3.1. Synthesis of DCPD in the Presence of PASP
The powder X-ray diffraction patterns of the products obtained from the synthesis of DCPD in the presence of increasing concentrations of PASP are reported in Figure 1
. The patterns show a series of diffraction reflections consistent with the presence of DCPD as unique crystalline phase up to a poly-amino acid concentration in solution of 0.8 mM. However, the significant decrease of the amount of precipitate on increasing PASP concentration (Table 1
) indicates an inhibitory role of the poly-amino acid on the crystallization of DCPD.
Increasing the concentration up to 1 mM provokes the appearance of a broad halo superimposed to the reflections of DCPD, suggesting the presence of amorphous material.
Further increase of PASP up to 1.5 and 2 mM provides products that show XRD patterns devoid of diffraction reflections and consistent with the presence of amorphous material (data not shown). The patterns of PASP02, PASP05, and PASP08 display a slightly modified distribution of the relative intensity of the diffraction peaks when compared to pure DCPD. The most evident variation is the increase of the relative intensity of the 040 reflection (2θ = 23.4°), which can be justified by the orientation effect and morphology variation [18
]. PASP has indeed a great effect on the morphology of the precipitates. Scanning Electron Microscopy (SEM) images reported in Figure 2
show that pure DCPD is constituted of plate like crystals with large (0k0) faces and sharp edges, whereas the crystals synthesized in the presence of PASP up to PASP08 exhibit indented edges and a strong tendency to aggregate into spherulites. The crystallization of the material precipitated in the presence of 1 mM PASP is so disturbed that the presence of crystals is barely appreciable in its SEM image (Figure 2
The plate-like DCPD crystals tend to lie flat on the sample holder of the diffractometer, with consequent enhancement of the relative intensity of the 0k0 reflections. Crystal aggregation of the products synthesized in the presence of PASP reduces this phenomenon and, as a consequence, the relative intensity of the relevant X-ray diffraction (XRD) reflections.
Possible interaction between PASP and the (0k0) faces of DCPD crystals would be favored by their good structural fit: The distances between neighboring calcium ions on the (0k0) faces along the crystallographic a
direction (6.23 Å) are commensurate to those between carboxylic groups in the polyaspartic β-sheet (6.90 Å). On considering that PASP in solution has not a rigid structure and the modest difference of distances, it is possible to suggest interactions between the carboxylic groups perpendicular to the crystal flat plane ((0k0) plane) and the calcium ions exposed on the large crystal face. This interaction model (Figure 3
) is much more convincing than that previously advanced on the basis of the distances between neighboring calcium ions from two adjacent layers within one Ca-HPO4 bilayer (parallel to the (0k0) plane), which were erroneously reported as 6.95 Å [15
The results of the Differential Scanning Calorimetry (DSC) analysis put into evidence the influence of the polyelectrolyte on the stability of DCPD. In fact, although the endothermic peak corresponding to the transformation of the DCPD into DCPA is displayed at about 190 °C for all the samples (Figure 4
), the values of ΔH associated to this process decreases significantly as a function of PASP concentration in solution, as shown in Table 1
. The data indicate that the energy required to transform DCPD into its anhydrous form decreases on increasing the presence of PASP in the reaction medium, which suggests a destabilization of DCPD structure provoked by the poly-amino acid.
The FT-IR spectrum of DCPD shows the presence of a number of absorption bands due to H2
O stretching (3000–3500 cm−1
) and bending (1651 cm−1
), PO stretching (900–1300 cm−1
) and bending (500–700 cm−1
) modes, as well as to P-O(H) stretching at about 876 cm−1
]. Sample PASP08 displays a FT-IR spectrum which shows, other than the characteristic bands of DCPD, a shoulder at about 1580 cm−1
and an absorption band at about 1410 cm−1
indicating the presence of the polyelectrolyte, as it can be inferred by comparing the spectrum with that of PASP (Figure S1
). The presence of the polyelectrolyte in the samples is further supported by the values of zeta potential, which are more negative for the products synthesized in the presence of PASP than that measured for pure DCPD (Table 1
Moreover, the results of elemental analysis indicate a significant content of carbon, which increases with PASP concentration in solution and allows us to calculate that the samples contain up to about 2.3 wt% of the poly-amino acid, as reported in Table 1
In agreement, after heat treatment at 300 °C, the PASP containing powders display a pale-yellow color, which becomes darker on passing from PASP02 to PASP08 (Figure S2
), due to the partial combustion of organic material and residual C remains.
After heat treatment, all the samples exhibit XRD patterns consistent with the presence of DCPA as unique crystalline phase (Figure 5
). In addition, after heat treatment the patterns of the PASP functionalized samples present a slightly different distribution of the relative intensity of the reflections in comparison with that of the sample obtained by heat treatment of pure DCPD.
The differences can be ascribed to the different morphology of the samples: 300DCPD maintains the characteristic plate-like morphology, which can result in crystal orientation, whereas 300PASP samples are still aggregated into spherulites (Figure 6
). Moreover, all the SEM images show the presence of a quantity of small particles on the surface of the crystals, most likely due to a partial crumbling of the crystal during dehydration caused by heat treatment.
3.2. Synthesis of DCPD in the Presence of ASP
In contrast with what was found for the influence of PASP on the synthesis of DCPD, all the results obtained on the products of synthesis in the presence of ASP indicate that the amino-acid does not cause any structural or morphological modification and its presence is not detectable in the precipitates. In fact, all these products exhibit XRD patterns consistent with the presence of DCPD as a unique crystalline phase (Figure 7
). The distribution of the relative intensity of the diffraction peaks appears similar to that of pure DCPD. In agreement, SEM images show the characteristic morphology of the DCPD crystals (Figure 2
Moreover, the values of ΔH associated to the phase transformation from DCPD to DCPA do not vary with ASP concentration and are very close to those of pure DCPD (Table 1
, Figure S3
The data of zeta potential further support the absence of the amino acid in the products of synthesis (Table 1
), which is confirmed also by the white color of the samples heat treated at 300 °C. The XRD patterns of heat-treated samples indicate that they are constituted by DCPA (Figure S4
In agreement with the results obtained on prepared samples, the morphology of the crystals and the relative intensity distributions of the XRD peaks recorded from 300ASP samples do not differ significantly from that of 300DCPD (Figure 6
and Figure S4
It was previously reported that aspartic acid can be incorporated into HA crystals if present in the synthesis solution [20
]. The different effect of ASP on the synthesis of HA and DCPD might be ascribed to a better structural interaction of the amino acid with HA as suggested by the variation of the mean dimensions of the perfect crystalline domains of HA, which indicates a preferential interaction of ASP with specific crystallographic faces of HA [20
The analysis of the samples stored in physiological solution for different periods of time up to 3 days puts into evidence the different roles played by the additives and by temperature. On comparing the results obtained when the hydrolysis is carried out at 37 °C with those at 60 °C, it is evident that increasing the temperature accelerates the transformation of DCPD into thermodynamically more stable phases, in agreement with previous data [21
At 37 °C pure DCPD is partially transformed into OCP after just 3 h (the conversion into OCP is complete after 2 days) and at 3 days HA appears as a secondary phase; at 60 °C HA is present since the first stages, and it is the only crystalline phase after 2 days (Table 2
and Table 3
The data obtained for PASP08 confirm the presence of the poly-amino acid in the sample. In fact, the phase conversion is delayed when compared to that observed for pure DCPD: After 3 days at 37 °C just a part of DCPD is converted into OCP, whereas at 60 °C the phases obtained after 3 days are OCP and HA. On the contrary, the hydrolysis of ASP10 proceeds with the same trend as that of pure DCPD, in agreement with the absence of aspartic acid in the sample.
Furthermore, the presence in the hydrolysis solution of PASP or ASP results in a strong inhibition of the conversion of DCPD, as shown by the data reported in Table 2
and Table 3
and by comparing the XRD patterns recorded from the different samples after three days at 60 °C (Figure 8
). PASP is even more effective than ASP in delaying the hydrolysis reaction, thus that the pattern still shows the presence of DCPD even after 3 days at 60 °C.
The presence of the different phases can be appreciated also by comparing the FT-IR spectra of the samples after 3 days of hydrolysis at 60 °C (Figure S5
). In particular, the absence of the bands at 3572 and 630 cm−1
, due to OH stretching and bending modes respectively, in the spectra of DCPD and ASP10 confirms the poor crystallinity of the apatitic phases obtained from their hydrolysis. The spectrum of inASP after hydrolysis shows the characteristic bands of OCP [23
], whereas that of inPASP exhibits a greater number of bands due to the co-presence of OCP and DCPD (Figure S5
). Moreover, the band at about 1410 cm−1
in the spectra of PASP08 and inPASP after hydrolysis indicates the presence of the polyelectrolyte in these samples.
The differences among the products of hydrolysis are well evident in comparing the SEM images reported in Figure 9
: The big crystals of DCPD are still evident in the image of inPASP08, whereas the samples constituted of OCP and/or HA show the presence of platelet-like and/or needle crystals.
The stronger inhibition exerted by the poly-amino acid is not surprising and can be ascribed to the cooperative action of the carboxylate groups and to its good structural fit with DCPD, which promotes PASP adsorption on the surface of the (0k0) faces [15
]. A similar mechanism was previously reported for the inhibition effect of PASP on OCP hydrolysis, where adsorption of the polyelectrolyte on the hydrated layer of the OCP (100) faces prevents OCP transformation into HA [24