Figure 1.
Formation of mineral particles with biomimetic morphologies in biological fluids.
The mineral particles shown here, which we refer to as bions, were prepared by adding 12 (A), NaCl (B), MgCl2 (C), AlCl3 (D), CoCl2 (E), NiCl2 (F), CuCl2 (G), ZnCl2 (H) or SrCl2 (I) in DMEM containing 10% FBS, followed by addition of 1 mM Na2HPO4 to induce mineral precipitation. After incubation overnight in cell culture conditions, bions were collected by centrifugation, washed with HEPES buffer and prepared for SEM as described in Materials and Methods. Bions formed round, bacteria-like structures at various degrees of aggregation and conversion to crystalline films.
Figure 2.
Bions form various biomimetic morphologies and complex structures in biological fluids.
Bions were prepared by adding 1(A), AlCl3 (B), BaCl2 (C, E, and F), or MnCl2 (D) in DMEM containing 10% FBS (A, C, and D) or 10% HS (B, E, and F), followed by addition of 1 mM Na2HPO4 to induce mineral precipitation. In (A), 1 mM Na2CO3 was also added before addition of Na2HPO4. Bions were incubated in cell culture conditions overnight, collected by centrifugation, washed with HEPES buffer, and prepared for SEM. Bions formed various morphologies, including shapes similar to bacillus bacteria (A), mushrooms (B), and flowers (C), as well as round, cell-like formations apparently formed of smaller, round NPs (D; inset, NPs denoted by white arrows). Complex structures similar to dumbbells and muscle fibers (E and F) were also observed in some bion samples. (G) Addition of 1 mM CoCl2 and Na2HPO4 each in water containing 10% FBS produced mineral NPs that quickly converted into crystalline biofilm-like structures. (H) When MnCl2 and Na2HPO4 were added at 1 mM each in DMEM alone without a body fluid, a mineral precipitate showing a flaky appearance and sharp edges was observed. (I) Addition of 1 mM FeCl2 and Na2HPO4 each in water formed round Fe-bions of small sizes. The samples shown in (G–I) were incubated overnight before preparation for SEM.
Figure 3.
Bions form in various human body fluids.
Bions were prepared by adding 12, MgCl2, MnCl2, SrCl2 or BaCl2 in DMEM containing 10% HS (A-E), saliva (F-J), or urine (K-O), followed by addition of 1 mM Na2HPO4. After incubation overnight in cell culture conditions, bions were collected by centrifugation, washed with HEPES buffer, and prepared for SEM. Bions produced in various body fluids displayed bacteria-like morphologies at different stages of aggregation and conversion to biofilm-like structures.
Figure 4.
Thin-section TEM observations of bions derived from biological fluids.
Bions were prepared by adding 12 (A), MgCl2 (B), MnCl2 (C), SrCl2 (D) or BaCl2 (E) in DMEM containing 10% FBS, prior to addition of 1 mM Na2HPO4. The particles were incubated overnight in cell culture conditions, collected by centrifugation, and washed with HEPES buffer. Thin-sections were observed under TEM without staining. Bions consisted of round bacteria-like formations with a wall and a central cavity. Solid, electron-dense, mineral particles were also noted.
Figure 5.
Elemental and chemical compositions of bions formed in biological fluids.
(A) EDX spectra of bions prepared as in Fig. 4 showed peaks of carbon, calcium, oxygen, and phosphorus, consistent with the presence of carbonate apatite. Peaks corresponding to the charged cations added were also noted (i.e., Mg2+, Mn2+, Sr2+, or Ba2+). The peaks present in every sample were labeled with black geometrical figures on top for clarity. The oxygen peak, which superimposed with the Mn peak in the spectrum of Mn-bions, was also found in all samples, and was labeled with an inverted white triangle. The following calcium:phosphorus atomic ratios were observed: Ca-bions, 1.54; Mg-bions, 0.76; Mn-bions, 0.11; Sr-bions, 0.61; Ba-bions, 0.37. The ratios of added cations to phosphorus were as follows: Mg:P, 0.63; Mn:P, 1.05; Sr:P, 0.41; Ba:P, 0.51. (B) FTIR analysis of bions prepared as in Fig. 4 revealed peaks of phosphate (575 cm−1, 605 cm−1, 960 cm−1, and 1,000−1,150 cm−1) and carbonate (875 cm−1 and 1,400−1,430 cm−1). Low peaks corresponding to amide I/H2O, amide II, and amide III at 1,660 cm−1, 1,550 cm−1, and 1,250 cm−1, respectively, were also observed in some samples due to the presence of water and serum proteins. Representative spectra of CaCO3, Ca3(PO4)2 and HAP controls were included for comparison.
Figure 6.
Amorphous-to-crystalline mineral conversion of bions in biological fluids.
Bions prepared as in Fig. 4 were incubated for either 1 day (A) or 1 week (B) in cell culture conditions, prior to preparation for XRD analysis. In (A), low, broad peaks corresponding to Ca10(PO4)6(OH)2 and Ca3Mg3(PO4)4 were noted for Ca-bions and Mg-bions, respectively, whereas the other bion samples produced amorphous spectra. (B) One-week old bions produced sharp peaks corresponding to Ca10(PO4)6(OH)2 for Ca-bions, Ca3Mg3(PO4)4 for Mg-bions, Mn3(PO4)2 for Mn-bions, Ca4Sr6(PO4)6(OH)2 for Sr-bions, and Ca4Ba2(HPO4)2(H2PO4)2 for Ba-bions. These results suggest that bions initially form an amorphous mineral phase that gradually converts to crystalline mineral during incubation.
Figure 7.
Serum produces a dual inhibition-seeding effect on bion formation.
Bions were prepared in 24-well plates by adding 10 mM CaCl2, MgCl2, MnCl2, SrCl2 or BaCl2 in DMEM containing 0.1–10% FBS, followed by addition of 10 mM Na2HPO4 to induce mineral precipitation. Each bion preparation consisted of a total of 1 ml. The plates were incubated in cell culture conditions for one day (A) or two weeks (B), prior to photography and spectrophotometry measurements at a wavelength of 650 nm (A650). While FBS inhibited bion formation in a dose-dependent manner at day 1, the inhibition was gradually overcome with time as seen by the additional bion precipitation observed after 2 weeks (compare “Day 1” with “2 Weeks”). *P < 0.05, versus well 1 of the same row; **P < 0.05, versus the same well at Day 1.
Figure 8.
Protein profiles of bions derived from serum.
Bions were prepared by adding 102, MnCl2, SrCl2 or BaCl2 in DMEM containing 5% FBS, followed by addition of 10 mM Na2HPO4 to induce bion formation. A final volume of 1 ml was used. Mg-bions were prepared by adding 20 mM MgCl2 due to the lower amount of precipitation observed for this cation. The particles were incubated in cell culture conditions for two days. Following incubation, the particles were washed and treated with 0.1 mM EDTA. The protein samples were prepared for SDS-PAGE under denaturing and reducing conditions (see Materials and Methods).
Table 1.
Bion-binding proteins identified by LC-MS/MS and ranked by spectral counting.
Figure 9.
Albumin and fetuin-A inhibit bion formation.
Bions were prepared by adding 32 (A) or BaCl2 (B) in DMEM containing albumin and/or fetuin-A, followed by addition of 3 mM Na2HPO4 to induce bion formation. When used alone, albumin was added at the concentrations shown on top whereas fetuin-A was used at the concentrations shown at the bottom. When albumin and fetuin-A were added together, both proteins were used at the concentrations written at the bottom. Bion formation was evaluated by monitoring absorbance at a wavelength of 650 nm (A650). Compared to albumin, fetuin-A showed a more potent inhibitory effect on the formation of both types of bions. A slight additive inhibitory effect was noted when both albumin and fetuin-A were added together.
Figure 10.
Increase in size and number, and sub-culture of bions.
(A) Bions increase in size during incubation. Bions were prepared by adding 1 mM CaCl2, 10 mM MgCl2, 1 mM MnCl2, 5 mM SrCl2 or 3 mM BaCl2 in DMEM containing 5% FBS, followed by addition of Na2HPO4 at the same concentration to induce precipitation. Particle size was determined by DLS during a 2-week incubation period. (B) Bions increase in number in culture. Bions were prepared and incubated as described in (A) and particle number was determined by DLS. (C) Bions are sub-culturable in fresh medium. Bions were subcultured by diluting a solution containing abundant, pre-formed bions (A650 of 0.7) in fresh DMEM containing 10% FBS at a ratio of 1∶10 using a final volume of 15 ml. Bions sub-cultures were incubated in cell culture conditions for 2 weeks and the amount of bions was monitored by light absorbance. (D) EDX analysis of bions after sub-culture and incubation for 2 weeks. Bions were retrieved by centrifugation, followed by washing steps and preparation for EDX analysis. See the text for more details.