Time-related changes of developing enamel crystals after exposure to the tissue fluid in vivo: analysis of a subcutaneously implanted rat incisor.

To investigate the effects of tissue fluid on the growth of enamel crystals, upper and lower incisors extracted from 3-week-old Wistar rats were removed of the enamel organ, implanted subcutaneously in the dorsal portion of animals from the same litter, and harvested at 72 h or 1 week after implantation. The grafts were chemically fixed with surrounding tissues and prepared for light and electron microscopy, X-ray microanalysis, or for the immunohistochemistry of amelogenin. Mineralization of implanted enamel layers was examined by contact X-ray microradiography. The immunoreactivities for 25 kD amelogenin in immature enamel decreased sequentially, starting from the surface to the deeper layers; by 1 week after implantation, no positive reactivities remained in the entire enamel layers at the stages of matrix formation and early maturation. In accordance with the loss of enamel proteins, immature enamel gained mineral density until it attained higher radio opacity than that of the adjacent dentin by 1 week. In contrast, the radio opacity of the full thickness of the enamel at early maturation remained low except for a superficial thin layer. Electron microscopy revealed no sign of growth of original enamel crystals, but showed heavy precipitation of electron-dense fine granules of calcium phosphate in all layers of the secretory enamel and the superficial layer of enamel at early maturation, which showed high radio opacity. The Ca/P ratio and electron diffraction patterns of the granular materials precipitated between intrinsic enamel crystals indicated the property of hydroxy apatite or octacalcium phosphate though a characteristic ribbon-like profile of enamel crystals was lacking. These data indicate that the enamel organ blocks exogenous mineral precipitates in growing enamel during the stage of matrix formation and plays an essential regulatory role for fine enamel crystallites to grow into large hexagonal crystals.