Influence of Sr2+ doping and phase composition on the physicochemical properties and cytocompatibility of biphasic calcium phosphate for bone graft applications
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Abstract
Objectives
To investigate how phase composition influences the physicochemical properties, Sr2+ ion release behavior, and cytocompatibility of Strontium (Sr)-doped calcium phosphate (CaP) materials, focusing on Sr-HA, Sr-β-TCP, and three Sr-BCP compositions.
Methods
This work focuses on Sr-HA, Sr-β-TCP, and Sr-BCP powders with HA/β-TCP ratios (60:40 (BCP1), 30:70 (BCP2), and 20:80 (BCP3)) that were synthesized by wet chemical precipitation followed by calcination. The effect of CaP phase compositions on physicochemical characteristics, Sr2+ release, and cytocompatibility was investigated by using ICP-OES, FTIR, XRD, SEM-EDX, and MTT assays.
Results
EDX confirmed the Ca/P ratios, and both FTIR and XRD indicated successful phase formation without secondary phases. The Sr-BCP samples demonstrated enhanced cell viability after 48 h in MTT assays, highlighting biological responses associated with the biphasic structure. ICP-OES analysis indicated composition-dependent Sr2+ release, with Sr-BCP1 showing the highest initial and sustained ion release.
Conclusions
Sr-BCP1 offers a promising balance between structural stability, favorable cytocompatibility, and controlled Sr2+ ion delivery, supporting its potential for bone applications.
Introduction
Bone diseases and injuries are highly prevalent clinical conditions affecting millions worldwide. They are often characterized by reduced bone mineral density and mass, which significantly increases the risk of fractures and impairs the natural healing process with age. Globally, over 200 million individuals are affected by osteoporosis, with 1 in 3 women over the age of 50 and 1 in 5 men experiencing the condition during their lifetime [1]. Although bone possesses an inherent capacity for self-renewal and regeneration, bone grafting is often required to support healing through osteoconduction, osteogenesis, and osteoinduction [2]. However, conventional bone grafting techniques such as autograft and allograft have important limitations, including donor-site morbidity with autografts and the risk of immune reactions with allografts.
As a result, synthetic bone graft materials have garnered significant interest, as they can overcome donor-site complications while reducing the risks of immune and disease transmission associated with allografts. Calcium phosphate (CaP)-based ceramics, particularly hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), are the most widely used synthetic materials due to their biocompatibility and structural similarity to natural bone [3]. Hydroxyapatite (Ca10(PO4)6(OH)2), which comprises approximately 50 % of bone’s inorganic matrix, is highly biocompatible, osteoconductive, and phase-stable [4]. Nevertheless, the slow resorption rate and limited osteoinductive potential limit its effectiveness in dynamic bone environments. Importantly, to improve the limited bioactivity and slow resorption of pure HA, it is frequently combined with the more soluble β-TCP to form biphasic calcium phosphate (BCP).
Mostly, the BCP powder, in the form of granules and blocks, has been used in clinical settings for bone graft substitutes due to its biocompatibility and osteoconductive properties [5]. The amount of HA in BCP affects its biological performance by altering degradation behavior and release profiles relative to β-TCP [5]. The Ca/P ratios of HA and β-TCP, which are 1.67 and 1.5, respectively, allowed the modification of BCP’s phase composition in tuning the biological properties [6]. By altering the BCP ratios, the biological requirements will be adjusted according to the bone regeneration applications. This biphasic state of composition helps to mimic natural bone minerals by having HA’s stable phase and β-TCP’s soluble phase in degradability to provide balance in crystalline phase, compatibility, and bioresorption.
A commonly used composition is 20:80 (HA: β-TCP), which promotes enhanced osteogenic differentiation and early bone formation by influencing osteoclast activity [5], 6]. Meanwhile, the intermediate composition 30:70 (HA:β-TCP), has shown optimal dissolution behavior and improved cellular responses when applied as coatings on implants [6]. In contrast, a higher HA content (60 %) combined with 40 % β-TCP provides a balance between mechanical support and bone regeneration, achieving complete calvarial defect repair within 60 days, aided by high porosity that facilitates vascularization [7]. Clinical studies supporting the relevance of phase balance by comparing 60:40 and 78:22 ratios for dental socket preservation have found that both were biocompatible. However, the 60:40 composition produced more new bones and left less remaining biomaterial after six months [8]. These findings indicate that each ratio offers advantages, but also carries limitations such as degrading too rapidly, limited mechanical properties, or excessive dissolution behavior. As a result, no clear consensus has been established regarding the most suitable phase ratio. Therefore, analyzing multiple phase compositions is essential to identify a formulation that provides a balance between stability, compatibility, and functional performance, particularly when doping is incorporated into the lattice to enhance bone regenerative potential.
Furthermore, trace elements such as iron (Fe), calcium (Ca), magnesium (Mg), and strontium (Sr) have been doped into the CaP structure to enhance the biological properties [9]. The trace elements influence bone metabolism, cellular activity, and bone regeneration at the defect site. By doping the elements into CaP, the trace elements were replacing Ca2+ or PO43− ions in the crystal lattice of CaP. Those dopants affect physicochemical properties and balanced ion release and influence biological responses such as osteogenesis, immunomodulation, antimicrobial, and angiogenesis.
Among these, strontium (Sr) has been used due to its strong affinity for bone tissue, where the majority of Sr in the body is localized in bones. Sr doping in CaP materials increases the structural and biological properties by changing the crystallinity, solubility, and degradation. The substitution of Ca2+ with Sr2+ also improves compatibility and bioactivity, thereby aligning material behavior with the physiological processes of bone remodeling [10], 11].
Strontium ions exhibit dual functionality, which promotes osteoblast proliferation and matrix synthesis while inhibiting osteoclast-mediated bone resorption [12]. This osteogenic and anti-resorptive synergy positions Sr-doped CaP as a promising biomaterial for enhanced bone regeneration, especially in conditions involving high osteoclastic [12], 13]. Sr-doped CaP materials have been widely studied despite most investigations focusing on single phases. The Sr2+ ion release behavior from BCP remains underexplored, despite its critical role in regulating osteogenesis, angiogenesis, and long-term bioactivity. Understanding how CaP phase composition influences Sr2+ release kinetics is essential for designing BCP powders that provide both early therapeutic effects and sustained biological support.
The formulation of CaP influences the resorption rate, ion release for specific defect environments, and healing duration. The ideal CaP with Sr doping enhances osteogenic activity and long-term bone regeneration, making it suitable for use in clinical conditions such as osteoporosis or large defect repair. The Sr-CaP powder under careful control of phase composition can be fabricated into injectable pastes, porous scaffolds, or implant coatings for further clinical applications.
Previous studies on Sr-CaP materials vary the Sr concentrations or evaluate only a single BCP composition, making it difficult to determine how phase composition alone influences Sr2+ ion release and biological compatibility. To address this limitation, the present study synthesizes Sr-HA, Sr-β-TCP and Sr-BCP at different formulations of HA/β-TCP while maintaining constant Sr doping levels at 5 mol%. This approach allows a comparison of how defined HA/β-TCP ratios affect morphology, crystallinity, and elemental distribution. The Sr2+ ion release concentration and cytocompatibility were analyzed to understand the biological performance of each composition. By varying the HA/β-TCP phase ratio, this study can identify which Sr-doped BCP formulation provides the most effective combination between structural stability, sustained ion release, and cellular viability as a bone graft material in bone regeneration.
Materials and methods
Preparation of BCP powder
The CaP powders were synthesized via a wet chemical precipitation method, followed by high-temperature calcination and incorporation of 5 mol% of Sr in all formulations. Single-phase Sr-HA and Sr-β-TCP powders were prepared by adjusting the precursor ratios and pH conditions. For Sr-BCP powders, the precursor concentration was further modified to obtain nominal HA/β-TCP phase ratios of 60:40 (BCP1), 30:70 (BCP2), and 20:80 (BCP3). Firstly, calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) and strontium nitrate (Sr (NO3)2) were dissolved in deionized water at 45 °C until a clear solution was formed. Diammonium hydrogen phosphate (NH4)2HPO4 was dissolved separately in deionized water at room temperature. The phosphate solution was then added dropwise to the calcium–strontium solution, with simultaneous addition of 1 M sodium hydroxide (NaOH) to adjust the pH. The mixture was maintained at 45 °C and stirred continuously for 3 h. After stirring, the solution was aged at room temperature for 24 h. The resulting slurry was filtered and washed multiple times with deionized water, followed by two ethanol washes, using centrifugation at 2,800 rpm for 5 min each. The precipitate was dried in an oven at 60 °C for 24 h and ground into a fine powder using a mortar. Finally, the powders were calcined at 1,000 °C for 2 h to eliminate organic residues and induce crystallization. After calcination, the sintered powders were gently milled using a mortar and pestle to obtain homogeneous Sr-CaP powders suitable for characterization.
Physicochemical characterization
The morphology of the synthesized Sr-CaP powders was analyzed using Scanning Electron Microscopy (SEM; S-4800, Hitachi, Japan) at 1,000 × magnification with an accelerating voltage of 15 kV. Prior to imaging, the samples were sputter-coated with gold to enhance conductivity. Energy-Dispersive X-ray Spectroscopy (EDX; S-4800, Hitachi, Japan) was conducted with a 15-s acquisition time and a 15 kV accelerating voltage to determine the elemental composition. Fourier Transform Infrared Spectroscopy (FTIR; Spectrum One, PerkinElmer, USA) was used to identify functional groups. The powder samples were mixed with potassium bromide (KBr) in a 1:100 ratio and pressed into pellets. Spectra were recorded in transmittance mode over the range of 4,000–400 cm−1. X-ray Diffraction (XRD; SmartLab, Rigaku Japan) was used to determine the phase composition and crystal structure of the powders. The XRD measurements were based on the parameters of Cu Kα radiation (λ=1.5406 Å) in θ–2θ configuration with a step interval of 0.02° and a scan rate of 2 min−1 over the 2θ range of 10–80°. The crystallite size (D) was estimated by the Debye-Scherrer Equation (1), from line broadening of the (211) reflection [14], 15]:
Where K=0.9 was the shape factor, λ=0.15406 nm (Cu Kα), β was the full width at half maximum (FWHM), and θ was the Bragg angle. The calculated FWHM and crystallite sizes were tabulated in Table 2.
Strontium release was evaluated using Sr-BCP1 as biphasic composition along with Sr-HA and Sr-β-TCP via Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES; Optima 8,000, PerkinElmer, USA). Powder samples were soaked in a pH 7.4 saline solution at 37 °C for various time intervals, and the ion concentrations were quantified using a standard calibration curve.
Cell culture
Human osteoblasts immortalized (iCell) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Gibco, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (both from Gibco, USA). Cells were maintained at 37 °C in a 5 % CO2 incubator. The medium was refreshed two to three times weekly based on nutrient depletion or pH shifts. Cells were used for assays at passage 5 when approximately 80 % confluency was reached.
MTT assay
The MTT assay (Sigma-Aldrich, USA) was used as an initial cytocompatibility screening method based on the indirect extract approach in accordance with ISO 10993-5. Leachates were prepared by immersing 2 mg of Sr-CaP powder in 2 mL of complete medium, corresponding to a solid-to-liquid ratio of 1 mg/mL, and incubated in a water bath at 37 °C for 24 h. The extracts were then collected, filtered through a sterile 0.22 μm polyethersulfone (PES) filter, and subsequently used for cell culture assays. Osteoblasts were detached by using 0.25 % trypsin-EDTA solution (Sigma-Aldrich, USA) and were centrifuged before being seeded into 96-well plates at a density of 5 × 103 cells/well. After 24 h of incubation for cell attachment, 150 µL of filtered leachate was added to each well and incubated for an additional 72 h. Subsequently, 50 µL of 0.5 mg/mL MTT solution (prepared in PBS) was added to each well and incubated in the dark for 4 h. The MTT solution was then removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals. After 15 min, absorbance was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, Multiskan FC, USA). Data were recorded and analyzed to quantify cell viability as a percentage relative to the untreated control cells. As shown in Equation (2), cell viability was calculated based on the absorbance of the treated sample compared to the absorbance of control cultures [16].
Results
SEM and EDX analysis
The surface morphology and microstructural characteristics of the synthesized Sr-CaP powders were examined using SEM to show the effect of varying phase compositions. Morphological analysis to understand particle distribution, agglomeration behavior, and crystallinity, which directly influence biological interactions and material performance in bone tissue engineering applications. In particular, the inclusion of Sr2+ and the ratio were expected to affect crystal formation and surface texture. Figure 1 presents the SEM micrographs of Sr-HA, Sr-βTCP, and Sr-BCP powders with varying ratios, providing insights into how phase composition influences the powder morphology.
SEM micrographs of Sr-doped calcium phosphate powders at × 10 k magnification. (a) Sr-HA, (b) Sr-β-TCP, (c) Sr-BCP1, (d) Sr-BCP2, and (e) Sr-BCP3.
Based on the SEM micrographs, Sr-HA exhibited an elongated, rod-like morphology with evenly distributed particles, though occasional agglomerates were observed. Sr-BCP2 demonstrated a similar morphology to Sr-HA despite its higher β-TCP content. In contrast, Sr-β-TCP and Sr-BCP3 displayed highly compacted fine particles with irregular shapes, uniformly distributed throughout the field. These powders showed a finer and more crystallized microstructure. Among all compositions, Sr-BCP1 presented a semi-dense and continuous microstructure, distinct from the β-TCP-rich samples. Its morphology appeared more balanced, suggesting a synergistic interaction between the HA and β-TCP phases. Although Sr-BCP1 contains a higher proportion of HA, its morphology differed from pure Sr-HA, likely due to increased β-TCP content affecting crystal packing and surface texture. Conversely, Sr-BCP2 appeared more like HA in morphology, potentially influenced by synthesis parameters or the dominant crystalline nature of HA despite its lower ratio. Clusters observed across all samples may indicate successful Sr incorporation into the CaP matrix. The elemental composition obtained from EDX was summarized in Table 1. The calculated Ca/P ratio ranged from 1.81 for Sr-HA to 1.34 for Sr-β-TCP, with the Sr-BCP samples showing values in between.
Results of the calculation of Ca/P ratio based on EDX analysis.
| Sample | Average weight percentage of Ca, % | Average weight percentage of P, % | EDX Ca/P ratio |
|---|---|---|---|
| Sr-HA | 19.094 | 10.564 | 1.81 |
| Sr-β-TCP | 17.088 | 12.759 | 1.34 |
| Sr-BCP1 | 17.660 | 10.240 | 1.72 |
| Sr-BCP2 | 13.967 | 8.687 | 1.61 |
| Sr-BCP3 | 13.406 | 8.821 | 1.52 |
FTIR analysis
The FTIR spectra of the synthesized powders are shown in Figure 2. The characteristic vibrational bands corresponding to both Sr-HA and Sr-β-TCP were observed across all compositions. The broad bands at 3,571 cm−1 and 3,645 cm−1 are attributed to the stretching and bending vibrations of hydroxyl (OH−) groups and adsorbed water, respectively. The symmetric stretching mode ν1 identified phosphate (PO43−) functional groups at 968 cm−1 and the bending mode ν2 at 473 cm−1. The asymmetric stretching vibrations ν3 appeared in the range of 1,009–1,095 cm−1, while bending modes ν4 were detected at 634, 603, and 570 cm−1. A distinct carbonate (CO32−) peak was observed at 1,421 cm−1 in the Sr-HA and Sr-BCP samples, indicating the possible substitution of carbonate into the apatite lattice during synthesis. The Sr-BCP samples with varying BCP composition exhibited the characteristic bands of both HA and β-TCP, confirming successful phase integration and retention of functional group identities from each component.
FTIR spectra of Sr-CaP powders (Sr-HA, Sr-β-TCP, and Sr-BCP1-3) showing the characteristic vibrational bands of hydroxyl (O–H) and phosphate (PO43−) groups of ν1, ν2, ν3, and ν4. The peak shifts indicate lattice distortion and possible Sr2+ substitution in the CaP lattice.
XRD analysis
The XRD patterns of the Sr-doped CaP powders were presented in Figure 3. By identifying peaks that correspond well to standard reference patterns from the Crystallography Open Database, it was confirmed that the presence of Sr-substituted HA and β-TCP phases. Major diffraction peaks for Sr-HA were located at 2θ values of 26.07°, 31.65°, 32.28°, 32.71°, 34.23°, 39.56°, 46.77°, 47.50°, and 49.63°. In contrast, Sr-β-TCP showed major peaks at 26.07°, 28.50°, 29.07°, 29.75°, 32.71°, 34.23°, 39.56°, 46.77°, and 49.56°. The Sr-BCP samples exhibited a combination of these characteristic peaks, with some overlapping reflections due to structural similarity, confirming the coexistence of both HA and β-TCP phases. Importantly, no additional peaks were detected, indicating the absence of secondary or impurity phases. The crystallite size and FWHM were calculated from the (211) reflection using the Scherrer equation as summarized in Table 2. The FWHM values increased from 0.070° for Sr-HA to 0.124° for Sr-β-TCP, while the corresponding crystallite size decreased with β-TCP. The result was consistent with previous studies in terms of lower crystalline order and higher solubility of β-TCP compared to HA [17]. The differences in peak intensity and width were due to variation in HA/β-TCP phase composition rather than Sr concentration. Among all compositions, Sr-BCP1 demonstrated the most intense and sharp diffraction peaks, signifying a higher degree of crystalline order. Sr-BCP2 showed slightly reduced peak intensity, while Sr-BCP3 exhibited the broadest and least intense peaks, reflecting lower crystalline order with increasing β-TCP content.
XRD patterns of Sr-CaP powders showing phase identification of Sr-HA, Sr-β-TCP, and Sr-BCP (1–3) compositions. The reference peaks are indicated (* for HA, ◆ for β-TCP). The coexistence of both HA and β-TCP shown in Sr-BCP confirms biphasic CaP formation after calcination at 1,000 °C for 2 h.
FWHM and crystallite size of Sr-HA, Sr-β-TCP, and Sr-BCP powders calculated from (211) using the Debye-Scherrer equation.
| Sample | FWHM, ° | Crystallite size, nm |
|---|---|---|
| Sr-HA | 0.070 | 108 |
| Sr-β-TCP | 0.124 | 70 |
| Sr-BCP1 | 0.083 | 104 |
| Sr-BCP2 | 0.090 | 95 |
| Sr-BCP3 | 0.105 | 83 |
MTT assay
The cytotoxicity of Sr-doped CaP powders was evaluated using the MTT assay, with absorbance measured at 570 nm after 24, 48, and 72 h of incubation. Significant differences in cell viability were observed between the samples and incubation times, as shown in Figure 4. All tested samples, such as Sr-HA, Sr-β-TCP, and Sr-BCP composites, exhibited an increase in absorbance, indicating progressive cell viability and proliferation over 72 h. These findings confirm that none of the Sr-incorporated CaP powders induced cytotoxicity during the testing period. Among the single-phase materials, Sr-β-TCP demonstrated the highest absorbance values at each time point, followed closely by Sr-HA, reflecting their favorable biological compatibility. Notably, all Sr-BCP compositions showed excellent biocompatibility, with increasing cell viability across all time points. The Sr-BCP1 sample has the highest viability overall from 24, 48, and especially at 72 h, showing enhanced cell proliferation due to the balance phase composition between HA and β-TCP. This composition provides structural stability and ion release conducive to cell compatibility.
Cell viability of osteoblasts treated with leachates of Sr-HA, Sr-β-TCP, and Sr BCP powders after 24, 48, and 72 h, analyzed using the MTT assay. Data are presented as mean ± SD (n=3). Statistical analysis was performed using two-way repeated-measures ANOVA followed by Tukey’s post-hoc test (*p<0.05, *p<0.01, *p<0.001).
ICP-OES analysis
The Sr2+ ion release from Sr-doped HA, β-TCP, and BCP1 powders over 1, 3, and 7 days is illustrated in Figure 5. Two-way ANOVA indicated the significant effects of biomaterial type, immersion timepoints, and Sr2+ ion release (p<0.0001). On day 1, Sr-BCP1 showed the highest Sr2+ ion released concentration (6.83 ± 0.04 mg/L), followed by Sr-β-TCP (4.80 ± 0.05 mg/L), and Sr-HA (3.11 ± 0.02 mg/L). The Sr-BCP1 concentration has significantly dropped around 42 % from (4.75 ± 0.02 mg/L) on day 3 to (3.94 ± 0.03 mg/L) of Sr2+ ion release on day 7. In contrast, Sr-β-TCP maintained a consistent concentration release of approximately 12 % between day 1 (4.80 mg/L) and day 7 (4.22 mg/L). This is due to the moderate solubility and ionic release of Sr from the β-TCP lattice. Besides that, Sr-HA exhibited the lowest Sr2+ concentrations and a gradual decline over the 7 days. The behavior shows the slow dissolution and high structural stability of the HA phase that inhibits the release of incorporated Sr2+ ions [18]. These analyses confirm that the Sr2+ release rate is also influenced by the material’s biphasic phase composition, enabling more controlled ion delivery for therapeutic applications in bone.
Sr2+ ion released concentration (mg/L) of Sr-HA, Sr-β-TCP, and Sr BCP powders measured by ICP-OES at 1, 3, and 7 days. Data are plotted as mean ± SD (n=3). Statistical analysis was performed using two-way ANOVA followed by Tukey’s post-hoc test (p<0.05).
Discussions
The rod-shaped morphology observed in the Sr-HA powders indicates anisotropic crystal growth along the c-axis, which has a feature of the hexagonal HA reported previously for undoped HA via surfactant-assisted routes [19]. The formation of nanofibrous and rod-like architectures in Sr-substituted HA demonstrates that Sr incorporation does not inhibit anisotropic crystal growth and allows the formation of highly ordered HA crystallites [20]. The β-TCP-rich powders (Sr-β-TCP and Sr-BCP3) showed fine, irregularly shaped granular particles that were more densely packed due to the phase transformation. This morphology shows the characteristic of β-TCP because of lower symmetry [21] and the tendency to form fragmented or irregular crystallites that occurred during the calcination process. This morphology corresponds to the Ca/P ratio, where Sr-HA (1.81) is due to the Ca-rich surface of the stable phase [22]. Sr-β-TCP showed the lowest (1.34), and the Sr-BCP powders presented intermediate values, possibly influenced by Sr incorporation and high temperature calcination. Besides that, the presence of Sr2+ doping causes structural effects because Sr2+ has a larger ionic radius than Ca2+. Substitution into the CaP lattice initiates local lattice distortions that can alter the nucleation and crystal growth directions. These lattice distortions influence the degree of crystallinity and may enhance particle agglomeration during calcination, where distorted grains tend to cluster together [23], 24].
The FTIR spectra in Figure 2 show the stretching and bending bands of the hydroxyl group (OH−) in HA around 3,570 cm−1 [25]. The OH− band was less intense and significant in Sr-substituted samples, indicating decreases in the density of OH− bonds within the unit cell due to Sr incorporation [26], 27]. In addition, literature reports that Sr substitution disrupts the local symmetry around Ca (II) sites, reducing the hydroxyl ion liberation and the stretching vibration intensity [28], 29]. The ν3(PO43−) functional group range for undoped HA and β-TCP typically lies between 1,045–1,144 cm−1 and 1,098–1,037 cm−1, respectively [30], 31]. In this study, Sr incorporation leads to a slight shift of these phosphate-related peaks to lower wavenumbers. The shift was modest and partially overlaps with HA and β-TCP bands but was correlated with lattice expansion of Ca2+ (∼0.99 Å) by Sr2+ (∼1.13 Å), which increases bond length and decreases vibration frequency, resulting in peak shifts [27]. Additionally, carbonate peaks observed in Sr-HA and Sr-BCP samples around 1,421 cm−1 suggest B-type carbonate substitution, where CO32− replaces PO43− in the HA lattice [26]. This substitution is enhanced by Sr incorporation, which induces local lattice distortion. Since β-TCP has less affinity for CO32− ions compared to HA, carbonate substitution is more commonly and stably integrated within HA-rich regions of BCP powders.
The diffraction peaks of Sr-HA at 26.07° (002), 31.65° (211), and 32.28° (112) matched well with standard HA reflections (JCPDS 09-0432). Meanwhile, the peaks at 33.02° (214), 34.23° (110) and 46.77° (205) align with significant Sr-β-TCP peaks (JCPDS 09-0169), validating the synthesis of Sr-CaP powders. Literature has shown that doping HA and BCP with Sr leads to a slight shift of XRD peaks toward lower 2θ angles, due to the substitution of Ca2+ by larger Sr2+ ions and resulting lattice expansion [25], 32], 33]. In this work, the Sr shift was very subtle and cannot be distinguished visually, as no undoped Sr reference is included. However, Joshi et al. and Baldassarre et al. reported that Sr substitution causes only a slight displacement towards lower 2θ values within (0.05–0.15)° for 2–8 mol% Sr [34], 35]. The consistent Sr content across samples confirms that the observed peak shifts were due to structural changes within the crystal lattice rather than variation in Sr concentration. The Sr was doped at 5 mol% in all BCP samples, ensuring that differences in peak sharpness and intensity were mainly attributed to BCP phase composition. The Sr-BCP powders were synthesized via a wet chemical precipitation method followed by sintering.
During high-temperature calcination, HA remained crystalline, while Ca-deficient HA (CDHA) partially decomposed into β-TCP, producing a BCP structure [36], 37]. Since β-TCP inherently has lower crystalline order and a smaller crystallite size, increasing its proportion led to broader and less intense diffraction peaks [37]. This trend aligns with previous findings that β-TCP shows broader peaks than HA, especially when Sr is incorporated, due to lattice distortion [38]. Although all samples contained a constant 5 mol% Sr doping, the varying HA/β-TCP phase compositions influenced XRD diffraction patterns. Samples with higher HA content, such as SrBCP1, displayed sharper and more intense peaks, indicating higher crystalline order and reduced HA decomposition during sintering [36]. In contrast, SrBCP2 showed β-TCP-dominant features, though some sharp peaks from residual HA remained, along with moderate formation of Sr-β-TCP. Meanwhile, Sr-BCP3, which contains the highest β-TCP content and shows the smallest crystallite size, displayed the lowest peak intensities, reflecting increased lattice disorder. These results confirm that partial HA to β-TCP transformation occurred during calcination, and Sr substitution slightly influenced the lattice structure by changing the overall phase assemblage.
As the control of the experiment, Sr-β-TCP and Sr-HA of single-phase show good cell viability, which supports the synergistic effect and gives a better response for different phase compositions of Sr-BCP powder. All the samples with Sr-doped BCP exhibited high absorbance values and did not show any toxicity over time. As the β-TCP composition increases, the material becomes more soluble than HA-rich compositions as β-TCP is known to dissolve faster than HA under physiological conditions [39]. The higher solubility can promote early ion exchange and increased ionic strength in the surrounding medium. Although such changes do not result in acute cytotoxicity, they may adversely affect cellular metabolic activity during prolonged exposure, which is consistent with the comparatively lower viability observed for Sr-BCP2 and Sr-BCP3. In addition, the reduced crystalline and higher defect density associated with higher β-TCP may further accelerate dissolution and reduce the surface stability during incubation [40], thereby influencing cell-material interactions. Among the biphasic compositions, Sr-BCP1 ensures homogenous phase distribution with higher crystalline order, providing improved structural stability and supporting reliable Sr2+ releases for bone regeneration [36]. Moreover, the Sr2+ concentrations remain within the biologically favorable window, making it a better composition to elucidate the Sr2+ release mechanism without drawbacks arising from microstructural instability or cytotoxicity. Also, the Sr release of BCP1 supports that the increasing release, especially faster in the early phase, supports early osteoblast activity, which enhances the osteoblast viability [38]. The sustained, non-toxic level of Sr further supports cell attachment and mitochondrial activity.
β-TCP is typically a more soluble phase than Sr-HA; however, soaking in NaCl solution, which lacks phosphate ions, limited β-TCP dissolution because phosphate ions facilitate its breakdown. As a result, Sr2+ release from Sr-β-TCP remained moderate and relatively stable over 7 days, rather than showing a rapid burst as would be expected in phosphate-containing media such as PBS or SBF. The NaCl medium creates a more controlled dissolution environment by preventing calcium phosphate reprecipitation, but it also results in a slower dissolution for Sr-β-TCP under these ionic conditions. In contrast, Sr2+ released from Sr-HA remains the lowest throughout due to the high crystallinity and structural stability of the HA lattice, which restricts diffusion of Sr2+ ions out of the lattice. Sr-BCP1, with the highest initial release, demonstrates the biphasic nature of the material. Early Sr2+ liberation is dominated by the more soluble β-TCP phase, whereas the subsequent release is increasingly controlled by the stable HA phase.
The Sr2+ ion release in this study exhibited a burst on day 1, followed by a slight decrease from day 3 to day 7. This pattern has been reported before and is known as a biphasic release. Zhao et al. stated that Sr-doped bone scaffolds release Sr2+ in a large amount at the early stage due to loosely attached surface ions, and release slows down as the available ions decrease [41]. He et al. also mentioned that HA becomes less stable and transforms into β-TCP when Sr is doped into the structure of calcium phosphates. This process provides a balance between dissolution and reprecipitation, where Sr2+ ions form into a stable phase [18]. Ye et al. found a two-way release pattern in Sr-CaP/PCL/CS membranes with fast release at first, followed by a slower and sustained release related to the dissolution behaviour of the material [42]. In the study, using NaCl solution without refreshing the medium could cause early ion saturation and Sr2+ release. Therefore, the results correspond to the previous findings and suggest that the drop of Sr2+ ion is due to the combination of phase structure, ion dynamics, and test conditions.
However, as the Sr2+ concentration in the soaking solution decreased between Day 1 and Day 3, cell viability, as indicated by the MTT assay, continued to increase from Day 1 to Day 3. This phenomenon may be attributed to early-stage Sr2+ uptake by cells or reprecipitation of Sr into mineral phases, as previously reported by [43], who noted that cell-mediated mineralization and Sr incorporation into calcium phosphate matrices can reduce measurable ion release despite ongoing biological effects [43]. Similarly, Mohapatra and Rautray reported that 5 mol% Sr-substituted BCP scaffolds supported osteoblast proliferation even under diminishing Sr2+ concentration, reinforcing the idea that early-phase Sr release initiates and sustains osteogenic responses [27]. A systematic review by Côrtes et al. emphasized that burst Sr2+ release, followed by gradual depletion, does not impair cell viability, as the initial release phase is sufficient to activate osteogenic signalling pathways [44]. These findings align well with the observed decoupling between Sr2+ release and MTT data in the present study. Moreover, the Sr2+ ion does not induce cytotoxicity even at 5 mol% doping. The presence of BCP powder reduces the potential toxicity of metals [27]. Sr further enhances the osteogenic potential of CaP powders in general through Sr ion substitution and does not induce cytotoxicity, supporting its biocompatibility for bone tissue engineering [45], 46].
Modulating the BCP composition and Sr doping enhances a biologically suitable environment that supports osteoblast viability and attachment, indicating a compatible material. The Sr’s osteogenic effect is dose-dependent, which low to moderate concentrations, promotes osteogenic marker expression and calcium deposition, while higher doses may trigger apoptosis in human adipose-derived stem cells (hASCs) [47]. In this study, the use of 5 mol% Sr resulted in non-toxic ion release and high cell viability, consistent with Oliveira et al. and Moghanian et al., showing improved compatibility, and the study further indicates may modulate into osteogenic signaling by Sr doping [48], 49].
This study demonstrates the novelty of using a direct comparison of Sr-doped HA, β-TCP, and three different biphasic ratios to establish how phase composition influences the relationship between crystallinity, Sr2+ ion release, and cytocompatibility. The larger ionic radius of Sr2+ compared to Ca2+ causes lattice expansion and structural distortion in Sr-doped HA and β-TCP [24], 33]. The structural disruption, shown through slight PO43− vibrational shifts, increased FWHM values, and reduced crystallite sizes, is observed in the high β-TCP composition, indicating possible structural disorder and higher solubility [23]. Such vibrational changes weaken the lattice cohesion and increase local free energy to promote enhanced dissolution [50]. In addition, the more soluble β-TCP releases the Sr2+ rapidly at early timepoints, while the highly crystalline HA provides slower and sustained release. Sr-BCP1 exhibits an optimal dissolution behavior, where the Sr2+ release concentration corresponds with the highest cell viability observed without causing any excessive material breakdown. Therefore, BCP1 composition is particularly promising due to its synergistic microstructure and controlled Sr2+ release.
Conclusions
In this study, 5 mol% of Sr-doped CaP (Sr-HA, Sr-β-TCP, and Sr-BCP) with HA/β-TCP ratios of 60:40, 30:70, and 20:80 were synthesized by wet chemical precipitation followed by calcination. The phase composition strongly influenced the microstructure, elemental distribution, crystallinity, Sr2+ release, and cytocompatibility. Among all compositions, Sr-BCP1 showed the most balanced combination of structural stability for HA and higher solubility of β-TCP. FTIR and XRD analyses confirmed the incorporation of Sr and high crystallinity, while MTT assays indicated enhanced cytocompatibility with the highest cell viability for Sr-BCP1. This biphasic composition yielded compact and highly crystalline particles, characterized by an initial burst of Sr2 ion release, followed by sustained release. These findings highlight the strong potential of Sr-BCP1 powder as a promising development prospect for future bone replacement grafts. Clinically, such controlled-release powders are ideal for early osteoinduction, followed by long-term regeneration support. This enables Sr-BCP1 to have a more balanced use in bone defects or orthopedic implants that require both initial bioactivity and sustained performance. Limitations of this study include the use of static in vitro conditions, which may not fully capture dynamic biological responses. Future work should incorporate simulated body fluid immersion and explore osteogenic differentiation studies to further validate the long-term biological performance of these materials.
Funding source: Universiti Teknologi Malaysia
Award Identifier / Grant number: Q.J130000.3023.04M47
Award Identifier / Grant number: R.J130000.7623.4B875
Acknowledgments
This work was supported by RUG-UTM Matching Grant Q. J130000.3023.04M47 and R.J130000.7623.4B875 from Universiti Teknologi Malaysia, Malaysia.
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Research ethics: Not applicable.
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Informed consent: Not applicable.
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Author contributions: Ravathi Marathandi – Writing, Methodology, Investigation. Adlisa Abdul Samad – Investigation. Murfiqah Taufiqiah Mohd Amin – Investigation. Zatul Faqihah Mohd Salaha – Investigation. Norhana Jusoh – Writing, Methodology, Resources. Ardiyansyah Syahrom – Resources. Dian Agustin Wahjuningrum –Resources.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The author states no conflict of interest.
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Research funding: This work was supported by RUG-UTM Matching Grant Q.J130000.3023.04M47 and R.J130000.7623.4B875 from Universiti Teknologi Malaysia, Malaysia.
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Data availability: Not applicable.
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