Abstract
The transport of mineral ions from the enamel organ-associated blood vessels to the developing enamel crystals involves complex cargo packaging and carriage mechanisms across several cell layers, including the ameloblast layer and the stratum intermedium. Previous studies have established PHOSPHO1 as a matrix vesicle membrane-associated phosphatase that interacts with matrix vesicles molecules phosphoethanolamine and phosphocholine to initiate apatite crystal formation inside of matrix vesicles in bone. In the present study, we sought to determine the function of Phospho1 during amelogenesis. PHOSPHO1 protein localization during amelogenesis was verified using immunohistochemistry, with positive signals in the enamel layer, ameloblast Tomes' processes, and in the walls of ameloblast secretory vesicles. These ameloblast secretory vesicle walls were also labeled for amelogenin and the exosomal protein marker HSP70 using immunohistochemistry. Furthermore, PHOSPHO1 presence in the enamel organ was confirmed by Western blot. Phospho1−/− mice lacked sharp incisal tips, featured a significant 25% increase in total enamel volume, and demonstrated a significant 2-fold reduction in silver grain density of von Kossa stained ground sections indicative of reduced mineralization in the enamel layer when compared to wild-type mice (p < 0.001). Scanning electron micrographs of Phospho1−/− mouse enamel revealed a loss of the prominent enamel prism “picket fence” structure, a loss of parallel crystal organization within prisms, and a 1.56-fold increase in enamel prism width (p < 0.0001). Finally, EDS elemental analysis demonstrated a significant decrease in phosphate incorporation in the enamel layer when compared to controls (p < 0.05). Together, these data establish that the matrix vesicle membrane-associated phosphatase PHOSPHO1 is essential for physiological enamel mineralization. Our findings also suggest that intracellular ameloblast secretory vesicles have unexpected compositional similarities with the extracellular matrix vesicles of bone, dentin, and cementum in terms of vesicle membrane composition and intravesicular ion assembly.
Highlights
Amelogenesis is a complex process that involves a multitude of proteins and proteinases to facilitate the orderly assembly of elongated calcium phosphate apatite crystals into enamel prisms
PHOSPHO1 expression levels were stronger in the mineralized enamel layer and at the ameloblast secretory pole (Figures 1A,C), while there was no staining in the control section using the same technique without primary antibody (Figure 1E)
In this study we compared the enamel layers of wild-type and Phospho1−/− mice using light and electron microscopy as well as elemental mapping and von Kossa staining
Summary
Amelogenesis is a complex process that involves a multitude of proteins and proteinases to facilitate the orderly assembly of elongated calcium phosphate apatite crystals into enamel prisms. The secretory cells of bone, dentin, and cartilage connective tissues contain small vesicles surrounded by a lipid bilayer that in addition to small calcium phosphate crystals contain a number of enzymes that are important for their function in tissue mineralization, including tissue nonspecific alkaline phosphate (TNAP), nucleotide pyrophosphatase phosphodiesterase (NPP1/PC-1), annexins (ANX), and other matrix metalloproteinases (MMPs; Hsu and Anderson, 1978; Anderson, 1984; Bonucci, 1992; Dean et al, 1992; Wuthier et al, 1992). These unique proteases have been implicated in the mineralization of bone and dentin (Golub, 2009)
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