Figure 2. Energy-dispersive X-ray spectroscopy of NLP and NB.

EDX results were selected to illustrate the wide spectrum of NLP compositions, with respect to the amounts of calcium and phosphorus incorporated, and their similarities to NB. (A) Elemental composition of NLP prepared by adding CaCl₂ and (NH₄)₂CO₃ at 50 mM into DMEM corresponding to calcium carbonate. (B and C) Amorphous CaCO₃ NLP produced as in (A) except that the precipitating reagents were added to DMEM and incubated at room temperature for 2 days (B) or one month (C), showing increased incorporation of P with prolonged incubation. Ca/P ratios are 5.09 for (B) and 1.60 for (C), with the latter close to the theoretical value of 1.67 associated with stoichiometric HAP. (D–H) Formation of NLP with various inputs of ions, including P, yielding different Ca/P ratios. (D) NLP formed from 0.25 M each of CaCl₂, Na₂CO₃, and NaH₂PO₄, mixed in with DMEM at vol. ratios of 2∶5∶5∶13 that had been incubated in DMEM for 2 days, revealing the presence of magnesium ions and a Ca/P ratio of 0.78. (E–H) NLP were prepared like in (D) at vol. ratios of (E) 1∶1∶1∶22, yielding a Ca/P ratio of 1.29; (F) 3∶2∶2∶43, Ca/P ratio of 1.49; (G) 9∶5∶5∶106, Ca/P ratio of 1.76; (H) 2∶1∶1∶21, Ca/P ratio of 2.03. NLP were submitted to EDX immediately after formation. (I–L) Elemental composition of various NB preparations. (I and J) NB obtained from one-month-old DMEM cultures containing (I) 10% HS or (J) 10% FBS. Ca/P ratios: 1.75 (I) and 1.53 (J). (K) NB strain DSM 5819, after one week in DMEM without serum, giving a Ca/P ratio of 1.45. (L) “Nanons” after one week in DMEM; Ca/P ratio: 1.66.