Materials Science & Engineering C 112 (2020) 110892

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Materials Science & Engineering C

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Effects of strontium amount on the mechanical strength and cell-biological T

performance of magnesium-strontium phosphate bioceramics for bone
regeneration
⁎,1 ⁎⁎
Fupo Hea,b, , Teliang Luc,1, Xibo Fanga, Yanhui Lia, Fei Zuoa, Xin Denga,b, Jiandong Yec,
a
School of Electromechanical Engineering, Guangdong University of Technology, Guangzhou 510006, People's Republic of China
b
Jihua Laboratory, Foshan 528200, People's Republic of China
c
School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, People's Republic of China

A R T I C LE I N FO A B S T R A C T

Keywords: Magnesium and strontium are able to enhance osteogenesis and suppress osteoclastic activities simultaneously,
Magnesium and they were nontoxic in wide concentration ranges; these make the magnesium-strontium phosphate bio-
Strontium ceramics suitable for treating osteoporotic bone defects. The aim of this study was to investigate the effects of
Bioceramic strontium amount on the mechanical strength and cell-biological performance of magnesium-strontium phos-
Bone regeneration
phate [MgxSr3-x(PO4)2; 3-x = 0, 0.1, 0.25, 0.5, 0.75, 1] bioceramics, which were sintered at 1100 °C. The results
Cell response
indicated that the magnesium-strontium phosphate bioceramics except Mg2.9Sr0.1(PO4)2 and Mg2.25Sr0.75(PO4)2
bioceramics had considerable compressive strength. The variation in magnesium and strontium contents did not
regularly affect the in vitro osteogenic differentiation and osteoclastic activities. The Mg2.75Sr0.25(PO4)2 bio-
ceramic had the most desirable overall performance, as reflected by considerably high compressive strength,
enhanced in vitro osteogenesis and inhibited osteoclastic activities. Therefore, the Mg2.75Sr0.25(PO4)2 bioceramic
is considered a promising biomaterial for osteoporotic bone regeneration.

1. Introduction
The mineral phase of natural bone is predominantly composed of
calcium and phosphorus. The synthetic bone repair biomaterials based
on calcium and phosphorus, namely calcium phosphates, are degrad-
able, excellently biocompatible and osteoconductive [1]. However, the
calcium phosphates lack the capacity of modulating biological func-
tions, such as enhancing osteogenesis and vascularization, and inhibi-
tion of bone resorption, and so on [2,3]. This fact hinders the appli-
cation of calcium phosphate for treating bone defects under some
specific pathological conditions, such as osteoporosis, etc. [4]. Osteo-
porosis features accelerated bone resorption determined by the osteo-
clastic activity, and damaged osteogenic capability depending on the
osteoblastic activity [5]. Bone grafts capable of enhancing osteogenesis
and suppressing osteoclastic activity are considered efficacious in
treating osteoporotic bone defects [6,7].
Other than calcium and phosphorus, the nutrient elements such as
magnesium (Mg), zinc, strontium (Sr), copper, and so on, play
important roles in the bone metabolism, homeostasis and health, and
bone repair process [8]. Of these nutrient elements, the magnesium and
strontium in wide concentration ranges do not generate distinct cyto-
toxicity [9–12]. Strontium is a non-essential but beneficial element, and
it is capable of improving osteoblast proliferation, promoting bone
mineralization, and suppressing osteoclast growth, differentiation and
resorption activity [13]. Magnesium is an essential and abundant ele-
ment existing in the human skeleton. Magnesium plays a vital role in
biological activities, including stabilization of DNA, cofactor and cata-
lyzer for 300 enzymes [14], and stimulation of cell growth and pro-
liferation [15]. The biomaterials incorporated with wide concentration
ranges of magnesium or strontium are able to distinctly promote os-
teogenesis and inhibit osteoclastic activities, while maintaining ex-
cellent cytocompatibility [10,16–22]. What is more, in recent years
increasing attention has been given to magnesium- and strontium-based
biomaterials [15]. Zhu et al. found that strontium-based Sr5(PO4)2SiO4
bioceramic scaffold presented more favorable cell proliferation and
osteogenesis in contrast to the β-tricalcium phosphate bioceramic

⁎
Correspondence to: F. He, School of Electromechanical Engineering, Guangdong University of Technology, Guangzhou 510006, People's Republic of China.
⁎⁎
Corresponding author.
E-mail addresses: fphe@gdut.edu.cn (F. He), jdye@scut.edu.cn (J. Ye).
1
Equal contribution to this paper.

https://doi.org/10.1016/j.msec.2020.110892
Received 20 August 2019; Received in revised form 20 January 2020; Accepted 20 March 2020
Available online 21 March 2020
0928-4931/ © 2020 Elsevier B.V. All rights reserved.
F. He, et al.
scaffold [11]. Ewald et al. and Ostrowski et al. reported that magnesium
phosphate showed higher osteoblast activity and suspended the dif-
ferentiation of monocytes into polynucleated osteoclasts, as compared
with the calcium phosphate [23,24].
In the light of biological effects of strontium and magnesium in wide
concentration ranges, in previous work we fabricated magnesium-
strontium phosphate [MgxSr3-x(PO4)2; x = 0, 1, 1.5, 2, 3] bioceramics
aimed to repair osteoporotic bone defects [25]. We found that mag-
nesium-strontium phosphate bioceramics were well biocompatible, and
increased alkaline phosphatase (ALP) activity of mouse bone me-
senchymal stem cells (mBMSCs), and suppressed osteoclastic activities-
related gene expressions of RAW264.7 cells; interestingly, compared
with Mg3(PO4)2, the strontium-containing bioceramics did not de-
monstrate remarkable effect in enhancing osteogenesis [25]. High
concentration of strontium was reported to suppress osteogenic differ-
entiation [9]. Hence, it is hypothesized that the biological performance
of magnesium-strontium phosphate bioceramics will be further im-
proved by decreasing the ratio of strontium to magnesium. Herein,
magnesium-strontium phosphate bioceramics, with Sr/Mg mole ratio in
a narrower range of 0 to 0.5, were prepared. The phase, microstructure,
mechanical strength, in vitro osteogenic differentiation and osteoclastic
activities of the magnesium-strontium phosphate bioceramics were
comprehensively characterized.
2. Materials and methods
2.1. Materials synthesis
Mg3(PO4)2 and Mg2Sr(PO4)2 powders were synthesized employing a
solid-state reaction method using NH4H2PO4, 4MgCO3·Mg(OH)2·5H2O
and SrCO3 as raw materials, as presented in our previous work [25].
Briefly, the raw materials were dispersed in the ethyl alcohol, then
grinded in a planetary muller (QM-2SP20, Nanjing University Instru-
ment Factory, China) with zirconia ceramic cylinders for 4 h. The mass
ratio of raw materials to ethyl alcohol was 1:1.5, and that of raw ma-
terials to zirconia cylinders was 1:4. After air-drying, the uniformly
mixed raw materials were calcined in a furnace at 1000 °C for 2 h. The
collected particles were further milled in the planetary muller for 10 h,
then air-dried, and the Mg3(PO4)2 and Mg2Sr(PO4)2 powders were ac-
quired.
2.2. Bioceramics preparation
Magnesium-strontium phosphates [MgxSr3-x(PO4)2; 3-x = 0, 0.1,
0.25, 0.5, 0.75, 1], which were synthesized by the use of solid-state
reaction, were composed of Mg3(PO4)2 and Mg2Sr(PO4)2 free of other
phase [26]. Therefore, the Mg2.9Sr0.1(PO4)2, Mg2.75Sr0.25(PO4)2,
Mg2.5Sr0.5(PO4)2 and Mg2.25Sr0.75(PO4)2 bioceramics were prepared
using Mg3(PO4)2 and Mg2Sr(PO4)2 as starting materials. The mass
fractions of Mg3(PO4)2 and Mg2Sr(PO4)2 for preparing various magne-
sium-strontium phosphate bioceramics are listed in Supplementary
Table S1. The magnesium-strontium phosphate powders were mixed
with 7.5% (w/w) paraffin solution, then dried at 65 °C for 24 h to re-
move the heptane, which was acted as solvent for the paraffin. The
mass fraction of paraffin as a binder in the dried mixtures was around
2 wt%. Subsequently, the dried mixtures were put into a cylindrical
mold, then subjected to axial pressure (5 MPa). The resultant pellets
(Φ50 mm × 7 mm) were isostatically pressed at 200 MPa for 2 min.
The obtained green parts were heated in a furnace at a heating rate of
1 °C min−1, and held at 420 °C for 1 h to remove the paraffin. After-
wards, the specimens were heated with a heating rate of 2 °C min−1 up
to 1100 °C, then dwelled for 2 h, and finally cooled down in the en-
closed furnace. As listed in Supplementary Table S1, the specimen
names of Mg3(PO4)2, Mg2.9Sr0.1(PO4)2, Mg2.75Sr0.25(PO4)2,
Mg2.5Sr0.5(PO4)2, Mg2.25Sr0.75(PO4)2 and Mg2Sr(PO4)2 bioceramics
were abbreviated as Mg3, Mg2.9Sr0.1, Mg2.75Sr0.25, Mg2.5Sr0.5,
2
Materials Science & Engineering C 112 (2020) 110892
Mg2.25Sr0.75 and Mg2Sr, respectively.
2.3. Materials characterization
The phase of bioceramic specimens was determined employing an
X-ray diffraction diffractometer (X'Pert PRO, PANalytical Co., the
Netherlands) using Cu-Kα radiation. The current and voltage were
40 mA and 40 kV, respectively. Data were obtained from 10° to 70° for
2θ, and the step size was 0.0131°. Microstructure of the bioceramic
specimens was observed using a field emission scanning electron mi-
croscope (SEM; MERLIN, Zeiss, Germany) with an accelerating voltage
of 10 kV. Before SEM observation, the bioceramic specimens were
subjected to sputter-coating with platinum.
Open porosity (Po) of the bioceramic specimens was determined
using the Archimedes method with ethyl alcohol being displacement
liquid [4]. Total porosity (Pt) and closed porosity (Pc) of the bioceramic
specimens were determined by the following equations:
Dt − Db
Pt = × 100%
Dt
Pc = Pt − Po
where Db denotes the bulk density of bioceramic specimens; Dt is the
theoretical density of fully dense bioceramic specimens. The procedures
of measuring Db and Dt are presented in the Supplementary materials.
Compressive strength of bioceramics (5 mm × 5 mm × 10 mm)
was tested by a universal material testing machine (Instron 5567,
Instron, USA), and the crosshead speed was 0.5 mm min−1. The max-
imum stress for breaking the bioceramic specimens was considered the
compressive strength of the bioceramics. 6 replicates were measured for
each bioceramic group.
2.4. Evaluation of in vitro cell response
2.4.1. Cell culture
In vitro osteogenic performances and osteoclastic activities were
evaluated using mBMSCs (ATCC, USA) and RAW264.7 murine mono-
cyte cell line (Cobioer, China), respectively. High-glucose Dulbecco's
modified eagle's medium (H-DMEM; Gibco, USA) containing 10 vol%
fetal bovine serum (FBS; Gibco, USA) was used to culture the mBMSCs
and RAW264.7 cells. The cell culture was conducted in a humidified
incubator with atmosphere of 5% CO2 at 37 °C. The medium was re-
freshed every 2 days. All the biology kits were used following the
manufacturers' instructions.
2.4.2. Preparation of bioceramic extracts
The bioceramic extracts were prepared in accordance to the
guidelines of International Standard Organization (ISO 10993-12). The
bioceramics were crushed, grinded into particles (300–450 μm), and
soaked in H-DMEM with a solid mass to volume ratio of 200 mg/mL.
After being incubated at 37 °C for 24 h, the mixtures were subjected to
centrifugation and the supernatant was collected. The obtained extracts
were sterilized by filtration through 0.2 μm filtering membranes. The
concentrations of calcium, phosphorus, magnesium and strontium in
the bioceramic extracts were determined with an inductively coupled
plasma mass spectrometer (Optimal 5300DV, Perkin Elmer, USA). The
calibration was conducted by plotting the standard curves using ICP-MS
standards (XNEF-54, SPEX CertiPrep, USA). The concentrations of cal-
cium, phosphorus, magnesium and strontium were measured at wave-
length of 318, 177, 280 and 422 nm, respectively.
2.4.3. Viability and proliferation of mBMSCs
2 × 103 mBMSCs were seeded into the 96-well plate. After culturing
for 1 day, the medium was replaced by the bioceramic extract con-
taining 10 vol% FBS. The viability of mBMSCs cultured in the extracts
was evaluated with a Live/Dead kit (calcein-AM, Biotium, USA). After
F. He, et al.
Live/Dead staining, the fluorescent photographs of mBMSCs were
captured with a fluorescence microscope (Zeiss Axioskop 40, Zeiss,
Germany). Proliferation of mBMSCs cultured in the bioceramic extracts
was assessed using a CCK-8 kit (Dojindo Laboratories, Japan).
2.4.4. Osteogenesis– and osteoclastic activities–related gene expressions
Osteogenesis–related gene expressions of mBMSCs and osteoclastic
activities–related gene expressions of RAW264.7 cells cultivated in the
bioceramic extracts were assessed employing a quantitative real-time
polymerase chain reaction (RT-PCR) assay. mBMSCs or RAW264.7 cells
were seeded into a 24-well plate at a density of 5 × 104 cells per well.
After being cultured for 1 day, the mBMSCs were cultivated with bio-
ceramic extracts containing osteoinductive supplements (82 mg/mL
vitamin, 10 mM sodium glycerophosphate and 10 nM dexamethasone);
the RAW264.7 cells were cultured with the bioceramic extracts con-
taining 50 ng mL−1 of receptor activator of nuclear factor kappa-B li-
gand (RANKL; R&D, USA). After cell culture for predetermined time,
total RNAs of mBMSCs or RAW264.7 cells were isolated with a HiPure
Total RNA Micro Kit (Magen, China). The total RNA was measured by
Nano-drop 2000 (Thermo Scientific, USA). First strand complementary
DNA was obtained from RNA using a Transcriptor First Strand cDNA
Synthesis Kit (Roche, Germany). PCR assay for determining osteogen-
esis–related and osteoclastic activities–related genes was performed
using the Applied Biosystems™ QuantStudio™ 6 Flex (Thermo Fisher
Scientific, USA). The osteogenesis–related genes were alkaline phos-
phatase (ALP), collagen type I (Col I), runt-related transcription factor 2
(Runx2), bone sialoprotein (BSP), osteopontin (OPN) and osteocalcin
(OCN); the osteoclastic activities–related genes included tartrate-re-
sistant acid phosphatase (TRAP), carbonic anhydrase II (Car2), cathe-
psin (Cath), matrix metalloproteinase 9 (MMP9), c-Fos and macrophage
colony-stimulating factor (MCSF). Glyceraldehyde phosphate dehy-
drogenase (GAPDH) was selected as a housekeeping gene. The primer
sequences for genes are tabulated in the Supplementary Table S2. The
RT-PCR reaction was performed using the following thermal profile:
30 s at 95 °C, followed by 5 s at 95 °C, 30 s at 60 °C for 40 cycles. The
relative quantity of target genes was determined using a ΔCt method,
and ΔCt = Ct (target gene) – Ct (GAPDH). The relative gene expression
was determined by calculation of 2−ΔΔCt.
2.4.5. ALP assay
2 × 104 mBMSCs were seeded into a 48 well-plate. The mBMSCs
were cultured with bioceramic extracts containing osteoinductive sub-
stances. After cell culture for scheduled time, the mBMSCs were lysed
using Triton X-100 solution (0.1 vol%). The lysates were collected, and
their total protein amount was determined using a Pierce BCA Protein
Assay Kit (Thermo Scientific, USA). The enzymatic activity of ALP was
determined using a p-nitrophenyl phosphate (p-NPP, Sigma–Aldrich,
USA) assay. The quantitative ALP activity of mBMSCs was determined
by normalizing the enzymatic activity relative to the total protein
amount. Additionally, the ALP of mBMSCs was stained employing an
Alkaline Phosphatase stain assay kit (Beyotime, China), and their
photographs were taken under the fluorescence microscope.
2.5. Statistical analysis
The data were expressed as means ± standard deviations, which
were obtained from at least 3 replicates. Student's t-test was utilized to
analyze differences between experimental groups. p value < 0.05 was
considered to have significant difference.
3. Results
3.1. Phase composition
Fig. 1 presents the XRD patterns of various magnesium-strontium
phosphate bioceramics (Mg3, Mg2.9Sr0.1, Mg2.75Sr0.25, Mg2.5Sr0.5,
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Materials Science & Engineering C 112 (2020) 110892
Fig. 1. XRD patterns of the magnesium-strontium phosphate bioceramics.
Mg2.25Sr0.75 and Mg2Sr). Characteristic diffraction peaks of
Mg3(PO4)2 (ICDD PDF # 33-0876) and Mg2Sr(PO4)2 (ICDD PDF # 52-
1590) appeared in the patterns of Mg3 and Mg2Sr, respectively. The
pattern of Mg2.9Sr0.1 bioceramic was dominated by the diffraction
peaks of Mg3(PO4)2, and a minor phase of Mg2Sr(PO4)2 was seen. The
peaks in the pattern of Mg2.25Sr0.75 bioceramic could not be identified
according to the PDF-2 2004 X-ray data cards. Although the amount of
Mg2Sr(PO4)2 for preparing Mg2.75Sr0.25 bioceramics was larger than
that for Mg2.9Sr0.1 bioceramic, the relative peak intensity of Mg2Sr
(PO4)2 in the pattern of Mg2.75Sr0.25 bioceramic was markedly
weaker than that of Mg2.9Sr0.1 bioceramic. The Mg2.75Sr0.25 and
Mg2.5Sr0.5 bioceramics mainly consisted of Mg3(PO4)2 and
Mg3Sr2P4O15 (ICDD PDF # 21-0964), demonstrating the reaction be-
tween Mg3(PO4)2 and Mg2Sr(PO4)2 during the sintering process. The
relative density of peaks for Mg3Sr2P4O15 in the pattern of Mg2.5Sr0.5
bioceramic was more intense than that of Mg2.75Sr0.25 bioceramic.
3.2. Microstructure
Fig. 2 exhibits the SEM images of external surface and fractured
surface of magnesium-strontium phosphate bioceramics. As shown in
Fig. 2A, the Mg2.9Sr0.1 had plenty of micropores with size up to 6 μm.
By contrast, other magnesium-strontium phosphate bioceramics are
characterized by much compact microstructure, and their grains were
lined tightly with each other. It was observed that the grains of Mg3,
Mg2.75Sr0.25 and Mg2Sr were in narrow size distribution. The Mg3
had the finest grains (< 3 μm), followed by Mg2.75Sr0.25 and MgSr
whose grains were in the range of 1–6 μm. The grain size of Mg2.5Sr0.5
was in the range of 1–10 μm. With respect to the Mg2.9Sr0.1 and
Mg2.25Sr0.75, some large grains (> 15 μm) were surrounded by dis-
tinctly smaller grains (< 2 μm); this demonstrated that abnormal grain
growth took place during the sintering process. The size of abnormally
grown grains in Mg2.25Sr0.75 was up to 40 μm. Substantial pores were
present in the interior of grains for all the bioceramics (Fig. 2B). The
pores inside the Mg2.25Sr0.75 were as large as 10 μm.
3.3. Porosity and compressive strength
Fig. 3 presents the porosity and compressive strength of magnesium-
strontium phosphate bioceramics. As indicated in Fig. 3A, the Mg3
bioceramic free of Sr was measured to have open porosity of 3.3%. The
Mg2.9Sr0.1 bioceramic with a small amount of Sr possessed the highest
open porosity (11.0%), followed by the Mg2.25Sr0.75 (5.9%). The
Mg2Sr had the lowest open porosity (0.1%). The open porosity of
F. He, et al.
Fig. 2. SEM images of the external surface (A) and fractured surface (B) of magnesium-strontium phosphate bioceramics.
Mg2.75Sr0.25 and Mg2.5Sr0.5 was 1.7% and 1.8%, respectively. The
closed porosity was markedly higher than open porosity for all the
bioceramics. The Mg2.9Sr0.1 and Mg2.25Sr0.75 had tremendously
higher closed porosity as compared with other bioceramics (Fig. 3B).
The order of total porosity was similar to that of closed porosity for the
bioceramics (Fig. 3C). As presented in Fig. 3D, the order of compressive
strength for magnesium-strontium phosphate bioceramics was almost
contrary to that of total porosity. The compressive strength of Mg3
bioceramic was 178.1 MPa. The Mg2Sr with the lowest porosity had the
highest compressive strength (440.0 MPa), followed by the
Mg2.75Sr0.25 (258.8 MPa). The compressive strength of Mg2.9Sr0.1
bioceramic was as low as 45.2 MPa, which was comparable with that of
Mg2.25Sr0.75 bioceramic (35.3 MPa).
3.4. In vitro cell response
3.4.1. Element concentrations of the bioceramic extracts
The concentrations of calcium, phosphorus, magnesium and stron-
tium in the bioceramic extracts are tabulated in the Table 1. The con-
centrations of calcium and magnesium in various bioceramic extracts
were in the range of 41–53 ppm and 14–18 ppm, respectively, dis-
tinctively lower than those in the complete H-DMEM. The magnesium
concentration in the bioceramic extracts was over three times as high as
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Materials Science & Engineering C 112 (2020) 110892
that in the H-DMEM. Unexpectedly, of the 6 bioceramic extracts, the
Mg3 extract was detected to have the lowest magnesium concentration.
The Sr concentration in the bioceramic extracts was dependent on the
Sr amount contained in the magnesium-strontium phosphate bio-
ceramics. That is to say, the Sr concentration of the bioceramic extracts
in increasing order was as follows: Mg3 < Mg2.9Sr0.1 <
Mg2.75Sr0.25 < Mg2.5Sr0.5 < Mg2.25Sr0.75 < Mg2Sr.
3.4.2. Viability, proliferation and osteogenic differentiation of mBMSCs
Fig. 4A gives the fluorescent photographs of Live/Dead staining of
mBMSCs cultured in the bioceramic extracts. Copious viable cells
(green fluorescence) were seen in all the bioceramic extracts, and few
compromised cells (red fluorescence) were found. Moreover, the viable
cells well spread in all the bioceramic extracts. Fig. 4B depicts the
proliferation profiles of mBMSCs cultivated in the bioceramic extracts.
It is clear that the cells gradually proliferated with prolongation of
culture time. Various bioceramic extracts did not show significant dif-
ference in the cell proliferation. These results indicated excellent cy-
tocompatibility of all the magnesium-strontium phosphate bioceramics.
Fig. 5A presents the quantitative ALP activity of mBMSCs cultured
in the bioceramic extracts. At the 7th day, the ALP activity of mBMSCs
cultured in Mg2.75Sr0.25 extract was higher than that in other bio-
ceramic extracts. The ALP activity was not significantly different among
F. He, et al. Materials Science & Engineering C 112 (2020) 110892

Fig. 3. Open porosity (A), closed porosity (B), total porosity (C) and compressive strength (D) of the magnesium-strontium phosphate bioceramics. n = 6; * denotes
significant difference between groups; & represents significant difference when compared with all the other groups; # denotes significant difference compared with
Mg3, Mg2.75Sr0.25, Mg2.5Sr0.5 and Mg2Sr.

Table 1 the extracts of Mg3, Mg2.9Sr0.1, Mg2.5Sr0.5, Mg2.25Sr0.75 and

Concentrations of calcium, phosphorus, magnesium and strontium in the ex- Mg2Sr. After cell culture for 14 days, higher ALP activity was found in
tracts of magnesium-strontium phosphate bioceramics (Mg3, Mg2.9Sr0.1, the mBMSCs treated with the extracts of Mg2.9Sr0.1, Mg2.75Sr0.25,
Mg2.75Sr0.25, Mg2.5Sr0.5, Mg2.25Sr0.75 and Mg2Sr); n = 3. Mg2.5Sr0.5 and Mg2.25Sr0.75, as compared with the extracts of Mg3
Specimen Calcium (ppm) Phosphorus Magnesium (ppm) Strontium and Mg2Sr; the ALP activity of mBMSCs cultured in the Mg3 extract
(ppm) (ppm) was comparable with that in the Mg2Sr extract; these results were
consistent with the photographs of ALP staining of mBMSCs cultured in
H-DMEM 71.3 ± 1.7 20.6 ± 0.1 20.2 ± 1.0 –
Mg3 52.7 ± 1.3 17.1 ± 0.1 72.9 ± 0.4 –
various bioceramic extracts for 14 days (Fig. 5B). Fig. 5C gives osteo-
Mg2.9Sr0.1 40.6 ± 0.2 18.0 ± 0.2 121.1 ± 1.6 0.3 ± 0.0 genesis-related expressions of mBMSCs cultured in various bioceramic
Mg2.75Sr0.25 40.6 ± 0.1 12.9 ± 0.1 114.3 ± 1.0 0.4 ± 0.0 extracts for 7 days. The Mg2.5Sr0.5 extract had the lowest level of ALP
Mg2.5Sr0.5 46.8 ± 0.2 17.7 ± 0.1 97.1 ± 3.6 0.7 ± 0.0 expression of mBMSCs, in contrast to other bioceramic extracts. The
Mg2.25Sr0.75 51.5 ± 0.6 16.6 ± 0.2 100.8 ± 0.9 0.9 ± 0.0
mBMSCs in Mg2.75Sr0.25 extract expressed the highest level of Col I,
Mg2Sr 52.2 ± 0.9 13.8 ± 0.2 102.4 ± 1.8 1.5 ± 0.1
followed by those in Mg2.25Sr0.75. As compared with Mg3 extract, the
extracts of Mg2.9Sr0.1, Mg2.5Sr0.5, Mg2.25Sr0.75 and Mg2Sr pro-
moted BSP and OPN expressions of mBMSCs. The cells in Mg2.9Sr0.1
extract expressed the highest level of BSP and OPN. The Mg2.9Sr0.1,

5
F. He, et al. Materials Science & Engineering C 112 (2020) 110892

Fig. 4. Fluorescent photographs of live/dead staining (A) and proliferation (B) of mBMSCs cultured in the bioceramic extracts. Green fluorescence denotes viable
cells; red fluorescence represents dead or compromised cells; n = 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Fig. 5. Quantitative ALP activity (A), images of ALP staining (B) and osteogenesis-related gene expressions (C) of mBMSCs cultured in the bioceramic extracts. n = 4;
* means significant difference between experimental groups; & denotes significant difference when compared with all the other groups.

6
F. He, et al.
Fig. 6. Osteoclastic activities-related gene expressions of RAW264.7 cells treated with the bioceramic extracts. n = 4; * means significant difference between
experimental groups; & denotes significant difference when compared with all the other groups.
Mg2.25Sr0.75 and Mg2Sr extracts upregulated Runx2 expression of
mBMSCs. Higher OCN expression was detected in the cells cultured in
Mg2.9Sr0.1, Mg2.75Sr0.25 and Mg2.25Sr0.75 extracts. It is inferred
that the presence of certain amounts of strontium in magnesium-
strontium phosphate bioceramics [MgxSr3-x(PO4)2; 3-x = 0.1, 0.25, 0.5,
0.75] accounted for the enhancement of osteogenesis-related gene ex-
pressions.
3.4.3. Osteoclastic activities of RAW264.7 cells
Fig. 6 delineates the osteoclastic activities-related gene expressions
of RAW264.7 cells cultured in the bioceramic extracts for 3 days. The
cells in the Mg2.75Sr0.25 extract showed the lowest TRAP expression,
while those in Mg2Sr extract expressed the highest level of TRAP. The
Cath expression of cells in Mg2Sr extract was higher than that in other
extracts. The c-Fos expression was higher in the Mg2.5Sr0.5 and
Mg2.25Sr0.75 extracts, and the lowest c-Fos expression was found in
the Mg2Sr extract. In comparison with Mg3 extract, the extracts of
Mg2.9Sr0.1, Mg2.75Sr0.25 and Mg2Sr starkly suppressed Car2 ex-
pression of RAW264.7 cells; the extracts of Mg2.75Sr0.25 and Mg2Sr
noticeably inhibited MMP9 expression; the Mg2.9Sr0.1 and Mg2.5Sr0.5
extracts expressed lower level of MCSF, while Mg2.25Sr0.75 and Mg2Sr
extracts upregulated MCSF expression of RAW264.7 cells.
4. Discussion
Both Mg3(PO4)2 and Mg2Sr(PO4)2 have been confirmed to be ex-
cellently biocompatible [25]. It has been well documented that mag-
nesium and strontium are capable of enhancing osteogenesis and
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Materials Science & Engineering C 112 (2020) 110892
suppressing osteoclastic activities [13,25]. However, compared with
Mg3(PO4)2, the Mg2Sr(PO4)2 did not have more desirable cell-biological
performances in promoting osteogenesis and inhibiting osteoclastic
activities [25]. Hence, the magnesium-strontium phosphate, whose
strontium amount is smaller than that of Mg2Sr(PO4)2, is expected to
have more favorable behaviors, such as enhancing osteoblastic activ-
ities and suppressing osteoclastic activities. In this study, magnesium-
strontium phosphate [MgxSr3-x(PO4)2; 3-x = 0.1, 0.25, 0.5, 0.75] bio-
ceramics were prepared by sintering the mixture blocks of Mg3(PO4)2
and Mg2Sr(PO4)2 at 1100 °C. That is to say, the strontium amount in the
magnesium-strontium phosphate bioceramics was larger than
Mg3(PO4)2 but smaller than Mg2Sr(PO4)2. According to the phase dia-
gram of Mg3(PO4)2-Mg2Sr(PO4)2 system, a solid-liquid coexistence zone
is present at temperatures ranging from 1015 °C to 1300 °C [26]. Hence,
Mg2.9Sr0.1, Mg2.75Sr0.25, Mg2.5Sr0.5 and Mg2.25Sr0.75 bioceramics
were sintered in the presence of liquid phase. It is shared that liquid-
phase sintering is more efficacious than solid-state sintering. As in-
dicated in Fig. 1, the Mg2.5Sr0.5 and Mg2.75Sr0.25 bioceramics were
dominated by the phases of Mg3(PO4)2 and Mg3Sr2P4O15, with minor
phase of Mg2Sr(PO4)2 detected; this indicated that most Mg2Sr(PO4)2
reacted with Mg3(PO4)2 when sintering the Mg2.5Sr0.5 and
Mg2.75Sr0.25 bioceramics. The X-ray scattering factors of Sr2+ is lar-
gely different from that of Mg2+. The increased amount of Sr2+ sub-
stituted for Mg2+ in the Mg3(PO4)2 leads to dramatical decease in the
reflection intensity; this results in difficulty in interpreting the XRD
pattern of Sr-substituted Mg3(PO4)2 [26]. It is deduced that greater
amount of Sr2+ was substituted for Mg2+ in the Mg2Sr(PO4)2 phase,
which was largely included in the Mg2.25Sr0.75 bioceramic; this led to
F. He, et al.
unidentifiable phase composition for the XRD pattern of Mg2.25Sr0.75
bioceramic.
The presence of liquid phase in the sintering process was responsible
for larger grains or abnormally grown grains in Mg2.9Sr0.1,
Mg2.5Sr0.5, and Mg2.25Sr0.75 bioceramics (Fig. 2). From the phase
diagram of Mg3(PO4)2-Mg2Sr(PO4)2 system, the amount of liquid
formed in Mg2.9Sr0.1 bioceramic was limited, while a large amount of
liquid was produced in the Mg2.25Sr0.75. The low liquid content
cannot efficiently promote densification, but leads to local abnormal
grain growth, resulting in increased porosity [27]; this explain why the
Mg2.9Sr0.1 bioceramic had the highest porosity (Fig. 3A–C). During the
sintering process, the greater amount of liquid in Mg2.25Sr0.75 re-
sulted in formation of large grains and substantial enclosed pores. The
mechanical strength of ceramic closely correlates with porosity. The
Mg2Sr with the lowest porosity possessed the highest compressive
strength, while the Mg2.9Sr0.1 and Mg2.25Sr0.75 with higher porosity
had lower compressive strength (Fig. 3). Moreover, the coarse grains
will compromise the mechanical strength of ceramics. In spite of similar
porosity, the Mg2.75Sr0.25 possessed higher compressive strength than
the Mg2.5Sr0.5, which was shown to have larger grains. The
Mg2.25Sr0.75 had not only high porosity but also noticeably larger
grains, in contrast to other magnesium-strontium phosphate bio-
ceramics (Fig. 2A); this fact was responsible for the deteriorated com-
pressive strength of Mg2.25Sr0.75 bioceramic.
The concentrations of calcium and phosphorus in the bioceramic
extracts were lower than those in the H-DMEM (Table 1), even though
the phosphorus in magnesium-strontium phosphate would be released
into the medium. Calcium ion was detected on the surface of bio-
ceramic, which was immersed in the calcium-containing H-DMEM for
24 h (Supplementary Fig. S1); this demonstrated that the ions in the
culture medium were deposited on the bioceramics. Interestingly, the
magnesium concentration in the Mg3 extract (73 ppm) was dramati-
cally lower than that in other bioceramic extracts (97–121 ppm). Pre-
vious study found that the dissolution rate of Mg2Sr(PO4)2 was higher
than Mg3(PO4)2. Therefore, the magnesium concentration in Mg2Sr
extract was higher than that in Mg3 extract [25]. The porosity of
Mg2.9Sr0.1 and Mg2.25Sr0.75 was tremendously higher than that of
other bioceramics (Fig. 3A–C). It is shared that the materials with
higher porosity provide larger surface in contact with the culture
medium, thereby accelerating material dissolution. Therefore, the pre-
sence of Mg2Sr(PO4)2 phase or higher porosity was responsible for
higher magnesium concentration in the extracts of Mg2.9Sr0.1,
Mg2.75Sr0.25, Mg2.5Sr0.5 and Mg2.25Sr0.75, in comparison with the
Mg3 extract. As listed in Table 1, the concentrations of calcium, phos-
phorus, magnesium and strontium in various bioceramic extracts were
in the range of 41–52, 14–18, 73–121 and 0.29–1.21 ppm, respectively.
The concentration difference in calcium and phosphorus was far less
than that in magnesium and strontium. It is inferred that the cell re-
sponse to bioceramic extracts mainly hinged on the concentrations of
magnesium and strontium rather than those of calcium and phosphorus.
The cells were compatible with magnesium in wide concentration range
[12]. Although the magnesium concentration in the bioceramic extracts
was over 3 times as high as that in H-DMEM, all the bioceramic extracts
showed excellent cytocompatibility. Both magnesium and strontium are
capable of enhancing osteogenesis and suppressing osteoclastic activ-
ities simultaneously [13,23]. The cell-biological performances are in-
fluenced by ions in a concentration-dependent manner [9,28,29]. The
combination of multiple ions may complicate the cell response [30,31].
On account of various affecting factors on cell response, none of the
bioceramics had the most desirable cell behavior in every cell activity
(Figs. 4–6). Among the 6 bioceramics, the Mg2.75Sr0.25 is the sole one
not only efficaciously promoting osteoblastic differentiation (ALP ac-
tivity, and expressions of Col I and OCN) and inhibiting osteoclastic
activities (expressions of TRAP, Car2, MMP9 and c-Fos), but also pos-
sessing considerable compressive strength. It is therefore concluded
that the Mg2.75Sr0.25 bioceramic is the most favorable biomaterial
8
Materials Science & Engineering C 112 (2020) 110892
used for treating osteoporotic bone defect.
5. Conclusion
All the magnesium-strontium phosphate bioceramics except
Mg2.9Sr0.1 and Mg2.25Sr0.75 had good sinterability, thereby condu-
cing to considerably high compressive strength. The cell response of
bioceramic was mainly dependent on the concentrations of released
magnesium and strontium. All the magnesium-strontium phosphate
bioceramics were well biocompatible. The bioceramics with increasing
strontium amount did not show regular variation trend in mediating
osteogenic differentiation and osteoclastic activities. The Mg2.75Sr0.25
bioceramic was characterized by considerably high compressive
strength and high efficiency in promoting osteogenesis and inhibiting
osteoclastic activities. Hence, the Mg2.75Sr0.25 bioceramic can serve
as a potential bone graft for treating bone defects under the osteo-
porosis pathological condition.
CRediT authorship contribution statement
Fupo He:Conceptualization, Writing - review & editing, Funding
acquisition.Teliang Lu:Methodology, Investigation.Xibo
Fang:Methodology, Investigation.Yanhui Li:Formal analysis.Fei
Zuo:Validation.Xin Deng:Funding acquisition.Jiandong
Ye:Supervision, Project administration.
Declaration of competing interest
All authors declare no conflict of interest associated with this paper.
Acknowledgments
This work was sponsored by the Pearl River S&T Nova Program of
Guangzhou under grant No. 201710010149, and the Jihua Laboratory
Project under grant No. X190061UZ190, and the Open Project of Key
Laboratory of Biomedical Engineering of Guangdong Province under
grant NO. KLBEMGD202001, and the Open Project of National
Engineering Research Center for Tissue Restoration & Reconstruction
under grant NO. NERC-TRR202001.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.msec.2020.110892.
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