Intravesicular Phosphatase PHOSPHO1 Function in Enamel Mineralization and Prism Formation

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.

Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation

“…While the Ca 2+ transport mechanism in these cells is relatively well understood ( Lacruz, 2017 ; Lacruz et al, 2017 ; Varga et al, 2018 ), much less is known about the mechanism of phosphate transport into the enamel matrix. Some possibilities include transport into the cells of the enamel via Na + -phosphate cotransporters of the SLC20 and SLC34 gene families ( Merametdjian et al, 2018 ; Beck, 2019 ) followed by secretion across the apical membrane or into matrix vesicles ( Pandya et al, 2017 ).…”

The Glycoprotein/Cytokine Erythropoietin Promotes Rapid Alveolar Ridge Regeneration In Vivo by Promoting New Bone Extracellular Matrix Deposition in Conjunction with Coupled Angiogenesis/Osteogenesis

“…After the samples were in 100% light cure technovit (Technovit 7200, EXAKT), the next step was to polymerize and embed the samples. Next, the samples were sectioned using a diamond bandsaw (EXAKT 300 CP), ground and polished to achieve 50–70 µm thin sections as described in our previous study [ 30 ].…”

How To Build a Bone: PHOSPHO1, Biomineralization, and Beyond

“…In enamel, Phospho1 -/mice exhibited a 25% increase in tissue volume, with a significantly reduced level of mineralization. (47) The authors furthermore reported that ablation of PHOSPHO1 caused loss of enamel prism morphology and an impaired crystal organization compared to wild-type mice.…”