Abstract
The present study aims to investigate the contribution of two biologically important cations, Mg2+ and Sr2+, when co-substituted into the structure of hydroxyapatite (Ca10(PO4)6(OH)2, HA). The substituted samples were synthesized by a hydrothermal method that involved the addition of Mg2+ and Sr2+ containing precursors to partially replace Ca2+ in the apatite structure. Four co-substituted HA samples with different concentrations of Mg2+ and Sr2+ ((Mg + Sr)/(Mg + Sr + Ca) = 30%) were investigated, and they were compared with pure HA. Experimental results showed that only a limited amount of Mg (Mg/(Mg + Ca + Sr) < 14%) could successfully substitute for Ca in HA. In addition, Mg substitution resulted in reduced crystallinity, thermal stability and lattice parameters of HA. In contrast, Sr could fully substitute for Ca. Furthermore, the addition of Sr increased the lattice parameters of HA. Here, we obtained the cation leach liquor by immersing the prepared samples in a culture medium for cell experiments. The in vitro study showed that 10Mg20Sr promoted better MG63 cell attachment, proliferation and differentiation than HA. Thus, the presence of an appropriate proportion of Mg and Sr could play a significant role in the increased biocompatibility of HA.
Introduction
Hydroxyapatite (Ca10(PO4)6(OH)2, HA) has been widely used in biomedical applications because of its chemical similarity to the mineral phase of human hard tissues as well as its excellent biocompatibility, bioactivity and osteoconductivity.1–4 However, owing to its poor in vivo degradation rate and low osseointegration ability, an increasing number of studies are now focusing on the modification of HA in order to improve its biological properties. As is well known, biological apatite contains trace ions such as Na+, Mg2+ and Zn2+, which are essential in biological processes. 5 Therefore, one of the most effective and feasible methods for modifying HA is to introduce extrinsic ions such as CO32−, 6 F−, 7 Zn2+, 8 and Co2+, 9 into its lattice; Mg2+ and Sr2+,10–13 are particularly preferable, being main-group elements along with Ca.
Strontium (Sr) is a bone-seeking element, of which 98% in the human body can be found in the skeleton. 14 In vitro experiments have proved that Sr not only increases the osteoblast-related gene expression and alkaline phosphatase (ALP) activity in mesenchymal stem cells (MSCs) but also inhibits the differentiation of osteoclasts.15,16 Sr has also been shown to stimulate bone formation and decrease bone resorption in both animal studies and clinical trials.17–19 Magnesium (Mg) is another essential element of great interest. It has been confirmed that Mg2+ plays a key role in bone metabolism, as it affects osteoblast and osteoclast activity, and thus, bone growth.20,21 Mg deficiency in bones has been suggested as a possible risk factor for osteoporosis in humans. Several magnesium-based cements have been approved by the FDA, while magnesium-based degradable stents are in advanced clinical trials and are being actively researched in orthopaedics. 22 For these reasons, Sr- and Mg-substituted HA have attracted considerable interest from researchers and clinicians.
Although many articles on the chemico-physical and biological properties of Sr- and Mg-substituted apatites have already been published, only a few studies have focused on careful investigation of Sr/Mg-co-substituted HA. A recent study by Kannan et al. 23 reported the synthesis and structural characterization of Sr/Mg-co-substituted β-tricalcium phosphate (β-Ca3(PO4)2, β-TCP). In another study, Aina et al. 24 investigated the contribution of Mg2+ and Sr2+ to the structure and chemico-physical properties of HA. Their results showed that Mg plays a major role in destabilising the phase compositions of the substituted apatites and that increased Mg concentration is related to the increased proportion of the β-TCP phase. However, to the best of our knowledge, no study has reported the co-substitution of Sr and Mg in HA by a hydrothermal method. Furthermore, existing biological studies on Sr/Mg-co-substituted HA are inadequate. Considering the importance of Sr and Mg, we report herein the synthesis, characterization and in vitro biological evaluation of Sr/Mg-co-substituted HA.
Experimental
Preparation of Mg/Sr-co-substituted HA samples
All the samples were synthesized by a one-step hydrothermal method as previously reported. 25 In summary, Ca(NO3)2ċ4H2O, Mg(NO3)2ċ4H2O, Sr(NO3)2, and Na3PO4 were used as the Ca, Mg, Sr, and P sources, respectively. For pure HA nanocrystallite, Ca(NO3)2ċ4H2O and Na3PO4 with a molar ratio of 10:6 were each dissolved in 80 mL of deionized water. Then, the Ca(NO3)2ċ4H2O solution was added dropwise into the Na3PO4 solution. After rigorous agitation for 10 min, the suspension was hydrothermally treated at 150℃ for 5 h. The precipitates were washed, respectively, with deionized water and dehydrated absolute ethanol twice. Then, the product was placed in an oven and heated at 70℃ for 24 h for drying. Subsequently, the dried powder was manually ground with a corundum mortar. Sr/Mg-co-substituted HA nanocrystallite with different Sr and Mg concentrations were synthesized by replacing Ca(NO3)2ċ4H2O with Sr(NO3)2 and Mg(NO3)2ċ4H2O, respectively. Four co-substituted HA samples were prepared, denoted by 5Mg25Sr, 10Mg20Sr, 20Mg10Sr and 25Mg5Sr. The samples are distinguished by the substitution ion concentration. For example, 5Mg25Sr indicates that the sample has a Mg/Sr/(Ca + Mg + Sr) concentration of 5/25 mol.%. Here, it is important to note that the X-ray powder diffraction (XRD) patterns showed a characteristic amorphous peak when the content of Mg was higher than 30% in our previous study. 25 Therefore, the concentrations of Mg2+ and Sr2+ ((Mg + Sr)/(Mg + Sr + Ca) = 30%) were selected in this experiment.
Characterization of the samples
Phase analysis of the synthesized powders was conducted via XRD and X-ray photoelectron spectroscopy (XPS). The XRD patterns were provided by a Bruker D8 ADVANCE X-ray diffractometer equipped with graphite-monochromatized Kα radiation (λ = 1.5418 Å). The diffractometer was operated at 40.0 kV and 30.0 mA with a 2θ range of 10–90° (step size, 0.02) and exposure of 50 s. XPS was conducted in a vacuum chamber at a base pressure of ∼3.5 × 10 − 8 Pa with a beam spot diameter of ∼500 µm (ESCALAB 250, VG Thermo, UK).
The atomic concentrations of elements (Ca, Mg, Sr and P) in the samples were quantified via inductively coupled plasma emission spectroscopy (ICP, VISTA-MPX).
The lattice parameters (c and a) were calculated from peaks (0 0 2) and (2 1 1), respectively, using the standard hexagonal close packed unit cell plane spacing relationship:26
The degree of crystallinity, corresponding to the fraction of crystalline phase present in the examined volume, was evaluated as:
27
In order to examine the functional groups of the obtained powder, infrared spectra of all samples were obtained using an infrared Fourier-transform spectrometer (FT-IR, Bruker TENSOR 27, Germany) in the range of 4000–400 cm−1. The powder was ground with KBr in the proportion of 1/150 (by weight) and pressed to a wafer of diameter of 13 mm using a hand press.
The powder morphology was respectively observed via scanning and transmission electron microscopy (SEM; TEM). An accelerating voltage of 5 kV was chosen for SEM analysis and the micrographs were captured using secondary electrons collected with an in-lens detector. TEM images were acquired using a JEM-2100 F microscope at 200 kV. High-resolution transmission electron microscopy (HRTEM) imaging and selected-area electron diffraction (SAED) patterns were obtained. In order to investigate the dopant distribution within the co-substituted HA, energy-dispersive X-ray spectrometer (EDS) analysis and elemental mapping were performed using a TEM equipped with an EDS analyser, operated at 200 kV.
Thermogravimetric analysis was carried out using TGA/DSC 1 (METTLER TOLEDO, Switzerland) to analyse the thermal behaviour of the powder during heating. The sample weight was 30 mg and heating was performed in an alumina crucible at a rate of 10℃ min−1 up to 1000℃.
Cell experiments
Preparation of biomaterial extracts
Cation leach liquor (CLL) was prepared according to methods described by the International Organization for Standardization (ISO 10993-12). Briefly, the ratio of each sample to the culture medium was 100 mg/mL. Each sample was incubated at 37℃ for three days in complete α-MEM (Hyclone; USA). The extracts were analysed via ICP to determine the elemental concentrations of Ca, Mg and Sr. More specifically, HA was collected with Ca2+ = 2.43 ± 0.17 mM and Mg2+ = 0.78 ± 0.09 mM; 5Mg25Sr was collected with Ca2+ = 2.34 ± 0.14 mM, Mg2+ = 0.93 ± 0.11 mM, and Sr2+ = 0.23 ± 0.02 mM; 10Mg20Sr was collected with Ca2+ = 2.27 ± 0.15 mM, Mg2+ = 1.17 ± 0.13 mM, and Sr2+ = 0.16 ± 0.015 mM; 20Mg10Sr was collected with Ca2+ = 2.54 ± 0.18 mM, Mg2+ = 1.84 ± 0.19 mM, and Sr2+ = 0.07 ± 0.008 mM; and 25Mg5Sr was collected with Ca2+ = 2.67 ± 0.16 mM, Mg2+ = 2.08 ± 0.22 mM, and Sr2+ = 0.02 ± 0.003 mM.
Cell culture
Osteoblast-like MG-63 cells (ATCC-1427, Experiment and Technology Center, China Medical University, Shenyang, China) were cultured at 37℃ in 5% CO2 and the aforementioned biomaterial extracts containing 10% foetal bovine serum (Invitrogen, Carlsbad, CA). The culture medium was changed every 2–3 days.
Cell morphology
For SEM observation, after one and three days of incubation, the cells were gently rinsed with PBS three times to remove unattached cells, and then fixed with 2.5% glutaraldehyde in PBS for 60 min at room temperature. After dehydration in graded series of ethanol (50%, 60%, 70%, 80%, 90% and 100%) for 20 min each and drying in hexamethyldisilazane (HMDS) solution, the samples were sputter-coated with gold.
Cell proliferation, adhesion and distribution
MTT assay was performed to estimate the cell proliferation. MG-63 cells were seeded in a 12-well plate and incubated for 1, 3 and 7 days. After the selected incubation periods, the samples were washed with phosphate-buffered saline (PBS) and transferred to a new 12-well plate. Then, 300 µL of the culture medium and 300 µL of MTT (3-[4, 5-dimethylthiazol-3-yl]-2, 5-diphenyl tetrazolium bromide) reagent (5 mg/mL in PBS, Sigma, USA) were added to each well. After 4 h of incubation in a 5% CO2 incubator at 37℃, the medium was replaced with 500 µL of dimethyl sulfoxide to dissolve formazan. The plate was shaken for 10 min, and then the solution in each well was transferred to a 96-well ELISA plate. The optical density (OD) of the dissolved solute was measured using an ELISA reader (Tecan, Austria) at 570 nm (n = 9 in each group). The common OD value of the blank group (n = 9) was subtracted from the OD value of each group at each time point. The blank group was treated with the same procedures and incubated for the same time as the above groups.
Representative fluorescence microscopy images of the MG63 cells were obtained to evaluate the cell adhesion and distribution. After seeding for one and three days, the samples were rinsed with PBS (Sigma, USA) and stained with Hoechst #33342, which stains the cell nuclei (excitation max., 346 nm; emission max., 460 nm; Sigma, USA; 5 µg/mL of PBS). This dye was applied for 2 h at room temperature. The microscopy images were acquired using an IX-51 microscope equipped with a digital camera (DP-70, Olympus, Japan).
ALP activity
MG-63 cells were seeded in a 12-well plate (approximately 10,000 cells/cm2) with 1 mL of the biomaterial extracts containing 10% foetal bovine serum. After culturing for 3, 7 and 14 days, the cultural medium was carefully removed and the plates were gently washed twice with PBS. Then, 500 µL of 0.2% (v/v) Triton X-100 (Sigma, USA) was added to each well. After lysis in a 5% CO2 incubator for 2 h, the solutions were transferred to a micro-centrifuge tube and frozen at −80℃ for 2 h. Three freeze–thaw cycles were completed to homogenize the solutions. Then, 3 mL of Coomassie Brilliant Blue staining solution, 0.6 mL of cell lysis solution, and 0.4 mL of double distilled water were mixed and allowed to stand for 10 min. The OD value of the mixed solution was measured using an ELISA reader (Tecan, Austria) at 595 nm. The protein concentration of the cell lysis solution was calculated on the basis of a standard curve obtained using bovine serum albumin as a standard. Then, 100 µL of the cell lysis solution and 100 µL of 25 µg/mL p-nitrophenyl phosphate disodium salt (PNPP) were added into each well of a 96-well plate (8 wells in each group). After 30 min, 50 µL of 3 mol/L NaOH was added to terminate the reaction. In the blank group, 100 µL of 0.2% (v/v) Triton X-100, 100 µL of 25 µg/mL PNPP and 50 µL of 3 mol/L NaOH were added. The OD values were measured at 405 nm. The OD per milligram of protein was calculated.
Osteogenesis-related gene expressions
The expressions of osteogenesis-related genes were evaluated on the basis of a real-time polymerase chain reaction (real-time PCR). The cells were seeded with 2 × 104 cells/well and cultured for 3, 7 and 14 days. The total RNA was isolated using the TRIzol reagent (Gibco). Here, 1 mg RNA from each sample was reversed transcribed into complementary DNA (cDNA) using the PrimeScript™ RT reagent kit (TaKaRa). The forward and reverse primers for the selected genes were the same as those described in Zhao et al. 28 The expressions of osteogenesis-related genes, including ALP, runt-related transcription factor 2 (Runx2) and osteocalcin (OCN), were quantified on the basis of real-time PCR (Bio-Rad iQ™5 multicolour real-time PCR detection system) with SYBR® Premix Ex™ Taq II (TaKaRa). Data analysis was carried out using the iQ™5 optical system software version 2.0 (Bio-Rad). The relative expression levels for each gene of interest were normalized to the level of the housekeeping gene GAPDH.
Statistical analysis
At least three samples were employed for each analysis. All data were statistically analysed using one-way analysis of variance (ANOVA) and Tukey’s post hoc comparison to evaluate statistically significant differences between the sample groups. The quantitative data was presented as mean ± standard deviation (SD). The value p ≤ 0.05 was considered significant. Dedicated software such as DigitalMicrograph 365 (for PC) (Gatan, Inc., Pleasanton, CA) and MicroCall Origin were employed for image processing and mathematical data computation.
Results
Results of sample characterization
The typical XRD patterns of all synthesized samples are shown in Figure 1. It is evident that the diffraction peaks shift to higher 2θ values and are considerably broadened and weakened with Mg addition, indicating a decrease in lattice parameters and crystallinity. The effect of Sr addition is just the opposite. This is due to the larger ionic radius of Sr (1.13 Å) and smaller ionic radius of Mg (0.72 Å) than that of Ca (0.99 Å).
22
XRD patterns of as-prepared pure and co-substituted HA samples.
Lattice parameters and crystallinity of the prepared samples.
Elemental composition of the synthesized samples measured via ICP (M = Ca + Mg + Sr).
Figure 2 shows the XPS spectra of the synthesized HA and various co-substituted HA samples. HA shows only Ca, P and O XPS peaks, whereas co-substituted HA samples with different Mg and Sr concentrations show the signals of Mg and Sr peaks. The high-resolution Ca spectra (Figure 2b) illustrate that the intensities of Ca peaks decline dramatically with the addition of Mg and Sr. Furthermore, the high-resolution Mg (Figure 2c) and Sr (Figure 2d) spectra indicate that the intensities of the Mg and Sr peaks are proportional to the element concentrations.
(a) XPS spectra of the as-synthesized samples. (b) High-resolution Ca spectra of all samples. (c) High-resolution Mg spectra of co-substituted HA samples. (d) High-resolution Sr spectra of co-substituted HA samples.
Figure 3 shows the FT-IR spectra of the prepared samples. The IR spectra of each of the prepared powders are characteristic of phosphate compounds. The band at 963 cm−1 is attributed to the υ1 vibration peak of FT-IR spectra of the prepared samples.
Figure 4 shows the morphology of synthesized samples with different Mg and Sr concentrations. The average size of HA was around 50 nm. HA crystallites formed greater agglomerates with Sr/Mg-co-substitution (Figure 4a–d). The agglomerates became bigger and formed a fusiform cluster structure with the increase in Mg concentration, which indicated that Mg plays a major role from the structural perspective. These agglomerates showed good dispersion.
SEM images of the synthesized samples: (a) 5Mg25Sr, (b) 10Mg20Sr, (c) 20Mg10Sr and (d) 25Mg5Sr. Scale bars: 500 nm.
Figure 5 shows the TEM and SAED patterns of the synthesized samples. It can be clearly seen that the morphology of pure HA consists of regular nanoparticles with good crystallinity (Figure 5a,b), whereas the co-substituted samples become irregular and form agglomerates (Figure 5c–f). When the Mg concentration is below 10%, each grain can be clearly distinguished (Figure 5c,d). However, the fine crystals form spindly aggregates with an average size of 200–400 nm and are difficult to distinguish in the case of samples having an Mg concentration of higher than 20% (Figure 5e,f). In addition, an increase in the Mg concentration leads to dimming of the corresponding SAED patterns, indicative of decreasing crystallinity. All the TEM results are in agreement with the SEM results.
TEM and SAED patterns of the synthesized samples: (a) pure HA, (b) HRTEM of pure HA, (c) 5Mg25Sr, (d) 10Mg20Sr, (e) 20Mg10Sr and (f) 25Mg5Sr. Scale bars: (a) and (c–f), 100 nm; (b), 5 nm.
The elemental distribution of the dopant in the co-substituted HA via EDS is shown in Figure 6. The elemental maps show homogeneous distribution of all elements in each sample. However, the dot densities of Ca and P are higher than those of Mg and Sr owing to the higher concentration and atomic number of these elements.
30
Thus, the dot densities of Sr and Mg are proportional to their concentrations.
Elemental mapping of the synthesized samples: (a) 5Mg25Sr, (b) 10Mg20Sr, (c) 20Mg10Sr and (d) 25Mg5Sr.
The TG plots shown in Figure 7 illustrate report the weight loss along the investigated temperature range for the precipitated powders with different concentrations of added Mg and Sr. Significant weight loss up to approximately 400℃ was due to the loss of adsorbed (up to 200℃) and lattice water.
31
The loss of carbonate ions could contribute to the total weight loss above 550℃.
32
TG curves of the prepared samples.
Cell adhesion, distribution and proliferation and ALP activity
Cell adhesion and distribution after one and three days of incubation are shown in Figure 8(a–j). Obviously, the cells differed in number for materials with different chemical compositions. At each time interval adopted in this study, the number of adherent cells on the HA, 10Mg20Sr and 20Mg10Sr samples is larger than that on the 5Mg25Sr and 25Mg5Sr samples. In addition, the number of cells on the 10Mg20Sr sample is slightly larger than that on the HA and 20Mg10Sr samples but the statistical difference is small (Figure 8k). The results from the ALP activity test indicate that MG63 cells cultured on the 10Mg20Sr samples have a significantly higher ALP activity than that on other samples (Figure 8 l). Moreover, MG63 cells cultured on the 5Mg25Sr, 20Mg10Sr and 25Mg5Sr samples show lower ALP activity than those on HA in the early stages. However, 5Mg25Sr and 20Mg10Sr show higher ALP activity than HA after incubation for 14 days (not significantly).
MG63 cell adhesion and distribution after 1 (a–e) and 3 (f–j) days of incubation. (a, f) pure HA, (b, g) 5Mg25Sr, (c, h) 10Mg20Sr, (d, i) 20Mg10Sr and (e, j) 25Mg5Sr. Scale bars, 200 µm. (k) Cell proliferation after 1, 3 and 7 days of incubation measured by colorimetric MTT assay. (l) ALP activity of MG63 cells after 3, 7 and 14 days of incubation. Statistically significant differences (*p < 0.05, **p < 0.01).
Cell morphology and differentiation
Figure 9(a–j) shows the morphologies of MG63 cells cultured on different samples after one and three days of incubation. The cells attach and spread well on all samples with no significant differences after culturing for one day. However, the cells grow and spread better on the HA, 10Mg20Sr and 20Mg10Sr samples after incubation for three days, almost completely covering the 10Mg20Sr and 20Mg10Sr surfaces and starting to grow to confluence.
SEM morphologies of MG63 cells cultured on different samples for 1 (a − e) and 3 (f − j) days. (a, f) HA, (b, g) 5Mg25Sr, (c, h) 10Mg20Sr, (d, i) 20Mg10Sr and (e, j) 25Mg5Sr. Scale bars, 100 µm. Gene expressions of MG63 cells cultured on different samples after incubation for 3, 7 and 14 days: (a) ALP; (b) Runx2; (c) OCN. Statistically significant differences (*p < 0.05, **p < 0.01).
The expressions of osteogenesis related genes, including ALP, Runx2 and OCN, in cells cultured on different samples for 3, 7 and 14 days are quantified by real-time PCR and the results are shown in Figure 9(k–m). In general, gene expressions for all samples show a time-dependent pattern. The gene expressions of ALP, Runx2 and OCN on 10Mg20Sr are the highest among the five groups at any point of time. In the early stages of incubation, the gene expressions on 5Mg25Sr, 20Mg10Sr and 25Mg5Sr are lower than those on pure HA, whereas the gene expressions on 5Mg25Sr and 20Mg10Sr are higher (not significantly) than those on pure HA after 7 and 14 days of incubation. The results indicate that Sr/Mg-co-substituted HA samples can stimulate the gene expressions of MG63 cells, especially for 10Mg20Sr, which presents a sustainable and significant promoting effect.
Discussion
The incorporation of ions into calcium phosphate cements is of great importance because many biological tissues, such as bone and teeth, are composed of apatite mineral phase containing tiny amounts of other elements such as magnesium and strontium as well as silicon. 33 In this study, a series of Sr/Mg-co-substituted apatites were synthesized by a hydrothermal process. The best advantage of the hydrothermal method is simple and efficient. Meanwhile, the crystallinity of the samples synthesized by one-step hydrothermal method is lower than that of prepared by heat treating, which had a good crystallinity (compared Figure 1 and Figure S1). Thus, the solubility of HA synthesized by hydrothermal method is higher than that of well crystallized HA. The higher solubility of HA would be more suitable for orthopaedic application as biodegradable bone grafts.
The results, which were consistent with previous studies,5,34 indicated that the lattice parameters decrease with the addition of Mg whereas they increase with the addition of Sr. This can be attributed to the larger (smaller) atomic radius of Sr (Mg) than that of Ca. Furthermore, the crystallinity decreases with the addition of Mg. Kanzaki et al.35,36 studied the effect of Mg2+ and Zn2+ on the growth kinetics of a HA c-plane in pseudophysiological solutions and found that these ions inhibited the growth rate of the c-plane via absorption at the kink sites of two-dimensional islands. Moreover, Bigi et al. 37 found that magnesium inhibited the crystallization of HA by reducing the Ca/P molar ratio and crystal sizes of apatite. Owing to the unique functions of Mg, its effects on both the structure and the stability of co-substituted HA were greater than those of Sr. An interesting aspect of this study is that uniform agglomerates were obtained with the addition of Mg. Further, the size of the agglomerates could be controlled by varying the Mg concentration.
The in vitro experimental results showed that the co-addition of Mg and Sr to HA could have important effects on cell growth. Both cell proliferation and bioactivity increased on 10Mg20Sr but decreased on 25Mg5Sr, as compared to HA. These results suggest that the co-addition of Mg and Sr has both advantages and disadvantages. Appropriate Ca, Mg and Sr concentrations can enhance cell growth; otherwise, the cell growth would be reduced. Previous studies have shown that Sr or Mg doping enhance cellular attachment and proliferation on HA.38,39 However, despite these positive effects, some studies have shown that the effects of Sr and Mg are complex and dose-dependent. 22 Studies based on primary osteoblasts isolated from foetal rat calvaria have shown that, at a low concentration (1 mg/L Sr in the culture medium), nodule formation is reduced while mineralization is not impaired; at an intermediate concentration (5 mg/L), there is no effect on either nodule formation or mineralization; and at high concentrations (20–100 mg/L), mineralization is reduced while there is no effect on nodule formulation. 40 Moreover, several in vivo studies have shown that Sr could cause rickets and induce defective bone mineralization by disrupting intestinal calcium absorption.41–44 With regard to biomedical applications, the most important issue is to achieve a trade-off between cell proliferation and cell differentiation. The cell differentiation results showed that the addition of Mg and Sr is beneficial for long-term cell differentiation, especially for 10Mg20Sr, which showed an accelerating effect throughout the experiment. Thus, it can be inferred that cations have a dose-dependent synergetic effect on cell growth. An appropriate proportion of Mg and Sr could play a significant role in the increased biocompatibility of HA. However, the detailed mechanism needs further investigation.
Conclusion
In this study, a series of Sr/Mg-co-substituted HAs were synthesized by a hydrothermal method. Magnesium was the destabilising factor in the phase compositions of the co-substituted HA, and increased Mg concentration was related to decreased crystallinity, thermal stability and lattice parameters of HA. Sr showed full substitution of Ca, whereas only partial substitution of Ca could be achieved by Mg. In vitro studies with MG63 cells cultured in CLL, which was derived from synthesized samples incubated in the culture medium, indicated that all samples showed good biocompatibility after 14 days of culture, especially 10Mg20Sr, which remarkably enhanced cell attachment, proliferation, and differentiation as compared to pure HA throughout the experiment. Our results indicated that HA substituted by an appropriate proportion of Mg and Sr has considerable potential for biomedical applications.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Natural Science Foundation of China (Grant No. 31370970), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130032110003), and Tianjin Research Program of Application Foundation and Advanced Technology (Grant No.13JCZDJC33300, 14JCZDJC38500).