Comparison of the Effect of the Amino Acids on Spontaneous Formation and Transformation of Calcium Phosphates

Abstract

Understanding the effect that specific amino acids (AA) exert on calcium phosphate (CaPs) formation is proposed as a way of providing deeper insight into CaPs’ biomineralization and enabling the design of tailored-made additives for the synthesis of functional materials. Despite a number of investigations, the role of specific AA is still unclear, mostly because markedly different experimental conditions have been employed in different studies. The aim of this paper was to compare the influence of different classes of amino acids, charged (aspartic acid, Asp and lysine, Lys), polar (asparagine, Asn and serine, Ser) and non-polar (phenylalanine, Phe) on CaPs formation and transformation in conditions similar to physiological conditions. The precipitation process was followed potentiometrically, while Fourier transform infrared spectroscopy, powder X-ray diffraction, electron paramagnetic spectroscopy (EPR), scanning and transmission electron microscopy were used for the characterization of precipitates. Except for Phe, all investigated AAs inhibited amorphous calcium phosphate (ACP) transformation, with Ser being the most efficient inhibitor. In all systems, ACP transformed in calcium-deficient hydroxyapatite (CaDHA). However, the size of crystalline domains was affected, as well as CaDHA morphology. In EPR spectra, the contribution of different radical species with different proportions in diverse surroundings, depending on the type of AA present, was observed. The obtained results are of interest for the preparation of functionalized CaPs’, as well as for the understanding of their formation in vivo.

1. Introduction

The biomimetic approach in the development and synthesis of novel materials, especially hard tissue regeneration biomaterials, is constantly gaining in importance due to the superior properties of natural biomaterials, which have not been achieved by any engineered material to date [1,2,3,4]. In addition, it enables a more environmentally friendly approach to advanced material production [5].

The key feature of the biomineralization, the process of hard tissue formation in organisms, is the strict control that the organic matrix exerts over precipitation of the inorganic component of the hard tissue in question [6,7,8]. In vitro investigations of the role that specific components of the organic matrix and/or their building blocks play in the formation of inorganic biominerals have been utilized as a successful tool in the elucidation of biomineralization and as a promising way of designing tailored additives for different applications in biomedicine and biotechnology [9,10,11,12,13].

Among the 60 different types of biominerals, the most interesting to humans are calcium phosphates (CaPs), the main inorganic component of vertebrate skeletons, which are also prominent in pathological biomineralization [8,14]. Although CaPs are less common than calcium carbonates (CaCO₃) or silicon dioxide (SiO₂), their role in vertebrates makes them particularly important in biomedicine and materials science [1]. Fourteen chemically different calcium phosphate compounds are known [14]. The main inorganic component of vertebrates’ hard tissue is poorly crystallized, nonstoichiometric hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂) or calcium-deficient hydroxyapatite (CaDHA, Ca_10−x(HPO₄)_x(PO₄)_6−x(OH)_2−x, 0 < x < 1), substituted with sodium, magnesium or carbonate ions, which is often called “biological apatite”. The similarity with the bone mineral makes other CaPs interesting for potential application as bone regeneration materials [14], e.g., octacalcium phosphate (OCP, Ca₈(HPO₄)₂(PO₄)₄·5H₂O), calcium hydrogen phosphate dihydrate (DCPD, CaHPO₄·2H₂O) and β – tricalcium phosphate (β-TCP, Ca₃(PO₄)₂) [15].

The inclusion of additives, organic molecules, macromolecules and surfactants in the precipitation system affects the rate and mechanism of CaP formation, as well as the properties of the formed solid phase(s) [15,16]. In vivo, proteins play a key role in controlling CaP formation. Although more than 200 proteins can be found in the bone, about 90% of bone protein content consists of collagen type I [4]. Investigating the influence of amino acids (AAs), the building blocks of proteins, is considered an important approach to understanding protein’s role in CaP formation in vivo. However, despite numerous investigations, the role that individual AAs exert on CaPs formation is still not completely clarified [17]. One of the key reasons for this is the diverse range of experimental conditions, which prevent a straightforward comparison of the results obtained in different studies [16,18]. In addition, AAs can modulate the growing crystalline through different types of interaction [18,19].

Constant composition experiments have shown that different classes of AAs, with polar, hydrophobic, acidic or basic side groups, inhibit the crystal growth of HA by adsorption at crystal growth sites [20,21,22,23,24,25]. However, in spontaneous precipitation, inhibition [26] and promotion [27,28,29] of, as well as no effect [30] on, the rate of CaP precipitation and/or transformation were observed. Similarly, contradictory results regarding AAs’ influence on crystal morphology were obtained [17,31,32].

In order to contribute to the elucidation of effects that specific AAs exert on the formation of CaPs at conditions similar to physiological conditions, the influence of different classes of AAs, i.e., charged (Asp, Lys) polar (Ser, Asn) and non-polar (Phe) (Figure S1), on the spontaneous precipitation of CaPs and properties of the formed solid phases was investigated in this work.

2. Materials and Methods

Analytical grade chemicals, calcium chloride dihydrate (CaCl₂·2H₂O), sodium hydrogenphosphate (Na₂HPO₄), sodium chloride (NaCl), L−lysine and L−asparagine were obtained from Sigma Aldrich (Darmstadt, Germany), hydrochloric acid (HCl) was obtained from Kemika (Zagreb, Croatia), while L−serine, L−phenylalanine and L−aspartic acid were purchased from Alfa Aesar (Tewksbury, MA, USA). Milli-Q water (Millipore, 0.05 µS cm⁻¹) was used in all experiments.

CaCl₂·2H₂O, Na₂HPO₄, NaCl and amino acid stock solutions were prepared from the corresponding chemicals, which were dried overnight in a vacuum desiccator. The pH of sodium hydrogenphosphate stock solution was adjusted to 7.4 using HCl.

2.1. Preparation of Precipitation Systems

Cationic and anionic reactant solutions were prepared by diluting the respective CaCl₂·2H₂O and Na₂HPO₄ stock solutions to a concentration of c(CaCl₂·2H₂O) = c(Na₂HPO₄) = 0.1 mol dm⁻³. Both reactant solutions contained 0.15 mol dm⁻³ NaCl to maintain the ionic strength of the precipitation system constant. The pH of the anionic solution was adjusted to 7.4 using 0.1 mol dm⁻³ HCl. Amino acids were added to the anionic reactant solution, and the pH was readjusted if needed.

The precipitation systems were prepared by the fast mixing of equal volumes (20 mL) of equimolar cationic and anionic reactant solutions. The initial reactant concentrations in precipitation systems were c(CaCl₂·2H₂O) = c(Na₂HPO₄) = 5 mmol dm⁻³, c(NaCl) = 0.15 mol dm⁻³, c(AA) = 1, 2.5 and 5 mmol dm⁻³ at pH = 7.4. The control precipitation system (CS) contained no amino acids. The scheme of the experimental set-up is shown in Figure S2, and the initial experimental conditions for precipitation experiments are given in Table S1.

All experiments were performed at (25 ± 0.1) °C in a thermostated double-walled vessel, with a total volume of 50 mL. The systems were magnetically stirred and the precipitation process was followed by constantly monitoring pH changes (Metrohm 701 pH/ion meter). Based on pH vs. time curves, induction times for ACP transformation were determined. The different rates of pH change in the stages in which ACP and crystalline CaP form enable the determination of the induction time for secondary precipitation (t_i), as the time at the intersection of the tangents drawn on the first two sections of the pH vs. time curve [33] (Figure S3). Samples for further analysis were taken after 10 and 60 min, except for the precipitation system containing Ser, for which samples were taken after 60 and 150 min. The chosen periods enabled the characterization of precipitates formed in different precipitation stages. Precipitates were filtered through a 0.45 μm Millipore filter paper, washed three times with Milli-Q water and once with ethanol, and dried in a stream of nitrogen. The samples were kept at 4 °C until further analysis.

2.2. Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra of formed precipitates were recorded on an FTIR spectrometer equipped with an attenuated total reflection module (Tensor II, Brucker, Ettlingen, Germany) in the range from 4000 to 400 cm⁻¹ with a resolution of 4 cm⁻¹. The recorded spectra are the average of 16 scans.

2.3. Powder X-ray Diffraction (PXRD)

Powder XRD patterns were obtained using a Rigaku Ultima IV diffractometer (Tokyo, Japan), operating at a voltage and current of 40 kV and 40 mA, respectively, in Bragg-Bretano geometry using CuK_α radiation and 5° Soller slits. XRD patterns were scanned in 0.02° steps (2θ) in the 2θ range from 3.25° to 60.00°, with a scan speed of 1° (2θ) min⁻¹. The baselines of powder XRD patterns were corrected and patterns smoothed using a 15-point Savitzky–Golay algorithm. The size of CaDHA crystalline domains along the [002] direction was calculated using the Scherrer Equation (1) [26,34]

$$\mathrm{crystallite}\mathrm{size}=\frac{k\lambda }{\mathrm{FWHM}\cos (\theta )}$$

(1)

where k is the shape factor (k = 0.9), λ is the wavelength of Cu K_α radiation (λ = 0.154056 nm), FWHM is full width at half maximum of the peak at 2θ 25.9° and θ is diffraction angle.

2.4. Scanning Electron Microscopy (SEM)

For SEM analysis, a small amount of dried sample was placed on double-sided carbon tape. The morphology was observed by FE-SEM (JEOL JSM-7000F microscope, Tokyo, Japan) and by a tungsten filament electron microscope (SEM; TESCAN VEGA 3 microscope, Fuveau, France).

2.5. Transmission Electron Microscopy (TEM)

For TEM analysis, a drop of the suspension was placed on a copper grid covered with a Formvar membrane. The excess solution was removed by filter paper and the precipitate was washed three times with a drop of Milli-Q Water. After removing the excess water, the samples were dried in a stream of nitrogen and kept at 4 °C until further analysis. Transmission electron microscopy images were obtained using a Zeiss TEM 902A (Oberkochen, Germany), operated at 80 kV. Particle sizes from the TEM micrograph were determined by means of the image analysis program ImageJ 1.48v (freely available at https://imagej.nih.gov/ij/index.html). At least 20 particles were measured for each sample.

2.6. Electron Paramagnetic Resonance Spectroscopy (EPR)

The EPR spectra were collected by a house-modified Varian E-109 spectrometer (Santa Clara, CA, USA) using a Bruker ER 041 XG microwave bridge working at X-band (i.e., a microwave frequency of 9.3 GHz) at room temperature (25 °C). The temperature in the EPR cavity was controlled by a Bruker ER 4111 temperature controller (Billerica, MA, USA) using a nitrogen gas flow with an accuracy of 0.2 °C. A Mn²⁺/MgO standard reference was used to calibrate the magnetic field of the EPR spectrometer. Radicals induced by gamma irradiation in the presence of air in all investigated samples, using a ⁶⁰Co gamma ray source of the Ruđer Bošković Institute [35], to a cumulative dose of 25 kGy, have been used to facilitate detection by EPR spectroscopy. No EPR signal could be detected for the empty sample tube or non-irradiated samples. The EPR spectra were simulated with a custom-built program in MATLAB (T he MathWorks Inc., Natick, MA, USA) using the EasySpin program package [36] to obtain the spectral parameters.

3. Results

3.1. Influence of Amino Acids on the Rate of CaP Transformation

In vitro, at conditions close to physiological (neutral or slightly basic solutions), CaPs usually precipitate in two steps [15,37,38,39]. The first step is the formation of amorphous calcium phosphate (Ca_xH_y(PO₄)_z·n H₂O (3 < n < 4.5), ACP). Depending on experimental conditions, ACP can be further transformed into OCP, CaDHA or HA [33,38,40]. Recent studies provided evidence that amorphous and metastable CaP phases have similar precursor roles in hard tissue formation [41,42].

During CaP precipitation, pH changes reflect different stages of the precipitation and transformation processes. Therefore the reaction progress can be followed by monitoring the pH changes in the system of interest [33,43,44,45,46]. Typically, sigmoidal pH vs. time curves are obtained, in which [33,43,44,45,46,47]:

−

In the initial precipitation stage (stage I), slight or negligible pH changes are observed, which correspond to the formation of ACP;

−

A subsequent rapid decrease in pH (stage II) is associated with secondary precipitation of crystalline phase upon the formation of ACP;

−

A final slight pH change (stage III) corresponds to the solution-mediated crystal growth and phase transformation of the crystalline phase formed in stage II.

In the pH vs. time curve of the control system (Figure 1) the three stages of the precipitation process can be clearly distinguished. However, in the presence of AAs, a difference in the shape of the stage I can be observed. Indeed, the pH changes in these systems are no longer continuous, as two regions of somewhat different rates of pH decrease can be observed. The difference from the behavior of the control system becomes more pronounced with an increase in the AAs concentration. Such behavior is usually ascribed to the difference in the pathway of ACP to crystalline phase transformation, caused by the change in the experimental conditions, e.g., the presence of additives [43,48,49].

The stability of ACP is reflected in the length of stage I, i.e., in the time elapsed from the initiation of the precipitation to the beginning of secondary precipitation, the so-called induction time. A longer induction time points to the greater stability of ACP, as its transformation to the crystalline phase is delayed [33]. The induction times obtained in the control system and in the presence of different AAs concentrations are given in

Table 1. Average induction times (t_i) and corresponding standard deviations obtained from pH vs. time curves from 3 measurements in the control system (CS) and systems containing amino acids. c(CaCl₂·2H₂O) = c(Na₂HPO₄) = 5 mmol dm⁻³, c(NaCl) = 0.15 mol dm⁻³, pH_initial = 7.4, ϑ = (25.0 ± 0.1) °C, magnetic stirring. Asp—aspartic acid, Lys—lysine, Asn—asparagine, Ser—serine, Phe—phenylalanine.

	(ti ± SD)/min
c(AA)/mmol dm−3	Asp	Lys	Asn	Ser	Phe
0 (CS)	18.2 ± 0.5
1.0	21.4 ± 1.5	17.4 ± 1.1	21.5 ± 0.7	20.3 ± 1.0	18.8 ± 1.1
2.5	16.4 ± 0.5	19.7 ± 0.3	22.2 ± 0.2	18.8 ± 1.6	20.8 ± 0.5
5.0	23.1 ± 0.2	19.3 ± 0.1	16.7 ± 0.9	113.7 ± 6.3	15.9 ± 0.1

. Although no straightforward correlation between induction times and AAs concentration is observed, it can be concluded that the dominant effect of the investigated AAs on ACP transformation at the highest concentrations applied is inhibition. Thus, the most pronounced inhibition was observed in the presence of 5 mmol dm⁻³ Ser. However, the promotion of ACP transformation was also observed, i.e., in the presence of 1.0 mmol dm⁻³ Lys, 2.5 mmol dm⁻³ Asp, and 5 mmol dm⁻³ Phe and Asn. The most effective promotor was nonpolar Phe.

Despite the generally recognized importance of revealing the specific amino acids’ influence on understanding proteins’ role in precipitation processes, there is still no clear conclusion regarding their inhibiting or promoting mechanism. AAs can either decrease the solution supersaturation through the complexation of calcium or phosphate ions or they can interact with the solid phase by non-specific adsorption at surfaces or at growing sites [20,21,22,23,24,25], which both intuitively lead to inhibition [18]. It should be noted that the AAs affinity towards the solid phases, which appear in biomineralization, is influenced by the overall charge and the charge of specific AAs segments, their ability to form chemical bonds, and their stereochemical or geometrical properties [18,20,21,31,50,51,52]. In addition to the complexity in the mechanism of action, the reason for contradictory results also lies in the fact that the employed experimental conditions (reactant concentration, temperature, mode of mixing) and experimental setup (seeded growth, constant composition, spontaneous precipitation) vary considerably between different studies. In a pH free-drift experimental setup, similar to the one employed in this study, Yang et al. [27] have shown that Asp, glycine (Gly) and Lys at a 1.0 mmol dm⁻³ concentration additionally reduce the induction and transformation times, even in the presence of known inhibitors, such as Mg²⁺ ions. The most efficient promotor was Lys. Contrary to this, Tavafoghi Jahormi et al. [26] have determined that positively charged arginine (Arg) more effectively inhibits HA nucleation than negatively charged glutamic acid (Glu), using a 10 times higher AAs concentration. The observed effect, determined by the change in turbidity, was attributed to the ability of Arg to interact more strongly with both calcium and phosphate ions than Glu. On the other hand, Ser present at low concentrations (up to 0.07 mmol dm⁻³) did not influence induction time [30]. In our recent study, we investigated the influence of different classes of amino acids on CaPs’ seeded growth [52]. In the case of OCP-seeded crystals, Ser was the most efficient inhibitor, while Phe was the most efficient promotor of CaP formation, similar to the behavior observed in this study. The effect of Phe could be ascribed to its molecular structure, i.e., the presence of an aromatic ring in the side group, which can act as an electron donor [20,21].

3.2. Influence of Amino Acids on the Properties of Formed CaPs

In order to gain additional insight into the observed behaviour of AAs, the precipitates formed in the presence of the highest AA concentration investigated in this study were analysed after 10 and 60 min (60 and 150 min in the case of Ser) reaction time.

PXRD patterns of the precipitates formed in the control system and systems containing 5 mmol dm⁻³ AAs (Figure 2a) contained only broad amorphous diffraction maxima in the 8–18° and 20–36° 2θ region, thus indicating that, in all systems, in the initial stage of the precipitation process, ACP was formed [53,54].

FTIR spectra (Figure 2b) confirmed the PXRD results. In the spectrum of the precipitate formed in the control system phosphate, the water bands characteristic of calcium phosphates [55,56] were observed: bands at 1283 cm⁻¹ and 859 cm⁻¹ characteristic of the ${\mathrm{HPO}}_4^{2-}$ group, ν_3c triply degenerate asymmetric stretching mode of ${\mathrm{PO}}_4^{3-}$ at 1059 cm⁻¹, ν_4b triply degenerate bending mode of ${\mathrm{PO}}_4^{3-}$ at 572 cm⁻¹, and a broad band between 3704 and 2797 cm⁻¹ and a band at 1644 cm⁻¹, characteristic of water. The formation of the ACP was confirmed by the absence of phosphate bands’ splitting, corresponding to asymmetric stretching and bending mode [53,57]. In the spectra of the precipitates formed in the presence of AAs, changes in the wavenumbers of ν_3c triply degenerate asymmetric stretching mode of ${\mathrm{PO}}_4^{3-}$ and the lower wavenumber of the ${\mathrm{HPO}}_4^{2-}$ group were observed. In addition, in the case of Lys and Phe, low-intensity broad bands characteristic of COO⁻ asymmetric stretching [32] were noted at around 1555 cm⁻¹ and 1444 cm⁻¹ (the latter observed only in the presence of Lysine). Additionally, in the FTIR spectra of the precipitate formed in the presence of Ser, the intensity of the water band at 1643 cm⁻¹ decreased, while the band at around 1283 cm⁻¹ was not observed.

In addition to changes in FTIR spectra, changes in the morphology of ACP particles were observed by TEM (Figure 3). In the control system, after 10 min reaction time (Figure 3a), chain-like aggregates of spherical particles typical of ACP were observed [33,40,54]. The average diameter of these particles was 143.5 ± 24.8 nm. A similar ACP morphology was observed in the presence of Ser (Figure 3e) as, after 60 min, the precipitation process was still at the beginning of the initial precipitation stage (Figure 1d). The average size of particles was 64.5 ± 9.9 nm. In the presence of other investigated AAs, more profound morphological changes were observed. In the presence of Asp, a denser precipitate with a still-visible, chain-like structure was observed (Figure 3b), while, in the presence of Lys and Asn, a denser precipitate in which needle-like crystals had begun to form was observed (Figure 3c,d). In the presence of Phe, a granular gel-like phase was formed (Figure 3f). The observed difference in the morphology of ACP formed in the presence of AAs confirms the results of potentiometric measurements, which indicated a change in transformation mechanism compared to the control system, i.e., the possible formation of another amorphous phase, ACP2 [48].

After 60 min (150 min in the case of Ser) reaction time, in all investigated precipitation systems, ACP transformed to CaDHA, as indicated by PXRD diffractograms and FTIR spectra (Figure 2c,d). In the diffractogram of the control system (Figure 2c), prominent reflections at 2θ 25.97° and 32.10° were observed, as well as low-intensity reflections at 2θ 28.52°, 39.53°, 46.62°, 49.51° and 53.25°, characteristic of CaDHA [32,53,58]. In the FTIR spectra, in comparison to the spectra of ACP, formed after 10 min, two new bands were observed: the ν_3a triply degenerate asymmetric stretching mode of ${\mathrm{PO}}_4^{3-}$at 1119 cm⁻¹ and ν_4a triply degenerate bending mode of ${\mathrm{PO}}_4^{3-}$at 606 cm⁻¹. In addition, splitting of the phosphate band at 1034 cm⁻¹, corresponding to the stretching mode of ${\mathrm{PO}}_4^{3-}$, and at 606 cm⁻¹, corresponding to the bending mode of ${\mathrm{PO}}_4^{3-}$, can be observed, confirming the formation of CaDHA.

In the presence of AAs, precipitates of lower crystallinity than seen in the CS were obtained. The most crystalline were the precipitates formed in the presence of Asn and Phe. Their diffractograms contained all reflections, as in the CS, except for the one at 2θ 39.53°. The diffractogram of precipitate formed in the presence of Ser contained only the most prominent reflections at 2θ 25.83° and 31.99°, indicating that poorly crystalline CaDHA had formed in this system. In order to additionally investigate the influence of AAs on the structure of CaDHA, the size of crystalline domains along the [002] direction was determined (

Table 2. Size of crystalline domains along the [002] direction of CaDHA formed after 60 min reaction time (150 min in the case of Ser) in the control system (CS) and in the presence of 5 mmol dm⁻³ aspartic acid (Asp), asparagine (Asn), serine (Ser), lysine (Lys), and phenylalanine (Phe). c(CaCl₂·2H₂O) = c(Na₂HPO₄) = 5 mmol dm⁻³, c(NaCl) = 0.15 mol dm⁻³, c(AAs) 5 mmol dm⁻³_, pH_initial = 7.4, ϑ = (25.0 ± 0.1) °C, magnetic stirring.

	D002/nm
CS	17.40
Asp	14.10
Lys	17.50
Asn	17.50
Ser	16.10
Phe	19.60

). Asp was the most effective in reducing the size of the crystalline domain. Such a strong Asp effect was observed in previous studies [17,32,59]. The crystalline domain size was also reduced in the presence of Ser, which is consistent with some other investigations [17,59]. On the contrary, in the presence of Phe, the size of the crystalline domain increased, while in the presence of Lys and Asn, it was not significantly different from that of the control system. The behavior of Asn is in contrast to that observed in the study of Gonzalez-McQuire et al. [17], where much higher reactant concentrations were used. Previous studies showed that AAs are able to inhibit HAP particle and crystal growth, with charged AAs being more effective than non-charged AAs [18].

In the FTIR spectra of precipitates formed in the presence of AAs after 60 min reaction time (150 min in the case of Ser, Figure 2d), the changes in wavenumbers of ν_3c triply degenerate asymmetric stretching mode of ${\mathrm{PO}}_4^{3-}$ and lower wavenumber band of the ${\mathrm{HPO}}_4^{2-}$ group observed after 10 min reaction time are still present. A band characteristic of the ν₁ nondegenerate symmetric stretching mode of ${\mathrm{PO}}_4^{3-}$at around 960 cm⁻¹ was detected. In contrast, this band was not present in the spectrum of the CS and the spectra of the precipitates formed in the presence of AAs after 10 min reaction time. No bands characteristic of AAs were observed, probably due to the greater amount of formed CaP.

To gain more insight into the influence of AAs on local ordering in the CaDHA, crystal lattice EPR spectroscopy was performed. No signal was detected in EPR spectra, before precipitate irradiation, confirming the purity of the applied preparation pathway, with no traces of metal or other impurities. Thus, radiation-induced radicals were used to monitor structural changes within different samples. In Figure 4. the experimental and simulated EPR spectra of irradiated CS and precipitates formed in the presence of AAs are shown. For irradiated samples, the main stable component can be assigned to ${\mathrm{CO}}_2^{-}$. In previous studies [60,61,62,63,64,65,66,67], it was shown that the EPR spectra of biological, as well as synthetic carbonated calcium apatites, exhibit a complex EPR signal at around g = 2, which comprises several powder spectrum contributions arising from different paramagnetic species (${\mathrm{CO}}_3^{-}$,${\mathrm{CO}}_3^{3-}$, ${\mathrm{CO}}_2^{-}$, ${\mathrm{O}}_3^{-}$, ${\mathrm{O}}^{-}$) stabilized in different locations (at hydroxyl, phosphate or surface sites) or from the same species, recognized by the difference in g anisotropy. However, as the EPR spectra of apatite are strongly dependent on the conditions of sample preparation (CO₂ flow, drying temperature, annealing procedure, etc.) [60,63,68], a further analysis was performed.

In our study, the total intensities in terms of the area of EPR absorption normalized to mass (i.e., proportional to radiation-induced radical concentration) were not different regarding the range of measurement error in the presence of AAs. However, by using phenomenological parameters, such as R-value [61,62], line-shape deviations due to the ${\mathrm{CO}}_3^{3-}$ centre, caused by the presence of different AAs during precipitation, can be observed, confirming that AAs indeed influence the local ordering. The definition of the R-value is given in Figure S4. The differences in obtained R-value are shown in

Table 3. R-values of the powdered irradiated precipitates formed after 60 min reaction time (150 min in case of Ser) in control system (CS) and in the presence of 5 mmol dm⁻³ aspartic acid (Asp), lysine (Lys), asparagine (Asn), serine (Ser) and phenylalanine (Phe). c(CaCl₂·2H₂O) = c(Na₂HPO₄) = 5 mmol dm⁻³, c(NaCl) = 0.15 mol dm⁻³, c(AAs) 5 mmol dm⁻³_, pH_initial = 7.4, ϑ = (25.0 ± 0.1) °C, magnetic stirring.

	R-Value
CS	0.5505
Asp	0.8535
Lys	0.5821
Asn	0.5714
Ser	0.6487
Phe	0.5477

. It should be noted that the R-value of the sample obtained in the presence of Asp is 55.03% higher than the R-value obtained for the CS. A similar increase was obtained for the system containing Ser, which was 17.85% higher than CS, in line with the decrease in the size of crystalline domains along [002]. On the contrary, in the presence of Phe, the obtained R-value is slightly smaller. The observed effect can be ascribed to the two ${\mathrm{CO}}_3^{3-}$^-radicals present in different proportions in diverse surroundings, depending on the type of AA present. A simulation of the experimentally recorded EPR spectra confirms these results, as the weight fraction of each spectral component depends on the environmental surroundings, i.e., the presence of different AA types, as shown in

Table 4. Radical species identified in the EPR spectra of samples with extracted EPR parameters and weight based on spectra simulation. Asp—aspartic acid, Lys—lysine, Asn—asparagine, Ser—serine, Phe—phenylalanine.

Sample	CS	Asp	Lys	Asn	Ser	Phe
Species
orth. $C{O}_2^{-}$ gx=2.0022 gy=1.9973 gz=2.0034	53.46%	44.63%	35.11%	13.82%	70.64%	75.51%
orth. $C{O}_2^{-}$ gx=2.0034 gy=1.9973 gz=2.0017	--	28.50%	9.79%	-	21.40%	--
iso. $C{O}_2^{-}$ go=2.0008	--	--	--	2.48%	--	11.35%
axial $C{O}_3^{3-}$  gx=2.0039 gy=2.0039 gz=2.0014	--	--	55.10%	45.52%	--	--
axial $C{O}_3^{3-}$ gx=2.0044 gy=2.0044 gz=2.0020	46.54%	26.87%	--	38.18%	7.96%	13.14%

. The spectra are mainly composed of a ${\mathrm{CO}}_2^{-}$ and axial ${\mathrm{CO}}_3^{3-}$ radical contributions. Furthermore, the EPR spectra of irradiated samples of Asp, Lys and Ser consist of two axial ${\mathrm{CO}}_2^{-}$ radical components, while Asn contains one axial and one isotropic contribution, which indicates that the ${\mathrm{CO}}_2^{-}$ radical is located in the crystal lattice in several slightly different environments, depending on the AA present [60]. At least one of the corresponding radicals is formed in the interior of the precipitate, while the other is created near the surface. Only Asn can be represented by two axial ${\mathrm{CO}}_3^{3-}$ radical contributions. One component can be assigned to ${\mathrm{CO}}_3^{3-}$ at a phosphate site, while the other can be assigned to the ${\mathrm{CO}}_3^{3-}$ on the surface or occupy a hydroxyl site [63].

SEM micrographs (Figure 5) show that AAs only slightly influence CaDHA morphology. Thus, in CS, after 60 min reaction time, irregular aggregates of large, thin, plate-like crystals are obtained. A precipitate of similar morphology was obtained in the presence of Phe. However, in the presence of the other amino acids applied, spherical aggregates of smaller and less-developed, thin, plate-like crystals were obtained.

Several studies have investigated the effect of AAs on HA morphology or transformation. Matsumoto et al. [59] reported that, in the presence of AAs such as Gly, Ser, Asp and Glu flake-like particles consisting of nano-sized platelets are formed. The flake-like morphology was also observed in their control sample. However, the platelets in this sample were much larger and thicker than those observed in the presence of AAs, which is consistent with our results. These AAs also reduce the degree of crystallinity in the HA [59]. The flake-like HA morphology was also reported by Eiden -Abmann et al. in the presence of AAs such as Asp, Glu or Ser [69].

4. Conclusions

The influence of different classes of AAs, namely, charged (Asp, Lys), polar (Asn, Ser) and non-polar (Phe), was investigated at conditions similar to physiological conditions. In the range of applied concentrations, AAs influence the ACP transformation mechanism, stability and morphology. All AAs except Phe inhibited the ACP transformation at higher concentrations, with Ser and Asp being the most efficient. Nevertheless, except for Ser, the investigated AAs influenced ACP morphology.

None of the investigated AAs influenced the composition of the CaDHA, which is formed in the later precipitation stage. However, the size of crystalline domains along the [002] direction decreased in the presence of Asp and Ser and increased in the presence of Phe. The observed changes were corroborated by EPR results, which indicate that the presence of diverse AAs differently affects the local microstructure of CaDHA. In addition, spherical aggregates of smaller, less-developed, thin, plate-like crystals, as compared to the control system, were obtained in the presence of Asp, Lys, Asn and Ser.

The observed differences in the effects that AAs exert on CaP precipitation, as well as the difference in the behavior of AAs of the same class, indicates that classification based on charge and polarity is not satisfactory to explain the different effects. Rather, these relatively simple molecules should be studied as specific entities.

The obtained results point to the likely complex role of AAs in biological mineralization, but also to a rather simple method of controlling CaPs properties, which are of importance in the preparation of multifunctional bone regeneration materials.