Journal of
Functional
Biomaterials
Article
Magnesium-Rich Calcium Phosphate Derived from Tilapia
Bone Has Superior Osteogenic Potential
Xiaxin Cao 1,† , Jiaqi Zhu 1,† , Changze Zhang 1 , Jiaru Xian 1 , Mengting Li 1, * , Swastina Nath Varma 2 , Ziyu Qin 1 ,
Qiaoyuan Deng 3 , Xinyue Zhang 1 , Wei Yang 1,4 and Chaozong Liu 1,2, *
1
2
3
4
*
†
Citation: Cao, X.; Zhu, J.; Zhang, C.;
Xian, J.; Li, M.; Nath Varma, S.; Qin, Z.;
Deng, Q.; Zhang, X.; Yang, W.; et al.
Magnesium-Rich Calcium Phosphate
Derived from Tilapia Bone Has
Superior Osteogenic Potential. J. Funct.
Biomater. 2023, 14, 390. https://
Hainan Provincial Fine Chemical Engineering Research Center, Hainan University, Haikou 570228, China;
20203105022@hainanu.edu.cn (X.C.); 22210710000051@hainanu.edu.cn (J.Z.);
20203100926@hainanu.edu.cn (C.Z.); 21210817000024@hainanu.edu.cn (J.X.); ziyuqin@hainanu.edu.cn (Z.Q.);
xinyuezhang@hananu.edu.cn (X.Z.); yw@esfish.com (W.Y.)
Institute of Orthopaedic & Musculoskeletal Science, University College London, Royal National Orthopaedic
Hospital, London HA7 4LP, UK; t.varma@ucl.ac.uk
Key Laboratory of Advanced Material of Tropical Island Resources of Educational Ministry School of
Materials Science and Engineering, Hainan University, Haikou 570228, China; qydeng@hainanu.edu.cn
Hainan Xiangtai Fishery Co., Ltd., South of Yutang Road, Industrial Avenue, Laocheng Development Zone,
Chengmai City 571924, China
Correspondence: limengting@hainanu.edu.cn (M.L.); chaozong.liu@ucl.ac.uk (C.L.)
These authors contributed equally to this work.
Abstract: We extracted magnesium-rich calcium phosphate bioceramics from tilapia bone using a
gradient thermal treatment approach and investigated their chemical and physicochemical properties.
X-ray diffraction showed that tilapia fish bone-derived hydroxyapatite (FHA) was generated through
the first stage of thermal processing at 600–800 ◦ C. Using FHA as a precursor, fish bone biphasic
calcium phosphate (FBCP) was produced after the second stage of thermal processing at 900–1200 ◦ C.
The beta-tricalcium phosphate content in the FBCP increased with an increasing calcination temperature. The fact that the lattice spacing of the FHA and FBCP was smaller than that of commercial
hydroxyapatite (CHA) suggests that Mg-substituted calcium phosphate was produced via the gradient thermal treatment. Both the FHA and FBCP contained considerable quantities of magnesium,
with the FHA having a higher concentration. In addition, the FHA and FBCP, particularly the FBCP,
degraded faster than the CHA. After one day of degradation, both the FHA and FBCP released Mg2+ ,
with cumulative amounts of 4.38 mg/L and 0.58 mg/L, respectively. Furthermore, the FHA and
FBCP demonstrated superior bone-like apatite formation; they are non-toxic and exhibit better osteoconductive activity than the CHA. In light of our findings, bioceramics originating from tilapia bone
appear to be promising in biomedical applications such as fabricating tissue engineering scaffolds.
doi.org/10.3390/jfb14070390
Academic Editor: Masakazu
Keywords: tilapia bone; gradient thermal sintering; calcium phosphate bioceramics; osteogenesis
Kawashita
Received: 20 June 2023
Revised: 19 July 2023
Accepted: 20 July 2023
Published: 24 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
In recent years, bone fracture has become increasingly frequent. In 2019 alone, there
were 178 million new cases of fracture all over the world, which is an increase of 33.4%
compared with 1990 [1]. Thus, the materials used for bone repair are in great demand. Autografting is the gold standard of bone grafting [2,3]. However, some doubts surrounding
autografting are associated with some complications, such as haematoma formation, blood
loss, nerve injury, hernia formation, and chronic pain at the donor site [3]. Furthermore, in
some circumstances, the autologous bone graft material may be deficient [4].
Owing to their excellent biocompatibility and bone conductivity, calcium phosphate ceramics such as hydroxyapatite (HA), biphasic calcium phosphate (BCP), and beta-tricalcium
phosphate (β-TCP) have been used as bone repair materials [5–7]. Synthetic calcium phosphate is the main source of calcium phosphate owing to its easily controlled chemical
J. Funct. Biomater. 2023, 14, 390. https://doi.org/10.3390/jfb14070390
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composition and phase composition; however, its biological activity is potentially limited
by its deficiency in trace elements and nanostructures compared to natural calcium phosphate materials [8,9]. To address these deficiencies, an increasing number of researchers
have attempted to incorporate trace elements such as magnesium, iron, and strontium into
hydroxyapatite [10–12]. Gong et al. synthesised Mg-doped hydroxyapatite whiskers which
promote cell proliferation and differentiation [13]. Qi et al. developed strontium-substituted
hydroxyapatite (SrHA) using a hydrothermal technique. The synthesised SrHA was then
employed in the 3D fabrication of PLLA/SrHA composite scaffolds. The PLLA/SrHA
scaffolds exhibited sustained Sr2+ release, with a cumulative concentration of 5.6 mg/L
over 28 days which significantly enhanced the bioactivity of the PLLA/HA [14]. However,
this form of doping cannot fully mimic the trace elements that exist in natural bone.
In the past, bovine bone was regarded as the optimal natural resource for producing
natural calcium phosphate bioceramics. It possesses exceptional bone-repair properties and
was used extensively in periodontal and implant surgery [15,16]. Due to mad cow disease
(BSE), however, the safety of bovine-derived bone was questioned [17]. Since the 1970s,
there has been study and application of the use of marine species as sources for biomaterials [18]. Early research studies indicate that corals [19], sponges [20], cuttlebone [21], and
sea urchin spines [22] possess great potential as bone substitutes. Three important reasons
why bioceramics derived from marine fish are being extensively researched for use as bone
fillers are as follows: (1) they are generally composed of calcium carbonate in the form of
aragonite or calcite, which are easily converted into calcium phosphates via hydrothermal
conversion; (2) they have unique porous structures that are useful for cell migration and
liquid penetration; and (3) they are rich sources of trace elements. According to Pujie Shi
et al.’s studies, salmon, cod, and rainbow trout bones were heated to 650 ◦ C for five hours
to produce pure HA, which had significantly improved biological properties compared to
other materials [9]. From tilapia bone, Modolon et al. successfully produced nanostructured
biological hydroxyapatite with a variety of trace elements [23]. Through the calcination
of various tilapia bones of varying ages, Weinand et al. were able to synthesise biphasic
calcium phosphate (BCP) ceramics, which exhibit impressive alveolar bone regenerating
capability and non-cytotoxicity [24].
In this study, fish bone hydroxyapatite (FHA) and biphasic calcium phosphate (FBCP)
were synthesised from tilapia bone using a simple and novel gradient calcination approach.
The physicochemical characteristics, including the morphology, components, degradation,
and biomineralisation properties, of the tilapia bone-derived bioceramics were studied
in depth. In vitro cytotoxicity and osteogenesis assessments were performed to evaluate
biocompatibility and osteoinductive activity.
2. Materials and Methods
2.1. Materials
Tilapia bones were collected from Hainan Xiangtai Fishery Co., Ltd., located in Chengmai, Hainan Province, China. They were used as raw materials to produce bioceramics.
Based on previous studies [25–27], the tilapia bones were boiled at 100 ◦ C for 30 min to
remove organic substances and the adherent fish meat. The bones were then cut into small
pieces, dried in a hot-air oven, and prepared for use after drying.
Commercial hydroxyapatite (CHA, purity 98%, RHAWN), which was employed as a
control, is a biomedically pure hydroxyapatite whisker.
2.2. Conversion of Tilapia Bone into Calcium Phosphate Bioceramics
The tilapia bones were placed in a muffle furnace for calcination and calcined at various
temperatures (600 ◦ C, 700 ◦ C, and 800 ◦ C) at a heating rate of 10 ◦ C/min in air. Once the
calcination temperature had been reached, the bones were maintained isothermally for
1 h to synthesise the FHA bioceramic. The tilapia bones were placed in a muffle furnace
for calcination at various temperatures (600 ◦ C, 700 ◦ C, and 800 ◦ C). To prepare the FHA
bioceramics, the tilapia bones were sintered at the designated temperature for 1 h at a
J. Funct. Biomater. 2023, 14, 390
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heating rate of 10 ◦ C/min, then cooled to room temperature at 10 ◦ C/min. Subsequently,
the obtained FHA was used as a precursor and calcined at various temperatures (900 ◦ C,
1000 ◦ C, 1100 ◦ C, and 1200 ◦ C) for 4 h to develop FBCP bioceramics [28]. Finally, the
obtained products were ground using an agate mortar and pestle, milled into a powder,
and further characterized [29].
2.3. Characterizations
2.3.1. X-ray Diffraction (XRD) Analysis
The phase compositions and crystal structures of the tilapia-derived bioceramics were
analysed via X-ray diffraction (XRD, Rigaku Smart Lab, Japan) with Cu Kα1 radiation.
The data were collected over an angular range from 10◦ to 80◦ , with a step size of 0.01◦
and a step time of 0.06 s at a voltage of 40 kV and a current of 30 mA. The phases were
identified by comparing the experimental X-ray diffractions with the standards compiled
by the International Centre for Diffraction Data (ICDD PDF No. 74-0566) and (ICDD PDF
No. 09-0169) [30]. We calculated the relative amount of HA using the relative areas of three
strong characteristic peaks. The formula is as follows [31]:
ω1 =
I1
I
where I1 is the reflection intensity of three characteristic peaks of HA or β-TCP, I is the sum
of the reflection intensity of the three characteristic peaks of both HA and β-TCP, and ω 1 is
the relative amount of HA or β-TCP in the sample.
2.3.2. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)
The morphologies and chemical compositions of the tilapia-derived bioceramics and
CHA were observed via the combination of scanning electron microscopy (SEM) (GeminiSEM 300, Zeiss, Oberkochen, Baden-Württemberg, Germany) and energy dispersive
spectroscopy (EDS) [28].The samples were deposited onto conductive adhesive to observe
their surface morphologies. The crystallography of various samples was investigated using
a transmission electron microscope (TEM, JEM-2100F, JEOL, Akishima-shi, Tokyo Metropolis, Japan) [32], and their lattice spacings were calculated using a digital micrograph. The
samples were ultrasonically dispersed in ethanol for 15 min before they were tested on the
copper grid [33].
2.3.3. Fourier Transform Infrared Spectroscopy (FTIR) Analysis
The functional groups of the samples were detected via Fourier transform infrared
spectroscopy (FTIR) (TENSOR27, Bruker, Karlsruhe, Baden-Württemberg, Germany) in
a wavenumber ranging from 400 cm−1 to 4000 cm−1 with a 4 cm−1 resolution [25]. The
samples were mixed with KBr and pressed to carry out the FTIR test [9].
2.4. Degradation Test In Vitro
The in vitro degradation of the samples was carried out by measuring the weight loss
of the samples after they were soaked in a PBS solution (phosphate-buffered solution).
The CHA was used as a control [28]. Powder (30 mg) was added into the PBS solution
(30 mL) in a centrifuge tube (1 mg/mL), and each centrifuge tube was then placed in a
shaking water bath at 37 ◦ C for 28 days. The in vitro degradation of the samples was tested
by measuring the weight loss of the samples after soaking them in a PBS solution for 7,
14, 21, and 28 days. At each predetermined time, the samples were dried at 60 ◦ C for
24 h, and the weight loss (%) was determined using the following formula: weight loss
(%) = (w0 − w1 )/w0 × 100%, where w0 and w1 represent the dry weight of the powder
before and after immersion, respectively. Additionally, approximately 0.5 mL of the PBS
solution was collected after 1 day and the concentrations of Ca2+ and Mg2+ were determined
via inductively coupled plasma–optical emission spectroscopy (ICP-OES) (Thermo Fisher
iCAP 7400) [34].
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2.5. Mineralization Test In Vitro
The in vitro mineralization of the samples was assessed by immersing the samples
(1 mg/mL) in an SBF solution (simulated body fluid) at 37 ◦ C for 14 days, and the SBF
solution was refreshed once every 3 days during the incubation period. After 14 days, the
samples were dried at 60 ◦ C for 24 h, and SEM was used to examine the growth of apatite
on the surface. In addition, the samples’ crystal phase changes were analysed via XRD,
and the differences in functional groups before and after mineralization were analysed
using FTIR.
2.6. Cytotoxicity Test In Vitro
L929 cells were purchased from the Shanghai cell bank of the Chinese Academy of
Sciences and used to evaluate the toxicity of the tilapia-derived bioceramics. L929 cells
were maintained in Dulbecco’s modified eagle medium (DMEM), which containing 10%
FBS and a 1% penicillin–streptomycin solution. The culture medium was incubated at
37 ◦ C with 5% CO2 -saturated humidity. All cells were cultured to reached 80% confluence
for the subsequent experiments.
The CCK-8 (Dojindo, Kumamoto, Japan) assay was used to assess the cytotoxicity of
the FHA and FBCP using the L929 cells [28]. In short, the cells were seeded into 24-well
plates at a density of 1 × 104 per well and incubated overnight at 37 ◦ C in a 5% CO2
incubator. The samples with different concentrations (200 µg/mL, 400 µg/mL, 600 µg/mL,
and 800 µg/mL) were added to the 24-well plate and incubated at 37 ◦ C for another 24 h.
After that, the culture medium was removed and washed 2–3 times with PBS. CCK-8 was
added and cultured in the incubator at 37 ◦ C for additional 2 h. Subsequently, the CCK-8
working solution was aspirated, and 400 µL of DMSO was added to each well in a 24-well
plate and incubated on a shaker for 10 min. The absorbance value at 450 nm was detected
using a microplate reader (Singapore, model number: Mutiskan Sky), and the cell viability
was expressed as a percentage of the control group without treatment. Meanwhile, cell
proliferation was further evaluated via live/dead staining. After being incubated with
different samples at a concentration of 800 µg/mL for 24 h, the L929 cells were washed
three times with PBS and stained for 10 min with acridine orange (AO; Sigma Aldrich,
Louis Missouri, DE, USA) and propidium iodide (PI; Sigma Aldrich) solutions. Finally, the
stained samples were observed under a fluorescence microscope (Leica DM50000B).
2.7. Osteogenic Differentiation Analyses
The osteogenic ability of the different samples (CHA, FHA, and FBCP) was measured
using alkaline phosphate staining [9]. Before extraction, the samples were sterilized in
70% ethanol and placed under ultraviolet (UV) light for 30 min. The sterilized samples
were immersed for 24 h in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher
Scientific, Waltham, MA, USA) in order to obtain the leaching liquid, called the conditioned
medium. BMSCs cells were seeded in a 6-well plate and cultured with a regular culture
medium. After 12 h, the medium was replaced with extracts containing 10% FBS. The
BMSCs cultured in the conventional medium were regarded as the control. The ALP
produced from the cells in the presence of extracts of the CHA, FHA and FBCP was
evaluated after 7 days of culture. The staining was measured with an ALP staining kit in
accordance with the manufacturer’s instructions, and the stained cells were photographed
under a microscope. The positive staining area was calculated using image J (V1.8.0.112)
software.
2.8. Statistical Analysis
All obtained results were subjected to statistical analysis using Prism Software version
8 (GraphPad). All data are expressed as the means ± standard deviations. Comparisons
between multiple groups were performed using a one-way analysis of variance (ANOVA)
with Bonferroni’s multiple comparison test [28]. Values of p < 0.05 were considered statistically significant.
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3. Results and Discussion
3.1. Analysis of the Morphologies and Compositions of FHA and FBCP
Tilapia-derived FHA and FBCP were prepared via a gradientβ thermal treatment
technology. The initial phase of the conversion of tilapia bone intoffhydroxyapatite (HA)
tt
when
occurs between 600 ◦ C and 800 ◦ C, and the HA began to decompose into β-TCP
ff
◦
the calcination temperature reached 900
tt C. Figure 1a shows the X-ray diffraction (XRD)
patterns of the FHA samples prepared at variousfftemperatures, and most of the diffraction
peaks matched with the characteristic HA pattern (ICDD PDF No. 74-0566). With the
increase in calcination temperature in the first stage, the diffraction peak of the FHA shows
a narrow and sharp changing trend, suggesting that the crystallinity of the HA increased
β
as the temperature rose. In the second stage, the FBCP was obtained by further calcining
◦
◦
the FHA between 900 C and 1200 C, which caused the HA to partially decompose into
β-TCP (ICDD PDF No. 09-0169) (Figure 1b). Furthermore, the typical peaks of FHA
(2theta = 31.82◦ , 32.22◦ , and 32.96◦ ) and FBCP (2theta = 27.91◦ , 31.13◦ ,tt31.81◦ , 32.20◦ , 32.95◦ ,
tt also displaced to higher angles, indicating that the lattice parameter was
and 34.50◦ ) are
reduced, and lattice contraction occurred [35].
Figure 1. (a) XRD of FHA obtained via thermal processing at the first stage at various calcination
temperatures (600 ◦ C, 700 ◦ C, and 800 ◦ C). (b) XRD of FBCP obtained via thermal processing at the
second stage at various calcination temperatures (900 ◦ C, 1000 ◦ C, 1100 ◦ C, and 1200 ◦ C).
The amount of ββ-TCP in the FBCP tended to increase from 16.6% to 28.8% as the
second gradient sintering temperature increased from 900 ◦ C to 1200 ◦ C. The relevant
contents
β of β-TCP and HA are presented in Table 1. According to previous studies,ββ-TCP
can be produced from the decomposition of HA when the calcination temperature is higher
than 1100 ◦ C [36,37]. However, in this study, β-TCP was generated when the second
β
gradient sintering temperature reached 900 ◦ C. This phenomenon may be attributed to
tt
the substitution of Ca2+ with Mg2+ , which has a smaller ionic radius in FHA, resulting in
crystal lattice distortion and thermal instability [36,38]. As a consequence, magnesium-rich
tt could decompose into β-TCP at a lower calcination temperature.
HA
The morphologiesβof tilapia bone-derived bioceramics and CHA were observed using
a scanning electron microscope (SEM). As shown in Figure 2a, the SEM images of the CHA
and FHA exhibit similar small granules. On the other hand, the FBCP grains are well
joined together and exhibit a porous surface structure, indicating that during the second
calcination process, small granules agglomerated and developed into larger ones with
larger particle sizes. The corresponding EDS analysis revealed that both the FHA and FBCP
contained Mg; however, the magnesium content of the FBCP (2.9 wt%) is lower than that
of the FHA (12.9 wt%) because the second gradient of high-temperature calcination of the
FBCP promoted the volatilisation of magnesium [39,40], thereby reducing the magnesium
content of the FBCP.
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Table 1. Relevant contents of FBCP and FHA.
Samples
FHA
FBCP
Calcination Condition
β-TCP%
HA%
◦C
0
0
0
16.6
18.0
20.7
28.8
100
100
100
83.4
82.0
79.3
71.2
600
1h
700 ◦ C 1 h
800 ◦ C 1 h
◦
800 C 1 h + 900 ◦ C 4 h
800 ◦ C 1 h + 1000 ◦ C 4 h
800 ◦ C 1 h + 1100 ◦ C 4 h
800 ◦ C 1 h + 1200 ◦ C 4 h
Figure 2. (a) SEM images of CHA, FHA, and FBCP and the corresponding EDS analyses (the white
crosses show the EDS analysis points). (b) High-resolution TEM analyses of CHA, FHA, and FBCP
(detected in the yellow box area of the inset TEM image).
The TEM results further confirmed the crystal phase of the magnesium-rich calcium
phosphate bioceramics derived from tilapia bone. As shown in Figure 2b, the lattice spacing
tt the (2 1
of the FHA is 0.279 nm, corresponding to the (2 1 1) plane, which is smaller than
1) lattice spacing of the pure HA (0.28147 nm). As we know, BCP is composed of HA and
tt this study, the lattice spacings of 0.1822 nm and 0.2598 nm are ascribed to the
β-TCP. In
β
(2 1 3) and (2 2 0) planes of HAttand β-TCP, respectively, which are also smaller than the
corresponding lattice spacings of pure HAβ(0.18403 nm) and pure β-TCP (0.2607 nm). This is
tt
β
in decreases
due to the partial substitution
of Ca2+ by Mg2+ in the FHA and FBCP, resulting
in the d-spacings and lattice parameters [41,42]. This phenomenon is in accordance with
the XRD results. Together with these findings, the gradient thermal treatment allowed
for the production of magnesium-rich bioceramics derived from tilapia bones, with FHA
having a greater magnesium content than FBCP.
3.2. In Vitro Study of the Degradation and Bioactivity of FHA and FBCP
The degradation behaviours of the CHA, FHA, and FBCP were evaluated by measuring their weight loss in PBS. We know that an ion exchange process in solution can result in
the formation of a biologically active carbonate apatite layer. This indicates that during the
process of degradation, sorption definitely occurred on the calcium phosphate bioceramics.
As a result, the weight loss of the samples due to degradation is greater than the sorption
of apatite layer from the solution. As shown in Figure 3a, the weight loss of the samples
increased with an increase in the immersion time. After immersion for 28 days, the weight
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losses of the CHA, FHA, and FBCP were 3.5%, 6.91%, and 16.45%, respectively. These
findings suggest that the FBCP presented the maximum mass loss. In addition, owing to the
high magnesium concentration in the FHA, the solubility of the FHA is enhanced [43,44].
Therefore, the FHA presented a higher degradation rate than the CHA. Furthermore, as
seen in Figure 3c, owing to the higher magnesium content in the FHA, the concentrations of
Mg2+ release from the FHA (4.38 mg/L) were higher than those from the FBCP (0.58 mg/L)
after 1 days. The bioinorganic magnesium cation (Mg2+ ), which is naturally present in
bone tissues and plays a crucial role in numerous cellular functions, is essential for the
regulation of protein synthesis, enzyme activation, and bone formation [45,46]. For instance,
magnesium can considerably promote the development of human marrow stromal cells
(MSCs) and the expression of endogenous bone morphogenetic protein [47–49]. Previous
studies suggested that the addition of Mg to biomaterials could improve their dissolubility
and promote osteogenic and angiogenic differentiation, making them more suitable for
use as a bone substitute [50–52]. Furthermore, magnesium also plays a role in calcification,
regulates the immune microenvironment, and alters pH when it comes to bone metabolism.
Herein, we believe that the magnesium cation produced by the FHA and FBCP may act
as a biochemical signal molecule to provide a superior microenvironment for bone tissue
regeneration.
Figure 3. (a) The degradation behaviours of CHA, FHA, and FBCP in PBS. (b) Cumulative release of
Ca2+ from CHA, FHA and FBCP after 1 day of degradation. (c) Cumulative release of Mg2+ from
FHA and FBCP after 1 day of degradation. (*** p < 0.001).
β
θ
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The bioactivity of FHA and FBCP was evaluated by investigating their phase compositions and surface morphologies after immersion in simulated body fluid (SBF) for
14 days (Figure 4a–d). The characteristic peaks of β-TCP in the FBCP were remarkably
reduced when compared with the FBCP without SBF immersion (Figure 4a). On the other
hand, the relative intensities of the characteristic peaks at 2θ = 31.76◦ , 32.18◦ , and 32.92◦ in
the FHA and FBCP increased, which can be attributed to the HA diffracting planes (211),
(112), and (300). FTIR was also employed to identify the functional groups present in the
various samples. As shown in Figure 4b,c, compared with Fourier transform infrared spectroscopy (FTIR) results before and after SBF immersion, new absorption peaks at 874 cm−1 ,
1464 cm−1 , and 1465 cm−1 emerged which corresponded to a typical CO3 2− vibration,
suggesting that carbonate hydroxyapatite had precipitated on the samples [9,53,54]. The
corresponding SEM images show that the surfaces of the FHA and FBCP became coarse
and covered with a newly formed dense, plate-like pattern arranged in a regular manner to
form the well-known cauliflower-shaped morphology. Therefore, the results confirmed
that tilapia bone-derived bioceramics show superior apatite formation capacity.
Figure 4. (a) XRD spectra after SBF immersion for 14 days. (b) FTIR spectrum before SBF immersion
for 14 days. (c) FTIR spectrum after SBF immersion for 14 days. (d) SEM images after SBF immersion
for 14 days.
ff
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3.3. Evaluation of Cell Proliferation and Differentiation of FHA and FBCP
The cytotoxicity of the FHA and FBCP was evaluated using L929 cells. The CCK-8
assay (Figure 5a) was used to quantitatively characterise cell proliferation capacity, and
the findings showed that the cells were metabolically active. Both the FHA and FBCP
specimens have demonstrated compatible viability to control group at concentrations of
200, 400, 600, and 800 µg/mL. It was observed that the cell viability percentage increased
consistently for the FHA samples in all concentration groups. Additionally, the live/dead
staining results (Figure 5b) agreed with the quantitative evaluation, demonstrating that
both the FHA and FBCP had negligible cytotoxicity. The aforementioned findings suggest
that bioceramics derived from tilapia bone have good biocompatibility and a wide range of
application possibilities in bone tissue engineering.
ff
Figure 5. (a) Viability of cells with different concentrations of FHA and FBCP. (b) Live/dead staining
of cells on FHA and FBCP.
ALP plays a critical role in early osteogenesis and hydrolyses various types of phosphates to promote cell maturation and calcification [55–58]. Therefore, in this study, the
ALP activities of the BMSCs’ indirect co-cultures with samples were investigated to evaluate the osteoinductive properties of the bioceramics derived from tilapia bone. As shown
in Figure 6a, although there were no significant differences
in the levels of ALP activity
ff
between the FHA and FBCP groups on day 7, both were higher than that of the CHA
group. Furthermore, in the FHA and FBCP groups, ALP staining was more intense than
in the CHA group, which is in accordance with the ALP expression results. These results
demonstrated that the FHA and FBCP were superior to the CHA in terms of osteogenesis.
In general, the natural tilapia bone-derived FHA and FBCP facilitated cell proliferation
and differentiation
and the formation of mineralised tissue. These improved osteogenic
ff
tt
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properties may be attributed to the increased degradation of the FHA and FBCP, which
resulted in a greater release of Ca2+ . Our ICP-OES results (Figure 3b,c) revealed that the
Mg2+ release concentration increased significantly from 0 to 4.38 mg/L on the first day.
According to previous studies, a moderate Mg2+ concentration (0–100 mg/L) could improve bone regeneration, whereas free Mg2+ and a high Mg2+ content (above 400 mg/L)
both inhibited cell adhesion, proliferation, and osteogenic differentiation [59,60]. Therefore,
we believe that the Mg2+ released from the FHA could also facilitate the proliferation and
differentiation of BMSCs. Further in vivo studies are required to confirm that magnesium
ions released by the FHA can effectively stimulate vascularisation and bone regeneration.
ff
Figure 6. (a) ALP activity and (b) ALP staining images at 7 days of indirect cell culture with different
samples (** p < 0.01 and *** p < 0.001).
Due to their porous structure, abundance of trace elements such as Mg2+ , Sr2+ , Na+ ,
Cl , and −F− , and the ease of their conversion into calcium phosphate, these marine fish
bones have shown great promise as bone substitutes. In recent years, marine fish bones have
been extensively examined. For instance, the bones of the Japanese sea bream [61], Brazilian
river fish [62], Atlantic swordfish [63], and Atlantic cod fish [64] were all successfully
employed to obtain calcium phosphates. However, the sea water living conditions of
marine fish will have a significant effect
on the types and amounts of trace elements in
ff
fish bones, leading to variations in the compositions of bioceramics derived from marine
fish [65]. Tilapia is one the most popular fish in China’s Hainan Province, and the Hainan
Xiangtai company supplied tilapia fish bones for this investigation. The tilapia were farmed
in net tanks under controlled environmental and feeding conditions. Therefore, the bone
composition of the tilapia used in this study is relatively stable. In this study, magnesiumrich calcium phosphate bioceramics (FHA and FBCP) were extracted from the tilapia bone
using a gradient thermal treatment approach. Although the gradient thermal treatment
allowed for the production of magnesium-rich FHA and FBCP, the second gradient of
high-temperature calcination may have resulted in the volatilisation of magnesium, thereby
reducing the magnesium content of the FBCP. Therefore, the magnesium content of the
FHA is substantially greater than that of the FBCP, and the release of Mg2+ from the FHA
was greater than that of the FBCP. On the other hand, the FBCP is composed of β-TCP
−−
β
ff
ff
J. Funct. Biomater. 2023, 14, 390
11 of 14
and HA, degrades faster and has a higher concentration of Ca2+ release than the FHA.
According to our research, the FBCP and FHA were both more effective at promoting cell
growth and the osteogenic differentiation of BMSCs than the commercial hydroxyapatite
product. This indicates that tilapia bone has a great potential for being converted into
highly valuable compounds that can be used as substitute materials for artificial bone.
4. Conclusions
In this study, natural magnesium-rich calcium phosphate bioceramics (FHA and
FBCP) were extracted from tilapia bones using a thermal gradient treatment strategy.
Together, these results confirm that Mg-substituted calcium phosphate was generated via
this gradient thermal treatment and that the FHA contains more magnesium than the FBCP.
Additionally, the FHA and FBCP have greater in vitro mineralisation and degradation
capacities than CHA, which can result in a more favourable microenvironment for the
proliferation and differentiation of BMSCs due to appropriate concentrations of Ca2+
and Mg2+ . In conclusion, the magnesium-rich calcium phosphate bioceramics generated
from tilapia are expected to have widespread application in bone repair and regeneration
owing to their favourable mineralisation capability, acceptable degradation characteristics,
continuous magnesium ion release, and good biological features.
Author Contributions: Conceptualization and writing—original draft preparation and editing: X.C.
and J.Z.; methodology: C.Z. and J.X.; validation: Z.Q. and S.N.V.; formal analysis: Q.D. and X.Z.;
resources: W.Y.; data curation, acquiring funding and project administration: M.L. and C.L. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the Natural Science Foundation of Hainan Province (GHYF2022001);
MRC-UCL Therapeutic Acceleration Support (TAS) Fund (project No. 564022), NIHR UCLH BRCUCL Therapeutic Acceleration Support (TAS) Fund (grant No. 564021), and the Engineering and
Physical Sciences Research Council via the DTP CASE Program (grant No. EP/T517793/1).
Data Availability Statement: The relevant data are already included in the article, and no additional
data are required.
Acknowledgments: We would like to acknowledge our team leader, Xueqiong Yin, for providing us
with laboratory instruments.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
GBD 2019 Fracture Collaborators. Global, regional, and national burden of bone fractures in 204 countries and territories,
1990–2019: A systematic analysis from the Global Burden of Disease Study 2019. Lancet Healthy Longev. 2021, 2, e580–e592.
[CrossRef] [PubMed]
Wang, C.-Z.; Wang, Y.-H.; Lin, C.-W.; Lee, T.-C.; Fu, Y.-C.; Ho, M.-L.; Wang, C.-K. Combination of a Bioceramic Scaffold and
Simvastatin Nanoparticles as a Synthetic Alternative to Autologous Bone Grafting. Int. J. Mol. Sci. 2018, 19, 4099. [CrossRef]
Giannoudis, P.V.; Dinopoulos, H.; Tsiridis, E. Bone substitutes: An update. Injury 2005, 36 (Suppl. 3), S20–S27. [CrossRef]
Zhou, J.; Mei, J.; Liu, Q.; Xu, D.; Wang, X.; Zhang, X.; Zhu, W.; Zhu, C.; Wang, J. Spatiotemporal On–Off Immunomodulatory
Hydrogel Targeting NLRP3 Inflammasome for the Treatment of Biofilm-Infected Diabetic Wounds. Adv. Funct. Mater. 2023,
33, 2211811. [CrossRef]
Emadi, R.; Esfahani, S.I.R.; Tavangarian, F. A novel, low temperature method for the preparation of ß-TCP/HAP biphasic
nanostructured ceramic scaffold from natural cancellous bone. Mater. Lett. 2010, 64, 993–996. [CrossRef]
Kumar, R.; Pattanayak, I.; Dash, P.A.; Mohanty, S. Bioceramics: A review on design concepts toward tailor-made (multi)-functional
materials for tissue engineering applications. J. Mater. Sci. 2023, 58, 3460–3484. [CrossRef]
Bouler, J.M.; Pilet, P.; Gauthier, O.; Verron, E. Biphasic calcium phosphate ceramics for bone reconstruction: A review of biological
response. Acta Biomater. 2017, 53, 1–12. [CrossRef] [PubMed]
Chesley, M.; Kennard, R.; Roozbahani, S.; Kim, S.M.; Kukk, K.; Mason, M. One-step hydrothermal synthesis with in situ milling
of biologically relevant hydroxyapatite. Mater. Sci. Eng. C 2020, 113, 110962. [CrossRef]
Shi, P.; Liu, M.; Fan, F.; Yu, C.; Lu, W.; Du, M. Characterization of natural hydroxyapatite originated from fish bone and its
biocompatibility with osteoblasts. Mater. Sci. Eng. C 2018, 90, 706–712. [CrossRef]
Laurencin, D.; Almora-Barrios, N.; de Leeuw, N.H.; Gervais, C.; Bonhomme, C.; Mauri, F.; Chrzanowski, W.; Knowles, J.C.;
Newport, R.J.; Wong, A.; et al. Magnesium incorporation into hydroxyapatite. Biomaterials 2011, 32, 1826–1837. [CrossRef]
J. Funct. Biomater. 2023, 14, 390
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
12 of 14
Dai, Y.; Mei, J.; Li, Z.; Kong, L.; Zhu, W.; Li, Q.; Wu, K.; Huang, Y.; Shang, X.; Zhu, C. Acidity-Activatable Nanoparticles with
Glucose Oxidase-Enhanced Photoacoustic Imaging and Photothermal Effect, and Macrophage-Related Immunomodulation for
Synergistic Treatment of Biofilm Infection. Small 2022, 18, e2204377. [CrossRef] [PubMed]
Landi, E.; Tampieri, A.; Celotti, G.; Sprio, S.; Sandri, M.; Logroscino, G. Sr-substituted hydroxyapatites for osteoporotic bone
replacement. Acta Biomater. 2007, 3, 961–969. [CrossRef] [PubMed]
Gong, L.; Zhang, W.; Shen, Y. Magnesium substituted hydroxyapatite whiskers: Synthesis, characterization and bioactivity
evaluation. RSC Adv. 2016, 6, 114707–114713.
Qi, F.; Wang, Z.; Shuai, Y.; Peng, S.; Shuai, C. Sr2+ Sustained Release System Augments Bioactivity of Polymer Scaffold. ACS Appl.
Polym. Mater. 2022, 4, 2691–2702. [CrossRef]
Baldini, N.; De Sanctis, M.; Ferrari, M. Deproteinized bovine bone in periodontal and implant surgery. Dent. Mater. 2011, 27,
61–70. [CrossRef]
Ooi, C.Y.; Hamdi, M.; Ramesh, S. Properties of hydroxyapatite produced by annealing of bovine bone. Ceram. Int. 2007, 33,
1171–1177. [CrossRef]
Kim, Y.; Nowzari, H.; Rich, S.K. Risk of prion disease transmission through bovine-derived bone substitutes: A systematic review.
Clin. Implant. Dent. Relat. Res. 2013, 15, 645–653. [CrossRef]
Liu, Y.; Puthia, M.; Sheehy, E.J.; Ambite, I.; Petrlova, J.; Prithviraj, S.; Oxborg, M.W.; Sebastian, S.; Vater, C.; Zwingenberger, S.;
et al. Sustained Delivery of a Heterodimer Bone Morphogenetic Protein-2/7 via a Collagen Hydroxyapatite Scaffold Accelerates
and Improves Critical Femoral Defect Healing. Acta Biomater. 2023, 162, 164–181. [CrossRef]
Barros, A.A.; Aroso, I.M.; Silva, T.H.; Mano, J.F.; Duarte, A.R.C.; Reis, R.L. In vitro bioactivity studies of ceramic structures
isolated from marine sponges. Biomed. Mater. 2016, 11, 045004. [CrossRef]
Ben-Nissan, B. Natural bioceramics: From coral to bone and beyond. Curr. Opin. Solid State Mater. Sci. 2003, 7, 283–288. [CrossRef]
Ivankovic, H.; Ferrer, G.G.; Tkalcec, E.; Orlic, S.; Ivankovic, M. Preparation of highly porous hydroxyapatite from cuttlefish bone.
J. Mater. Sci. Mater. Med. 2009, 20, 1039–1046. [CrossRef] [PubMed]
Vecchio, K.S.; Zhang, X.; Massie, J.B.; Wang, M.; Kim, C.W. Conversion of sea urchin spines to Mg-substituted tricalcium
phosphate for bone implants. Acta Biomater. 2007, 3, 785–793. [CrossRef] [PubMed]
Modolon, H.B.; Inocente, J.; Bernardin, A.M.; Montedo, O.R.K.; Arcaro, S. Nanostructured biological hydroxyapatite from Tilapia
bone: A pathway to control crystallite size and crystallinity. Ceram. Int. 2021, 47, 27685–27693. [CrossRef]
Weinand, W.R.; Cruz, J.A.; Medina, A.N.; Lima, W.M.; Sato, F.; da Silva Palacios, R.; Gibin, M.S.; Volnistem, E.A.; Rosso, J.M.;
Santos, I.A.; et al. Dynamics of the natural genesis of beta-TCP/HAp phases in postnatal fishbones towards gold standard
biocomposites for bone regeneration. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2022, 279, 121407. [CrossRef] [PubMed]
Boutinguiza, M.; Pou, J.; Comesaña, R.; Lusquiños, F.; de Carlos, A.; León, B. Biological hydroxyapatite obtained from fish bones.
Mater. Sci. Eng. C 2012, 32, 478–486. [CrossRef]
Terzioğlu, P.; Öğüt, H.; Kalemtaş, A. Natural calcium phosphates from fish bones and their potential biomedical applications.
Mater. Sci. Eng. C 2018, 91, 899–911. [CrossRef]
Aydin, G.; Terzioğlu, P.; Öğüt, H.; Kalemtas, A. Production, characterization, and cytotoxicity of calcium phosphate ceramics
derived from the bone of meagre fish, Argyrosomus regius. J. Aust. Ceram. Soc. 2020, 57, 37–46. [CrossRef]
Wu, W.; Zhou, Z.; Sun, G.; Liu, Y.; Zhang, A.; Chen, X. Construction and characterization of degradable fish scales for enhancing
cellular adhesion and potential using as tissue engineering scaffolds. Mater. Sci. Eng. C 2021, 122, 111919. [CrossRef]
Maidaniuc, A.; Miculescu, F.; Ciocoiu, R.C.; Butte, T.M.; Pasuk, I.; Stan, G.E.; Voicu, S.I.; Ciocan, L.T. Effect of the processing
parameters on surface, physico-chemical and mechanical features of bioceramics synthesized from abundant carp fish bones.
Ceram. Int. 2020, 46, 10159–10171. [CrossRef]
Goto, T.; Sasaki, K. Effects of trace elements in fish bones on crystal characteristics of hydroxyapatite obtained by calcination.
Ceram. Int. 2014, 40, 10777–10785. [CrossRef]
Hardy, M. X-ray diffraction measurement of the quartz content of clay and silt fractions in soils. Clay Miner. 1992, 27, 47–55.
[CrossRef]
Deb, P.; Barua, E.; Lala, S.D.; Deoghare, A.B. Synthesis of hydroxyapatite from Labeo rohita fish scale for biomedical application.
Mater. Today Proc. 2019, 15, 277–283. [CrossRef]
Bee, S.-L.; Bustami, Y.; Ul-Hamid, A.; Lim, K.; Hamid, Z.A.A. Synthesis of silver nanoparticle-decorated hydroxyapatite
nanocomposite with combined bioactivity and antibacterial properties. J. Mater. Sci. Mater. Med. 2021, 32, 106. [CrossRef]
[PubMed]
Fernández-Arias, M.; Álvarez-Olcina, I.; Malvido-Fresnillo, P.; Vázquez, J.A.; Boutinguiza, M.; Comesaña, R.; Pou, J. Biogenic
Calcium Phosphate from Fish Discards and By-Products. Appl. Sci. 2021, 11, 3387. [CrossRef]
Koo, K.; Shen, B.; Baik, S.-I.; Mao, Z.; Smeets, P.J.M.; Cheuk, I.; He, K.; Reis, R.D.; Huang, L.; Ye, Z.; et al. Formation mechanism
of high-index faceted Pt-Bi alloy nanoparticles by evaporation-induced growth from metal salts. Nat. Commun. 2023, 14, 3790.
[CrossRef]
Zhu, Q.; Ablikim, Z.; Chen, T.; Cai, Q.; Xia, J.; Jiang, D.; Wang, S. The preparation and characterization of HA/β-TCP biphasic
ceramics from fish bones. Ceram. Int. 2017, 43, 12213–12220. [CrossRef]
Jahangir, M.U.; Islam, F.; Wong, S.Y.; Jahan, R.A.; Matin, M.A.; Li, X.; Arafat, M.T. Comparative analysis and antibacterial
properties of thermally sintered apatites with varied processing conditions. J. Am. Ceram. Soc. 2020, 104, 1023–1039. [CrossRef]
J. Funct. Biomater. 2023, 14, 390
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
13 of 14
Cho, J.S.; Um, S.H.; Yoo, D.S.; Chung, Y.C.; Chung, S.H.; Lee, J.C.; Rhee, S.H. Enhanced osteoconductivity of sodium-substituted
hydroxyapatite by system instability. J. Biomed. Mater. Res. B Appl. Biomater. 2014, 102, 1046–1062.
Rahman, M.; Li, Y.; Wen, C. HA coating on Mg alloys for biomedical applications: A review. J. Magnes. Alloys 2020, 8, 929–943.
[CrossRef]
Ren, M.; Cai, S.; Xu, G.; Ye, X.; Dou, Y.; Huang, K.; Wang, X. Influence of heat treatment on crystallization and corrosion behavior
of calcium phosphate glass coated AZ31 magnesium alloy by sol–gel method. J. Non-Cryst. Solids 2013, 369, 69–75. [CrossRef]
Geng, Z.; Cui, Z.; Li, Z.; Zhu, S.; Liang, Y.; Lu, W.W.; Yang, X. Synthesis, characterization and the formation mechanism of
magnesium- and strontium-substituted hydroxyapatite. J. Mater. Chem. B 2015, 3, 3738–3746. [CrossRef] [PubMed]
Gallo, M.; Le Gars Santoni, B.; Douillard, T.; Zhang, F.; Gremillard, L.; Dolder, S.; Hofstetter, W.; Meille, S.; Bohner, M.; Chevalier, J.;
et al. Effect of grain orientation and magnesium doping on beta-tricalcium phosphate resorption behavior. Acta Biomater. 2019, 89,
391–402. [CrossRef] [PubMed]
Shan, H.; Zhou, X.; Tian, B.; Zhou, C.; Gao, X.; Bai, C.; Shan, B.; Zhang, Y.; Sun, S.; Sun, D.; et al. Gold nanorods modified by
endogenous protein with light-irradiation enhance bone repair via multiple osteogenic signal pathways. Biomaterials 2022, 284,
121482. [CrossRef] [PubMed]
Kazakova, G.; Safronova, T.; Golubchikov, D.; Shevtsova, O.; Rau, J.V. Resorbable Mg2+ -Containing Phosphates for Bone Tissue
Repair. Materials 2021, 14, 4857. [CrossRef] [PubMed]
Schatkoski, V.M.; Montanheiro, T.L.D.A.; de Menezes, B.R.C.; Pereira, R.M.; Rodrigues, K.F.; Ribas, R.G.; da Silva, D.M.; Thim, G.P.
Current advances concerning the most cited metal ions doped bioceramics and silicate-based bioactive glasses for bone tissue
engineering. Ceram. Int. 2021, 47, 2999–3012. [CrossRef]
Chen, Z.; Zhang, W.; Wang, M.; Backman, L.J.; Chen, J. Effects of Zinc, Magnesium, and Iron Ions on Bone Tissue Engineering.
ACS Biomater. Sci. Eng. 2022, 8, 2321–2335. [CrossRef]
Galli, S.; Stocchero, M.; Andersson, M.; Karlsson, J.; He, W.; Lilin, T.; Wennerberg, A.; Jimbo, R. The effect of magnesium on early
osseointegration in osteoporotic bone: A histological and gene expression investigation. Osteoporos. Int. 2017, 28, 2195–2205.
[CrossRef]
Sekiya, I.; Colter, D.C.; Prockop, D.J. BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells. Biochem.
Biophys. Res. Commun. 2001, 284, 411–418. [CrossRef]
Kugimiya, F.; Kawaguchi, H.; Kamekura, S.; Chikuda, H.; Ohba, S.; Yano, F.; Ogata, N.; Katagiri, T.; Harada, Y.; Azuma, Y.;
et al. Involvement of endogenous bone morphogenetic protein (BMP) 2 and BMP6 in bone formation. J. Biol. Chem. 2005, 280,
35704–35712. [CrossRef]
Du, Z.; Leng, H.; Guo, L.; Huang, Y.; Zheng, T.; Zhao, Z.; Liu, X.; Zhang, X.; Cai, Q.; Yang, X. Calcium silicate scaffolds promoting
bone regeneration via the doping of Mg2+ or Mn2+ ion. Compos. Part B Eng. 2020, 190, 107937. [CrossRef]
Kanasan, N.; Adzila, S.; Koh, C.T.; Rahman, H.A.; Panerselvan, G. Effects of magnesium doping on the properties of hydroxyapatite/sodium alginate biocomposite. Adv. Appl. Ceram. 2019, 118, 381–386. [CrossRef]
Tabia, Z.; El Mabrouk, K.; Bricha, M.; Nouneh, K. Mesoporous bioactive glass nanoparticles doped with magnesium: Drug
delivery and acellular in vitro bioactivity. RSC Adv. 2019, 9, 12232–12246. [CrossRef]
Koutsopoulos, S. Synthesis and characterization of hydroxyapatite crystals: A review study on the analytical methods. J. Biomed.
Mater. Res. 2002, 62, 600–612. [CrossRef]
Kim, S.-C.; Heo, S.-Y.; Oh, G.-W.; Yi, M.; Jung, W.-K. A 3D-Printed Polycaprolactone/Marine Collagen Scaffold Reinforced with
Carbonated Hydroxyapatite from Fish Bones for Bone Regeneration. Mar. Drugs 2022, 20, 344. [CrossRef]
Shokri, M.; Kharaziha, M.; Tafti, H.A.; Eslaminejad, M.B.; Aghdam, R.M. Synergic role of zinc and gallium doping in hydroxyapatite nanoparticles to improve osteogenesis and antibacterial activity. Biomater. Adv. 2022, 134, 112684. [CrossRef]
[PubMed]
Maleki-Ghaleh, H.; Siadati, M.H.; Fallah, A.; Koc, B.; Kavanlouei, M.; Khademi-Azandehi, P.; Moradpur-Tari, E.; Omidi, Y.;
Barar, J.; Beygi-Khosrowshahi, Y.; et al. Antibacterial and Cellular Behaviors of Novel Zinc-Doped Hydroxyapatite/Graphene
Nanocomposite for Bone Tissue Engineering. Int. J. Mol. Sci. 2021, 22, 9564. [CrossRef]
Zhang, J.; Zhang, W.; Dai, J.; Wang, X.; Shen, S.G. Overexpression of Dlx2 enhances osteogenic differentiation of BMSCs and
MC3T3-E1 cells via direct upregulation of Osteocalcin and Alp. Int. J. Oral Sci. 2019, 11, 12. [CrossRef]
Deligianni, D.D.; Katsala, N.D.; Koutsoukos, P.G.; Missirlis, Y.F. Effect of surface roughness of hydroxyapatite on human bone
marrow cell adhesion, proliferation, differentiation and detachment strength. Biomaterials 2000, 22, 87–96. [CrossRef] [PubMed]
Shen, J.; Chen, B.; Zhai, X.; Qiao, W.; Wu, S.; Liu, X.; Zhao, Y.; Ruan, C.; Pan, H.; Chu, P.K.; et al. Stepwise 3D-spatio-temporal
magnesium cationic niche: Nanocomposite scaffold mediated microenvironment for modulating intramembranous ossification.
Bioact. Mater. 2020, 6, 503–519. [CrossRef] [PubMed]
Shi, H.; Hong, L.; Pan, K.; Wei, W.; Liu, X.; Li, X. Biodegradable polyacrylate copolymer coating for bio-functional magnesium
alloy. Prog. Org. Coat. 2021, 159, 106422. [CrossRef]
Ozawa, M.; Suzuki, S. Microstructural Development of Natural Hydroxyapatite Originated from Fish-Bone Waste through Heat
Treatment. J. Am. Ceram. Soc. 2004, 85, 1315–1317. [CrossRef]
Yang, X.; Huang, J.; Chen, C.; Zhou, L.; Ren, H.; Sun, D. Biomimetic Design of Double-Sided Functionalized Silver Nanoparticle/Bacterial Cellulose/Hydroxyapatite Hydrogel Mesh for Temporary Cranioplasty. ACS Appl. Mater. Interfaces 2023, 15,
10506–10519. [CrossRef] [PubMed]
J. Funct. Biomater. 2023, 14, 390
63.
64.
65.
14 of 14
Boutinguiza, M.; Lusquiños, F.; Riveiro, A.; Comesaña, R.; Pou, J. Hydroxylapatite nanoparticles obtained by fiber laser-induced
fracture. Appl. Surf. Sci. 2009, 255, 5382–5385. [CrossRef]
Piccirillo, C.; Silva, M.F.; Pullar, R.C.; da Cruz, I.B.; Jorge, R.; Pintado, M.M.E.; Castro, P.M.L. Extraction and characterisation of
apatite- and tricalcium phosphate-based materials from cod fish bones. Mater. Sci. Eng. C 2013, 33, 103–110. [CrossRef]
Scopelliti, G.; Di Leonardo, R.; Tramati, C.D.; Mazzola, A.; Vizzini, S. Premature aging in bone of fish from a highly polluted
marine area. Mar. Pollut. Bull. 2015, 97, 333–341. [CrossRef]
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