Materials Today Chemistry 29 (2023) 101410

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Materials Today Chemistry

journal homepage: www.journals.elsevier.com/materials-today-chemistry/

Enhancing osteoinduction and bone regeneration of biphasic calcium

phosphate scaffold thought modulating the balance between
pro-osteogenesis and anti-osteoclastogenesis by zinc doping
T. Lu a, b, X. Yuan a, b, L. Zhang a, b, F. He d, X. Wang c, J. Ye a, b, e, *
a
School of Materials Science and Engineering and Key Laboratory of Biomedical Materials of Ministry of Education, South China University of Technology,
Guangzhou 510641, PR China
b
National Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, PR China
c
Medical Research Center, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080, China
d
School of Electromechanical Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
e
Key Laboratory of Biomedical Engineering of Guangdong Province and Innovation Center for Tissue Restoration and Reconstruction, South China
University of Technology, Guangzhou 510641, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Biphasic calcium phosphate (BCP) is a promising bone regeneration material with an adjustable
Received 23 November 2022 degradation rate, and ions doping is an effective method to improve its bone repair effect. However, few
Received in revised form studies have focused on the role of ions doping-mediated osteogenesis and osteoclastogenesis balance in
20 January 2023
bone regeneration. In order to improve the bone repair effect of BCP and emphasize the role of osteo-
Accepted 20 January 2023
genesis and osteoclastogenesis balance regulated by ions doping to promote bone regeneration. Here,
Available online 18 February 2023
zinc (Zn), an essential trace element with pro-osteogenesis and anti-osteoclastogenesis, was dopped into
BCP scaffolds, and its effects on the physicochemical properties, in vitro cytological responses, in vivo
Keywords:
Biphasic calcium phosphate
osteoinduction, and bone defect regeneration of BCP were systematically investigated. Results showed
Zinc doping that Zn was successfully introduced into the crystal lattice of BCP. The differentiation of both osteoblasts
Osteoclastogenesis and osteoclasts showed a doping content-dependent behavior. By increasing the doping content (0
Osteogenesis e5 mol.%), the anti-osteoclastogenesis effect of BCP gradually increased, while its pro-osteogenesis
performance first increased and then decreased. ThoughBCP with a doping content of 5 mol.% could
promote osteogenic differentiation of stem cells and reduced fibrous capsule encapsulation, it could not
enhance ectopic bone formation or bone regeneration due to its excessive anti-osteoclastogenesis. BCP
with a doping content of 2.5 mol.% remarkably enhanced the osteoinduction activity and accelerated new
bone formation, which were associated with its highest pro-osteogenesis and appropriate anti-
osteoclastogenesis activity. This study highlights on regulating the balance between pro-osteogenesis
and anti-osteoclastogenesis by Zn doping and paves the way for the development of a more osteoin-
ductive calcium phosphate-based material for bone regeneration.
© 2023 Elsevier Ltd. All rights reserved.

1. Introduction b-tricalcium phosphate (b-TCP) and hydroxyapatite (HA) are most

Bone defect is a common clinical disease, usually caused by
functional degeneration, tumor, and trauma, and brings great
tribulation to patients. Calcium phosphate-based bone regenera-
tion biomaterials have attracted much attention due to their
excellent bioactivity and perfect osteoconductivity [1,2]. Thereinto,
* Corresponding author.
E-mail address: jdye@scut.edu.cn (J. Ye).
extensively studied. HA and b-TCP can not only be used as the
matrix of bone repair materials, but can also be used alone or in
combination with nanomaterials to modify metal or other matrix
materials for preparing intelligent antibacterial or antitumor im-
plants and endow the implants with good angiogenesis [3] or
osteogenesis [4e6]. The mixture of b-TCP and HA named biphasic
calcium phosphate (BCP) with an adjustable degradation rate is
considered to be a promising bone regeneration material [7,8].
Furthermore, according to the literature, it is found that a BCP
ceramic possessed an apparent osteoinduction activity compared

https://doi.org/10.1016/j.mtchem.2023.101410
2468-5194/© 2023 Elsevier Ltd. All rights reserved.
T. Lu, X. Yuan, L. Zhang et al.
to a pure HA ceramic and b-TCP ceramic [9]. In addition, it was also
found that osteoinductive materials possessed better bone defect
repair effects than non-osteoinductive materials [10].
Bone defect healing is a temporally coordinated and highly
complex biological process that requires a series of interactions
among a range of different cell types [11]. When a bone is injured,
damaged blood vessels lead to the formation of blood clots to
provide hemostasis. The clotting is followed by an inflammatory
response. Next is the phase of fibrovascular healing, when
mesenchymal stem cells (MSCs) are recruited into the temporary
callus. These recruited MSCs subsequently differentiate into oste-
oblasts and/or chondrocytes, which eventually form new bones
through the mechanism of entochondrostosis and/or intra-
membranous ossification. Finally, the newly forming bones are
remodeled by the coordination between osteoclasts to resorb
woven bones and osteoblasts to form mature lamellar bones. This
remodeling stage eventually leads to bone regeneration. It would
be feasible to target any of these healing stages to enhance bone
regeneration [12].
Zinc (Zn) is one of the essential trace elements, which could
inhibit the activities of osteoclasts while stimulating the osteogenic
differentiation of stem cells [13]. Zn doping has been used to
improve the bone defect repair effects of calcium phosphate ce-
ramics [14e16]. However, previous research mainly focused on
promoting the osteogenic differentiation of MSCs [14,15] or inhib-
iting the formation of osteoclasts as well as their bone resorption
activity [17]. Few studies were concerned about the balance be-
tween osteogenesis and osteoclastogenesis in bone regeneration
mediated by bioactive ions doping. According to bone healing
processes, osteoclasts also play vital roles in bone healing, espe-
cially in a bone remolding phase. In addition, the effects of Zn on
osteogenesis and osteoclastogenesis are concentration dependent
[18e20]. Hence, we speculated that designing a kind of BCP ceramic
scaffold with the balance of promoting osteogenesis and inhibiting
osteoclastogenesis by Zn doping at an appropriate content would
facilitate bone defect healing.
In the present study, Zn doping was used to improve the bone
defect repair effects of BCP ceramic scaffolds by mediating the
balance between osteogenesis and anti-osteoclastogenesis. The
effects of Zn doping on the physicochemical properties, in vitro
cytological responses and in vivo osteoinduction as well as bone
defect regeneration of BCP were systematically investigated. We
aim to explore the effect of Zn doping content on the osteogenic
and osteoclastic properties of BCP, and to further screen out the
appropriate doping content of Zn, which endows BCP with an
excellent bone defect repair effect. This study would pave the way
for the development of a more practical calcium phosphate-based
material for bone regeneration in the clinic.
2. Materials and method
2.1. Raw materials and reagents
NH4OH solution (25%), (NH4)2HPO4, Ca(NO3)2$4H2O, and
Zn(NO3)2$6H2O were commercially obtained from Guangzhou
Chemical Reagent Factory (China). A DAPI staining solution, a BCIP/
NBT alkaline phosphatase (ALP) color development kit and a RIPA
lysis buffer were purchased from Biyotime (China). A Live/Dead
staining solution was offered by Biotium (USA). An F-actin Labeling
Kit was offered by AAT Bioquest (USA). A receptor activator of nu-
clear factor-kappa B ligand (RANKL) was offered by R&D System
(USA). Disodium 4-nitrophenylphosphate, sodium b-glycer-
ophosphate, vitamin C, and dexamethasone were bought from
Sigma-Aldrich (USA). A micro-BCA protein assay kit was purchased
from Thermo Scientific (USA). A high-glucose Dulbecco's modified
2
Materials Today Chemistry 29 (2023) 101410
eagle's medium (H-DMEM) was offered by Gibco (USA). Fetal
bovine serum (FBS) was bought from Biological Industries (Israel).
A cell counting kit-8 solution was purchased from Dojindo (Japan).
The rabbit-anti-mouse iNOS primary antibody, rabbit-anti-mouse
CD163 primary antibody, Cy3-labeled anti-rabbit secondary anti-
body, and 488-labeled anti-rabbit secondary antibody were offered
by Abcam (UK).
2.2. Preparation of BCP powder
A BCP powder was synthesized by a chemical precipitation
method, as described in our previous work [14]. Briefly, Ca(N-
O3)2$4H2O and (NH4)2HPO4 were separately dissolved in deionized
water to prepare solution A (1 mol/L) and B (0.6 mol/L), respec-
tively. Then, solution B was dropwise added to solution A by a
peristaltic pump under stirring. The pH of the reaction solution was
adjusted to 10.2 by NH4OH after finishing the addition. The reaction
solution was stirred for 2 h and then aged for 24 h. After that, the
reaction product was washed, centrifuged, frozen, and lyophilized.
Then, the obtained powder was treated at 900
C for 2 h. For the
preparation of the Zn-doped BCP(Zn-BCP) powder, the Ca(N-
O3)2$4H2O solution was exchanged by a mixed solution (1 mol/L)
composed of Zn(NO3)2$6H2O and Ca(NO3)2$4H2O. The molar ratio
of (Ca þ Zn) to P was 1.67 in the initial reaction solution. The initial
doping content of Zn was in the range of 0e6 mol.%. Zn-BCP
powders were denoted as ZnX (X ¼ Zn/(Zn þ Ca)  100). The in-
gredients and initial reaction solutions of Zn-BCP powders are
shown in Table S1.
2.3. Fabrication of Zn-BCP scaffolds
Zn-BCP scaffolds were fabricated by an extrusion 3D printing
method. A ceramic paste was prepared by mixing 8 g of the Zn-BCP
powder with 7 g of a 8 wt% polyvinyl alcohol solution under me-
chanical stirring for 3 min. The scaffold models were designed by
CAD. The prepared paste was extruded through a plastic nozzle
(inside diameter ¼ 400 mm), with an interval between adjacent
fibers of 800 mm at about 20
C, and successively dried at 37
C and
60
C for 2 days and 1 day, respectively. The green scaffolds were
sintered at 1100
C for 2 h to obtain the final scaffolds. Zn-BCP
scaffolds were denoted as ZnX-S.
2.4. Phase composition and morphology characterization
X-ray diffraction (X'Pert, PANalytical, the Netherlands) with a
CuKa radiation was used to analyze the phase of Zn-BCP scaffolds.
Rietveld refinements were performed with the program of General
Structure Analysis System to calculate the phase composition of Zn-
BCP scaffolds. A refinement strategy was carried out according to
the guidelines for structure refinement by using the Rietveld
method [21]. The morphology and structure of scaffolds were
observed by a field emission scanning electron microscope (Merlin,
Carl Zeiss AG, Germany). Scanning electron microscope was
coupled with an energy dispersive spectrometer (EDS), which was
used to analyze the distribution of elements. The determination of
Ca, P, O, and Zn elements were also analyzed by using an X-ray
photoelectron spectroscope (Thermo Scientific K-Alpha, USA) with
Al Ka radiation.
2.5. In vitro degradation and ions release behaviors
An in vitro degradation experiment was conducted under acidic
conditions (pH ¼ 4.5) because in a bone remodeling process, the
ruffled border of osteoclasts has a pH in the range of 4.0e5.0 [22].
Each Scaffold (weight ¼ 0.2 g, diameter ¼ 7.5 mm, and
T. Lu, X. Yuan, L. Zhang et al.
height ¼ 1.5 mm) was placed in a container with 10 mL of an ac-
etate-sodium acetate buffer solution (pH ¼ 4.5) and incubated in a
table concentrator (37
C, 60 rpm) for 1e12 weeks. The buffer so-
lution was exchanged by new ones every week. After immersion for
setting time points (1, 4, 8, and 12 w), scaffolds were taken out and
dried at 80
C for 3 days and weighted. The ion concentrations of
the soaking solution immersion with scaffolds for 1, 4, 8, and 12 w
were measured by an inductively coupled plasma atomic emission
spectroscope (Optimal 5300DV, Perkin Elmer, USA).
2.6. In vitro cell responses evaluation
2.6.1. Cell cultivation
RAW264.7 (Cobioer, China) and mouse bone mesenchymal stem
cells (mBMSCs, ATCC, USA) were used as cell models in this study. A
cell culture medium was composed of 10 vol% fetal bovine serum
and 90 vol% high-glucose Dulbecco's modified eagle's medium . The
osteoclast differentiation culture medium and osteogenic inductive
medium were obtained by further adding an osteoclast induction
factor (50 ng/mL of RANKL) and osteogenic inductive components
(0.1 mM of dexamethasone, 50 mg/L of vitamin C, and 10 mM of
sodium b-glycerophosphate), respectively.
2.6.2. Cell proliferation
2  104 of mBMSCs in 500 mL of the culture medium were
cultured with scaffolds (diameter ¼ 7.5 mm and height ¼ 1.5 mm)
in a 48-well plate. After being cultured for 1, 3, and 7 days, 250 mL of
a cell counting kit-8 working solution was added to each well and
incubated in a cell incubator for 1 h. Then, 100 mL of the reaction
solution of each well was exchanged to a 96-plate well. Finally, its
optical density value at 450 nm wave length was measured by an
ELISA reader (Thermo 3001, Thermo, USA) to determine the num-
ber of cells.
2.6.3. Cell activity and adhesion
After culturing for 3 days, the cells on scaffold were stained by
the Live/Dead staining kit for 15 min in a dark place and observed
by a fluorescence inverted microscope (Axio Observer 7, ZEISS,
Germany). For the observation of cell morphologies, the cells
cultured for 1 and 7 days were stained with the F-actin Labeling Kit
and DAPI for 8 h and 5 min, respectively, before observing by a laser
scanning confocal microscope (TCPS SP8, Leica, Germany).
2.6.4. In vitro osteogenesis
The effects of Zn doping on the in vitro osteogenesis were
evaluated by ALP staining, ALP activities, and osteogenesis-related
genes expression of mBMSCs cultured with scaffolds for 3, 7, and
14 days. The genes expression was determined by a quantitative
real-time polymerase chain reaction. GAPDH was used as a
housekeeping gene. The osteogenesis-related genes included ALP,
collagen type I (Col-I), bone sialoprotein (BSP), osteocalcin (OCN),
and runt-related transcription factor 2 (Runx2). The primer se-
quences are summarized in Table S2.
2.6.5. In vitro osteoclastogenesis
Raw 264.7 at a cell density of 4  104 cell/mL was cultured with
a scaffold in a 48-well plate with a 500 mL osteoclast differentiation
culture medium. The osteoclasts differentiation-related genes used
in this study included c-Fos, tartrate-resistant acid phosphatase
(TRAP), and nuclear factor of activated T-cells 1 (NFATc1). The
primer sequences are provided in Table S2. Cells cultured in the
osteoclast differentiation culture medium for 5 days were stained
with DAPI and F-actin Labeling Kit and observed by a laser scanning
confocal microscope. To further evaluate the osteoclastogenesis,
3
Materials Today Chemistry 29 (2023) 101410
TRAP staining of the cells cultured for 5 days was also conducted
and observed by a fluorescence inverted microscope.
2.6.6. In vitro immune osteogenesis
Raw 264.7 was cultured with scaffolds for 3 days, then collected
the culture medium. The collected medium was filtrated by a
0.2 mm filter membrane, followed by mixing with a fresh culture
medium (25 vol% vs 75 vol%), and a macrophage-conditioned me-
dium was obtained. mBMSCs were first cultured for 12 h in a 48-
well plate, then the culture medium was replaced by the macro-
phage-conditioned medium mentioned above and refreshed every
other day. After culturing for 7 and 14 days, the osteogenic differ-
entiation of mBMSCs tests including ALP activity, ALP staining, and
genes expression were conducted to evaluate the in vitro immune
osteogenesis.
2.7. In vivo experiments
2.7.1. In vivo angiogenesis and macrophage polarization assessment
Animal experiments were approved by the South China Univer-
sity of Technology Ethics Committee. Zn0-S, Zn2.5-S, and Zn5.0-S
were used in this experiment. A total of 18 rats were divided into
three groups, and scaffolds (diameter ¼ 6 mm and height ¼ 3 mm)
were implanted into the abdominal skin. After seven days and 14
days after implantation, the scaffolds with surrounding tissues were
retrieved, fixed, decalcified, and embedded in paraffin. Samples were
sectioned with a thickness of 5 mm. The generation and development
of blood vessels were assessed by the immunohistochemical staining
of CD31. The effects of the scaffolds on macrophage polarization were
investigated using immunofluorescent staining. Cell nuclei were
stained by DAPI. M1 macrophages were labeled by iNOS primary
antibody and Cy3-labeled secondary antibody. M2 macrophages
were labeled by CD163 primary antibody and 488-labeled secondary
antibody. The stained samples were then captured by a digital pa-
thology system (P250 Flash, 3D Histech, Hungary).
2.7.2. In vivo osteoinduction estimation
Zn0-S, Zn2.5-S, and Zn5.0-S were used for the in vivo osteoin-
duction experiment. Four healthy adult mongrel dogs (male,
20e25 kg) were offered by the Guangdong Medical Laboratory
Animal Center. Incisions (8 mm) over the paraspinal muscle were
first made on both sides of the spine along the dorsal midline, then
scaffolds (diameter ¼ 6 mm and height ¼ 3 mm) were implanted.
After the implantation for 4 and 8 weeks, the dogs were sacrificed
and the scaffolds within them were harvested and immersed in
paraformaldehyde. Tissue sections were prepared as described
above. The sections were conducted by TRAP staining, toluidine
blue staining, Masson's Trichrome staining as well as hematoxylin
and eosin (HE) staining.
2.7.3. In vivo bone regeneration evaluation
Zn0-S, Zn2.5-S, and Zn5.0-S were used for the in vivo bone
regeneration experiment. A total of 27 New Zealand white rabbits
(male, 2.5e3.0 kg) were used for this experiment. Scaffolds
(diameter ¼ 6 mm and height ¼ 8 mm) were implanted into the
critical-size bone defect (diameter ¼ 6.2 mm and height ¼ 8 mm) in
the condyles of the femur. After the implantation for 4, 8, and 12
weeks, the femora together with the implants of the sacrificed
rabbits was retrieved. Then, samples were evaluated by micro
computed tomography imaging (Aloka Latheta LCT-200, HITACHI,
Japan). Next, tissue sections were prepared as described above. The
obtained sections were stained with HE and Masson's trichrome
and examined by a digital pathology system. The bone regeneration
ratio according to the photographs of the HE staining sections was
quantitatively analyzed by an Image-Pro Plus 6.0 software.
T. Lu, X. Yuan, L. Zhang et al.
2.8. Statistical analysis
Quantitative data are presented as mean ± standard deviations.
A comparison between two means was made using the student's t-
test, with statistical significance set as p<0.05 (‘*’ p<0.05, ‘#’ p<0.01,
and ‘$’ p<0.001).
3. Results and discussion
3.1. Phase composition, morphology and in vitro degradation of Zn-
BCP scaffolds
Fig. 1A shows the X-ray diffraction patterns of Zn-BCP scaffolds.
Only the diffraction peaks corresponding to b-TCP (JCPDS #090169)
Fig. 1. X-ray diffraction patterns (A), phase compositions (B), X-ray photoelectron spectroscope survey spectra (C) and macroscopic features (D) of scaffolds. D1: fiber diameter, D2:
fibers distance in X- and Y-direction, D3: fibers distance in Z-direction.
4
Materials Today Chemistry 29 (2023) 101410
and HA (JCPDS #090432) can be observed, manifesting the suc-
cessful synthesis of BCP. The diffraction peaks of b-TCP shifted to
higher angles, while those of HA did not deviate with Zn doping.
This indicated that in the presence of both b-TCP and HA, Zn
preferred entering the lattice structure of b-TCP. Zn ions with
smaller radius substituted for Ca ions with larger radius [23],
resulted in the lattice contraction of b-TCP and led to the shift of
diffraction peaks to higher angles. The phase composition calcu-
lated by Rietveld refinements using the program of General
Structure Analysis System is showed in Fig. 1B. The mass ratio of b-
TCP and HA of all scaffolds fluctuated around 0.5 with the Zn doping
content increase, and it was in the range of 0.4e0.6. This indicated
that we had successfully prepared BCP scaffolds with a gradient
variation of Zn doping but a uniformly phase ratio. Because the
T. Lu, X. Yuan, L. Zhang et al.
phase ratio of BCP plays a vital role in the osteoinduction perfor-
mance and bone defect repair effect of BCP [24,25], it is necessary to
control the phase ratio of BCP. X-ray photoelectron spectroscope
and EDS were added to analyze the element composition and dis-
tribution of the scaffold, and the results are shown in Fig. 1C and
Fig. S1, respectively. According to Fig. 1C, we could find that several
deconvolution peaks were identified at 532, 350, 285, and 132 eV,
which were attributed to O1s, Ca2p, C1s, and P2p, respectively. New
peaks attributed to Zn2p (1045 eV and 1022 eV) were found on
Zn2.5-S and Zn5.0-S, and the peaks of Zn2p in Zn5.0-S were more
obvious than those in Zn2.5-S. Similar results can be seen in EDS
spectra (Fig. S1). In addition, the results of element distribution
show that Zn ions were uniformly distributed.
The fabricated scaffolds were three-dimensionally connected
(Fig. 1D) with a fiber diameter of about 350 mm. The fiber distance in
XY-direction and Z-direction was about 400 mm and 170 mm,
respectively (Fig. 1D, Table S3). As the sintering temperature
increased, the scaffolds became denser, and fewer micropores on
free surfaces and facture surfaces can be observed (Fig. S2). When
the sintering temperature rose to 1150
C, the micropores almost
disappeared. A study by Yuan et al. [26] found that micropores
played a dominant role on ectopic bone formation. However, the
existence of micropores was harmful to the mechanical strength of
scaffolds. Hence, taking the osteoinductivity and the mechanical
strength into consideration, the sintering temperature of BCP
scaffolds was set as 1100
C.
Scanning electron microscope images of Zn-BCP scaffolds after
sintering are exhibited in Fig. S3. Many micropores can be observed
on the free surfaces and fracture surfaces of scaffolds. Compared to
a bare BCP scaffold, the crystal grains on free surfaces of Zn-BCP
scaffolds bonded more tightly with each other, especially for the
scaffolds with a doping content 2.0 mol.%. This indicated that Zn
doping promoted the sintering and densification of BCP scaffolds
sintered at 1100
C for 2 h, which was consistent with the result
found by Sopyan et al. [27]. The denser structure of Zn-BCPs
resulted in their lower degradation rate than a bare BCP scaffold.
The in vitro degradation behaviors of Zn-BCP scaffolds immersed in
an acetic acid/sodium acetate buffer solution (pH ¼ 4.5) for 12
weeks are shown in Fig. S4. The weight loss of all scaffolds
increased as the immersion time prolonged. Compared to a bare
BCP scaffold (Zn0-S), Zn-BCP scaffolds showed lower weight loss,
whose values were about 15 wt% after immersion for 12 weeks
(Fig. S4A). With the increase of the Zn doping content, the Ca and P
concentrations released by BCP scaffolds showed a decreasing
trend in the first four weeks of immersion, while the Zn concen-
trations showed an increasing trend (Fig. S4BeD). The decrease of
the Ca and P concentrations was related to the decreased degra-
dation rate of BCP scaffolds caused by Zn doping, while the increase
of the Zn release concentration was related to the increase of the Zn
doping content. It should be noticed that Zn-BCP scaffolds with a
doping content 2.5 mol.% could release Zn ions for a long time (at
least for 12 weeks, Fig. S4D).
3.2. In vitro cell compatibility and osteogenic differentiation
The optical density value of mBMSCs cultured on the surfaces of
scaffolds increased as time prolonged, except for those cultured
with Zn6.0-S. Zn-BCP scaffolds with an appropriate Zn doping
content (1.0e3.5 mol.%) could promote the proliferation of mBMSCs
at both day 3 and day 7 (Fig. 2A). This was consistent with the live/
dead cell staining results for three days exhibited in Fig. 2B, for
more green live cells can be observed on the surfaces of Zn-BCP
scaffolds, with a doping content of 1.0e3.5 mol.%. The cellular
cytoskeleton staining for 1 day in Fig. S5 shows that mBMSCs could
adhere and spread well on the surfaces of scaffolds doped with
5
Materials Today Chemistry 29 (2023) 101410
Zn  5 mol.%. After seven days of culture, stem cells have covered
the whole scaffolds, indicating a long-term biocompatibility of BCP
scaffolds. Fig. 2CeE exhibit the ion concentration of the cell culture
medium immersed with scaffolds for seven days. Zn and Ca con-
centrations in the culture medium increased as the Zn doping
content increased. The concentration of Zn showed a decreasing
trend as the immersion time prolonged, while the concentration of
Ca showed an opposite trend. The P concentrations in the culture
medium of all scaffolds were in the range of 5e10 ppm, except for
Zn6.0-S. It is obvious that the ion concentrations of Zn6.0-S were
remarkably higher than those of the others, and its Zn concentra-
tion on day 1 was about 37 ppm, which was significantly higher
than the toxic concentration (9.75 ppm) for cells [28]. This would be
the reason for the apoptosis of mBMSCs cultured on Zn6.0-S (Fig. 2A
and B).
Fig. 3A and B show the ALP activity and ALP staining of mBMSCs
cultured on scaffolds for 7 and 14 days, respectively. Zn doping can
significantly up-regulate the ALP activity of mBMSCs both on days 7
and 14, and cells cultured with Zn2.0-S and Zn2.5-S had the highest
ALP activity (Fig. 3A). The ALP activity analysis agreed with the ALP
staining result. ALP was stained in blue, and the deeper staining
color on Zn2.0-S and Zn2.5-S indicated a more secretion content of
the ALP by the cells cultured on them (Fig. 3B).
The expression level of the osteogenesis-related genes of
mBMSCs cultured with scaffolds for 3, 7 and 14 days is exhibited in
Fig. 3CeG. For Runx2, only Zn2.5-S significantly enhanced its
expression on day 3. Zn-BCP scaffolds doped with Zn  1.0 mol.%
remarkably promoted the expression level of Runx2 on day 7. Zn-
BCP scaffolds doped with Zn  2.0 mol.% could still enhance the
expression level of Runx2 on day 14. It should be noted that, cells
cultured with Zn2.5-S expressed the highest level of Runx2 on
every detected time point. For Col-I and ALP, Zn-BCP scaffolds with
a doping range of 1.0e3.5 mol.% significantly promoted their
expression on day 3 and day 7. In addition, Zn1.5-S, Zn2.0-S, Zn2.5-
S, Zn3.0-S, and Zn3.5-S still could enhance the expression level of
ALP and Col-I on day 14. It is worth noting that, Zn5.0-S down-
regulated the expression level of Col-I on day 14 as well as the
expression level of ALP on day 3 and day 7. For BSP and OCN, cells
cultured on Zn-BCP scaffolds with a doping range of 1.5e3.5 mol.%
expressed remarkably a higher level of BSP and OCN than those
cultured on Zn0-S (control group) at every detected time point.
Cells cultured on Zn2.5-S expressed the highest level of BSP and
OCN on day 14. Taking the expression level of all these
osteogenesis-related genes into consideration, we could find that:
1) Zn0.5-S did not significantly enhance the osteogenic differenti-
ation of mBMSCs; 2) Zn-BCP scaffolds with a doping range of
1.0e5.0 mol.% could effectively promote osteogenic differentiation
compared to bare BCP scaffolds; 3) Zn2.0-S and Zn2.5-S showed the
most effective osteogenic differentiation accelerated ability.
The ions, especially Zn, released from scaffolds played vital roles
in stem cells survival, proliferation, and differentiation. In addition,
many bioactive ions, including Zn, mediated cell actions in a
concentration-dependent manner [18e20]. Taking the prolifera-
tion, ALP activity, and the expression level of osteogenesis-related
genes into consideration, we could find that Zn doping mediated
the activity and osteogenic differentiation of mBMSCs in a doping
content-dependent manner because the released concentration of
Zn depended on its doping content (Fig. 2C). Although we have not
thoroughly explored the molecular mechanism of the Zn-doped
BCP regulating the osteogenic differentiation of stem cells, a large
number of studies have proved that Zn may promote osteogenic
differentiation of BMSCs through various cellular molecular path-
ways, such as MAPK pathway, cAMP pathway, and TGF-b/BMP
pathway [29]. Gao et al. [30] found that extracellular Zn entered
BMSCs by endocytosis, after internalization, activating the MAPK/
T. Lu, X. Yuan, L. Zhang et al. Materials Today Chemistry 29 (2023) 101410

Fig. 2. cell compatibility and ions concentration. The cell proliferation (A; on days 1, 3 and 7) and live/dead staining (B; on day 3) of mouse bone mesenchymal stem cells cultured
on the surfaces of scaffolds; the concentrations of Zn (C), Ca (D) and P (E) in a cell cultured medium.

6
T. Lu, X. Yuan, L. Zhang et al.
Fig. 3. alkaline phosphatase activity (A), alkaline phosphatase staining (B) and osteogenesis-related genes expression (CeG) of mouse bone mesenchymal stem cells cultured with
scaffolds.
ERK pathway and eventually promoting osteogenesis. A study by
Zhu et al. [31] confirmed that extracellular Zn was transferred into
cells by cellular receptors, and then activated the cAMP-PKA
pathway and triggered Ca responses in the cytoplasm, leading to
the activation of the MAPK pathway. Zn was also found to promote
the differentiation of BMSCs to mature matrix-secreting osteoblasts
through the TGF-b/BMP signaling pathway [32]. Our in vitro oste-
ogenic differentiation results illustrated that the released Zn con-
centration from scaffolds doped with Zn  0.5 mol.% was not
enough to stimulate osteoblast differentiation, while that from
scaffolds doped with Zn  6 mol.% was exorbitant and resulted in
cell apoptosis. Zn-BCP scaffolds with a doping content of 2.5 mol.%
possessed the highest osteogenic differentiation property. Hence,
we chose Zn0-S, Zn1.0-S, Zn2.5-S, and Zn5.0-S to further explore
the effect of Zn doping on osteoclast differentiation.
3.3. In vitro osteoclastogenesis
Bone matrix can be absorbed by multinucleated cells named
osteoclasts [33], which differentiate from the macrophage/
monocyte lineage when stimulated by RANKL [34]. Fig. 4 shows
the osteoclast differentiation of Raw 264.7 cultured with scaffolds
7
Materials Today Chemistry 29 (2023) 101410
with RANKL (50 ng/mL). NFATc1 serves as a master regulator of
osteoclast differentiation. The activation of c-Fos and NFATc1 is
necessary for the sufficient differentiation of osteoclast [35,36].
For the osteoclast-related genes expression (Fig. 4AeC), Zn doping
significantly inhibited the expression level of NFATc1 on day 3.
While on day 5, the expression level of NFATc1 in cells cultured
with different scaffolds showed no statistically significant differ-
ence but decreased in the order of Zn0-S, Zn1.0-S, Zn2.5-S, and
Zn5.0-S. Zn-BCP scaffolds could inhibit the expression level of c-
Fos on day 3 and day 5, and thereinto, Zn2.5-S and Zn5.0-S
remarkably down-regulated the expression level of c-Fos on day
5. NFATc1 regulates a number of osteoclast specific genes such as
TRAP through cooperation with c-Fos [37]. Zn2.5-S and Zn5.0-S
inhibited the expression level of TRAP both on days 3 and 5, and
Zn1.0-S down-regulated the expression level of TRAP only on day
5.
The monocyte/macrophage lineage fuses to form TRAP-positive
multinucleated cells (osteoclasts), and cells that have two or more
nuclei are considered to be osteoclasts. According to cytoskeleton
staining images (Fig. 4D), we could easily find that there were
multinuclei cells which were osteoclasts, generating when cultured
with Zn0-S and Zn1.0-S. Not only that, the size of osteoclasts on
T. Lu, X. Yuan, L. Zhang et al. Materials Today Chemistry 29 (2023) 101410

Fig. 4. osteoclast differentiation of RAW 264.7. The osteoclast-related genes expression of RAW 264.7 co-cultured with scaffolds (AeC); the cytoskeleton staining (D) and tartrate-
resistant acid phosphatase staining (E) of RAW 264.7 cultured with scaffolds and in scaffold extracts for 5 days, respectively. The red circles in Fig. 4D and E labeled multinuclear
osteoclasts.

Zn0-S was larger than that on Zn1.0-S. However, there were few
osteoclasts generating on the surfaces of Zn2.5-S and Zn5.0-S. TRAP
staining of cells cultured in the extracts of scaffolds, showed in
Fig. 4E, also indicated that there were fewer TRAP positive cells in
the Zn-doped scaffold extracts, especially in the extracts of Zn2.5-S
and Zn5.0-S.
Concerning the genes expression level of TRAP, c-Fos, and NFATc1
as well as the fusion and formation of osteoclasts, we concluded that
Zn inhibited osteoclastogenesis in a doping content-dependent
manner. Higher Zn doping content resulted in a stronger inhibition
of osteoclastogenesis. Park et al. [38] found that Zn treatment during
osteoclast differentiation decreased RANKL-induced osteoclast for-
mation in a dose-dependent manner by suppressing the Ca2þ-
Calcineurin-NFATc1 signaling pathway. An animal study by Hie et al.
[39] showed that Zn-administration decreased osteoclastogenesis by
inhibiting RANK expression through the suppression of the pro-
duction of reactive oxygen species and ERK activation in Zn-adequate
rats. Further, Yamaguchi et al. [40] revealed that Zn suppressed
osteoclast differentiation and promoted osteoblast mineralization
and did indeed act as a potent NF-kappa B activation antagonist in
both osteoclast and osteoblast precursors. Our in vitro osteoclasto-
genesis results revealed that Zn doping with a content 2.5 mol.%
could effectively suppress osteoclast differentiation. Hence, we chose
Zn0-S, Zn2.5-S, and Zn5.0-S to further explore the effects of Zn
doping on the in vivo osteoinduction and bone regeneration effects of
BCP scaffolds.

8
T. Lu, X. Yuan, L. Zhang et al. Materials Today Chemistry 29 (2023) 101410

Fig. 5. polarization of macrophages surrounding scaffolds implanted for 14 days (A) and the osteogenesis of mouse bone mesenchymal stem cells cultured in the macrophage-
conditioned medium for 7 and 14 days (BeE). alkaline phosphatase activity (B), alkaline phosphatase staining images (C) and osteogenesis-related genes expression at day 7
(D) and day 14 (E).
9
T. Lu, X. Yuan, L. Zhang et al.
3.4. Immunomodulation and angiogenesis
It was found that immune system also played vital roles in bone
defect repair process [41,42], especially in the inflammation phase.
Much literature indicated that promoting macrophages polariza-
tion into M2 phenotype, which could secrete anti-inflammatory
and pro-healing factors, was beneficial to the reduction of inflam-
mation and the enhancement of defect repair [43]. Fig. 5A displays
the macrophage phenotypes around scaffolds after implanted for
14 days. iNOS and CD163 positive cells were M1 and M2 macro-
phages, respectively. It was obvious that, the distribution density of
M2 around Zn-BCP scaffolds, especially Zn2.5-S, was much higher
than that around bare BCP scaffolds (Zn0-S). However, lower dis-
tribution density of M1 was found around Zn2.5-S and Zn5.0-S.
These indicated that Zn doping contributed to the M2 polarization
while inhibited M1 polarization. This would result in reducing
inflammation and facilitating defects repair. On the one hand, the
thickness of fibrous capsules around Zn2.5-S and Zn5.0-S was
thinner than that around Zn0-S (Fig. S6). On the other hand, the
macrophage-conditioned medium extracted from Zn2.5-S and
Zn5.0-S was beneficial to the osteogenic differentiation of mBMSCs.
The macrophage-conditioned medium of Zn2.5-S and Zn5.0-S
effectively enhanced the ALP activity of cells (Fig. 5B and C).
Furthermore, the conditioned medium of Zn2.5-S up-regulated the
expression level of osteogenesis-related genes like ALP, BSP and
OCN both on days 7 and 14 (Fig. 5D and E). Though the conditioned
medium of Zn5.0-S down-regulated the expression level of Runx2,
Col-I, ALP and OCN on day 7, it up-regulated the genes expression
level of Col-I, ALP, BSP and OCN on day 14 (Fig. 5D and E). Therefore,
Zn-doped BCP scaffolds, especially Zn2.5-S, showed the potential to
promote osteogenesis by providing an anti-inflammation and pro-
healing microenvironment for cells involved in defect repair
through promoting M2 macrophage polarization.
Vascularization is necessary for bone defect repair. Blood
vessels not only can bring oxygen and nutrients to cells and
remove metabolic waste but also transport the cells involved in
repair to the bone defect sites. Hence, promoting early vascular-
ization can accelerate the repair process of bone defect [44]. Fig. 6
shows the CD31 staining images (Fig. 6A) and the relevant sta-
tistical analysis of the amount (Fig. 6B) and diameter (Fig. 6C and
D) of vessels around scaffolds after implanted for 7 and 14 days.
The CD31 positive brown circles were blood vessels. On day 7,
numerous new blood vessels could be seen generating around all
scaffolds, and few blood vessels distributing in the macropores in
the interior of Zn0-S as well as Zn5.0-S. However, a few vessels
had infiltrated into the macropores of Zn2.5-S. In addition, the
number of blood vessels around of Zn2.5-S was remarkably larger
than that of Zn0-S (Fig. 6B). When after implanted for 14 days,
blood vessels started to infiltrate into the inner macropores of
Zn0-S and Zn5.0-S (Fig. 6A). More new blood vessels could be
found around all scaffolds, and there were still more blood vessels
distributing on the periphery as well as in the interior of Zn2.5-S
(Fig. 6A and B). Although the size of vessels around Zn2.5-S
showed no significant difference from those around Zn0-S on
postoperative day 7 (Fig. 6C), we could easily see some larger
vessels around Zn2.5-S (Fig. 6A). On postoperative day 14, the size
of blood vessels around Zn2.5-S was significantly larger than
those around Zn0-S (Fig. 6D). However, vessels around Zn5.0-S
were comparable to those around Zn0-S, both in number and
size (Fig. 6BeD). Recently, extracellular Zn has been shown to
promote an endothelial activity by triggering the upregulation of
pro-angiogenic cytokines, such as PDGF-BB and VEGF, in a Zn-
sensing receptor ZnR/GPR39-dependent manner and through the
downstream Gaq-PLC pathways [45]. A study by Sai A et al. [46]
showed that an appropriate concentration of Zn treatment was
10
Materials Today Chemistry 29 (2023) 101410
beneficial to the migration, tube formation and angiogenesis
during a chicken embryo development. However, a high concen-
tration of Zn treatment had no positive effect on the endothelial
activity and even had side-effects. Their results were similar to
ours. Therefore, it is reasonable to believe that Zn2.5-S could
promote angiogenesis in vivo by releasing an appropriate con-
centration of Zn, while the excessive concentration of Zn released
by Zn5.0-S had no promoting effect on angiogenesis.
3.5. In vivo osteoinduction
When implanted in the mongrel dog dorsal muscle for four
weeks, numerous connective tissues were filled into the macro-
pores of all scaffolds (Fig. S7). There were multinuclear macro-
phages spreading on the interface between tissues and macropores.
Inside the macropores, new blood vessels appeared. However, few
materials degraded and few ectopic bones generated. TRAP staining
images (Fig. 7A) show that numerous TRAP positive cells (osteo-
clasts) spread on the interface between tissues and macropores in
Zn0-S, and its distribution density was higher in the place closer to
the margins of macropores. Fewer osteoclasts were observed in
Zn2.5-S, and hardly any TRAP positive cells could be found in Zn5.0-
S. This manifested that Zn doping could also inhibit the osteoclasts
formation in vivo, and the inhibition effect showed a doping
content-dependent manner, which agreed with the in vitro exper-
iments (Fig. 4).
When implanted for eight weeks, newly forming osteoids could
be observed in all groups (Fig. 7B). The osteoids were mainly
distributed around the macropores of scaffolds. Osteoblasts with
stringy shape were lined in the interface between the osteoids and
inner tissues, while osteocytes with circular shape were imbedded
in the osteoids. These observations indicated that the osteoids
initiated around the margins of macropores and infiltrated into the
internal gradually with the passage of implantation time. Toluidine
blue staining images in Fig. S8 show that at week four, there were
toluidine blue positive chondrocytes, indicating that the formation
of osteoids included entochondrostosis. The stained chondrocytes
were mainly distributed in the margins of macropores, which also
manifested that the osteoids initiated from the margins of mate-
rials. The newly osteoids formation ratio in Zn0-S was about 12%,
while it increased to about 25% in Zn2.5-S and dropped to 10% in
Zn5.0-S (Fig. S9). According to the results of the Masson staining
shown in Fig. S10, we found that the maturity of the generated
osteoids decreased in the order of Zn0-S, Zn2.5-S, and Zn5.0-
S because the osteoids in Zn0-S showed comparable characteris-
tics of mature trabecular bone (lamellar bone), while the new bones
in Zn2.5-S and Zn5.0-S, especially Zn5.0-S, showed higher sectors of
fibroreticular bone (woven bone).
The inhibition of osteoclasts formation and activity by Zn doping
would result in the reduction of the active degradation rate of
materials mediated by osteoclasts. Furthermore, Zn doping could
also reduce the passive degradation rate of BCP ceramic scaffolds
(Fig. S4). These two statuses led to a lower release concentration of
Ca and P ions around scaffolds when implanted in vivo. The released
Ca and P ions from materials were necessary for the formation of an
ectopic bone. When the concentration of Ca and P was higher than
their corresponding saturation concentration, ectopic bones would
more easily generate [47]. In addition, the formation of an ectopic
bone and osteoclastogenesis would be blocked by macrophage
depletion [48]. These all manifested that the material active
degradation mediated by osteoclasts and the material passive
degradation in body fluid by dissolving were both essential to the
induction of ectopic bones. We thought that, the higher degrada-
tion rate of Zn0-S made ectopic bones initiate in it earlier and
resulted in more mature ectopic bones. Zn5.0-S had adverse effects
T. Lu, X. Yuan, L. Zhang et al.
Fig. 6. blood vessel analysis on 7- and 14-day after surgery. CD31 immunohistochemical staining images of tissues around samples (A). The statistical analysis of the amount (B) and
diameter (CeD) of vessels. M: materials; CT: connective tissues; blue arrows: blood vessels.
on ectopic bone formation, which probably due to its obvious
inhibitory effect on osteoclastogenesis and low dissolution. While
Zn2.5-S could effectively promote osteogenesis, facilitating the
formation and infiltration of blood vessels, which eventually made
it possess the highest ectopic new bone ratio among all scaffolds.
3.6. In vivo bone regeneration
Fig. 8 exhibits the micro computed tomography images of rabbit
femur bones implanted with scaffolds. We can see that the
implanted scaffolds degraded gradually as implanted time went
on as the diameter of scaffolds decreased gradually. The degrada-
tion of scaffolds did not damage the structure of scaffolds. This
made scaffolds still in a whole after implantation for 12 weeks.
With the degradation of materials, new bones (stained in red) were
generated and infiltrated into the inner pore of scaffolds through
11
Materials Today Chemistry 29 (2023) 101410
interconnected pores. Much more new bones could be found
generating and infiltrating in Zn2.5-S. New bones were fully filled
in the macropores of Zn2.5-S at week 8 and week 12, while the new
bones in the inner pores of Zn0-S and Zn5.0-S were under filled and
mainly spread around the margins of pores.
Fig. 9A displays the HE staining images of rabbit femur bones
implanted with scaffolds. When implanted for four weeks, the
macropores of all scaffolds were filled with tissues, with a few new
bones which appeared around the margins of macropores. New
bones gradually infiltrated into the inner macropores of Zn0-S and
Zn5.0-S at week 8 and week 12. It should be noticed that new bones
not only distributed around the margins of pores but also filled in
some of the macropores of Zn2.5-S at week 4 and week 8. At week
12, with the degradation of the material, new bones further entered
the interior of Zn2.5-S through the degradation pores/channels. In
addition, the new bone area in Zn2.5-S was obviously larger than
T. Lu, X. Yuan, L. Zhang et al. Materials Today Chemistry 29 (2023) 101410

Fig. 7. tartrate-resistant acid phosphatase (A; 4 weeks) and hematoxylin and eosin (B; 8 weeks) staining images of scaffolds implanted in a mongrel dog dorsal muscle. In Fig. 7A,
while arrows: multinuclear osteoclasts. In Fig. 7B, M: materials; NB: new bones (osteoids); blue arrows: osteoblasts; black arrows: osteocytes.

Fig. 8. micro computed tomography images of the femur bone defect of rabbits after the implantation of scaffolds for 4, 8, and 12 weeks (New bones were staining in red).

12
T. Lu, X. Yuan, L. Zhang et al.
Fig. 9. hematoxylin and eosin staining images (A) and the new bone ratio (B) of scaffolds after implantation in the femur bone defect of rabbits for 4, 8, and 12 weeks. HB: host
bones, NB: new bones, CT: connective tissues, M: materials.
that in Zn0-S and Zn5.0-S. The new bone ratio calculation according
to the HE staining results is shown in Fig. 9B, it increased with the
passage of implanted time in all scaffolds. The new bone ratio of
Zn2.5-S was the highest at all-time points. At weeks 4 and 8, the
new bone ratio of Zn5.0-S was slightly higher than that of Zn0-S but
with no statistical difference.
Bone remodeling has been considered to consist of four
sequential processes [49], including: 1) osteoclast progenitors are
recruited; 2) osteoclast progenitors differentiate into mature oste-
oclasts and resorb damaged bones; 3) osteoclasts apoptosis and
osteoblast progenitors are recruited; 4) osteoblast progenitors
differentiate into mature osteoblasts, and produce new bone matrix
and then mineralize [50,51]. Almost all new bone formation was
observed in areas where bone resorption had previously occurred,
known as basic multicellular units in different anatomical struc-
tures [52]. Under homeostatic conditions, the balance between
osteoblast-mediated bone formation and osteoclast-mediated bone
resorption was strictly regulated without a major alteration.
Disruption of this balance would result in abnormal bone regen-
eration. Most anti-resorptive agents suppress osteoclasts formation
and activity while inhibiting bone formation simultaneously.
Analogously, the induced bone formation activity of anabolic
agents is modulated by increasing bone resorption at the same time
[53].
Zn5.0-S could promote osteogenic differentiation, relieve
inflammation, and reduce fibrous capsule formation, making it
promote new bone formation at early implantation stages (4 w and
8 w). While its excessive suppression effect on osteoclastogenesis
was not conducive to bone remolding, and its low dissolution rate
also did not match with the infiltration rate of new bone tissues.
These two factors made Zn5.0-S not facilitate new bone formation
13
Materials Today Chemistry 29 (2023) 101410
at the late implantation state (12 w). Zn2.5-S could effectively
promote osteoblast differentiation, facilitate new blood vessels
formation and infiltration, promote M2 polarization, and reduce
fibrous encapsulation, which made sure it possessed the highest
new bone ratio among all scaffolds at early implantation stages. In
addition, Zn2.5-S inhibited osteoclasts differentiation and activity
to an appropriate degree, and we thought that this appropriate
inhibition on osteoclastogenesis would not be harmful to bone
remolding. Namely, the balance between the pro-osteogenesis and
anti-osteoclastogenesis mediated by Zn2.5-S made it possess the
highest new bone ratio at late implantation stages.
4. Conclusion
In this study, BCP scaffolds with different Zn doping contents but
similar phase compositions were prepared. The effects of Zn doping
on the osteogenesis and osteoclastogenesis of BCP scaffolds were
systematically investigated in vitro and in vivo. The osteoblast dif-
ferentiation of mBMSCs and osteoclast differentiation of RAW264.7
both showed a Zn doping content-dependent behavior. By
increasing the Zn doping content (0e5 mol.%), the anti-
osteoclastogenesis effect of Zn-BCP scaffolds gradually increased.
While the pro-osteogenesis property of Zn-BCP scaffolds first
increased and then decreased, and the most effective doping con-
tent of Zn for promoting osteoblast differentiation was 2.5 mol.%.
The BCP scaffold with a Zn doping content of 2.5 mol.% possessed
the highest osteoinduction activity and fastest new bone formation,
which would be associated with the balance between pro-osteo-
genesis and anti-osteoclastogenesis mediated by the appropriate
Zn doping content. Hence, Zn2.5-S would be a promising bioma-
terial for bone defect regeneration.
T. Lu, X. Yuan, L. Zhang et al.
Author contributions
Teliang Lu: conceptualization, methodology, investigation, and
writing e original draft.
Xinyuan Yuan: investigation and formal analysis.
Luhui Zhang: formal analysis.
Fupo He: writing e review & editing and validation.
Xiaolan Wang: validation and formal analysis.
Jiandong Ye: conceptualization, supervision, writing - review &
editing, and funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgments
This work was supported by the National Key R&D Program of
China (Grant No. 2016YFB0700803) and Guangdong Basic and
Applied Basic Research Foundation (Grant No. 2022A1515110274).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.mtchem.2023.101410.
References
[1] S. Samavedi, A.R. Whittington, A.S. Goldstein, Calcium phosphate ceramics in
bone tissue engineering: a review of properties and their influence on cell
behavior, Acta Biomater. 9 (9) (2013) 8037e8045, https://doi.org/10.1016/
j.actbio.2013.06.014.
[2] N. Eliaz, N. Metoki, Calcium phosphate bioceramics: a review of their history,
structure, properties, coating technologies and biomedical applications, Ma-
terials 10 (4) (2017), https://doi.org/10.3390/ma10040334.
[3] Y. Li, X.M. Xu, X.M. Liu, B. Li, Y. Han, Y.F. Zheng, D.F. Chen, K.W.W. Yeung,
Z.D. Cui, Z.Y. Li, Y.Q. Liang, S.L. Zhu, X.B. Wang, S.L. Wu, Photoelectrons
mediating angiogenesis and immunotherapy through heterojunction film for
noninvasive disinfection, Adv. Sci. 7 (17) (2020), https://doi.org/10.1002/
advs.202000023.
[4] K. Zhang, Y. Zhou, C. Xiao, W.L. Zhao, H.F. Wu, J.Q. Tang, Z.T. Li, S. Yu, X.F. Li,
L. Min, Z.T. Yu, G. Wang, L. Wang, K. Zhang, X. Yang, X.D. Zhu, C.Q. Tu,
X.D. Zhang, Application of hydroxyapatite nanoparticles in tumor-associated
bone segmental defect, Adv. Sci. 5 (8) (2019), https://doi.org/10.1126/
sciadv.aax6946.
[5] J.N. Fu, X.M. Liu, L. Tan, Z.D. Cui, Y.F. Zheng, Y.Q. Liang, Z.Y. Li, S.L. Zhu,
K.W.K. Yeung, X.B. Feng, X.B. Wang, S.L. Wu, Photoelectric-responsive extra-
cellular matrix for bone engineering, ACS Nano 13 (11) (2019) 13581e13594,
https://doi.org/10.1021/acsnano.9b08115.
[6] J.N. Fu, W.D. Zhu, X.M. Liu, C.Y. Liang, Y.F. Zheng, Z.Y. Li, Y.Q. Liang, D. Zheng,
S.L. Zhu, Z.D. Cui, S.L. Wu, Self-activating anti-infection implant, Nat. Commun.
12 (1) (2021), https://doi.org/10.1038/s41467-021-27217-4.
[7] S.E. Kim, K. Park, Recent advances of biphasic calcium phosphate bioceramics
for bone tissue regeneration, in: H.J. Chun, R.L. Reis, A. Motta, G. Khang (Eds.),
Biomimicked Biomaterials: Advances in Tissue Engineering and Regenerative
Medicine, vol. 1250, 2020, pp. 177e188.
[8] J.M. Bouler, P. Pilet, O. Gauthier, E. Verron, Biphasic calcium phosphate ce-
ramics for bone reconstruction: a review of biological response, Acta Bio-
mater. 53 (2017) 1e12, https://doi.org/10.1016/j.actbio.2017.01.076.
[9] J. Wang, Y. Chen, X. Zhu, T. Yuan, Y. Tan, Y. Fan, X. Zhang, Effect of phase
composition on protein adsorption and osteoinduction of porous calcium
phosphate ceramics in mice, J. Biomed. Mater. Res. Part A 102 (12) (2014)
4234e4243, https://doi.org/10.1002/jbm.a.35102.
[10] H. Yuan, C.A. van Blitterswijk, K. de Groot, J.D. de Bruijn, A comparison of bone
formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA)
implanted in muscle and bone of dogs at different time periods, J. Biomed.
Mater. Res. Part A 78A (1) (2006) 139e147, https://doi.org/10.1002/
jbm.a.30707.
14
Materials Today Chemistry 29 (2023) 101410
[11] K.D. Hankenson, G. Zimmerman, R. Marcucio, Biological perspectives of
delayed fracture healing, Injury 45 (Suppl 2) (2014) S8eS15, https://doi.org/
10.1016/j.injury.2014.04.003.
[12] K.D. Hankenson, K. Gagne, M. Shaughnessy, Extracellular signaling molecules
to promote fracture healing and bone regeneration, Adv. Drug Delivery Rev.
94 (2015) 3e12, https://doi.org/10.1016/j.addr.2015.09.008.
[13] J.P. O'Connor, D. Kanjilal, M. Teitelbaum, S.S. Lin, J.A. Cottrell, Zinc as a ther-
apeutic agent in bone regeneration, Materials 13 (10) (2020), https://doi.org/
10.3390/ma13102211.
[14] T. Lu, X. Yuan, L. Zhang, F. He, X. Wang, Y. Zhang, J. Ye, High throughput
synthesis and screening of zinc-doped biphasic calcium phosphate for bone
regeneration, Appl. Mater. Today 25 (2021), 101225, https://doi.org/10.1016/
j.apmt.2021.101225.
[15] X. Huang, D.H. Huang, T. Zhu, X.H. Yu, K.C. Xu, H.Y. Li, H. Qu, Z.Y. Zhou,
K. Cheng, W.J. Wen, Z.M. Ye, Sustained zinc release in cooperation with CaP
scaffold promoted bone regeneration via directing stem cell fate and trig-
gering a pro-healing immune stimuli, J. Nanobiotechnol. (1) (2021) 19,
https://doi.org/10.1186/s12951-021-00956-8.
[16] R. Cruz, J. Calasans-Maia, S. Sartoretto, V. Moraschini, A.M. Rossi, R.S. Louro,
J.M. Granjeiro, M.D. Calasans-Maia, Does the incorporation of zinc into cal-
cium phosphate improve bone repair? A systematic review, Ceram. Int. 44 (2)
(2018) 1240e1249, https://doi.org/10.1016/j.ceramint.2017.10.157.
[17] D.V. Shepherd, K. Kauppinen, R.A. Brooks, S.M. Best, An in vitro study into the
effect of zinc substituted hydroxyapatite on osteoclast number and activity,
J. Biomed. Mater. Res. Part A 102 (11) (2014) 4136e4141, https://doi.org/
10.1002/jbm.a.35089.
[18] S. An, Q. Gong, Y. Huang, Promotive effect of zinc ions on the vitality,
migration, and osteogenic differentiation of human dental pulp cells, Biol.
Trace Elem. Res. 175 (1) (2017) 112e121, https://doi.org/10.1007/s12011-
016-0763-7.
[19] J.M. Yu, L.Z. Xu, N. Xie, K. Li, Y.H. Xi, X.L. Liu, X.B. Zheng, X.S. Chen, X.J. Ye,
D.X. Wei, Optimal Zn-modified Ca-Si-based ceramic nanocoating with Zn ion
release for osteoblast promotion and osteoclast inhibition in bone tissue en-
gineering, J. Nanomater. 2017 (2017), https://doi.org/10.1155/2017/7374510.
[20] H.J. Seo, Y.E. Cho, T. Kim, H.I. Shin, I.S. Kwun, Zinc may increase bone for-
mation through stimulating cell proliferation, alkaline phosphatase activity
and collagen synthesis in osteoblastic MC3T3-E1 cells, Nutr. Res. Pract. 4 (5)
(2010) 356e361, https://doi.org/10.4162/nrp.2010.4.5.356.
[21] L.B. McCusker, R.B. Von Dreele, D.E. Cox, D. Louer, P. Scardi, Rietveld refine-
ment guidelines, J. Appl. Crystallogr. 32 (1999) 36e50, https://doi.org/
10.1107/s0021889898009856.
[22] N.C. Andres, N.L. D'Elia, J.M. Ruso, A.E. Campelo, V.L. Massheimer, P.V. Messina,
Manipulation of Mg2þ-Ca2þ switch on the development of bone mimetic
hydroxyapatite, ACS Appl. Mater. Interfaces 9 (18) (2017) 15698e15710,
https://doi.org/10.1021/acsami.7b02241.
[23] E. Boanini, M. Gazzano, A. Bigi, Ionic substitutions in calcium phosphates
synthesized at low temperature, Acta Biomater. 6 (6) (2010) 1882e1894,
https://doi.org/10.1016/j.actbio.2009.12.041.
[24] Z.R. Tang, Y.F. Tan, Y.L. Ni, J. Wang, X.D. Zhu, Y.J. Fan, X.N. Chen, X. Yang,
X.D. Zhang, Comparison of ectopic bone formation process induced by four
calcium phosphate ceramics in mice, Mater. Sci. Eng. C 70 (2017) 1000e1010,
https://doi.org/10.1016/j.msec.2016.06.097.
[25] C.S. Shao, L.J. Chen, R.M. Tang, B. Zhang, J.J. Tang, W.N. Ma, Degradation and
biological performance of porous osteomimetic biphasic calcium phosphate
in vitro and in vivo, Rare Met. 41 (2) (2022) 457e468, https://doi.org/10.1007/
s12598-021-01814-0.
[26] H.P. Yuan, K. Kurashina, J.D. de Bruijn, Y.B. Li, K. de Groot, X.D. Zhang,
A preliminary study on osteoinduction of two kinds of calcium phosphate
ceramics, Biomaterials 20 (19) (1999) 1799e1806, https://doi.org/10.1016/
s0142-9612(99)00075-7.
[27] I. Sopyan, Gunawan, Q.H. Shah, M. Mel, Fabrication and sintering behavior of
zinc-doped biphasic calcium phosphate bioceramics, Mater. Manuf. Process.
31 (6) (2016) 713e718, https://doi.org/10.1080/10426914.2015.1048361.
[28] W. Watjen, H. Haase, M. Biagioli, D. Beyersmann, Induction of apoptosis in
mammalian cells by cadmium and zinc, Environ. Health Perspect. 110 (s5)
(2002) 865e867, https://doi.org/10.1289/ehp.110-1241262.
[29] H.Y. Li, M.Z. Li, X. Ran, J.C. Cui, F. Wei, G.L. Yi, W. Chen, X.L. Luo, Z.W. Chen, The
role of zinc in bone mesenchymal stem cell differentiation, Cell Reprogram. 24
(2) (2022) 80e94, https://doi.org/10.1089/cell.2021.0137.
[30] X.M. Gao, Y.Y. Xue, Z. Zhu, J.Y. Chen, Y.H. Liu, X.T. Cheng, X. Zhang, J. Wang,
X.B. Pei, Q.B. Wan, Nanoscale zeolitic imidazolate framework-8 activator of
canonical MAPK signaling for bone repair, ACS Appl. Mater. Interfaces 13 (1)
(2021) 97e111, https://doi.org/10.1021/acsami.0c15945.
[31] D.H. Zhu, Y.C. Su, M.L. Young, J. Ma, Y.F. Zheng, L.P. Tang, Biological responses
and mechanisms of human bone marrow mesenchymal stem cells to Zn and
Mg biomaterials, ACS Appl. Mater. Interfaces 9 (33) (2017) 27453e27461,
https://doi.org/10.1021/acsami.7b06654.
[32] K. Yusa, O. Yamamoto, M. Iino, H. Takano, M. Fukuda, Z.W. Qiao, T. Sugiyama,
Eluted zinc ions stimulate osteoblast differentiation and mineralization in
human dental pulp stem cells for bone tissue engineering, Arch. Oral Biol. 71
(2016) 162e169, https://doi.org/10.1016/j.archoralbio.2016.07.010.
[33] B.J. Kim, J.M. Koh, Coupling factors involved in preserving bone balance, Cell.
Mol. Life Sci. 76 (7) (2019) 1243e1253, https://doi.org/10.1007/s00018-018-
2981-y.
T. Lu, X. Yuan, L. Zhang et al.
[34] H. Takayanagi, RANKL as the master regulator of osteoclast differentiation,
J. Bone Miner. Metabol. 39 (1) (2021) 13e18, https://doi.org/10.1007/s00774-
020-01191-1.
[35] J.H. Park, N.K. Lee, S.Y. Lee, Current understanding of RANK signaling in
osteoclast differentiation and maturation, Mol. Cells 40 (10) (2017) 706e713,
https://doi.org/10.14348/molcells.2017.0225.
[36] J.Y. Kang, N. Kang, Y.M. Yang, J.H. Hong, D.M. Shin, The role of Ca2þ-NFATc1
signaling and its modulation on osteoclastogenesis, Int. J. Mol. Sci. 21 (10)
(2020), https://doi.org/10.3390/ijms21103646.
[37] J.H. Kim, N. Kim, Regulation of NFATc1 in osteoclast differentiation, J. Bone
Metabol. 21 (4) (2014) 233e241, https://doi.org/10.11005/jbm.2014.21.4.233.
[38] K.H. Park, B. Park, D.S. Yoon, S.H. Kwon, D.M. Shin, J.W. Lee, H.G. Lee, J.H. Shim,
J.H. Park, J.M. Lee, Zinc inhibits osteoclast differentiation by suppression of
Ca2þ-Calcineurin-NFATc1 signaling pathway, Cell Commun. Signal. 11 (2013),
https://doi.org/10.1186/1478-811x-11-74.
[39] M. Hie, I. Tsukamoto, Administration of zinc inhibits osteoclastogenesis
through the suppression of RANK expression in bone, Eur. J. Pharmacol. 668
(1e2) (2011) 140e146, https://doi.org/10.1016/j.ejphar.2011.07.003.
[40] M. Yamaguchi, M.N. Weitzmann, Zinc stimulates osteoblastogenesis and
suppresses osteoclastogenesis by antagonizing NF-kappa B activation, Mol.
Cell. Biochem. 355 (1e2) (2011) 179e186, https://doi.org/10.1007/s11010-
011-0852-z.
[41] T. Zhang, Y. Yao, Effects of inflammatory cytokines on bone/cartilage repair,
J. Cell. Biochem. 120 (5) (2018) 6841e6850, https://doi.org/10.1002/
jcb.27953.
[42] H. Liu, D. Li, Y. Zhang, M. Li, Inflammation, mesenchymal stem cells and bone
regeneration, Histochem. Cell Biol. 149 (4) (2018) 393e404, https://doi.org/
10.1007/s00418-018-1643-3.
[43] W. Liu, J. Li, M. Cheng, Q. Wang, K.W.K. Yeung, P.K. Chu, X. Zhang, Zinc-
modified sulfonated polyetheretherketone surface with immunomodulatory
function for guiding cell fate and bone regeneration, Adv. Sci. 5 (10) (2018),
1800749, https://doi.org/10.1002/advs.201800749.
[44] A.R. Armiento, L.P. Hatt, G.S. Rosenberg, K. Thompson, M.J. Stoddart, Func-
tional biomaterials for bone regeneration: a lesson in complex biology, Adv.
15
Materials Today Chemistry 29 (2023) 101410
Funct. Mater. 30 (44) (2020), 1909874, https://doi.org/10.1002/
adfm.201909874.
[45] D.H. Zhu, Y.C. Su, Y.F. Zheng, B.M. Fu, L.P. Tang, Y.X. Qin, Zinc regulates vascular
endothelial cell activity through zinc-sensing receptor ZnR/GPR39, Am. J.
Physiol. 314 (4) (2018) C404eC414, https://doi.org/10.1152/ajpcell.00279.2017.
[46] S.A. Sreenivasamurthy, F.F. Akhter, A. Akhter, Y.C. Su, D.H. Zhu, Cellular
mechanisms of biodegradable zinc and magnesium materials on promoting
angiogenesis, Biomater. Adv. (2022) 139, https://doi.org/10.1016/
j.bioadv.2022.213023.
[47] A.M.C. Barradas, H.P. Yuan, C.A. van Blitterswijk, P. Habibovic, Osteoinductive
biomaterials: current knowledge of properties, experimental models and
biological mechanisms, Eur. Cells Mater. 21 (2011) 407e429, https://doi.org/
10.22203/eCM.v021a31.
[48] N.L. Davison, A.-L. Gamblin, P. Layrolle, H. Yuan, J.D. de Bruijn, F. Barrere-de
Groot, Liposomal clodronate inhibition of osteoclastogenesis and osteoin-
duction by submicrostructured beta-tricalcium phosphate, Biomaterials 35
(19) (2014) 5088e5097, https://doi.org/10.1016/j.biomaterials.2014.03.013.
[49] R. Hattner, B.N. Epker, H.M. Frost, Suggested sequential mode of control of
changes in cell behaviour in adult bone remodelling, Nature 206 (983) (1965)
489e490, https://doi.org/10.1038/206489a0.
[50] B. Langdahl, S. Ferrari, D.W. Dempster, Bone modeling and remodeling: po-
tential as therapeutic targets for the treatment of osteoporosis, Ther. Adv.
Musculoskel. Dis. 8 (6) (2016) 225e235, https://doi.org/10.1177/
1759720x16670154.
[51] J.C. Crockett, M.J. Rogers, F.P. Coxon, L.J. Hocking, M.H. Helfrich, Bone
remodelling at a glance, J. Cell Sci. 124 (7) (2011) 991e998, https://doi.org/
10.1242/jcs.063032.
[52] A.M. Parfitt, The coupling of bone formation to bone resorption: a critical
analysis of the concept and of its relevance to the pathogenesis of osteopo-
rosis, Metab. Bone Dis. Relat. Res. 4 (1) (1982) 1e6, https://doi.org/10.1016/
0221-8747(82)90002-9.
[53] J.-M. Kim, C. Lin, Z. Stavre, M.B. Greenblatt, J.-H. Shim, Osteoblast-osteoclast
communication and bone homeostasis, Cells 9 (9) (2020), https://doi.org/
10.3390/cells9092073.