Previous work has proposed that cartilage and/or bone mineralization utilizes either a single vesicle population enriched in both calcium and phosphorus, or, two vesicle populations separately enriched in calcium or phosphorus (Figure 1) (Anderson, 1967; Bonucci, 1967; Arsenault and Ottensmeyer, 1984). In order to clarify this issue, the solubility of these ions necessitates the use of additional methods such as high pressure freezing and freeze substitution to avoid loss during specimen fixation, embedding, and sectioning. Since most prior studies have not consistently avoided water during specimen processing, the true impact of pseudo non-aqueous processing on the process of osteoblast-mediated mineralization is difficult to assess.
The goal of our study was to use the synchronized UMR106-01 culture model to devise and validate a pseudo nonaqueous processing method to image the distribution of calcium and phosphorus ions within BMF immediately prior to nucleation of the fi rst hydroxyapatite crystals therein (Figure 1). We believe this method should also be applicable to investigations of temporal changes in calcium ion distributions in other cells such as muscle.
Fig. 1: The single and dual vesicle models of extracellular mineralization. Upper panel, single matrix vesicle model. Calcium and phosphorus ions are progressively concentrated within a single population of matrix vesicles through the proposed actions of Ca+2-pumping ATPase, Na+-phosphate co-transporter, and phosphatases acting on phospholipids. Once Ca+2 and phosphate ions reach a threshold concentration (yellow), nucleation of initial mineral crystals occurs leading to breakage of the vesicle and release of crystals which can propagate additional crystals within the surrounding extracellular collagenous matrix. Lower panel, dual vesicle model. Calcium and phosphorus are progressively concentrated within two different populations of vesicles which biochemically display distinct functional distributions including Ca+2 pumping ATPase and Na+-phosphate co-transporter activities, respectively. Subsequently, these calcium and phosphate enriched vesicle populations fuse and nucleate mineral crystals leading to breakage of the vesicle and release of crystals which can propagate additional crystals within the surrounding extracellular collagenous matrix.
Use of high pressure freezing and freeze-substitution is not sufficient to retain labile calcium and phosphorus in mineralizing osteoblastic cell cultures
Calcium and phosphorus can be lost from samples either after high pressure freezing and freeze-substitution or during conventional fixation. To evaluate the effectiveness of our pseudo non-aqueous method, we chose to stop the cultures at 76 h, twelve hours after adding the ß-glycerol phosphate mineralization stimulus, but 2 h before the appearance of the first crystals of mineral within BMF (Midura et al., 2004; Wang et al., 2009). Some cultures were randomly chosen to be processed for conventional fixation (Figure 2) while others were processed by high pressure freezing (Leica EM PACT) and freeze-substitution (Figure 3). Comparison of these paired cultures support several conclusions. Large areas contain a somewhat homogeneous particulate organic matrix after conventional fixation. These areas seem to exclude membrane limited vesicles from their volume. In contrast, high pressure frozen cultures contain numerous almost "white" extracellular areas, roughly 0.5–1 μm in diameter, within BMF (arrows, Figure 3).
In higher power views, it is evident that despite the low contrast these "white" regions do possess an underlying detail which represents a range of irregularly shaped spherical bodies from about 50 to 800 nanometers in diameter (not shown). However, the presence of these “white” areas raised immediate concerns regarding the loss of inorganic or organic substances. In particular, when compared with similar BMF regions from paired, conventionally fixed cultures (Figure 2), we hypothesized that "white" spots represented materials which were preferentially retained by high pressure freezing but which were subsequently lost upon further processing.
Side-by-side comparison shows dry sectioning in combination with high-pressure freezing and freeze-substitution is required to observe extracellular vesicle populations enriched in calcium and phosphorus within mineralizing osteoblastic cultures
Notably, subsequent electron spectroscopic imaging of calcium and phosphorus was not possible in either conventionally fixed nor in freeze-substituted samples which were sectioned on water regardless of whether specimens were post-stained or not (results not shown). We therefore substituted dry sectioning for wet sectioning of high pressure frozen, freeze substituted cultures. Calcium and phosphorus retention is improved in dry sectioned high pressure frozen, freeze-substituted cultures. It is clear that substitution of dry sectioning leads to a dramatic increase in retention of calcium and phosphorus within biomineralization foci [compare Figures 3 (wet sectioning) and 4 (dry sectioning)].
Focal 0.5–1 μm diameter areas which appeared as “white” spots in Figure 3 after wet sectioning, now appear dark (Figure 4A) in the zero loss energy image. The fact that UMR106-01 cultures mineralize in a reproducible, temporally synchronous manner facilitates these direct comparisons among different cultures (Wang et al., 2009).In addition, electron spectroscopic imaging demonstrates that the dark appearing areas are enriched in calcium and phosphorus (compare Figures 4A, B, and C). Finally, as shown in the overlay image in Figure 4D, the calcium (red) and phosphorus (green) signals largely overlap each other in the 76 h cultures as shown by the presence of yellow. Since other studies have shown that 76 h UMR106-01 cultures do not contain detectable mineral crystals (Huffman et al., 2007; Wang et al., 2009), the enriched contents of calcium and phosphorus observed here could represent amorphous calcium phosphate (Driessens et al., 1978) and/or labile organic forms of phosphorus such as polyphosphates (Omelon et al., 2009).
Importantly, a functional role for the observed enriched focal contents of calcium and phosphorus in mineralization is supported by analyses of un-mineralized control cultures. When a similar high pressure freezing, freeze substitution, and dry sectioning approach was applied to un-mineralized UMR106-01 cultures, few dark (calcium and/or phosphorus enriched) vesicles or particles were detected (not shown). Finally, an additional advantage to use of the oscillating knife was that it reduced compression during sectioning and reduced wrinkling of resultant sections.
In summary, our results show that high pressure freezing and pseudo non-aqueous processing are required to detect extracellular sites of early calcium and phosphorus enrichment in mineralizing osteoblastic cultures. Use of dry sectioning proved to be a critical step in the preservation of calcium and phosphorus. Also, electron spectroscopic imaging demonstrated that darkly stained vesicles within extracellular biomineralization foci are enriched in calcium and phosphorus prior to the detection of crystalline mineral (Wang et al., 2009). We now plan to use this method to determine if osteoblastic cells in vitro and in vivo utilize a single or dual vesicle mineralization mechanism (Gorski et al., 2004; Midura et al., 2009).
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