Mechanism by which MLO-A5 late osteoblasts/early osteocytes mineralize in culture: similarities with mineralization of lamellar bone

Abstract

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray analysis, and atomic force microscopy. At 3-4 days of culture, spheres of approximately 20-50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5-6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoid-osteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization.

Fig. 1

Time course for mineral formation by MLO-A5 cells as detected using von Kossa stain. MLO-A5 cells were cultured with β GP and ascorbic acid as described previously [23]. After 3 days, cells become confluent; at 6 days, a ‘‘fractal’’ or ‘‘honeycomb’’ pattern was observed that continued to increase in staining until the mineral covered the entire well by 12 days. This process is delayed approximately 3 days in the absence of β GP (data not shown) [23]. The honeycomb pattern of mineralization is different from nodules formed by FRC cells and the ones formed by the osteoblast cell line 2T3, which require BMP-2 for mineralization to occur [32]. Images provided by Dr. Sarah Dallas (FRC nodules) and Wuchen Yang (2T3 nodules) in the laboratory of Dr. Steve Harris, University of Missouri at Kansas City.

Fig. 2

Visualization and quantitation of mineral formation by MLO-A5 cells using AR-S staining. A significant amount of staining is observed using either quantification by area or quantification by solubility. ^## Significantly different from days 0, 3 and 6; ⁺⁺ significantly different from day 0, 3, 6 and 9; ** significantly different from all other groups, all using oneway ANOVA followed by Tukey past test, P<0.001.

Fig. 3

SEM showing the mineralized honeycombed matrix formed by MLO-A5 cells at day 9. Comparative secondary (A, B) and backscatter (C) images. Magnification is × 300 (A) × 1,000 (B, C). The cells appear to be sending (or leaving) projections along the mineralizing structures (arrow).

Fig. 4

Elemental analysis of the mineralized ‘‘honeycomb’’ matrix formed by MLO-A5 cells at 12 days of culture. The calcium component (A) forms a pattern that overlays the phosphate component (C), which is consistent with the SEM image (B). EDS analysis shows a ratio of calcium to phosphorus that is similar to normal hydroxyapatite (D).

Fig. 5

Western blot showing that the osteocyte-specific antigen E11 is increased at 6 and 9 days of culture, suggesting that these cells are becoming more osteocyte-like. Ponceau S stain shows relative amounts of loaded protein. MLO-Y4 cells are shown as a positive control.

Fig. 6

Immunofluorescent staining for collagen type I expression in MLO-A5 cells. At day 0, when the cells become confluent, expression is only observed intracellularly, inside the cytoplasm of MLO-A5 cells. At 3 days, collagen is secreted into the ECM, forming bundles that continue to form through 6–9 days. At 12 days, the collagen appears more localized to the cytoplasm, again suggesting a layer of cells growing over the previously mineralized matrix.

Fig. 7

Visualization of collagen type I fibrils in MLO-A5 cells using TEM (A), AFM (B), and SEM (C, D). The collagen type I fibrils in these cultures show the characteristic 69 nm banding. Mineralized collagen fibrils are shown in D.

Fig. 8

Mineral is associated with collagen as shown by TEM of MLO-A5 cells at 12 days in culture. Magnification is ×3,000 (A) and ×35,000 (B) of the same section. At the same time, mineralized spheres of 100–250 nm (arrows) are associated with the cellular processes of MLO-A5 cells at 6 days of culture as determined by TEM (C).

Fig. 9

The spheres contain calcium and phosphorus whether associated with the cell membrane (day 6) or with collagen fibers (day 9). At day 0, no spheres (arrows) are observed in confluent cells. By 6 days they are highly associated with cellular processes and mineralized. By day 9, they are also associated with collagen fibers. Elemental analysis using EDS shows that these structures contain both calcium and phosphorus, whether associated with the cell process or with collagen. Note the budding spheres (arrows) in the magnified area from day 6 cultures. The upper budding structure appears to be mineralizing, as indicated by the white on the tip of the bud. ♦, areas not containing spheres; ★, areas over spheres.

Fig. 10

Comparative secondary and backscatter (A, B) images by SEM of cultures at day 6 showing the association of spheres with collagen fibrils at 6 days and still some association with cellular processes. Magnification is ×2,000 (A, B). Note the spherical structure that appears to be budding (arrow) from the cell process in A. Note other spherical structures under the cell process, which are only visible with backscatter imaging (arrows) in B. (C) A sphere that appears to be engulfing collagen fibrils (9 days). These spheres appear to increase in size, engulf collagen fibers, and coalesce to form larger mineralized structures (arrow) as shown in 15-day culture (D). The sizes of these structures range from 50 nm to 1 μm.

Fig. 11

AFM height map of MLO-A5 cells at 12 days of culture showing spherical structures intercalated between collagen (A). The sizes of the spherical structures are 50 nm to 1 μm, consistent with those in Figure 10 (B).

Fig. 12

SEM of FRC cells at 20 days of culture. Note in A that the mineralized spheres are only associated with areas of collagen production. A magnified image of the collagen fibrils, spheres, and potentially budding spheres is shown (B).

Fig. 13

TEM of osteoid-osteocytes in murine long bone. Note the small mineralized spheres in the osteoid, which appear to increase in size, coalesce, and eventually surround the cell (A). These mineralized structures are similar in size to those observed in MLO-A5 cells (arrowhead) and appear to be associated with dendritic processes (arrow) (B).

Fig. 14

SEM of the ontogeny of osteoid-osteocytes in murine lamellar bone. (A) A cell newly embedding in osteoid. Dendritic processes extend toward the mineralization front. The front contains mineralized structures of similar size to the mineralized spheres observed in MLO-A5 and appear to increase in size. (B, C) Osteoid-osteocytes beginning to be surrounded by mineral. (D) Fully embedded osteocyte surrounded by mineral and a cell partially surrounded by mineral.

Fig. 15

Our hypothesis is that as the osteoid-osteocyte becomes embedded in the nonmineralized matrix and converts from a polygonal matrix-producing osteoblast to a dendritic osteocyte, it begins to generate branching processes. As it forms these processes, small calcified spheres are formed along the cell membrane toward the mineralization front that eventually become associated with the collagen fibers that are simultaneously being produced by the cell. This occurs as the cytoplasm of the cells is shrinking and forming thinner and thinner processes. Therefore, a gradient in size of the mineralized spheres is observed. We propose that the osteoid-osteocyte initiates this gradient.