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Review
. 2017 May;53(2):34-45.
doi: 10.1016/j.jdsr.2016.09.002. Epub 2016 Nov 5.

Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization

Tomoka Hasegawa  1 , Tomomaya Yamamoto  1 , Erika Tsuchiya  1   2 , Hiromi Hongo  1 , Kanako Tsuboi  1   2 , Ai Kudo  1 , Miki Abe  1 , Taiji Yoshida  1 , Tomoya Nagai  1   3 , Naznin Khadiza  1   4 , Ayako Yokoyama  1   5 , Kimimitsu Oda  6 , Hidehiro Ozawa  7 , Paulo Henrique Luiz de Freitas  8 , Minqi Li  9 , Norio Amizuka  1
Affiliations
Review

Ultrastructural and biochemical aspects of matrix vesicle-mediated mineralization

Tomoka Hasegawa et al. Jpn Dent Sci Rev. 2017 May.

Abstract

Matrix vesicle-mediated mineralization is an orchestrated sequence of ultrastructural and biochemical events that lead to crystal nucleation and growth. The influx of phosphate ions into the matrix vesicle is mediated by several proteins such as TNAP, ENPP1, Pit1, annexin and so forth. The catalytic activity of ENPP1 generates pyrophosphate (PPi) using extracellular ATPs as a substrate, and the resultant PPi prevents crystal overgrowth. However, TNAP hydrolyzes PPi into phosphate ion monomers, which are then transported into the matrix vesicle through Pit1. Accumulation of Ca2+ and PO43- inside matrix vesicles then induces crystalline nucleation, with calcium phosphate crystals budding off radially, puncturing the matrix vesicle's membrane and finally growing out of it to form mineralized nodules.

Keywords: ENPP1; Matrix vesicle; Mineralization; Mineralized nodule; TNAP.

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Figures

Figure 1
Figure 1
TEM observation of matrix vesicles and mineralized nodules in osteoid. (A) TEM image of ultratrsucture of osteoid underlying mature osteoblasts (OB). Notice many mineralized globular structures referred to as mineralized nodule (MN) in the osteoid. (B–E) TEM images of matrix vesicles (MV) (B), appearance of mineral crystals in the matrix vesicles (C), exposure of mineral crystals out of the matrix vesicles (D) and mineralized nodule (MN) formation (E). An inset demonstrate a highly-magnified image of needle-shape of mineral crystals. Panel A is derived from Ref. (Amizuka and Ozawa), while panels B–F are modified from Ref. (Amizuka et al.).
Figure 2
Figure 2
Highly-magnified images of matrix vesicles and elemental mapping of calcium and phosphate ions. (A–C) TEM images of matrix vesicles (MV). (A) amorphous electron-dense structure (double arrows, black) are shown to be associated with plasma membrane of the matrix vesicle. (B) The grown mineral crystals (black structures) are seen inside the matrix vesicles. (C) Mineral crystals are getting out of the matrix vesicles. (D–F) TEM image (D) and elemental mapping of calcium (Ca, panel E) and phosphorus (P, panel F) assessed by electron energy loss spectroscopy. Note that calcium (Ca) was evenly distributed in the peripheral region of matrix vesicles, while phosphorus (P) is predominant in collagen fibrils. The images of A–C are modified from Ref. (Ozawa et al.), and D–F are from Ref. (Hoshi et al.).
Figure 3
Figure 3
TEM observation on mineralized nodules of normal rats and warfarin-treated rats. Panels A and C demonstrate the ultrastructure of mineralized nodules in the osteoid of normal and warfarin-administered rat bones. In control group, globular mineralized nodules (MN) are shown to be composed of many needle-shaped mineral crystals (A). However, the warfarin-administered osteoid shows dispersed mineral crystals throughout the osteoid (C). (B and D) Immunoelectron microscopy for osteocalcin localization. Osteocalcin immunoreactivity (black particles) can be seen on the mineralized nodules (MN, grey colored globular structures) of the control osteoid (B), while little immunoreactivity for osteocalcin is seen in the warfarin-administered osteoid (D). The images are derived from Ref. (Amizuka et al.).
Figure 4
Figure 4
Histochemical localization of alkaline phosphatase in bone. (A and B) Enzyme histochemistry of alkaline phosphatase (ALPase, red color) and tissue nonspecific ALPase (TNAP, brown color) immunohistochemistry. Both histochemical technique consistently reveal an intense enzymatic activity of ALPase and immunoreactivity of TNAP in the regions of perosteoblasts (arrows, in A and B), rather than mature osteoblast (OB) located on bone matrix (BM). (C) TEM image of ALPase enzyme cytochemistry. Note the ALPase activity (black) can be seen on cell membranes of preosteoblasts (pre-OB) and mature osteoblasts (OB) on the bone surfaces. Insets demonstrate the ALPase enzyme activity on matrix vesicle (MV) and mineralized nodule referred to as calcified nodules (black, CN). Panels A and B are derived from Ref. (Amizuka et al.), while panel C is from Ref. (Amizuka and Ozawa).
Figure 5
Figure 5
Domain organization of mouse ENPP1. Modified from Ref. (Kato et al.).
Figure 6
Figure 6
Schematic design of matrix vesicle-mediated mineralization. Matrix vesicles provide adequate micro-circumstance for initiation of mineralization. Membrane transporters and enzymes including TNAP, ENPP1, annexins, ANK and Pit1 equipped on matrix vesicles play a pivotal role in Ca2+ and PO43− transport into the vesicles. Phosphatidylserine and so forth in the plasma membrane has a high affinity to produce a stable calcium phosphate–phospholipid complex associated with the inner leaflet of the vesicle’s membrane. Thereafter, amorphous calcium phosphates develop hydroxyapatite to form needle-shaped mineral crystals. Many mineral crystals penetrate the vesicles’ membranes to form a globular assembly of numerous mineral crystals, i.e., mineralized nodules.
Figure 7
Figure 7
TEM images of the contact between collagen fibrils and mineralized nodules. (A) In osteoid, several mineralized nodules (MN) are shown to make contact with surrounding collagen fibrils (Co) (arrows). (B) In other region, mineralized nodules spread out minerals to neighboring collagen fibrils. Thus, collagen mineralization seems to be associated with mineralized nodules. In contrast, however, collagen striation (short arrows) do not show any mineral deposition. (C) At a higher magnification, laddering structures of mineral deposition are parallel to the longitudinal axis of the collagen fibrils, and the length of mineral crystal seems to be identical to that of superhelix. Panel A is from Ref. (Amizuka et al.), and panels B and C are modified from Ref. (Ozawa et al.).
Figure 8
Figure 8
Matrix vesicles and collagen mineralization in ascorbic acid insufficient circumstance. In a normal state, TEM observations demonstrate numerous matrix vesicles (A) and mineralized nodules (B). At a higher magnification, mineral crystals extending from the mineralized nodules ran along the collagen fibrils (B). In an insufficient circumstance of ascorbic acid, which is necessary for collagen synthesis, TEM observations verified the presence of matrix vesicles and mineralized nodules (C). At a higher magnification, however, collagen fibrils are shown be very fine, but, the fine mineral crystals from the mineralized nodule extended along fine fibrillar structures of collagen fibrils (D and E). All the images are modified from Ref. (Hasegawa et al.).
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