PMC:2779105 / 16132-24443 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2779105","sourcedb":"PMC","sourceid":"2779105","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2779105","text":"Slow and Spontaneous Formation of NB-Like Precipitations by Serum through a Dual Seeding-Inhibitory Mechanism\nAll previous NB studies have used a source of proteins like serum, followed by prolonged incubation in cell culture conditions in order to demonstrate the formation of NB-like particles [4]–[9]. This kind of slow culturing process gives at best marginal particle production over a period of several weeks, making the results difficult to quantify or interpret. To address these concerns, we have developed a simple culture methodology that allows for the quantitative and reproducible reconstitution of nanoparticles as well as the simultaneous comparison of a large number of samples [2], [3].\nFigure 1 illustrates the phenomenon of NB-like particle formation using this culture system and in response to the simple inoculation of serum as a source of proteins and other organic compounds. Here, the formation of precipitable complexes resembling NB was monitored in 24-well plates containing Dulbecco's modified Eagle's medium (DMEM) by means of turbidity measurements as captured visually through photography or by means of light spectroscopy reading at a wavelength of 650 nm (A650). Turbidity was seen to gradually increase with the addition of either FBS or HS followed by incubation at 37°C for several weeks (Fig. 1). Notably, two types of dose-dependence could be discerned depending on the concentration of serum used. Addition of low concentrations of either FBS or HS produced a dose-dependent increase in turbidity while levels of serum exceeding 0.3% for FBS and 5% for HS generally gave an increasingly lower amount of precipitation or turbidity measurement (Fig. 1A and B, respectively).\n10.1371/journal.pone.0008058.g001 Figure 1 Slow formation of NB-like particles by serum inoculated into DMEM.\n(A) Formation of NB-like particles by FBS. FBS was inoculated into DMEM to the indicated % by volume. Visual inspection and A650 turbidity reading (insets) were performed within 1 hour following inoculation (top panel, “Day 1”) as well as after incubation at 37°C in cell culture conditions for “1 Month” (middle panel) or “2 Months” (bottom panel). Notice the gradual increase in turbidity observed with time as well as the bell-shaped increase in turbidity as a function of the amount of serum inoculated, with maximum turbidity reached at 0.3% FBS, while higher concentrations of FBS resulted in increasingly lower turbidities. (B) Formation of NB-like particles by HS, performed as described in (A), except that higher serum concentrations varying from 1 to 10% were used. Notice the bell-shaped increase in turbidity, with peak reached at 1-to-3% HS at “1 Month” and 3% at “2 Months.” This dual dose-dependent response could be illustrated further by fine-tuning the range of serum used. For FBS, we have found that the use of a 100-fold difference in the amount of serum inoculated, varying between 0.1 and 10%, generally encompassed the entire range of dose-dependence observed (Fig. 1A). That is, precipitation could be seen to increase steadily in proportion to the amount of serum added, but only up to 0.3%, as illustrated both by Figure 1A (well 3) shown here as well as by Figure 1A (well 2) shown earlier [2]. Above 0.3% FBS, turbidity decreased proportionally with the amount of serum inoculated, presumably due to calcification-inhibitory influences exerted by the serum (Fig. 1A, wells 4–6). While the peak precipitation seen with 0.3% FBS varied with the source and batch of serum used, a bell-shaped relationship was observed however with all of the FBS samples tested.\nFor HS, on the other hand, we have noticed generally a rightward shift of this same dose-dependent response when compared to FBS that nonetheless retained its modal or dual nature (Fig. 1B, compare also with Fig. 1A of ref. [2]). A 10-fold HS concentration range was used in the experiment illustrated in Figure 1B to illustrate this characteristic dual dose-dependence and to better highlight the decrease of turbidity associated with higher amounts of serum. Here, peak turbidity in DMEM was seen at 1-to-3% serum after 1 month, and 3% at the end of 2 months (Fig. 1B, middle and bottom rows). This slight rightward shift of the serum concentration associated with culture aging was consistently more noticeable with HS than with FBS. As seen earlier with FBS, while the different HS samples tested gave variable precipitation amounts as well as peak turbidities, the dual or bell-shaped dose-dependence was generally observed with all of the serum samples tested to date.\nThe bell-shaped serum-dependence for turbidity was interpreted earlier as supporting a dual seeding-inhibitory concept for the formation of NB in the presence of serum, in which calcification inhibitors are seen both as interceptors of calcification as well as seeds for the formation of NB-like calcium granules—a dual role that is modulated by the amounts of calcium and phosphate present in the medium [2], [3]. That is, according to this model, the same calcification inhibitors become calcification-seeding agents when overcome by excess calcium and phosphate. Still in line with this interpretation and in the context of our experiments, low amounts of serum tended to seed NB-like precipitations (due to calcium and phosphate exceeding the inhibitory activity of serum) while higher serum amounts (exceeding 0.3% for FBS and 3% for HS, as determined from the batches of serum tested here) became increasingly inhibitory. This same inhibition appeared to last for several weeks and was evident even after 2 months (Fig. 1A and B, bottom rows). With this prolonged incubation, however, there were signs of partial reversal of the inhibitory activity associated with the serum, which was generally more evident in the case of HS after 2 months (Fig. 1B, note the slight rightward shift for the peak turbidity as compared to the peak seen at 1 month; several other experiments, not shown, have given even more prominent rightward shifts of the peak turbidity as a result of prolonged incubation). That is, although higher amounts of HS still exerted inhibitory influences on the observed precipitation, this same inhibitory effect became much less apparent after an incubation of 2 months (Fig. 1B, compare the middle and bottom rows, wells 5 and 6). For FBS, on the other hand, the inhibitory patterns tended to always last longer, and in this particular experiment, inhibition could still be clearly seen after 2 months with FBS at 1% or higher concentrations (Fig. 1A, bottom row, wells 4–6). This consistent difference between FBS and HS supports the notion that the same calcification-inhibitory influences exerted by HS are probably more easily overcome with incubation than those associated with FBS. This same difference can be further explained by the significantly larger amounts of fetuin-A—one of the more potent calcification inhibitors—found in FBS as compared with HS (10–21 mg/ml in FBS vs. 0.7–0.8 mg/ml in HS; see more detailed discussion in ref. [2] along with the many reference citations therein).\nIt is further apparent from Figure 1 and from experiments reported elsewhere [2], [3] that the formation of NB-like precipitations is a slow process that takes at least several weeks to develop. The results seen here with both FBS and HS resulting in marginal and barely visible precipitations are in fact representative of hundreds of seeding experiments that we have conducted over a period of two years using a variety of serum batches and different culture conditions. This slow deposition of mineral complexes may explain the earlier lack of reproducible and quantifiable data associated with NB that may have in turn given rise to many erroneous interpretations in the past. Besides, we have noticed a progressive dehydration of the medium used during the course of the prolonged incubation needed for the demonstration of NB, a process that may take upwards to 2 months, and this gradual change in fluid conditions by itself can be shown to lead to nanoparticle precipitation if not properly controlled (data not shown). Nonetheless, at least in our hands, the gradual deposition of mineral complexes in DMEM in response to the presence of serum can indeed be verified as well as quantified.","divisions":[{"label":"Title","span":{"begin":0,"end":109}},{"label":"Figure caption","span":{"begin":1714,"end":2716}},{"label":"Title","span":{"begin":1758,"end":1824}}],"tracks":[{"project":"2_test","denotations":[{"id":"19956594-12669155-88162750","span":{"begin":301,"end":302},"obj":"12669155"},{"id":"19956594-19198665-88162751","span":{"begin":696,"end":697},"obj":"19198665"},{"id":"19956594-19412552-88162752","span":{"begin":701,"end":702},"obj":"19412552"},{"id":"19956594-19198665-88162753","span":{"begin":3247,"end":3248},"obj":"19198665"},{"id":"19956594-19198665-88162754","span":{"begin":3841,"end":3842},"obj":"19198665"},{"id":"19956594-19198665-88162755","span":{"begin":4997,"end":4998},"obj":"19198665"},{"id":"19956594-19412552-88162756","span":{"begin":5002,"end":5003},"obj":"19412552"},{"id":"19956594-19198665-88162757","span":{"begin":7060,"end":7061},"obj":"19198665"},{"id":"19956594-19198665-88162758","span":{"begin":7191,"end":7192},"obj":"19198665"},{"id":"19956594-19412552-88162759","span":{"begin":7196,"end":7197},"obj":"19412552"},{"id":"T41987","span":{"begin":301,"end":302},"obj":"12669155"},{"id":"T18429","span":{"begin":696,"end":697},"obj":"19198665"},{"id":"T93848","span":{"begin":701,"end":702},"obj":"19412552"},{"id":"T85726","span":{"begin":3247,"end":3248},"obj":"19198665"},{"id":"T32679","span":{"begin":3841,"end":3842},"obj":"19198665"},{"id":"T18900","span":{"begin":4997,"end":4998},"obj":"19198665"},{"id":"T12526","span":{"begin":5002,"end":5003},"obj":"19412552"},{"id":"T55536","span":{"begin":7060,"end":7061},"obj":"19198665"},{"id":"T36616","span":{"begin":7191,"end":7192},"obj":"19198665"},{"id":"T92852","span":{"begin":7196,"end":7197},"obj":"19412552"}],"attributes":[{"subj":"19956594-12669155-88162750","pred":"source","obj":"2_test"},{"subj":"19956594-19198665-88162751","pred":"source","obj":"2_test"},{"subj":"19956594-19412552-88162752","pred":"source","obj":"2_test"},{"subj":"19956594-19198665-88162753","pred":"source","obj":"2_test"},{"subj":"19956594-19198665-88162754","pred":"source","obj":"2_test"},{"subj":"19956594-19198665-88162755","pred":"source","obj":"2_test"},{"subj":"19956594-19412552-88162756","pred":"source","obj":"2_test"},{"subj":"19956594-19198665-88162757","pred":"source","obj":"2_test"},{"subj":"19956594-19198665-88162758","pred":"source","obj":"2_test"},{"subj":"19956594-19412552-88162759","pred":"source","obj":"2_test"},{"subj":"T41987","pred":"source","obj":"2_test"},{"subj":"T18429","pred":"source","obj":"2_test"},{"subj":"T93848","pred":"source","obj":"2_test"},{"subj":"T85726","pred":"source","obj":"2_test"},{"subj":"T32679","pred":"source","obj":"2_test"},{"subj":"T18900","pred":"source","obj":"2_test"},{"subj":"T12526","pred":"source","obj":"2_test"},{"subj":"T55536","pred":"source","obj":"2_test"},{"subj":"T36616","pred":"source","obj":"2_test"},{"subj":"T92852","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93e7","default":true}]}]}}