vitro imaging of pore morphologies in teeth

The presence of a natural pore morphology is an essential factor in chemical and mechanical stability of teeth. Common histological methods give only an insufficient picture of the three-dimensional pore distribution in sound or carious teeth. This paper describes a new technique to obtain complete images of the pore structure in teeth or other biological hard tissues. Whole extracted human teeth from orthodontic therapy are mechanically cleaned and organic remnants removed chemically. After being (partly) dried, the teeth are penetrated by a freshly prepared and colored (rhodamine B dye) two-component epoxy resin. The resin is pressed into the pores and cavities of the teeth with pressures of up to 2000 bar in a high-pressure vessel by means of a manually driven piston screw pump for pressure generation. The resin fills all hollow spaces of the tooth down to sizes below 0.1 micron. The pulp and the root canals are cast in massive forms of the hardened epoxy resin, giving an exact replica of the natural structure. The penetrated samples in the form of whole, intact teeth are investigated microscopically so that the pore morphology of the tooth surface, including carious defects, can be examined. The structure of the pores extending into the interior of the tooth can be made visible by dissolution of the hard tissue--for example, in acid solutions. Micro-cavities filled with the resin are observed in thin, ground, and polished cross- and longitudinal sections cut from the teeth. The colored resin induces a high contrast to the dental apatite material. In fluorescence microscopy, only the resin structures are visible.