reSolution_LNT_No1_en - Leica Microsystems

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BIOLOGY Dry Ultrathin Sectioning Combined With High Pressure Freezing/Freeze-substitution Improves Retention and Visualization of Calcium and Phosphorus Ions Prior to Nucleation of Mineral Crystals Within Osteoblastic Cultures Jeff P. Gorski1 , N.T. Huffman1 , T. Hillman-Marti2 , and Daniel Studer2 1Department of Oral Biology and the UMKC Center of Excellence in Mineralized Tissue Research, School of Dentistry, Univ. Missouri-KC, Kansas City, Kansas City, MO and 2Institute of Anatomy, University of Bern, Bern, Switzerland 12 reSOLUTION We have used cultured UMR106-01 osteoblastic cells to investigate the process of bone mineralization. UMR106- 01 cells as well as primary calvarial bone cells assemble spherical extracellular supramolecular protein-lipid complexes, termed biomineralization foci (BMF), in which the fi rst crystals of hydroxyapatite mineral are deposited (Midura et al., 2004; Wang et al., 2004). A major difference between these culture models is the speed with which mineralization occurs, ranging from 12-16 days after plating for primary osteoblastic cells to 88h for UMR106-01 cells. If mineralization is blocked by omission of phosphate source or by addition of serine protease inhibitor AEBSF, BMF complexes are formed but no mineralization occurs. Interestingly, ultra structural studies have shown that prior to mineralization BMF contain numerous membrane limited vesicles ranging in size from 50 nm to 2 microns in diameter. However, the fi rst mineral crystals are not detected until 78 h after plating of UMR106-01 cells and are localized within spherical sites presumed to be vesicles. Specifi cally, confocal Raman spectral analyses have shown that mineralization within BMF is a progressive, multi-step process occurring simultaneously in all BMF within a culture fl ask (Wang et al., 2009). Importantly, several protein spectral changes are detectable within each BMF prior to the deposition of poorly crystalline hydroxyapatite and when mineralization was blocked, these changes did not Jeff P. Gorski Daniel Studer Thérèse Hillmann occur. Thus, mineralization within BMF is a temporally synchronized process. However, understanding the biochemical mechanism of mineralization requires a detailed appreciation of calcium and phosphorus ion handling prior to crystal nucleation within BMF. 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 (Fig. 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 fi xation, 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 diffi cult 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 (Fig. 1). We believe this method should also be applicable to investigations of temporal changes in calcium ion distributions in other cells such as muscle.
Calcium and phosphorus can be lost from samples either after high pressure freezing and freeze-substitution or during conventional fi xation. 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 fi rst crystals of mineral within BMF (Midura et al., 2004; Wang et al., 2009). Some cultures were randomly chosen to be processed for conventional fi xation (Fig. 2) while others were processed by high pressure freezing (Leica EM PACT) and freeze-substitution (Fig. 3). Comparison of these paired cultures support several conclusions. Large areas contain a somewhat homogeneous particulate organic matrix after conventional fi xation. 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, Fig. 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 fi xed cultures (Fig. 2), we hypothesized that “white” spots represented materials which were preferentially retained by high pressure freezing but which were subsequently lost upon further processing. Notably, subsequent electron spectroscopic imaging of calcium and phosphorus was not possible in either conventionally fi xed 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 Fig. 3 after wet sectioning, now appear dark (Fig. 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 Figs. 4A, B, and C). Finally, as shown in the overlay image in Fig. 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. BIOLOGY 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. Instrument related to this sample preparation: Leica EM PACT2 High Pressure Freezer with Rapid Transfer System NANOTECHNOLOGY 13
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