Formation of Amorphous Calcium Phosphate on Strontium-Containing Calcium Carbonate (Aragonite)

Abstract

A method for preparing micrometer-sized particles coated with amorphous calcium phosphate (ACP) was developed. The composite particles were synthesized using (Ca, Sr)CO₃ (aragonite) particles as a substrate. Immersion of these aragonite particles in a dilute phosphate aqueous solution at pH 4 led to the formation of strontium-containing ACP. This occurred through the reaction of Ca²⁺ and Sr²⁺ ions with phosphate ions. The incorporation of strontium into the aragonite effectively moderated the rapid dissolution of aragonite, facilitating the deposition of an ACP layer on the aragonite particles. Subsequent immersion of the composite particles in a tris-HCl buffer solution at pH 7.4 resulted in the slow release of therapeutic ions such as calcium, phosphate, and strontium for up to 72 h. Thereafter, a novel amorphous phase, distinct from the original composition and incorporating strontium and sodium, precipitated.

1. Introduction

Calcium phosphates, such as hydroxyapatite and β-tricalcium phosphate, are renowned for their excellent biocompatibility and osteoconductivity, making them highly suitable as implant materials in bone defects [1]. Amorphous calcium phosphate (ACP) is particularly notable for its solubility, biocompatibility, and bioresorbability [2, 3]. The provision of adequate amounts of Ca and phosphate ions to the body is anticipated to significantly promote bone formation, making ACP a promising candidate for bone repair materials.

The traditional method of ACP synthesis involves the spontaneous precipitation of nano-sized particles by mixing aqueous solutions of calcium and orthophosphate ions [2, 3]. However, when used in composites with polymers, the agglomeration of ACP particles can adversely affect the material’s mechanical properties [4]. The synthesis of micro-sized ACP particles could potentially facilitate easier handling.

Given that ACP is thermodynamically unstable [2, 3], enhancing its stability during synthesis is crucial. The introduction of inorganic ions such as Mg²⁺ [5], carbonate [6], and P₂O₇⁴⁻ [7] has been shown to stabilize ACP formation, suggesting that their presence during preparation is beneficial.

Calcium carbonate, with its micro-sized particles and provision of Ca²⁺ and carbonate ions, emerges as a viable substrate for ACP synthesis. To achieve this, a reaction with phosphate ions is necessary, requiring careful control over the solubility of calcium carbonate through the introduction of inorganic ions or pH adjustment of the phosphate solution.

In aragonite, which is one of the polymorphs of calcium carbonate, strontium and calcium share similar chemical and physical properties, and incorporating Sr²⁺ ions into biomaterials has been shown to positively influence bone formation [8, 9]. Given these properties, aragonite, which can partially substitute calcium with strontium [10], appears to be a more suitable candidate for Sr²⁺ ion introduction. This substitution might enable the control of aragonite’s solubility due to the dilation [10] and distortion [11] of its lattice sites.

The pH adjustment is crucial for ACP preparation, typically conducted under basic conditions to avoid the precipitation of crystalline calcium orthophosphates, which occurs in acidic environments [2, 3]. Considering aragonite’s resistance to dissolution in basic conditions, ACP formation must be explored under acidic conditions. The preparation of ACP in acidic conditions (approximately pH 6), facilitated by stabilizers such as Mg²⁺ ions, has been documented [12]. However, there are no reports of ACP preparation under more acidic conditions (e.g., below pH 6), to the best of our knowledges. We conceived that an acidic environment below pH 6 would enhance the dissolution of aragonite and allow, the released carbonate and Ca²⁺ ions to play a role in increasing supersaturation for stable ACP formation.

Considering this background, the aim of this study was to develop a method for coating micro-sized particles with ACP using (Ca, Sr)CO₃ (aragonite) as a template under acidic conditions. For comparative analysis, CaCO₃ (aragonite) particles were synthesized using the same methodology to assess the impact of Sr²⁺ ions on ACP formation.

2. Material and Methods

2.1 Preparation of (Ca, Sr)CO₃ (aragonite) particles

(Ca, Sr)CO₃ (aragonite) (SrA) was synthesized using a carbonation process. A 0.75 M MgCl₂ aqueous solution was prepared by dissolving MgCl₂·6H₂O (98.0% purity, Kishida Chemical) in 100 mL of distilled water. To this solution, 0.0125 mol of SrCl₂ (95.0% purity, FUJIFILM Wako Pure Chemical) and 0.025 mol of Ca(OH)₂ (96.0% purity, Kishida Chemical) were added, forming a MgCl₂-SrCl₂-Ca(OH)₂ suspension. CO₂ gas was introduced into the suspension at a flow rate of 0.5 L/min while maintaining the temperature at 80°C and stirring at 250 rpm for 90 min. The resultant precipitates were washed five times with distilled water to remove any residual ions and dried at 100°C for 24 h before being stored under vacuum conditions. The molar ratio of calcium to strontium in the final product was 2:1. For comparative purposes, CaCO₃ (aragonite) (denoted as “A”) was prepared using an identical method, excluding the addition of SrCl₂. There was almost no difference between the yields of samples “SrA” and “A”.

The morphology of the particles was analyzed using field emission scanning electron microscopy (SEM, JSM-7800F, JEOL) at an accelerating voltage of 5 kV. Prior to observation, samples were coated with amorphous osmium to improve conductivity. Elemental mapping for calcium and strontium was performed using an integrated energy-dispersive X-ray spectrometer (EDS). The crystal and chemical structures of the particles were characterized by X-ray powder diffraction (XRD, X’pert, Philips) and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR, FT-IR-4100, JASCO), respectively.

Lattice parameters for (Ca, Sr)CO₃ (aragonite) were determined using the Le Bail method [13], as implemented in RIETAN-FP [14]. Reference lattice parameters for aragonite and strontianite (SrCO₃) were obtained from Crystallographic Information Files, as established by the International Union of Crystallography.

The designation “A” or “SrA” was used for the sample codes to indicate the absence or presence of strontium, respectively, in the aragonite samples prepared.

2.2 Conversion of (Ca, Sr)CO₃ (aragonite) into ACP

A 1.5 M NaH₂PO₄ aqueous solution was prepared by dissolving NaH₂PO₄ (FUJIFILM Wako Pure Chemical, 99.0% purity) in distilled water. A desktop pH meter (F-73, HORIBA) showed pH 4.00 in this solution at 37°C. Subsequently, 0.15 g of the prepared particles was immersed in 20 mL of this phosphate solution at 37°C for 24 h. Post-immersion, the solution was filtered by suction, washed with ethanol (Kishida Chemical, 99.5% purity), and stored under vacuum. This procedure is hereinafter referred to as the “Phosphate solution (PS) treatment”.

Chemical structures of the treated particles were examined using XRD and ATR-FTIR. Particle morphologies were analyzed using SEM at an accelerating voltage of 5 kV, with samples coated in amorphous osmium to enhance conductivity. EDS was employed for elemental mapping (Ca, P) on the surface of sample A post-PS treatment.

Cross-sections of sample SrA post-PS treatment were prepared using a focused ion beam method (FIB, EM-9320FIB, JEOL) for examination under a transmission electron microscope (TEM, JEM-2100Plus, JEOL) at an accelerating voltage of 200 kV. Elemental mapping (Ca, Sr, Na, P) of the cross-sections was performed using EDS. Selected area electron diffraction (SAED) images were taken to observe the internal structure of the particles and the new products formed on the particle surface.

For dissolution studies, 5 mg of sample SrA post-PS treatment was immersed in 20 mL of a tris-HCl buffer solution (pH 7.4) and stirred at 37°C in a shaking incubator (KS4000i, IKA) for a set period (n = 3). The mixture was filtered, and the retained sample was dried at 37°C in a low-temperature incubator (FMU-133I, Fukushima). Concurrently, the concentrations of Ca²⁺, Sr²⁺, Na⁺, and phosphate ions in the solution were determined using inductively coupled plasma optical emission spectrometry (ICP-AES, ICPS-7510, Shimadzu). In this study, a tris-HCl buffer solution, which does not contain calcium and phosphate ions, was chosen because the precipitation of calcium phosphates would obscure the exact amount of calcium and phosphate ions released from the particles in fluids such as some simulated body fluids (SBFs) or culture media.

The resulting particles prepared from A and SrA are denoted as “A-PS” and “SrA-PS”, respectively.

3. Results

3.1 Structure of (Ca, Sr)CO₃ (aragonite) particles

SEM images (Fig. 1) revealed that the synthesized particles were rod-like, with lengths varying from several to tens of micrometers. EDS analysis confirmed the uniform distribution of calcium and strontium within the particles. The XRD pattern (Fig. 2(a)) indicated the formation of aragonite particles; however, a calcite peak was also detected at 2θ ∼ 29°. Notably, the intensity of this peak was higher in sample A compared to sample SrA. The ATR-FTIR spectra (Fig. 2(b)) exhibited characteristic peaks of CO₃²⁻ at 1455, 1082, 856, 713, and 700 cm⁻¹ [15, 16].

Fig. 1

SEM images of (Ca, Sr)CO₃ (aragonite) (sample SrA), along with EDS mapping for sample SrA showing the distribution of elements.

Fig. 2

(a) XRD patterns and (b) ATR-FTIR spectra for the synthesized particles.

Given that Sr²⁺ ions (1.13 Å, 6 coordination) possess a larger ionic radius than Ca²⁺ ions (1.00 Å, 6 coordination) [17], the lattice parameters of SrCO₃ (strontianite) are expected to exceed those of aragonite. Consequently, the lattice parameters of SrA should approach those of strontianite as the strontium content increases. The lattice parameters, determined using the Le Bail method, are summarized in Table 1. The lattice parameters for SrA were found to be larger than those for pure aragonite. The strontium content, estimated from the lattice parameters, was 33.8%, closely aligning with the theoretical value of 33.3%.

Table 1 Lattice parameters for sample SrA, aragonite, and strontianite.

3.2 Structure of composite particles

The XRD analysis of particles after PS treatment (Fig. 3(a)) revealed a reduction in peak intensities associated with aragonite, accompanied by the emergence of a broad peak around 2θ ∼ 30°, indicative of an amorphous phase. The ATR-FTIR spectrum of sample A-PS (Fig. 3(b)) demonstrated a decrease in the intensity of CO₃²⁻ related peaks, with the appearance of PO₄³⁻ related peaks near 1150, 1108, 1038, 1003, and 962 cm⁻¹ [18], P-O related peaks near 900 cm⁻¹ [19] and P-O-P related peaks near 745 cm⁻¹ [20]. Additionally, OH related peaks were observed around 3400 and 1650 cm⁻¹ [21, 22]. Conversely, in the spectrum of sample SrA-PS, CO₃²⁻ related peaks were absent, while phosphate and OH related peaks appeared in similar positions.

Fig. 3

(a) XRD patterns and (b) ATR-FTIR spectra for the particles following phosphate solution (PS) treatment.

SEM images of sample A before and after PS treatment are depicted in Fig. 4. Sample A consisted of rod-like particles, and sample A-PS showed aggregation, forming spherical aggregates several tens of micrometers in size. EDS analysis of sample A-PS (Fig. 4(b)) indicated uniform distribution of calcium, while phosphorus distribution was uneven. In contrast, no aggregates were observed in sample SrA-PS; instead, new products appeared on the particle surfaces (Fig. 5(a)). The aspect ratio of sample SrA-PS was smaller than that of SrA. This change is considered to be attributed to the dissolution and re-precipitation reaction of the particles.

Fig. 4

SEM images of (a) CaCO₃ (aragonite) (sample A) and (b) sample A-PS, and EDS spectrum and mapping of sample A-PS, illustrating the morphology and elemental distribution.

Fig. 5

(a) SEM image of sample SrA-PS, (b) cross-sectional TEM image and EDS mapping for sample SrA-PS, highlighting the layer structure and elemental composition.

Cross-sectional TEM images and EDS mapping of sample SrA-PS (Fig. 5(b)) revealed a layer on the particle surface, approximately 0.5 to 1 µm thick, thought to be new products, with numerous fine pores within this layer. EDS analysis of the cross-section showed both calcium and strontium present in the particles and the new products, whereas phosphorus was almost exclusively found in the new products. SAED patterns of the particle interiors (Fig. 6(a)) exhibited some spots, while those of the particle surfaces (Fig. 6(b)) displayed a halo pattern, suggestive of an amorphous phase.

Fig. 6

SAED patterns of sample SrA-PS, showing (a) the internal structure and (b) the surface amorphous layer.

Ion release behavior from sample SrA-PS in a tris-HCl buffer solution was investigated (Fig. 7(a)). Ca²⁺, Sr²⁺, Na⁺, and phosphate ions were released into the solution. Ion concentration decreased between 72 and 120 h post-immersion. The XRD pattern of particles after 120 h of immersion (Fig. 7(b)) displayed a halo peak attributed to the amorphous phase, rather than to apatite.

Fig. 7

(a) Concentration of ions in a tris-HCl buffer solution after immersion of sample SrA-PS, and (b) XRD patterns of sample SrA-PS before and after 120-hour immersion.

4. Discussion

Mg²⁺ ions have been shown to inhibit calcite growth and promote the stabilization of aragonite formation [23, 24]. In this study, Mg²⁺ and Sr²⁺ ions, sourced from MgCl₂·6H₂O and SrCl₂, respectively, coexisted during the preparation of the target particles. EDS analysis revealed negligible peaks for magnesium and chlorine, suggesting their presence as ions in the suspension, with the majority being removed during the particle cleaning process.

It has been reported that the adsorption of strontium on aragonite surfaces leads to lattice distortion, hindering the transformation of aragonite into calcite by increasing the energy required for transformation [10]. This effect likely contributed to the suppression of calcite formation in sample SrA.

The analysis of strontium content in the particles confirmed that sample SrA was synthesized in accordance with the intended composition, indicating the successful incorporation of strontium into the aragonite structure.

The discussion now turns to the formation and ion release behavior of the composite particles. XRD patterns confirmed the presence of ACP, while ATR-FTIR spectra revealed a water-containing phosphate phase on the particle surfaces. The distinct morphologies of samples A-PS and SrA-PS could be attributed to the altered solubility of aragonite upon strontium incorporation. Given that strontianite exhibits lower solubility than aragonite [25, 26], it is plausible that the introduction of strontium reduces the solubility of aragonite. In sample A, Ca²⁺ ions might disperse throughout the PS, reacting with phosphate ions to form a phosphate phase around the particles. Conversely, in sample SrA, the reaction of Ca²⁺ and Sr²⁺ ions with phosphate ions occurs on the particle surface, leading to the formation of a phosphate phase exclusively on the surface. This may be related to the ion dissolution rate from the SrA particle. It may be also necessary to further discuss the possibility that the difference in the morphology of each particle after PS treatment is due to the difference in their isoelectric points.

From the cross-sectional TEM images, the presence of fine pores within the newly formed products was noted, likely resulting from the dissolution of aragonite particles and the subsequent release of carbonic acid. EDS elemental mapping suggested that these new products formed as a result of the dissolution of aragonite in the phosphoric acid solution and the interaction between Ca²⁺ and Sr²⁺ ions with phosphate ions. The SAED patterns further supported the conclusion that the new products on the particle surfaces were strontium-containing ACP. PS treatment at pH 4 would have achieved the preparation of composite particles coated with ACP by successfully balancing the rate of the dissolution of sample SrA and the reaction of the dissolved Ca²⁺ and Sr²⁺ ions with phosphate ions.

The investigation into ion release behavior in the tris-HCl buffer solution revealed the release of Na⁺ ions, likely originating from the PS. This finding suggests the potential for incorporating additional therapeutic ions into the amorphous phase by including them in the PS. Moreover, a decrease in ion concentration was observed between 72 and 120 h post-immersion, possibly due to precipitate formation. Given that no crystalline calcium phosphate precipitation was detected in the XRD patterns of the particles 120 h post-immersion, it is proposed that the ACP re-precipitated as an amorphous phase distinct from the original composition, incorporating both strontium and sodium following dissolution. Its composition needs to be investigated further. Sr²⁺ ions have been shown to stabilize ACP [7, 27], suggesting their role in the initial stabilization of ACP and in controlling ion release and reprecipitation as an amorphous phase. Further kinetic discussion on the mechanism would be needed. This study indicates the contribution of Sr²⁺ ions to the formation of ACP composite particles during the conversion of aragonite to ACP.

Recent research has highlighted the beneficial effects of Sr²⁺ ions on immune cells and bone formation [28]. The release of several mM of Sr²⁺ ions at the beginning of the immersion is required to stimulate immune cells. In addition, the releases of 0.6–0.8 mM of Ca²⁺ and 0.5 mM of phosphate ions from poly(lactic acid)-composites containing ACP has been reported to induce biocompatible feature with pre-osteoblast cells and significant osteogenesis in calvarial bone defect [29]. The amounts of Ca²⁺ and phosphate ions released from sample SrA-PS could be adjusted to these amounts, which is expected to contribute to bone repair. Consequently, this material holds promise as a dispersant for delivering inorganic ions and may exhibit exceptional osteogenic potential, promoting bone formation through the activation of osteoblasts and the immune response.

5. Conclusions

This study aimed to develop a method for preparing micro-sized particles coated with ACP using (Ca, Sr)CO₃ (aragonite) particles as a substrate.

First, (Ca, Sr)CO₃ (aragonite) particles were synthesized by introducing carbon dioxide gas into a suspension containing Mg²⁺ and Sr²⁺ ions. Analysis confirmed that the particles were aragonite with strontium partially substituting for calcium (33.8%).

Subsequent immersion of these particles in a NaH₂PO₄ aqueous solution at pH 4 led to the formation of ACP on their surfaces. Notably, (Ca, Sr)CO₃ (aragonite) particles did not aggregate, and new amorphous products formed on their surfaces. These products resulted from the dissolution of aragonite in the PS and the reaction of Ca²⁺ and Sr²⁺ ions with phosphate ions. PS treatment at pH 4 contributed to adjusting the balance between the dissolution of (Ca, Sr)CO₃ (aragonite) and the precipitation of ACP, ACP formation under acidic conditions was found for the first time in this study. Ion release behavior in a tris-HCl buffer solution indicated the release of therapeutic ions for up to 72 h from the particles, followed by reprecipitation of the new product as an amorphous phase distinct from its original composition, incorporating both strontium and sodium. The findings suggest that Sr²⁺ ions played a crucial role in the formation of ACP composite particles during the conversion of aragonite to ACP. The newly developed composite particles, coated with ACP on the surface of (Ca, Sr)CO₃ (aragonite), hold potential for use as dispersants to enhance bone formation.

Acknowledgement

Funding: This study was supported in part by a JSPS KAKENHI [20H00304].

REFERENCES

• 1)   W.R.  Moore,  S.E.  Graves and  G.I.  Bain: Synthetic bone graft substitutes, ANZ J. Surg. 71 (2001) 354–361. doi:10.1046/j.1440-1622.2001.02128.x
• 2)   S.V.  Dorozhkin: Amorphous calcium orthophosphates: nature, chemistry and biomedical applications, Int. J. Mater. Chem. 2 (2012) 19–46. doi:10.5923/j.ijmc.20120201.04
• 3)   S.V.  Dorozhkin: Synthetic amorphous calcium phosphates (ACPs): preparation, structure, properties, and biomedical applications, Biomater. Sci. 9 (2021) 7748–7798. doi:10.1039/D1BM01239H
• 4)   J.N.R.  O’Donnell,  J.M.  Antonucci and  D.  Skrtic: Illuminating the role of agglomerates on critical physicochemical properties of amorphous calcium phosphate composites, J. Compos. Mater. 42 (2008) 2231–2246. doi:10.1177/0021998308094797
• 5)   A.L.  Boskey and  A.S.  Posner: Magnesium stabilization of amorphous calcium phosphate: a kinetic study, Mater. Res. Bull. 9 (1974) 907–916. doi:10.1016/0025-5408(74)90169-X
• 6)   D.J.  Greenfield and  E.D.  Eanes: Formation chemistry of amorphous calcium phosphates prepared from carbonate containing solutions, Calcif. Tissue Res. 9 (1972) 152–162. doi:10.1007/BF02061953
• 7)   M.J.  Root: Inhibition of the amorphous calcium phosphate phase transition reaction by polyphosphates and metal ions, Calcif. Tissue Int. 47 (1990) 112–116. doi:10.1007/BF02555994
• 8)   X.  Sheng,  C.  Li,  Z.  Wang,  Y.  Xu,  Y.  Sun,  W.  Zhang,  H.  Liu and  J.  Wang: Advanced applications of strontium-containing biomaterials in bone tissue engineering, Mater. Today Bio 20 (2023) 100636. doi:10.1016/j.mtbio.2023.100636
• 9)   G.  Borciani,  G.  Ciapetti,  C.  Vitale-Brovarone and  N.  Baldini: Strontium functionalization of biomaterials for bone tissue engineering purposes: a biological point of view, Materials 15 (2022) 1724. doi:10.3390/ma15051724
• 10)   A.A.  Finch and  N.  Allison: Coordination of Sr and Mg in calcite and aragonite, Mineral. Mag. 71 (2007) 539–552. doi:10.1180/minmag.2007.071.5.539
• 11)   N.  Wada and  T.  Umegaki: Effect of cation (Sr, Pb and Ba) on calcium carbonate polymorphs under diffusional conditions, Gypsum Lime 245 (1993) 211–219 (in Japanese). doi:10.11451/mukimate1953.1993.211
• 12)   C.  Holt,  M.J.J.M.  Van Kemenade,  L.S.  Nelson, Jr.,  D.W.L.  Hukins,  R.T.  Bailey,  J.E.  Harries,  S.S.  Hasnain and  P.L.  De Bruyn: Amorphous calcium phosphates prepared at pH 6.5 and 6.0, Mater. Res. Bull. 24 (1989) 55–62. doi:10.1016/0025-5408(89)90008-1
• 13)   A.  Le Bail,  H.  Duroy and  J.L.  Fourquet: Ab-initio structure determination of LiSbWO₆ by X-ray powder diffraction, Mater. Res. Bull. 23 (1988) 447–452. doi:10.1016/0025-5408(88)90019-0
• 14)   F.  Izumi and  K.  Momma: Three-dimensional visualization in powder diffraction, Solid State Phenom. 130 (2007) 15–20. doi:10.4028/www.scientific.net/SSP.130.15
• 15)   M.B.  Toffolo,  L.  Regev,  S.  Dubernet,  Y.  Lefrais and  E.  Boaretto: FTIR-based crystallinity assessment of aragonite-calcite mixtures in archaeological lime binders altered by diagenesis, Minerals 9 (2019) 121. doi:10.3390/min9020121
• 16)   K.N.  Islam,  M.Z.B.A.  Bakar,  M.M.  Noordin,  M.Z.B.  Hussein,  N.S.B.A.  Rahman and  M.E.  Ali: Characterisation of calcium carbonate and its polymorphs from cockle shells (Anadara granosa), Powder Technol. 213 (2011) 188–191. doi:10.1016/j.powtec.2011.07.031
• 17)   I.  Sunagawa,  Y.  Takahashi and  H.  Imai: Strontium and aragonite-calcite precipitation, J. Mineral. Petrol. Sci. 102 (2007) 174–181. doi:10.2465/jmps.060327a
• 18)   C.  Rey,  M.  Shimizu,  B.  Collins and  M.J.  Glimcher: Resolution-enhanced fourier transform infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase of calcium phosphate in bone and enamel and their evolution with age: 2. Investigations in the v₃ PO₄ domain, Calcif. Tissue Int. 49 (1991) 383–388. doi:10.1007/BF02555847
• 19)   Y.  Kim,  S.Y.  Lee,  Y.  Roh,  J.  Lee,  J.  Kim,  Y.  Lee,  J.  Bang and  Y.J.  Lee: Optimizing calcium phosphates by the control of pH and temperature via wet precipitation, J. Nanosci. Nanotechnol. 15 (2015) 10008–10016. doi:10.1166/jnn.2015.10636
• 20)   V.A.  Sinyayev,  E.S.  Shustikova,  D.  Griggs and  D.V.  Dorofeev: The nature of P-O bonds in the precipitated amorphous calcium phosphates and calcium magnesium phosphates, Glass Phys. Chem. 31 (2005) 671–675. doi:10.1007/s10720-005-0112-y
• 21)   P.  Layrolle and  A.  Lebugle: Characterization and reactivity of nanosized calcium phosphates prepared in anhydrous ethanol, Chem. Mater. 6 (1994) 1996–2004. doi:10.1021/cm00047a019
• 22)   A.C.  Tas: X-ray-amorphous calcium phosphate (ACP) synthesis in a simple biomineralization medium, J. Mater. Chem. B 1 (2013) 4511–4520. doi:10.1039/c3tb20854k
• 23)   K.J.  Davis,  P.M.  Dove and  J.J.  De Yoreo: The role of Mg²⁺ as an impurity in calcite growth, Science 290 (2000) 1134–1137. doi:10.1126/science.290.5494.1134
• 24)   W.K.  Park,  S.J.  Ko,  S.W.  Lee,  K.H.  Cho,  J.W.  Ahn and  C.  Han: Effects of magnesium chloride and organic additives on the synthesis of aragonite precipitated calcium carbonate, J. Cryst. Growth 310 (2008) 2593–2601. doi:10.1016/j.jcrysgro.2008.01.023
• 25)   E.  Busenberg,  L.N.  Plummer and  V.B.  Parker: The solubility of strontianite (SrCO₃) in CO₂-H₂O solutions between 2 and 91°C, the association constants of SrHCO+ 3 (aq) and SrCO0 3 (aq) between 5 and 80°C, and an evaluation of the thermodynamic properties of Sr²⁺ (aq) and SrCO₃ (cr) at 25°C and 1 atm total pressure, Geochim. Cosmochim. Acta 48 (1984) 2021–2035. doi:10.1016/0016-7037(84)90383-1
• 26)   J.Y.  Gal,  J.C.  Bollinger,  H.  Tolosa and  N.  Gache: Calcium carbonate solubility: a reappraisal of scale formation and inhibition, Talanta 43 (1996) 1497–1509. doi:10.1016/0039-9140(96)01925-X
• 27)   C.B.  Tovani,  A.  Gloter,  T.  Azais,  M.  Selmane,  A.P.  Ramos and  N.  Nassif: Formation of stable strontium-rich amorphous calcium phosphate: possible effects on bone mineral, Acta Biomater. 92 (2019) 315–324. doi:10.1016/j.actbio.2019.05.036
• 28)   A.M.  Negrescu and  A.  Cimpean: The state of the art and prospects for osteoimmunomodulatory biomaterials, Materials 14 (2021) 1357. doi:10.3390/ma14061357
• 29)   X.  Niu,  Z.  Liu,  F.  Tian,  S.  Chen,  L.  Lei,  T.  Jiang,  Q.  Feng and  Y.  Fan: Sustained delivery of calcium and orthophosphate ions from amorphous calcium phosphate and poly (L-lactic acid)-based electrospinning nanofibrous scaffold, Sci. Rep. 7 (2017) 45655. doi:10.1038/srep45655