Figure 2.

Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) (insets) analyses of mineral phases formed in the presence of 1 mg/mL P173 and P148 with added MMP20 (200:1), respectively, examined at selected times: 4 h (A, G), 12 h (B, H), 24 h (C, I), and 48 h (D, E, F, J, K), as described in Experimental Procedures. In the presence of P173 with added MMP20, as shown in A–C, amorphous calcium phosphate (ACP) was initially formed and predominately observed up to 24 h based on the SAED patterns (insets), although hydroxyapatite (HA) crystals started to appear at 24 h (data not shown). ACP particle size was found to vary somewhat due to growth and/or aggregation (Kwak et al. 2009). After 48 h, however, well-aligned bundles of needle-like HA crystals were readily observed (D), along with remaining ACP particles (E). Extended networks of dense and well-aligned HA crystals could also be seen at 48 h (F), in the presence of P173 with added MMP20. SAED analyses (D and F, insets) are consistent with the formation of HA crystals coaligned along their c-axes, as indicated by the narrow arc patterns at 002 and 004 reflections (arrows). In the presence of P148 with added MMP20, as shown in G–J (insets), ACP nanoparticles were observed throughout the first 24 h and again at 48 h, along with predominant protein aggregates with dark contrast. At 48 h, some agglomerates of disordered plate-like HA crystals were also observed (K, inset). In the presence of 2 mg/mL P148 with MMP20 (200:1) at 48 h (L), however, only ACP particles were found. The capacity of P148 to stabilize ACP in the presence of MMP20 was also examined using a higher P148 concentration (Appendix Fig. 2B).