2.3. Local Synthesis of PO
43−
by the Activities of TNAP and ENPP1
One of the most important enzymes enabling mineralization is TNAP, a glycosylphosphatidylinositol anchor enzyme associated with the cell membranes of matrix vesicles and osteoblastic cells. TNAP can hydrolyze various phosphate esters, especially pyrophosphates (PPi), and is broadly recognized as a hallmark of osteoblastic cells. However, the method of PPi supplementation is important. Currently, it is believed that ENPP1 mainly supplies PPi. ENPP1 is composed of two N-terminal somatomedin B-like domains, a catalytic domain, and a nuclease-like domain. Crystalline structure analysis of ENPP1 demonstrated that the nucleotides are accommodated in a pocket formed by an insertion loop in the catalytic domain of ENPP1, implying a preference for an ATP substrate
[7]. Therefore, in bone mineralization, the catalytic activity of ENPP1 may generate PPi, presumably using ATPs in the extracellular fluid. The resultant PPi is then hydrolyzed by TNAP into PO
43−. However, PPi is also known to inhibit mineralization by binding to nascent hydroxyapatite crystals, thereby preventing crystal overgrowth
[50,51,52][44][45][46]. Hence, a balance between PPi and PO4
3− is important for normal bone mineralization. TNAP is not uniformly distributed on the cell membranes of osteoblasts; it was distinctly observed on the basolateral sides rather than the secretory (osteoidal) domains
[37,53][37][47].
2.4. Transport of PPi and PO
43−
via ANK and Pit1/Pit2
ANK, encoded by the progressive ankylosis gene (
Ank), can serve as a non-enzymatic PPi channel, allowing PPi to pass through the plasma membrane to the outside of the cell
[16,17][16][17]. As shown in
ourthe recent reports, the immunoreactivity of ENPP1 was detected not only in the cell membranes but also in the cytoplasmic region of osteoblasts and osteocytes, indicating the presence of both extracellular and intracellular PPi in these cells
[54][48]. It is therefore likely that the ANK-mediated outflow of intracellular PPi may be involved in the dynamic equilibrium between intra- and extracellular levels of PPi. After the outflow of PPi to the extracellular region, TNAP hydrolyzes PPi into PO
43−.
Extracellular PO
43− may pass through the plasma membrane of the matrix vesicles by Pit1 and Pit2 mediation. Pit1 and Pit2 are type III sodium-inorganic phosphate (Pi) co-transporters encoded by Slc20a1 and Slc20a2
[8,9,10,11,12][8][9][10][11][12]. Recently, it has been reported that Pit1 and Pit2 form heterodimers, sense extracellular PO
43− concentrations, and increase the expression of matrix Gla protein (MGP) and osteopontin via the extracellular signal-regulated kinase (ERK) pathway
[55,56,57][49][50][51].
2.5. PHOSPHO1 for PO
43−
Production inside Matrix Vesicles
Alternative to the biological function of ENPP1/TNAP, PHOSPHO1 is an enzyme highly expressed in mineralizing osteoblasts and hypertrophic chondrocytes
[60][52]. This enzyme has been implicated in bone and cartilage formation and is thought to function inside cells and matrix vesicles to generate PO
43− using phosphocholine and phosphoethanolamine, which are components of the lipid bilayers of matrix vesicles
[13,14,15][13][14][15].
3. Development of Mineralized Nodules and Collagen Mineralization
3.1. Growth of Mineralized Nodules
The calcium phosphate crystals that are nucleated inside the matrix vesicles grow in all directions and then penetrate the plasma membrane to exit the vesicles, eventually forming mineralized nodules, which are also referred to as calcifying globules [1,3,4][1][3][4]. Under TEM observation, mineralized nodules appear as globular structures composed of radially assembled hydroxyapatite crystals [44,68][53][54]. It seems likely that the growth of mineralized nodules is regulated by non-collagenous proteins in the osteoid. Among these materials, osteopontin is especially suited to regulating mineralization because it is a negatively charged and highly phosphorylated molecule that can effectively inhibit hydroxyapatite formation and growth [6,69][6][55]. Osteocalcin is another important bone matrix protein subjected to vitamin K-dependent carboxylation at its glutamate residues.
3.2. Collagen Mineralization
Collagen mineralization begins at the point of contact with mineralized nodules. TEM observations demonstrated that mineralization spreads from the contact point of the mineralized nodules toward the periphery of the collagen fibrils
[2]. This finding suggests that collagen mineralization orderly progresses from the contact points with mineralized nodules, presumably allowing the regular deposition of calcium phosphate crystals onto the collagen fibrils. At a higher magnification, the spicules of calcium phosphate crystals can be seen on the fibrillar structures identical to the superhelix (tropocollagen) of collagen fibrils, thus indicating that mineral crystals are deposited on the superhelix, which serves as a scaffold for collagen mineralization. After contact with the mineralized nodules, the collagen fibrils eventually become completely mineralized.
Proteoglycans such as decorin and biglycan, which directly bind the collagen surface through GAG chains, inhibit the growth of mineral crystals
[74,75,76][56][57][58]. Collagen mineralization in the osteoid increases proportionally based on the distance from the osteoblasts, whereas the amount of decorin in the osteoid decreases further away from the bone surface
[77][59]. In the osteoid close to the osteoblasts, proteoglycans combined with the surface of newly formed collagen fibrils are localized to the large space between collagen fibrils.
4. Osteocyte Network and the Biological Function of Regulating Bone Mineralization
4.1. Distribution of the Osteocyte Network
Osteoblasts secrete bone matrix proteins and can become embedded in the bone matrix, where they differentiate into osteocytes. Immediately before becoming embedded into the bone matrix, osteoblasts rearrange the actin filament assembly along the cell membranes and the cytoplasmic processes, which resemble that of embedded osteocytes
[24]. This implies that the osteoblasts approaching osteocytic differentiation and the newly-differentiated osteocytes decide the geometrical structure of the cellular network of their cytoplasmic processes.
4.2. Osteocyte-Derived Molecules Involved in Peripheral Mineralization
Osteocytes physiologically synthesize several important molecules, e.g., dentin matrix protein (DMP) 1, matrix extracellular phosphoglycoprotein (MEPE), osteopontin, and Phex, for regulating surrounding bone mineralization. DMP1 has a high Ca
2+-binding capacity and, therefore, is postulated to play a role in bone mineralization in the vicinity of osteocytes
[33]. DMP1 belongs to the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, which also includes MEPE, osteopontin, bone sialoprotein, and dentin sialo-phosphoprotein, and is encoded by a gene located on human chromosome 4q21 and mouse chromosome 5q21
[92,93][60][61].
Since osteocytes express abundant MEPE
[96][62], DMP1
[33], and osteopontin, especially in
Hyp mice fed a high-phosphate diet
[97][63], it can be easily assumed that osteocyte-derived SIBLINGs would regulate peripheral bone mineralization by the osteocytes. This postulation is evidenced by the report that a DMP1 absence results in rickets or osteomalacia in mice
[98][64] and by autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR) in human patients
[99][65]. Hence, osteocytes seem to be involved in the regulation of the surrounding mineralization. However, Phex/SIBLINGs are usually associated with the congenital deformities rickets and osteomalacia. Therefore, it is necessary to elucidate whether SIBLINGs play an important role in the physiological regulation of bone mineralization in a normal state (
Figure 41).
Figure 41. A schematic design of matrix vesicle-mediated mineralization and subsequent osteocytic maturation of mineralization.
5. Cellular Interplay between Osteoblasts and Osteocytes in Bone Mineralization
Osteoblasts secrete matrix vesicles, which provide initiation sites for mineralization during primary mineralization, while osteocytes appear to regulate bone mineralization through Phex/SIBLINGs. Taking these findings into account, the interplay between osteoblasts and osteocytes in the regulation of bone mineralization seems likely. Matrix vesicles secreted by osteoblasts grow into globular assemblies of needle-like calcium phosphate crystals, called mineralized nodules, which then induce collagen mineralization. During nucleation and subsequent growth inside the vesicles, the influx of Ca
2+ and PO
43− is promoted by many enzymes and membrane transporters located on the matrix vesicles and mineralized nodules (particularly, they are located on the ruptured membranes of the vesicles).
However, the growth of large, terminal mineralized nodules that are distant from osteoblasts, as well as collagen mineralization, may be regulated by a mechanism other than enzymes associated with matrix vesicles secreted by the osteoblasts. In the osteoid, there seem to be two possible pathways that supply Ca
2+ and PO
43− to terminal mineralized nodules and collagen mineralization: one is from the osteoblast-covered bone surface, and the other is from osteocytic canaliculi, which are opened to the osteoid.
6. Conclusions
Primary mineralization in bone is achieved by matrix vesicle-mediated mineralization; matrix vesicles contain a variety of membrane transporters and enzymes involved in the nucleation and subsequent growth of crystalline calcium phosphates inside the vesicles. For proper mineralization, the biological accumulation of Ca
2+ and PO
43− in the vesicles is necessary. Of particular importance is the influx of PO
43− into matrix vesicles, which involves a complex interplay among ENPP1, ANK, TNAP, and Pit1. Crystalline calcium phosphates grow radially, penetrate the vesicle membranes, and then exit the vesicles to form mineralized nodules, which are globular assemblies of needle-shaped mineral crystals. In contrast to primary mineralization, secondary mineralization increases bone mineral density, presumably due to osteocytic functions. Osteocytes appear to regulate bone mineralization, which is mediated by Phex/SIBLINGs. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.