Regulation of calcium phosphate phase transition kinetics in aqueous solution via additives

Abstract

Additive-mediated regulation of calcium phosphate phase transitions is critical for synthesizing bone-like mineral structures in vitro. The main calcium phosphate phases involved in mineralization include amorphous calcium phosphate (ACP), dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP), and hydroxyapatite (HAp). This paper reviews the role of additives in these phase transitions. Additives are often adsorbed onto calcium phosphate surfaces, inhibiting transitions from ACP, DCPD, and OCP to HAp. Additives can act as nucleation templates or reduce particle size, promoting the transition from ACP to HAp. The concentration and addition timing of additives significantly influence their role in the ACP-to-HAp transition. Surface energy, incorporation of additives, and interactions with ions in solution also play an important role in calcium phosphate phase transitions.

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1. Introduction

Human bone possesses a complex structure with exceptional mechanical properties, primarily consisting of hydroxyapatite (HAp) and collagen.¹ Bone mineralization was once believed to follow the classical nucleation and growth pathway, wherein HAp forms directly on collagen scaffolds. Nevertheless, merely incorporating collagen into HAp crystallization solutions fails to replicate the natural bone structure.² Subsequent studies have revealed that bone mineralization involves a nonclassical crystallization pathway, which includes precursors such as amorphous calcium phosphate (ACP).^3–6 By regulating this ACP-to-HAp transition through the use of physiological additives, researchers have successfully synthesized mineralized collagen that closely mimics natural bone at the nanoscale.^2,7 These findings underscore the necessity of understanding the role of additives in calcium phosphate phase transitions.

Under physiological conditions, metastable phases such as ACP, OCP, and DCPD exist.^8–11 However, research on the in vivo crystallization process of calcium phosphate within the human body remains scarce and technically challenging. Consequently, the phase transition process of calcium phosphate is typically investigated in an in vitro solution environment, often utilizing additives to simulate ions, proteins, and other substances present in the body.^12,13 This review summarizes recent advances in the regulation of these phase transitions, with a specific focus on the ACP-to-HAp transition.

2. Amorphous calcium phosphate

Amorphous calcium phosphate (ACP), represented by the general formula Ca_xH_y (PO₄)z·nH₂O, is subject to compositional variation based on formation conditions.^14,15 Its formation mechanism in a pure supersaturated calcium phosphate solution at 25 °C involves cluster aggregation, leading to its persistence.¹³ Due to its thermodynamic metastability, ACP undergoes a solution-mediated phase transformation to hydroxyapatite (HAp), driven by the solution supersaturation with respect to HAp. This process, which typically involves the release of H⁺ ions, can proceed via two distinct mechanisms—dissolution–reprecipitation or solid-state reconstruction—with the specific pathway governed by the solution chemistry.^16,17 In aqueous environments, this transformation occurs in three stages: an induction period, a crystallization period, and a post-crystallization period. Under alkaline conditions at room temperature and a fixed Ca/P molar ratio of 2.5, the transformation of ACP to HAp was governed by solution supersaturation (ln SHAp), which ranged from approximately 19.25 to 28.36 across systems with calcium concentrations of 0.01–1 mol L⁻¹ and phosphate concentrations of 0.002–0.2 mol L⁻¹. This process exhibited three distinct stages: an induction period (0–4 h) at lower supersaturations, a crystallisation period (4–10 h), and a post-crystallisation stage (>10 h) at peak supersaturation.¹⁸

ACP crystallization kinetics are influenced by multiple factors, including solution supersaturation, pH, and ionic strength. A high thermodynamic driving force, such as elevated temperature and higher initial ion concentration, triggers and accelerates ACP crystallization.⁸ The effects of pH and the Ca/P molar ratio are more dependent on the specific solution environment.^19–22 In the context of bone mineralization and bone repair, the presence of additives such as ions and proteins further regulates the phase transformation process across different timescales, exerting a significant influence on ACP transformation (Tables 1–4).

Table 1 Additives that inhibit or promote the ACP-to-HAp transition (ions)

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
a Premix is defined as the addition and thorough mixing of additives with either the calcium or phosphate solution before their combination to form calcium phosphate phases. b Postmix refers to the introduction and mixing of additives into the reaction system after the calcium phosphate phases have formed.
Li^+	Premix^a	Inhibit	Reduced ACP particle concentration	Initial pH = 7.4, 25 °C, 50 mM, initial [Ca] = 10 mM, [P] = 10 mM^14
NO3^−
NH4^+	Postmix^b	Promote	Reduced ACP size
Cl^−
Na^+	Premix	Inhibit	Reduced ACP particle concentration	≥10 mM^14
				∼1.81 mM (concentration based on [Ca] = 12.09 mM), initial calcium–phosphorus concentration product: 14.6–131.6 mM^2 (ref. 20)
	Postmix	Promote	Reduced ACP size	10–70 mM^14
		Inhibit	Insufficient nucleation sites	≥180 mM^14
F^−	Postmix	Inhibit	Absorbed on ACP or HAp surface to inhibit aggregation, nucleation, and growth	4.5 mM^14
				pH = 7.0–8.0, 4–37 °C, ∼1.09 mM, ∼2.72 mM (concentration based on [Ca])^20
				pH = 7.4 ± 0.05, 19 ± 1 °C, 0.1–1 mM, the theoretical concentrations of calcium and phosphate after mixing were 5.88 and 4.12 mM^29
				pH = 7.4 ± 0.05, 25 ± 1 °C, 0–0.4 mM, initial [Ca] = 5.88 mM, [P] = 4.12 mM^30
K^+	Premix	Inhibit	Reduced ACP particle concentration	50 mM^14
				∼0.073 mM (concentration based on [Ca])^20
	Postmix	Promote	Reduced ACP size	50 mM^14
Mg^2+	Premix	Inhibit	Adsorbed on ACP surface; incorporated into ACP structure; competed for PO4^3− binding	4.5 mM^14
				∼0.30 mM, ∼1.21 mM (concentration based on [Ca])^20
				pH = 7.4, 37 °C, 0.99 × 10^−5–9.9 × 10^−5 M, initial [Ca] = 4.0–10.0 × 10^−4 M, [P] = 2.4–6.0 × 10^−4 M^31
				pH = 10.0 ± 0.2, 30.0 ± 1.0 °C, 0–1.5 mM, initial [Ca] = 2.5 mM, [P] = 1.5 mM^23
				7.4, 37 °C, 1.25 mM and 2.50 mM, the calcium and phosphate ions were present in a stoichiometric ratio of 1.67 ([Ca] = 3.16 mM, [P] = 1.9 mM; [Ca] = 2.52 mM, [PO4] =1.52 mM)^32
				pH = 7.4, 25 °C, 4.0 × 10^−5–2.0 × 10^−4 M, initial [Ca] = 4.0 mM, [P] = 4.0 mM^33
				pH = 7.4, 37 °C, 6 mM, initial [Ca] = 10 mM, [P] = 6 mM^34
	Postmix		Incorporated into ACP structure	4.5 mM^14
				0.99 × 10^−5–9.9 × 10^−5 M^31
				0–1.5 mM^23
				pH = 7.4, 30 °C, 5.7 × 10^−5, ACP addition quality: 100 mg/(120 ml of water or additive solution)^35
CO3^2−	Premix	Inhibit	Induced HAp lattice distortion and defects	∼2.18 mM, ∼6.53 mM (concentration based on [P] = 10.89 mM)^20
	Postmix		Replaces PO4^3−, incorporated into HAp structure	pH = 7.4, 25 °C, 0.0026 M, 0.026 M and 0.26 M, ACP addition quality: 200 mg/(25 ml reaction medium)^36
Zn^2+	Premix	Inhibit	Adsorbed on ACP and HAp surfaces	3.8 × 10^−6–1.53 × 10^−5 M^31
				4.0 × 10^−5–2.0 × 10^−4 M^33
				Initial (Ca/Zn) pH = 5.7–5.86, 25 °C, 0, 10, 25, 50, 100 and 200 mM, initial [Ca] = 0.5 mM, [P] = 0.3 mM^25
	Postmix		Adsorbed on HAp surfaces	3.8 × 10^−6–1.53 × 10^−5 M^31
Sr^2+	Premix	Inhibit	Adsorbed on HAp surfaces; reduced ACP solubility	4.0 × 10^−5–2.0 × 10^−4 M^33
				Initial pH = 3.5, final pH ≈ 11, 25 °C, Sr^2+ molar percentage (relative to total divalent cations Ca^2+ + Sr^2+): 0%, 5%, 10%, 25%, 50%, 75% and 100%, initial [Ca] = 110 mM, [P] = 33 mM^37
Ga^2+	Premix	Inhibit	Adsorbed on HAp surfaces, reduced HAp solubility	pH = 7.4, 37 °C, 0.0125 mM–1 mM, initial [Ca] = 2.79 mM, [P] = 1.87 mM (the direct precipitation of HA); ACP addition concentration: 1 mg ml^−1 (postmix, the transformation of ACP to HAp); initial [Ca] = 1.55 mM, [P] = 1.07 mM, HAp addition quality: 7.5 mg (postmix, the growth of HAp seeds.)^38
	Postmix
Gd^2+	Postmix	Inhibit	Interacted with PO4^3−	P/Gd molar ratio 1 : 1–1 : 4, ACP addition quality: 0.36 g (to obtain the ACP, initial [Ca] = 0.75 M, [P] = 0.5 M)^39
HAsO4^2−	Postmix	Inhibit	Incorporated into ACP structure	pH = 8, 25 °C, 0.1–1000 mg l^−1, ACP addition quality: 2 g L^−1, 4 g L^−1 (to obtain the ACP, initial [Ca] = 250 mM, [P] = 58 mM)^40

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Table 2 Additives that inhibit or promote the ACP-to-HAp transition (amino acids)

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
Aspartic acid (Asp)	Premix	Inhibit	Reduced ACP particle concentration; adsorbed on ACP surfaces	50 mM^14
				pH = 7.4, 25 ± 0.1 °C, 1.0, 2.5, 5.0 mM, initial [Ca] = 5 mM, [P] = 5 mM^41
		Promote	Reduce nucleation energy barrier	pH = 7.4, 37 °C, 1 mM^32
	Postmix	Promote	Reduced ACP size; displaced hydration water	<75 mM^14
Arginine (Arg)	Premix	Inhibit	Reduced ACP particle concentration	≥50 mM^14
		Promote	Reduce nucleation energy barrier	1 mM^32
	Postmix	Promote	Reduced ACP size; displaced hydration water; interacted with Ca^2+	<100 mM^14
Glycine (Gly)	Premix	Inhibit	Reduced ACP particle concentration	≥50 mM^14
		Promote	Reduce nucleation energy barrier	1 mM^32
Lysine (lys)	Premix	Inhibit	Amino-phosphate interactions	1.0, 2.5, 5.0mM^41
		Promote	Reduce nucleation energy barrier	1mM^32
Phenylalanine (phe)	Premix	Promote	Reduce nucleation energy barrier	1mM^32
				1.0, 2.5, 5.0 mM^41
Serine (Ser)	Premix	Inhibit	Adsorbed on ACP surfaces	1.0, 2.5, 5.0 mM^41
Asparagine	Premix	Inhibit	Polar amide group interactions	1.0, 2.5, 5.0 mM^41
Phosphatidyl serine (PS)	Premix	Inhibit	Adsorbed on ACP surfaces; interacted with Ca^2+ and PO4^3−	0.2 mg ml^−1 (ref. 20)
	Postmix	Inhibit	Adsorbed on ACP surfaces	pH = 7.4, 22 °C, 0.2, 0.5, 1.0 mg ml^−1, initial calcium–phosphorus concentration product: 18–125 mM^2 (ref. 42)

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Table 3 Additives that inhibit or promote the ACP-to-HAp transition (biological small molecules and polymers)

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
Citrate	Premix	Inhibit	Adsorbed on ACP surfaces	∼0.40 mM (concentration based on [Ca])^20
	Postmix			pH = 7.4, 0.5–3.0 mM, initial [Ca] = 8.0 mM, [P] = 4.8 mM^43
Ammonium iron citrate (AIC)	Premix	Inhibit	Adsorbed on ACP surfaces	pH = 5, 25 °C, 0.007, 0.02, 0.04, 0.1, 0.2 M, initial [Ca] = 0.5 mM, [P] = 0.3 mM^44
Pyrophosphate (PP)	Premix	Inhibit	Adsorbed on ACP and HAp surfaces	∼0.109 mM (concentration based on [P])^20
				4.0 × 10^−5 M^33
				pH = 12.3, 60 °C, 0–0.01 M, initial [Ca] = 0.2 mM, [P] = 0.12 mM^45
Tripolyphosphate (TPP)	Premix	Inhibit	Adsorbed on ACP surfaces	4.0 × 10^−5M^33
Alginate	Premix	Inhibit	Adsorbed on ACP surfaces; interacted with Ca^2+	pH = 7.4 ± 0.02, 25 °C, 1 mg l^−1, initial [Ca] = 50 mM, [P] = 2.4 mM^46
				pH = 10–11, 25 °C, 0.06 g ml^−1, initial [Ca] = 0.4 mM, [P] = 4 mM^47
Poly glutamic acid (PGA)	Premix	Inhibit	Adsorbed on surfaces, impedes ion transport	0.5 mg ml^−1 (ref. 20)
				pH = 7.4, 25 ± 0.1 °C, low molecular weight (LMw) PGA: >∼4.0 × 10^−4 mol l^−1, high molecular weight (HMw) PGA: >∼3.0 × 10^−4 mol l^−1, initial [Ca] = 6 mM, [P] = 6 mM^48
		Promote	Adsorbed on surfaces, forming a highly charged surface	LMw PGA: 1.0 × 10^−4–4.0 × 10^−4 mol l^−1, HMw PGA: 1.0 × 10^−4–3.0 × 10^−4 mol l^−1 (ref. 48)
	Postmix	Inhibit	Adsorbed on surfaces	5 × 10^−6 mol L^−1–7 × 10^−3 mol L^−1 (ref. 48)
Poly acrylic acid (PAA)	Premix	Inhibit	Adsorbed on ACP surface; interacted with Ca^2+	0.5 mg mL^−1 (ref. 20)
				pH = 9, PAA-2000: 100–5000 ppm, PAA-450000: 50–90 wt% (concentration relative to CDHA particles), 0.015, 0.03 and 0.045 mole of (CH3COO)2Ca·xH2O, 0.01, 0.02 and 0.03 mole of H3PO4 (ref. 49)
				pH = 7.4 ± 0.04, 37 ± 0.5 °C, PAA (Mw 2000): 60, 80, 90 μg ml^−1, initial [Ca] = 5.0 mM, [P] = 3.0 mM^50
		Promote	Enter the ACP interior, reduced ACP size	20, 40, 50 μg ml^−1 (ref. 50)
	Postmix	Inhibit	Adsorbed on ACP surface	3, 20, 80 μg ml^−1 (ref. 50)
				PAA-2000: 100–5000 ppm, PAA-450000: 50–90 wt% (concentration relative to CDHA particles)^49
Poly aspartic acid (pAsp)	Premix	Inhibit	Interacted with Ca^2+ and PO4^3−	pH = 7.4, 37 °C, 15, 75 μg ml^−1, initial [Ca] = 4.5 mM, [P] = 2.1 mM^51
Poly-L-lysine (PLL)	Premix	Inhibit	Adsorbed on surfaces, impedes ion transport	LMw PLL: >∼4.0 × 10^−3 mol L^−1, HMw PLL: >∼4.0 × 10^−3 mol L^−1 (ref. 48)
				25 °C, 8 mg ml^−1, initial [Ca] = 14.5 mM, [P] = 5.8 mM^52
		Promote	Adsorbed on surfaces, forming a highly charged surface	LMw PLL: 1.0 × 10^−4–4.0 × 10^−3 mol L^−1, HMw PLL: 1.0 × 10^−4–4.0 × 10^−3 mol L^−1 (ref. 48)
	Postmix	Inhibit	Adsorbed on surfaces	5 × 10^−6 mol L^−1–7 × 10^−3 mol L^−1 (ref. 48)
Poly ethylene glycol (PEG)	Premix	Inhibit	Adsorbed on ACP surfaces	5 °C, initial [Ca] = 0.1 mM, [P] = 0.133 mM^53
				pH = 10, ice water bath, PEG/Ca molar ratio: 1 : 8, 1 : 4, 1 : 1, 4 : 1, 8 : 1, 12 : 1, 16 : 1, initial [Ca] = 0.1 mM, [P] = 0.15 mM^54
Chitosan/ carboxymethyl chitosan	Premix	Inhibit	Interacted with PO4^3−	0.03 g ml^−1 (1 wt% acetic acid solution)^47
				25 °C, 10 mM, initial [Ca] = 20 mM, [P] = 10 mM^55
Poly styrene sulfonate (PSS)	Premix	Inhibit	Adsorbed on surfaces, impedes ion transport	>∼1.0 × 10^−3 mol l^−1 (ref. 48)
		Promote	Adsorbed on surfaces, forming a highly charged surface	∼1.0 × 10^−5–1.0 × 10^−3 mol l^−1 (ref. 48)
	Postmix	Inhibit	Adsorbed on surfaces	5 × 10^−6 mol L^−1–7 × 10^−3 mol L^−1 (ref. 48)
Polyvinylpyrrolidone (PVP)	Postmix	Inhibit	Adsorbed on ACP surfaces	pH = 10, room temperature, 3 g L^−1, initial [Ca] = 18 mM, [P] = 10.8 mM^56
Polyethyleneimine (PEI)
Poly propylene fumarate (PPF)	Premix	Inhibit	Adsorbed on ACP surfaces	pH = 7.4, 8.8, 10, PPF: HAp theoretical mass ratio 4 : 1, initial [Ca] = 0.4 mM, [P] = 0.156 mM^57
Adenosine triphosphate (ATP)	Postmix	Inhibit	Adsorbed on ACP surfaces	0.36 × 10^−5–1.44 × 10^−5 mol l^−1 (ref. 35)

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Table 4 Additives that inhibit or promote the ACP-to-HAp transition (proteins)

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
Phosvitin (yolk high phosphate protein)	Premix	Inhibit	Adsorbed on ACP surfaces	0.2, 0.5 mg ml^−1 (ref. 20)
Proteoglycans	Premix	Inhibit	Electrostatic and steric effects	3 mg/ml (ref. 20)
Casein/modified casein	Premix	Inhibit	Adsorbed on ACP surfaces; interacted with Ca^2+ and PO4^3−	0.5 mg/ml (ref. 20)
				pH = 9, 25 °C, 10 g l^−1, initial [Ca] = 100 mM, [P] = 60 mM (ref. 58)
				pH = 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 25 °C, 100 mg ml^−1, initial [Ca] = 10 mM, [P] = 6 mM^59
Serum proteins	Postmix	Inhibit	Adsorbed on ACP surfaces, reduced ACP solubility	Physiological concentration^36
Glycochenodeoxycholic acid	Premix	Inhibit	Competitively bound HPO4^2− sites	Glycochenodeoxycholic acid (2 mM), taurocholic acid (2–12 mM)^60
Osteonectin (ON)	Premix	Inhibit	Adsorbed on ACP surfaces to inhibit dissolution and nucleation	pH = 7.4, 37 °C, 0.4 μM and 0.8 μM, solution I: initial [Ca] = 1.33 mM, [P] = 1.0 mM; solution II: initial [Ca] = 2.43 mM, [P] = 1.87 mM; solution III: initial [Ca] = 1.87 mM, [P] = 1.64 mM; solution IV: initial [Ca] = 0.33 mM, [P] = 0.23 mM^61
Osteopontin (OPN)	Premix	Inhibit	Coated ACP particles	pH = 7.4, 25 ± 0.1 °C, 78–234 nM, calcium phosphate solution supersaturation σHAp = 29.9, ionic strength (IS) = 0.15 M^26
Silk fibroin (SF)	Premix in collagen solution	Inhibit	Cp: Neutral/hydrophobic interactions	pH = 7.4, 37 °C, SF was added at a ratio of 10% dry weight to collagen, initial [Ca] = 2.5 mM, [P] = 1.0 mM^62
		Promote	CS: Electronegative surface interactions
Salivary proteins	Postmix	Inhibit	Adsorbed on ACP surfaces	pH = 7.4, 37 ± 0.5 °C, PRP1: 0.0247–0.298 μM, PRP3: 0.0239–0.299 μM, Statherin: 0.16–0.781 μM, initial [Ca] = 1.06 mM, [P] = 0.63 mM^63
Albumin	Premix	Inhibit	Adsorbed on HAp surfaces; interacted with Ca^2+	20 mg ml^−1 (ref. 52)
	Postmix		Adsorbed on HAp surfaces	pH = 7.4, 37 °C, 75–250 μg cm^3, the degree of supersaturation was comparable to that in biological fluids and was such that all HAp precipitated would be expected to deposit on the seeds^64
Chondroitin sulfate	Premix	Inhibit	Interacted with Ca^2+	pH = 7.3, 25 °C, 1, 2, 3, 5, 10 mg ml^−1, initial [Ca] = 7 mM, [P] = 4.2 mM^65
Fetal bovine serum (FBS)	Premix	Inhibit	Adsorbed on ACP surfaces; prevented aggregation	37 °C, 10% FBS, initial [Ca] = 4.04 mM, [P] = 4.72 mM (the total concentrations of calcium and phosphate in rRPMI1640 media with 10% FBS were 0.385 and 5.12 mM)^66
Fetuin-A	Premix	Inhibit	Interacted with Ca^2+	pH = 7.4, 37 °C, 0.5 μM–2 μM, initial [Ca] = 4.8 mM, [P] = 1.6 mM^67
				15μM^34
				pH = 7.4, 37 °C, 1, 5 and 15 μM, initial [Ca] = 10 mM, [P] = 6 mM^68
Whey protein isolate	Premix	Inhibit	Adsorbed on calcium–phosphate phases	pH = 5–9, 15, 25, 38, 50 °C, 0.08 wt% WPI, initial [Ca] = 4.0–16.0 mM, [P] = 4.0–16.0 mM (Ca/P molar ratio = 1)^69
Peptide motifs present in dentin matrix protein 1 (DMP1)	Premix	Promote	Templated nucleation	pH = 8, 25 °C, 0.2–125 μg ml^−1, initial [Ca] = 2.0 mM, [P] = 4.5 mM^27
Amelotin (AMTN)	Postmix	Promote	Interacted with Ca^2+ to promote ACP dissolution	pH = 7.3, 25 °C, 2.5 μM, initial [Ca] = 0.5 mM, [P] = 3.9 mM^70
Soluble-matrix proteins from Lingula Anatina shells	Premix	Promote	Enhanced solubilization; templated nucleation	pH = 7.5, 25 °C, 0.02–2 μg ml^−1, initial [Ca] = 25 mM, [P] = 15 mM^71

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Additives can be categorized into two types based on the strength of their interaction with calcium phosphate: strongly interacting additives and weakly interacting additives, each exerting distinct effects on the transformation of ACP.

Weakly interacting additives (e.g., NaCl, l-aspartic acid) can either promote or inhibit the transformation of amorphous calcium phosphate (ACP), with their regulatory effects being both time- and concentration-dependent. For instance, when 70 mM NaCl was introduced before ACP formation, the lifetime of ACP increased from 13 minutes to approximately 27 minutes. Conversely, when the same concentration of NaCl was added after ACP had formed, the lifetime decreased to 3 minutes. However, introducing 200 mM NaCl after ACP formation extended the lifetime to 44 minutes. Similar effects were observed with other additives such as KCl, NaNO₃, l-Asp, and l-Arg.¹⁴

The underlying mechanism may be explained as follows: When additives are introduced before ACP formation, they reduce the solution supersaturation, thereby inhibiting crystallization. In contrast, when additives are introduced after ACP has formed, they exert two competing effects: they decrease the overall supersaturation of the solution—inhibiting the transformation—while simultaneously increasing local supersaturation by promoting the formation of small, high-surface-energy ACP particles. At relatively low additive concentrations (from zero to several tens of millimolar), the increase in supersaturation may dominate, promoting the transformation. At higher concentrations, however, the reduction in supersaturation becomes more significant, thus inhibiting the transformation. Control experiments using water as a diluent provided support for the proposed mechanism.¹⁴

Strongly interacting additives, such as NaF and MgCl₂, which exhibit strong interactions with calcium phosphate, generally suppress the crystallization of ACP in solution through mechanisms that include surface adsorption and incorporation into the ACP structure. By covering the surface of ACP, additives affect the active sites for nucleation and growth, thereby yielding significant inhibitory effects even at low concentrations. Mg²⁺ was found to adsorb onto the ACP surface, effectively delaying crystallization. This was demonstrated in systems containing 2.5 mM Ca²⁺ and 1.5 mM P at pH ∼10.0, where 1.5 mM Mg²⁺ extended the ACP stability time from 16 minutes to nearly 4 hours.²³ In a separate system with a higher cation concentration (0.25 M P, cation/P = 1.67, and pH ∼10–11), a Mg/Ca molar ratio of 1 : 4 (150 mM Mg²⁺), the amorphous state of ACP was maintained for over 145 days.²⁴ Due to its smaller ionic radius compared to Ca²⁺, Zn²⁺ at concentrations of 50–100 mM could substitute for calcium ions within the ACP structure; this substitution induced structural distortion and increased local disorder, thereby raising the nucleation energy barrier.²⁵ A 14-amino-acid peptide derived from osteopontin (OPN; sequence: DDVDDTDDSHQSDE) adsorbed onto ACP clusters via its phosphate and carboxyl groups. This adsorption increased the interfacial energy, elevated the nucleation barrier, and furthermore suppressed particle aggregation and dissolution through combined electrostatic and steric effects.²⁶ Typically, high concentrations of calcium and phosphate can increase supersaturation and accelerate HAp formation. But the inhibitory effects of strongly interacting additives often cannot be simply overcome by increasing ion concentrations. Through mechanisms such as surface adsorption or structural incorporation, these additives create a relatively isolated barrier from the solution, reducing the influence of calcium and phosphate concentrations on phase transformation.

However, a minority of strongly interacting additives can serve as templates to promote ACP crystallization. For example, engineered protein peptides with specific motifs and spatial arrangements were shown to provide nucleation sites and reduce interfacial energy, thereby facilitating ACP crystallization.²⁷ Similarly, molecular dynamics simulations revealed that the bone sialoprotein (BSP) peptides in the α-helical conformation could serve as templates to guide the arrangement of Ca²⁺ ions, matching the hydroxyapatite (HAp) crystal lattice structure and lowering the nucleation energy barrier.²⁸

3. Dicalcium phosphate dihydrate

The solubility product of DCPD (CaHPO₄·2H₂O) is 2.34 × 10⁻⁷ at 37 °C, and its transition to other calcium phosphate phases typically follows a dissolution–reprecipitation mechanism.⁷² Studies show that when DCPD transforms into HAp, there is a well-defined heteroepitaxial relationship between them, meaning HAp nuclei can form oriented on the DCPD substrate.^73,74 It is noteworthy that a similar directional transformation mechanism had also been reported in fluorinated hydroxyapatite (FHA). A crystallographic correlation exists between DCPA and FHA, demonstrating the pivotal role of fluorine in regulating phase transitions.⁷⁴

Similar to the crystallization pathway of ACP, the phase transition behavior of DCPD in aqueous solution is regulated by additives (Tables 5 and 6). Inhibitors mainly retard the transition by adsorption onto crystal surfaces, blocking the dissolution process, or decreasing ionic activity, while promoters accelerate the transition by lowering interfacial energy barriers or providing nucleation templates. The interfacial energy between DCPD and the solution is 0.20 mJ m⁻², while that between HAp and the solution is as high as 9.0 mJ m⁻², a difference of 8.80 mJ m⁻²,⁷⁵ constituting a significant phase transition energy barrier.

Table 5 Additives that inhibit or promote the DCPD-to-HAp transition

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
Zn^2+/Cu^2+/Fe^2+/Fe^3+/Al^3+/Cr^3+/Mn^2+/Co^2+/Mg^2+/Sr^2+/Ba^2+/Cd^2+/Ni^2+	Premix	Inhibit	Adsorbed on HAp nuclei	Crystallization experiment: 25 °C, 1 mM, initial [Ca] = 50 mM, [P] = 50 mM
	Postmix
Pb^2+	Premix	Promote	Formed more stable heterogeneous nucleation centers	Hydrolysis experiment: 37 °C, 5 μM and 10 μM, DCPD addition quality: 0.5 g/250 ml 0.1 mM H3PO4 (ref. 77)
	Postmix
La^3+	Premix	Inhibit	Prevented DCPD dissolution	After the reaction, pH = 5.8 and adjusted pH = 7.4, 25 ± 0.3 °C, 4 mmol l^−1, initial [Ca] = 40 mM, [P] = 24 mM^78
Aspartic acid (Asp)	Postmix	Promote	Reduced the interfacial barrier for the transition	pH = 8.45 ± 0.02, 25 °C, Asp: 0.05, 0.1, 0.2 mM
Glutamic acid (Glu)				Glu: 0.05, 0.20, 0.50 mM, DCPD addition quality: 50 mg/100 ml reaction solution^75
Citrate	Postmix	Inhibit	Adsorbed on HAp nuclei	pH = 6.70, 22 ± 0.5 °C, 10.0 mM, initial [Ca] = 30 mM, [P] = 19.5 mM^79
		Promote	Reduced the interfacial barrier for the transition	pH = 8.45 ± 0.01, 25 °C, 0.1, 0.4, 1.0, >1.0 mM, DCPD addition concentration: 0.5 g L^−1, initial [P] = 10 mM^76
Alendronate sodium (AS)	Premix	Inhibit	Adsorbed on DCPD to inhibit dissolution and reorganization	pH = 5.0, 60 °C, 2, 4, 8 μM, initial [Ca] = 40 mM, [P] = 30 mM^80
		Promote	Formed calcium–AS complex; induced lattice defect	16, 32 μM^80
Poly aspartic acid (pAsp)	Premix	Inhibit	Adsorbed on DCPD surfaces	pH = 4.9, 37 °C, pAsp: 0.2, 0.5, 0.8, 1.0 mM; Asp: 1, 2, 5, 10 mM, initial [Ca] = 50 mM, [P] = 50 mM^81
	Postmix
Pyruvate lactic acid	Postmix	Promote	Enhanced DCPD solubility	Pyruvate: 1.5 mM; lactic acid: 10 mM, DCPD/apatite/mixture of apatite and DCPD (70 : 30, 30 : 70) addition concentration: 50 mg ml^−1, initial [Ca] = 1.3 mM, [P] = 2.0 mM^82
Bovine serum albumin (BSA)	Postmix	Inhibit	Adsorbed on DCPD surfaces	Buffer HBSS: pH = 7.4, non-buffered environment: Initial, pH = 7.4, 25 °C, 1, 10, 30 mg l^−1, modified DCPD coating deposited on titanium plates, with a Ca/P molar ratio of 1 : 1 (ref. 83)
Non-collagenous proteins of bone	Postmix	Inhibit	Adsorbed on DCPD and HAp surfaces	pH = 7.4, 37 °C, 0.25–1 mg ml^−1 (ref. 82)
Osteocalcin (OCN)	Postmix	Promote	Adsorbed on DCPD surfaces to template HAp nucleation	pH = 7.4, 25 °C, 0.25 mg ml^−1, adding drops of OCN solution to the surface of the DCPD crystal (to obtained DCPD: initial [Ca]= 7.5 mM, [P] = 5.0 mM)^84

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Table 6 Additives that inhibit the DCPD-to-OCP transition

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
Poly aspartic acid (pAsp)	Premix	Inhibit	Adsorbed on DCPD	pH = 4.9, 37 °C, pAsp: 0.2, 0.5, 0.8, 1.0mM^81
	Postmix
Isophthalic acid (Isp)	Postmix	Promote	Caused lattice distortion	pH = 5.5, 60 °C, 0.1 M, DCPD addition quality: 4.13 g/0.3 L solution; 1.38 g/0.1 L solution (to obtained DCPD: initial [Ca]= 100 mM, [P] = 100 mM)^85
Acetic acid (ace)	Postmix	Promote	Adsorbed at crystal growth sites
Suberic acid sub	Postmix	Promote	Disrupted OCP hydration layers to expand interlayer space
Succinic acid (Suc)

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Acidic amino acids (e.g., aspartic acid [Asp] and glutamic acid [Glu]) possess a dicarboxyl group, enabling them to strongly adsorb onto the surfaces of DCPD and HAp. This adsorption increases the energy barrier at the DCPD-solution interface while lowering that at the HAp-solution interface, thereby effectively reducing the energy barrier required for heterogeneous nucleation and promoting the DCPD-to-HAp transition. Similarly, citrate adsorbs onto crystal surfaces via its carboxylate groups to modulate interfacial properties; this brings the interfacial energies of the two phases closer, which in turn reduces the nucleation barrier and drives the phase transition process.⁷⁶

4. Octacalcium phosphate

Octacalcium phosphate (OCP, chemical formula: Ca₈(HPO₄)₂(PO₄)₄·5H₂O) has a solubility product constant of 1.26 × 10⁻⁹⁶, which is significantly higher than that of HAp; therefore, it often exists as a thermodynamically unstable intermediate phase during calcium phosphate crystallization in aqueous solutions. Its crystal structure consists of alternating apatitic layers and hydrated layers,⁷² and it can undergo a topotactic transition to transform into the more stable HAp. Studies indicated that this transformation process usually begins with dissolution from the (100) crystal plane of OCP, accompanied by gradual expansion of the interlayer gap along the c-axis.⁸⁶ Furthermore, HAp nuclei formed on the OCP surface often maintained specific crystallographic orientation relationships with the substrate,⁸⁷ and HAp could also form via particle collision or topographical pathways.^88,89

Additives can inhibit OCP dissolution by adsorbing onto its surface, thereby delaying the phase transition process; some promoting additives can increase the thickness of the hydrated layer by incorporating into the OCP structure and enhance HPO₄²⁻ mass transfer efficiency, which in turn accelerates the transition to HAp (Table 7). The (100) crystal plane of OCP is the main surface exposed by the hydrated layer, rich in calcium ions, hydrogen phosphate ions, and water molecules. This plane provides abundant sites for electrostatic interactions and hydrogen bonding, enabling efficient adsorption of various additives. Inhibitory additives such as polyglutamic acid (PGLU) and polyaspartic acid (PASP) can bind to this surface via carboxylate side chains, inhibiting OCP dissolution, hindering crystal splitting along the c-axis, and disrupting its structural integrity—thus delaying the topotactic transition process of OCP to HAp.⁹⁰ Promoting additives such as the CEMP1-p3 peptide, upon adsorption on the OCP surface, could increase local HAp supersaturation and lower the heterogeneous nucleation barriers, thereby promoting HAp precipitation.⁹¹

Table 7 Additives that inhibit or promote the OCP-to-HAp transition

Additives	Adding time	Effect	Mechanism	Reaction conditions (pH, temperature, additive concentration, initial calcium and phosphorus concentration)
Sr^2+	Postmix	Inhibit	Caused lattice distortion	pH = 7.4 ± 0.1, 37 ± 0.1 °C, 0.1, 0.25, 0.5, 1 mM, OCP crystal growth: initial [Ca] = 1.2 mM, [P] = 0.9 mM
				HAp crystal growth: initial [Ca] = 1 mM, [P] = 0.6 mM; initial [Ca] = 1.5 mM, [P] = 0.9 mM^92
Sodium polyacrylate (NaPA)	Postmix	Inhibit	Adsorbed on OCP (100)	Crystallization experiment: initial pH = 5, 60 °C, 0.375–5 μM, initial [Ca] = 10 mM, [P] = 10 mM
				Hydrolysis experiment: pH = 7.4, 60 °C, 0.001–5 mM, OCP addition quality: 100 mg/100 ml solution^93
				pH = 7.4, 60 °C, 0.001 mM–5 mM, OCP addition quality: 100 mg/100 ml solution^94
Poly aspartate	Postmix	Inhibit	Adsorbed on OCP (100)	Hydrolysis experiment: initial pH = 7.4, 70 °C, 0–28 mM, OCP addition quality: 100 mg/100 ml solution^90
Poly glutamate (PGA)
Gelatin	Premix	Inhibit	Adsorbed on OCP (100)	pH = 4.7, 95 °C, the urea concentration was fixed at 20 mM, the gelatin concentration was 0.25, 0.5, 1, 5 g L^−1, initial [Ca] = 20 mM, [P] = 20 mM^95
Urea	Premix	Promote	Provided OH^−	pH = 4.7, 95 °C, the gelatin concentration was fixed at 1 g l^−1, the urea concentration was 5, 10, 20, 100 mmol^95
Succinate ions	Premix	Promote	Doped into OCP to increase water layer thickness	60 °C, 100 mM in a 200 mL solution system, OCP addition quality:10 mg/10 ml solution (to obtained OCP: initial [Ca] = 80 mM, [P] = 50 mM)^96
Suberate ions
Cementum protein 1-derived peptide (CEMP1-p3)	Postmix	Promote	Adsorbed on ACP surfaces to increase local HAp supersaturation	Octacalcium phosphate constant composition seeded growth (CCSG) assays: pH = 7.4, 37 °C, 0.6, 0.7, 1.25, 1.75, 2.75, 3.75 μg mL^−1, OCP addition quality: 2 mg, initial [Ca] = 1.7 mM, [P] = 1.7 mM^91

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5. New insights into bone growth and healing mechanisms and novel designs for bionic bone regeneration materials

Additives can bidirectionally regulate phase transitions in a concentration-, time-dependent manner: low concentrations may promote transitions while high concentrations inhibit them; inhibition may occur before precursor formation, whereas promotion may follow formation. This indicates that organisms can precisely control mineralization sites and rates by modulating the local concentration, spatiotemporal distribution, and structure of additives—thereby preventing ectopic mineralization and ensuring orderly bone growth and repair. The coexistence and progressive transformation of different calcium phosphate phases in physiological environments suggest that bone growth and remodeling involve the dynamic equilibrium of multiple mineral phases. This provides a new perspective for understanding bone metabolic diseases, implying that imbalances in certain physiological components may lead to abnormal phase transitions.

By drawing inspiration from the mechanisms of additives in biological systems, functional molecules (e.g., polyelectrolytes, peptide segments, and ions) have been incorporated into bone repair materials. This approach constructs diverse biomimetic additive systems to simulate the natural mineralization microenvironment, enabling precise guidance of the mineralization process within collagen fibers. Specifically, polyphenol-modified hyaluronic acid (HAD) was employed to construct biomimetic molecular bridges; through multifunctional synergistic action, these bridges formed dynamic hydration layers that reduced the nucleation energy barrier for ACP, while electrostatic attraction captured calcium ions to promote oriented ACP deposition along the collagen long axis.⁹⁷ A peptide with strong negative charge and calcium-binding ability—mimicking the phosphorylated fragment of OPN—was designed and covalently grafted onto the collagen surface to dual-regulate the mineralization process: first, by forming stable pre-nucleation clusters (PNCs) through calcium binding, and second, by reducing collagen interfacial energy via grafting to enhance ACP binding to collagen and promote intrafibrillar mineralization.⁹⁸ To simulate the high citrate concentration in the natural bone regeneration environment, citrate was pre-adsorbed onto collagen to improve the wettability of ACP on the collagen surface; this lowered the interfacial energy between collagen and the ACP precursor and synergized with polyaspartic acid to promote intrafibrillar mineralization within collagen fibers.⁹⁹ Additionally, a synergistic regulation strategy using bovine serum albumin (BSA) and polylysine (PLL) was constructed to stabilize the metastable calcium phosphate cluster (MCPC) structure, prolonging its penetration within dentinal tubules while appropriately promoting its mineralization process.⁵²

Previous research on 3D bioprinting technology has primarily focused on regenerating bone or cartilage structures.¹⁰⁰ However, the technology still faces challenges in achieving precise reconstruction of irregular, personalized bone defects.^101,102 To address this, four-dimensional (4D) printing introduces the dimension of time, allowing for pre-programmed changes in material shape or function,¹⁰³ which aligns with our research. Based on this concept, the time-dependent effects of additives are incorporated into the design of 4D bone repair materials: during the early bone repair phase, drugs are released to suppress infection and promote wound healing; subsequently, osteogenic bioactive substances and ACP are released to guide osteoblast migration, accelerate the ACP-to-HAp phase transition, and enhance bone mineralization—thereby accelerating the overall bone repair process. In summary, the effects of these additives not only mirror the sophisticated control mechanisms in biomineralization but also provide a foundational principle for designing advanced bone regeneration materials.

6. Conclusion

The regulation of calcium phosphate phase transitions by additives has been extensively studied, with mechanisms such as surface adsorption, surface energy modulation, and particle size effects well established. The efficacy of additives is highly dependent on their concentration, timing of introduction, and interaction strength with calcium phosphate phases. These insights have not only advanced the understanding of biomineralization mechanisms but also provided strategic guidance for the design of biomimetic bone materials. However, predicting when, at what concentration, and which additive combinations will exert quantitative effects on specific phase transition processes remains challenging, due to high characterization costs, substantial experimental workload, and pathway complexity. Therefore, developing precise, high-throughput, and low-cost characterization methods—coupled with accumulating sufficient data to train AI models for predicting additive synergies—may significantly enhance the understanding of bone mineralization and accelerate the development of more efficient and biocompatible bone regeneration materials.

Author contributions

Zhiyu Liu and Dongyue Yin: conceptualization, investigation, visualization, writing – original draft, writing – review & editing. Chunlin Deng: writing – review & editing, funding acquisition, supervision, project administration.

Conflicts of interest

There are no conflicts to declare.

Data availability

No primary research results, software or code have been included and no new data were generated or analysed as part of this review. The data supporting this review can be found in the references.

Acknowledgements

This work was financially supported by the Department of Natural Resources of Guangdong Province (GDNRC [2023]35).

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Footnote

† These authors contributed equally to this work and share first authorship.

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