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Citation: Drobyshev, A.; Komissarov,
A.; Redko, N.; Gurganchova, Z.;
Statnik, E.S.; Bazhenov, V.; Sadykova,
I.; Miterev, A.; Romanenko, I.;
Yanushevich, O. Bone Remodeling
Interaction with Magnesium Alloy
Implants Studied by SEM and EDX.
Materials 2022,15, 7529. https://
doi.org/10.3390/ma15217529
Academic Editor: Mirco Peron
Received: 29 August 2022
Accepted: 25 October 2022
Published: 27 October 2022
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materials
Article
Bone Remodeling Interaction with Magnesium Alloy Implants
Studied by SEM and EDX
Alexey Drobyshev 1, Alexander Komissarov 2,1 , Nikolay Redko 1, * , Zaira Gurganchova 1, Eugene S. Statnik 3,
Viacheslav Bazhenov 4, Iuliia Sadykova 3, Andrey Miterev 1, Igor Romanenko 1and Oleg Yanushevich 1
1Laboratory of Medical Bioresorption and Bioresistance, Moscow State University of Medicine and Dentistry,
127473 Moscow, Russia
2Laboratory of Hybrid Nanostructured Materials, National University of Science and Technology “MISiS”,
119049 Moscow, Russia
3HSM Laboratory, Center for Energy Science and Technology, Skoltech, 121205 Moscow, Russia
4Casting Department, National University of Science and Technology “MISiS”, 119049 Moscow, Russia
*Correspondence: dr.redko@mail.ru; Tel.: +7-916-954-44-44
Abstract:
The development direction of bioresorbable fixing structures is currently very relevant
because it corresponds to the priority areas in worldwide biotechnology development. Magnesium
(Mg)-based alloys are gaining high levels of attention due to their promising potential use as the
basis for fixating structures. These alloys can be an alternative to non-degradable metal implants
in orthopedics, maxillofacial surgery, neurosurgery, and veterinary medicine. In our study, we
formulated a Mg-2Zn-2Ga alloy, prepared pins, and analyzed their biodegradation level based on
SEM (scanning electron microscopy) and EDX (energy-dispersive X-ray analysis) after carrying out
an experimental study on rats. We assessed the resorption parameters 1, 3, and 6 months after surgery.
In general, the biodegradation process was characterized by the systematic development of newly
formed bone tissue. Our results showed that Mg-2Zn-2Ga magnesium alloys are suitable for clinical
applications.
Keywords: bioresorption; magnesium alloys; bioresorbable materials
1. Introduction
The use of magnesium alloys as a basis for manufacturing biodegradable elements
in medicine is promising [
1
]. Magnesium alloys have a high specific strength, and their
density is similar to human bone [
2
]. In modern clinical practice, substances for man-
ufacturing fixing and supporting structures for the human body include bone grafting
materials, titanium alloys, cobalt–chromium, stainless steel, etc. [
3
,
4
]. However, allergic
or inflammatory reactions are possible in the body when they are used because of their
foreign nature. Using non-resorbable structures in pediatric practice is restricted because of
child-body growth and the need to replace supporting elements when making a prosthetic
appliance, especially in the temporomandibular joint [
5
]. Removing non-resorbable ele-
ments or revising the area in question entails traumatic operations associated with pain, a
decrease in life quality, and an increase in treatment costs [
6
]. However, using magnesium
alloy-based materials would solve these problems [
2
,
7
]. Magnesium alloys are preferred
to existing resorbable systems because of their high Young’s modulus values and greater
elasticity compared with polymer screws.
However, the clinical use of magnesium alloys in the human body may be limited
due to inhomogeneous biodegradation, a high corrosion rate, and hydrogen release in
the first 2–4 months, which may adversely affect the healing process [
8
–
11
]. Composition
modification and alloy surface treatment can be used to solve these problems [
7
,
11
,
12
].
There is a significant number of studies devoted to additionally alloying magnesium by
rare earth or other metallic elements to improve corrosion resistance [13–15].
Materials 2022,15, 7529. https://doi.org/10.3390/ma15217529 https://www.mdpi.com/journal/materials
Materials 2022,15, 7529 2 of 14
Previously, Mg-Zn-Ga alloys were proposed as materials for osteosynthesis applica-
tions [
16
]. Zn and Ga exhibit the same high solution strengthening effect on Mg due to their
similar atomic radii and high solubility in Mg [
17
,
18
]. Mg alloys can obtain considerable
mechanical properties upon Zn and Ga additions after deformation processing [
17
–
19
].
Ga’s effectiveness in treating disorders associated with accelerated bone loss, and its an-
tibacterial properties, underline the potential of Ga as an alloying element for biodegradable
Mg alloys [20–24].
Our aim in this work was to record the resorption levels of fixing elements based
on magnesium alloys in the body of a rat. In our experiment, we used energy-dispersive
X-ray spectroscopy and scanning electron microscopy to quantitatively and qualitatively
determine the distribution of elements in the screw implantation bed.
2. Materials and Methods
2.1. Biomaterial Preparation
To prepare the alloy of Mg—2 wt.% Zn—2 wt.% Ga, we used high-purity metals:
magnesium Mg (99.95 wt.%), Zn (99.995 wt.%), and Ga (99.9999 wt.%). We carried out
melting in a resistance furnace in a steel crucible coated with BN in a protective atmosphere
of Ar + 2 vol.% SF
6
. We purged the resulting melt with Ar at 730–750
◦
C for 3 min and held
it for 10 min before pouring it into a mold. Next, we cast an ingot with a diameter of 60 mm
and a height of 200 mm into an aluminum mold. We heat treated the ingot: 300
◦
C for 15 h
+ 400
◦
C for 30 h. We machined a 145 mm-high and 50 mm-in-diameter cylindrical billet
from the ingot. Then, we subjected the billet to hot extrusion on a vertical hydraulic press
with a pressing force of 300 tons using the direct extrusion method at a speed of 1 mm/s
and an extrusion ratio of 6. We obtained a cylindrical extruded rod with a diameter of
20 mm and a length of ~1 m. The temperatures of the die and the extruded billet were
200 and 150
◦
C, respectively. We cut pins with a diameter of 1.5 mm and a height of 5 mm
from the rod using electro-erosion cutting. We cleaned the pins’ surfaces with emery bars.
2.2. Surgical Procedure
We conducted
in vivo
experiments in compliance with the rules of ethics and animal
welfare. We used both sexes of Wistar-line white laboratory rats from 6 months old with
average body weights of 340–400 g. The study protocol was approved by the interuniversity
ethics committee (No. 04 of 15 April 2021), and also complied with the principles of the
“European Convention for the Protection of Vertebrate Animals Used for Experiments or
for Other Scientific Purposes” dated 18 March 1986.
We performed the operation under general anesthesia with an intramuscular injec-
tion of Flexoprofen (2.5% 10 mg per kg; Vic, Belarus) and Zoletil (20 mg per kg; Vir-
bac, France). We used Brilocaine (1:200,000; Ferein, Russia) for local anesthesia. Under
the conditions of the experimental operating room and in compliance with the rules of
asepsis and antisepsis, we performed a skin incision in the femur area from the outside
and isolated the bone. Each animal underwent an installation of three 1.5 mm-diameter
and 5 mm-long femur-body implants. We sutured the wound in layers with Vicryl 4-0
(
Ethicon, Raritan, NJ, USA
). We treated the postoperative area with an antibacterial aerosol
Terramycin (
Zoetis, Germany
). We carried out postoperative antibiotic therapy using an
intramuscular injection of Convenia (Zoetis, Italy). The experiment duration was 6 months.
We bred animals at 1, 3, and 6 months after surgery by intramuscular injection and overdose
of Telazol (Zoetis, Parsippany-Troy hills, NJ, USA).
2.3. Bioresorption Monitoring
We used a scanning electronic microscope (SEM) and energy-dispersive X-ray spec-
troscopy (EDX) to study the microstructure of the installed implants. We carried out the
SEM study using an electron microscope VEGA 3 LMU (TESCAN ORSAY HOLDING, Brno,
Czech Republic). We carried out energy-dispersive X-ray spectroscopy using an Oxford
Instruments spectrometer integrated into the TESCAN microscope and AZtec control pro-
Materials 2022,15, 7529 3 of 14
gram. All images obtained with SEM are in secondary electrons. The signal of secondary
electrons is sensitive to the topography of the sample surface, so we used the SE detectors
(secondary electrons) when studying surface morphology. For example, to understand
the general appearance of biological samples, an SE detector is needed to observe their
fractures, pores and surface roughness.
We carried out biopsy preparation before our study. First, we cleaned by freeze-drying.
This is a dehydration process used to maintain the physical and biological integrity of
biological materials after storage for months or even years. The lyophilization process
is based on freezing the material, which is followed by a decrease in external pressure,
thereby allowing water to directly pass from a solid to a gaseous state. We deposited a
thin conductive gold film on the surface of a nonconductive sample. This is necessary for
obtaining better SEM images because non-conductive samples are charged when scanned
with an electron probe, which overexposes the image.
We calibrated the EDX detector before specimen studies by standard reference material
(SRM) 2910b obtained from the National Institute of Standards and Technology (NIST).
Usually, this calibrant is used for evaluating the physical and chemical properties of HAp
with biological, geological, or synthetic origins [
25
]. The Ca/P molar ratio of a SRM 2910b
is 1.67, which corresponds to the theoretical Ca/P value of HAp with a composition of
Ca
10
(PO
4
)
6
(OH)
2
. We extracted the quantitative information of elements distribution at
locations where the bone surface was appreciably flat.
3. Results
3.1. Scanning Electron Microscopy (SEM) of Biopsy Preparations
We captured all SEM images with corresponding elemental maps near the location
where the pin was implanted. For this reason, the results are comparable. We paid special
attention to the area of bone-to-implant contact when analyzing SEM data. After 1 month,
the implant is visualized, and is partially covered with the products of the interaction
of its resorption and bone tissue (the so-called “interface”) (Figure 1). However, on the
implant’s right side, there are fewer of these products; therefore, a surface more similar to
the original implant is visible. A layer of corrosion products is formed on the surface of the
implant as a result of the interactions between body fluids and implant metals. The formed
layer of corrosion products gradually dissolves in body fluids (Figure 2). A larger layer of
bone–implant junctions exists on the left side of the implant specimen; therefore, these sites
were closer in appearance to the original bone.
Materials 2022, 15, x FOR PEER REVIEW 4 of 16
Figure 1. SEM image of magnesium implant surface obtained by reflection of secondary electrons 1
month after installation.
Figure 2. Close-up SEM image of implant site.
The physical bioresorption of the implant occurred after 3 months, and it was impos-
sible to visualize it after this period. The implant remained within the bone and we found
no loss of stability. (Figure 3). We observed pores and spherical discharges in the proposed
installation site (Figure 4).
Figure 1.
SEM image of magnesium implant surface obtained by reflection of secondary electrons
1 month after installation.
Materials 2022,15, 7529 4 of 14
Materials 2022, 15, x FOR PEER REVIEW 4 of 16
Figure 1. SEM image of magnesium implant surface obtained by reflection of secondary electrons 1
month after installation.
Figure 2. Close-up SEM image of implant site.
The physical bioresorption of the implant occurred after 3 months, and it was impos-
sible to visualize it after this period. The implant remained within the bone and we found
no loss of stability. (Figure 3). We observed pores and spherical discharges in the proposed
installation site (Figure 4).
Figure 2. Close-up SEM image of implant site.
The physical bioresorption of the implant occurred after 3 months, and it was impossi-
ble to visualize it after this period. The implant remained within the bone and we found no
loss of stability. (Figure 3). We observed pores and spherical discharges in the proposed
installation site (Figure 4).
Materials 2022,15, 7529 5 of 14
Materials 2022, 15, x FOR PEER REVIEW 5 of 16
.
Figure 3. SEM image of bone area after 3 months at different magnifications: (a) small, (b) me-
dium, and (c) large, respectively. Arrows in picture show pores associated with release of hydro-
gen during biodegradation of magnesium alloy.
a)
b) c)
Figure 3.
SEM image of bone area after 3 months at different magnifications: (
a
) small, (
b
) medium, and (
c
) large,
respectively. Arrows inpicture show pores associated withreleaseofhydrogen during biodegradation of magnesium
alloy.
Materials 2022, 15, x FOR PEER REVIEW 6 of 16
Figure 4. SEM image of bone area at proposed implant site. Spherical discharges are determined on
surface of bone tissue. Similar results are found with a bone sample at 6 months (Figure 5). There
are no visible traces of implant at the location of implant, and spherical discharges are present (Fig-
ure 6).
Figure 5. SEM image of sample after 6 months.
Figure 4.
SEM image of bone area at proposed implant site. Spherical discharges are determined on
surface of bone tissue. Similar results are found with a bone sample at 6 months (Figure 5). There are no
visible traces of implant at the location of implant, and spherical discharges are present (Figure 6).
Materials 2022,15, 7529 6 of 14
3.2. EDX Analysis
Without taking gold into account (because it was deposited on the surface of the
samples), oxygen, phosphorus, carbon and magnesium (and, to a lesser extent, calcium)
form a large proportion (in wt.%) of the released materials (Figure 7, Table 1).
After 1 month, an oxygen-containing area with organic compounds (C) formed around
the implant. However, the surface topography shows that the compounds, including C, Ca,
O, and P, are closer to the bone, and Mg is present in relatively small amounts compared
with deeper areas. C, Ca, O, P, and Mg are located in approximately the same areas.
Therefore, we traced initial-stage osteoconduction within a month in one bone sample with
an implant installed.
This image shows discharges close to a spherical shape, which are located relative to
the implant closest to the bone (Figure 8). According to the composition, EDX found that
they mainly consist of Mg, O, and P, and to a much lesser extent Na and C. With a larger
increase in these spherical precipitates, it becomes clear that they consist of many sharp
plates (Figure 9). At a magnification of 20 microns, we visualized a spiral structure, which
consisted of many crystals of a certain shape (presumably HA crystals) growing from a
common center and forming a cellular structure and certain relief, which were layering on
each other. We hypothesize that under these conditions, in the interval from 1 to 3 months,
a new bone is formed, and the structure of the new bone formation is cellular, consisting of
fractal clusters of a certain spiral device.
When studying the chemical composition, it becomes clear that they consist of in-
clusions of Mg, O, P and, to a lesser extent, Na. These crystals signal the passage of
magnesium bioresorption, forming an interface from compounds with oxygen and phos-
phorus (probably from interaction with hydroxyapatite and calcium phosphate—the main
mineral components of bone tissue). However, this process is incomplete, and the elemental
distribution is uneven and represented by different morphologies.
Materials 2022, 15, x FOR PEER REVIEW 6 of 16
Figure 4. SEM image of bone area at proposed implant site. Spherical discharges are determined on
surface of bone tissue. Similar results are found with a bone sample at 6 months (Figure 5). There
are no visible traces of implant at the location of implant, and spherical discharges are present (Fig-
ure 6).
Figure 5. SEM image of sample after 6 months.
Figure 5. SEM image of sample after 6 months.
Table 1. Total spectrum of map and distribution of elements after 1 month.
Element Line Type Weight % Sigma Weight % Atom %
C K-series 15.58 0.09 25.60
O K-series 41.46 0.06 51.14
Mg K-series 11.80 0.02 9.58
P K-series 13.46 0.02 8.58
Ca K-series 5.32 0.01 2.62
Materials 2022,15, 7529 7 of 14
Materials 2022, 15, x FOR PEER REVIEW 7 of 16
Figure 6. Enlarged SEM image of spherical discharge at implant site after 6 months.
3.2. EDX Analysis
Without taking gold into account (because it was deposited on the surface of the sam-
ples), oxygen, phosphorus, carbon and magnesium (and, to a lesser extent, calcium) form
a large proportion (in wt.%) of the released materials (Figure 7, Table 1).
Figure 7. EDX image of surface relief of implant site after 1 month: (a) SEM image of bone surface;
(b–f) elemental maps.
Figure 6. Enlarged SEM image of spherical discharge at implant site after 6 months.
Materials 2022, 15, x FOR PEER REVIEW 7 of 16
Figure 6. Enlarged SEM image of spherical discharge at implant site after 6 months.
3.2. EDX Analysis
Without taking gold into account (because it was deposited on the surface of the sam-
ples), oxygen, phosphorus, carbon and magnesium (and, to a lesser extent, calcium) form
a large proportion (in wt.%) of the released materials (Figure 7, Table 1).
Figure 7. EDX image of surface relief of implant site after 1 month: (a) SEM image of bone surface;
(b–f) elemental maps.
Figure 7.
EDX image of surface relief of implant site after 1 month: (
a
) SEM image of bone surface;
(b–f) elemental maps.
Materials 2022,15, 7529 8 of 14
Materials 2022, 15, x FOR PEER REVIEW 8 of 16
Table 1. Total spectrum of map and distribution of elements after 1 month.
Element Line Type Weight % Sigma Weight % Atom %
C K-series 15.58 0.09 25.60
O K-series 41.46 0.06 51.14
Mg K-series 11.80 0.02 9.58
P K-series 13.46 0.02 8.58
Ca K-series 5.32 0.01 2.62
After 1 month, an oxygen-containing area with organic compounds (C) formed
around the implant. However, the surface topography shows that the compounds, includ-
ing C, Ca, O, and P, are closer to the bone, and Mg is present in relatively small amounts
compared with deeper areas. C, Ca, O, P, and Mg are located in approximately the same
areas. Therefore, we traced initial-stage osteoconduction within a month in one bone sam-
ple with an implant installed.
This image shows discharges close to a spherical shape, which are located relative to
the implant closest to the bone (Figure 8). According to the composition, EDX found that
they mainly consist of Mg, O, and P, and to a much lesser extent Na and C. With a larger
increase in these spherical precipitates, it becomes clear that they consist of many sharp
plates (Figure 9). At a magnification of 20 microns, we visualized a spiral structure, which
consisted of many crystals of a certain shape (presumably HA crystals) growing from a
common center and forming a cellular structure and certain relief, which were layering
on each other. We hypothesize that under these conditions, in the interval from 1 to 3
months, a new bone is formed, and the structure of the new bone formation is cellular,
consisting of fractal clusters of a certain spiral device.
Figure 8. Spherical discharges located on site of bone implant.
Figure 8. Spherical discharges located on site of bone implant.
Materials 2022, 15, x FOR PEER REVIEW 9 of 16
Figure 9. SEM image of multiple lamellae making up spherical highlights.
When studying the chemical composition, it becomes clear that they consist of inclu-
sions of Mg, O, P and, to a lesser extent, Na. These crystals signal the passage of magne-
sium bioresorption, forming an interface from compounds with oxygen and phosphorus
(probably from interaction with hydroxyapatite and calcium phosphate—the main min-
eral components of bone tissue). However, this process is incomplete, and the elemental
distribution is uneven and represented by different morphologies.
After 3 months, we observed pores and rounded organic discharges at the intended
installation site. However, when examining the surface using EDX, we found no traces of
the Mg compound in three areas of the bone. All three sites contain: C, O, Na, P, Ca, and
Au (Figure 10). We also found Al, Fe, and S in extremely small volumes (<1 wt. %). We
did not detect Mg, which leads us to conclude that it is not present on the surface of this
3-month-old bone sample implant. It was likely compl etely resorbe d and r eplaced by bon e
tissue (Table 2).
Figure 9. SEM image of multiple lamellae making up spherical highlights.
After 3 months, we observed pores and rounded organic discharges at the intended
installation site. However, when examining the surface using EDX, we found no traces of
the Mg compound in three areas of the bone. All three sites contain: C, O, Na, P, Ca, and
Au (Figure 10). We also found Al, Fe, and S in extremely small volumes (<1 wt. %). We
Materials 2022,15, 7529 9 of 14
did not detect Mg, which leads us to conclude that it is not present on the surface of this
3-month-old bone sample implant. It was likely completely resorbed and replaced by bone
tissue (Table 2).
Materials 2022, 15, x FOR PEER REVIEW 10 of 16
Figure 10. Layered image of EDX sample after 3 months: (a) SEM image of bone surface; (b–e) ele-
mental maps.
Table 2. Total spectrum of map and distribution of elements after 3 months.
Element Line Type Weight % Sigma Weight % Atom %
C K-series 60.09 0.07 82.36
O K-series 12.35 0.05 12.71
P K-series 1.95 0.01 1.04
Ca K-series 3.93 0.01 1.62
We found similar results with a bone sample at 6 months. There are no visible traces
of the implant at the implant site, and only spherical-shaped organic discharges are pre-
sent. We did not find Mg in the elemental distribution map and, consequently, its com-
pounds were also absent. Again, we detected C, O, Na, P, Ca, and Au, among which the
highest in weight percent are C and O. These indicators possibly indicate the standard
mineral components of bone—hydroxyapatite (Ca
5
(PO
4
)
3
(OH)) and calcium phosphate
(Ca
3
(PO
4
)
2
) (Figure 11). Moreover, the elemental distribution relative to previous samples
is quite uniform, which may indicate a completely replaced and homogeneous bone tissue
(Table 3). However, the data differ in the literature, and further experiments are required
[10].
Figure 10.
Layered image of EDX sample after 3 months: (
a
) SEM image of bone surface;
(b–e) elemental maps.
Table 2. Total spectrum of map and distribution of elements after 3 months.
Element Line Type Weight % Sigma Weight % Atom %
C K-series 60.09 0.07 82.36
O K-series 12.35 0.05 12.71
P K-series 1.95 0.01 1.04
Ca K-series 3.93 0.01 1.62
We found similar results with a bone sample at 6 months. There are no visible traces
of the implant at the implant site, and only spherical-shaped organic discharges are present.
We did not find Mg in the elemental distribution map and, consequently, its compounds
were also absent. Again, we detected C, O, Na, P, Ca, and Au, among which the highest
in weight percent are C and O. These indicators possibly indicate the standard mineral
components of bone—hydroxyapatite (Ca
5
(PO
4
)
3
(OH)) and calcium phosphate (Ca
3
(PO
4
)
2
)
(Figure 11). Moreover, the elemental distribution relative to previous samples is quite
uniform, which may indicate a completely replaced and homogeneous bone tissue (Table 3).
However, the data differ in the literature, and further experiments are required [10].
Table 3. Total spectrum of map and distribution of elements after 6 months.
Element Line Type Weight % Sigma Weight % Atom %
C K-series 78.78 0.05 86.39
O K-series 15.83 0.05 13.03
P K-series 0.23 0.01 0.10
Ca K-series 0.35 0.00 0.12
Materials 2022,15, 7529 10 of 14
Materials 2022, 15, x FOR PEER REVIEW 11 of 16
Figure 11. Layered image of EDX sample after 6 months: (a) SEM image of bone surface; (b–e) ele-
mental maps.
Table 3. Total spectrum of map and distribution of elements after 6 months.
Element Line Type Weight % Sigma Weight % Atom %
C K-series 78.78 0.05 86.39
O K-series 15.83 0.05 13.03
P K-series 0.23 0.01 0.10
Ca K-series 0.35 0.00 0.12
4. Discussion
Several million people suffer annually from bone fractures caused by accidents (car
accidents, industrial disasters, etc.) or various diseases [26]. The treatment of such patients
is impossible without the use of fixation structures, such as plates, screws, and meshes
[27,28]. In Russia, on average, 400,000 operations are performed per year using metal
structures [28]. The financial volume of the global market for fracture fixation systems is
more than USD 5 billion [29]. However, using titanium systems has a number disad-
vantages, such as a limitation of bone growth, which is extremely important in pediatric
practice; problems associated with radiation therapy (oncology treatment); tactile sensi-
tivity of the plate; and the limitation of limb movement [30–34]. In 2018, more than 170,000
operations were performed in Germany to remove titanium structures [35]. In Europe and
the USA, 80% of patients undergo removal of metal structures after osteosynthesis [36–
38]. In Germany in 2007, the estimated yearly costs of these procedures exceeded EUR 430
million, and in Russia they amounted to about RUB 6 billion [31,38]. Reducing the number
of such operations will benefit the patients themselves, as well as reducing the financial
burden on the global healthcare system.
Due to their mechanical and biocompatible properties, Mg-based connectors repre-
sent a promising technology compared with conventional materials used for the manu-
facture of plates and screws for osteosynthesis [39,40]. Mg-based alloys have the ability to
decompose under physiological conditions and demonstrate compatibility with living tis-
sues without any toxic, destructive, or negative immunological reactions [41–45]. Mg-
Figure 11.
Layered image of EDX sample after 6 months: (
a
) SEM image of bone surface;
(b–e) elemental maps.
4. Discussion
Several million people suffer annually from bone fractures caused by accidents (car ac-
cidents, industrial disasters, etc.) or various diseases [
26
]. The treatment of such patients is
impossible without the use of fixation structures, such as plates, screws, and meshes [
27
,
28
].
In Russia, on average, 400,000 operations are performed per year using metal structures [
28
].
The financial volume of the global market for fracture fixation systems is more than USD
5 billion [
29
]. However, using titanium systems has a number disadvantages, such as a
limitation of bone growth, which is extremely important in pediatric practice; problems
associated with radiation therapy (oncology treatment); tactile sensitivity of the plate; and
the limitation of limb movement [
30
–
34
]. In 2018, more than 170,000 operations were
performed in Germany to remove titanium structures [
35
]. In Europe and the USA, 80%
of patients undergo removal of metal structures after osteosynthesis [
36
–
38
]. In Germany
in 2007, the estimated yearly costs of these procedures exceeded EUR 430 million, and
in Russia they amounted to about RUB 6 billion [
31
,
38
]. Reducing the number of such
operations will benefit the patients themselves, as well as reducing the financial burden on
the global healthcare system.
Due to their mechanical and biocompatible properties, Mg-based connectors represent
a promising technology compared with conventional materials used for the manufacture of
plates and screws for osteosynthesis [
39
,
40
]. Mg-based alloys have the ability to decompose
under physiological conditions and demonstrate compatibility with living tissues without
any toxic, destructive, or negative immunological reactions [
41
–
45
]. Mg-based alloys
promoted the formation of bone tissue during
in vivo
experiments on large and small
animals [
8
,
46
]. However, the biodegradation of magnesium alloys leads to the release of
gaseous hydrogen, which, if not controlled, can have a negative impact on regeneration
processes [
47
–
49
]. The high degradation rate, uneven distribution of resorption, and
localized corrosion of magnesium alloys may hinder the further development of this
technology [50,51].
Materials 2022,15, 7529 11 of 14
Recently, researchers have investigated the addition of Zn to the alloy, which can
correct the rate and volume of hydrogen released. The results of studies have shown that
Mg-Zn alloys have excellent mechanical properties, biocompatibility, and higher corrosion
resistance [
52
]. Additionally, the addition of Zn to Mg alloys can significantly reduce
H2 emission [
53
,
54
]. However, depending on the zinc content in binary Mg–Zn alloys
and phase distribution, the corrosion resistance of Mg–Zn alloys varies greatly. Zhang
et al. implanted Mg-6Zn alloy rods into the body of rabbits, and their results, which were
obtained by the weight loss method, showed that Mg alloy can be gradually absorbed
in vivo
with a decomposition rate of 2.32 mm/year without heart, liver, kidney, and
spleen disorders. In addition, six weeks after implantation, the subcutaneous H2 gas that
accumulated as a result of alloy decomposition disappeared without noticeable adverse
effects [55].
Our SEM and EDX study results highlight that the corrosion front is composed of
calcium and phosphate, most likely in the form of an oxide layer. Similar results are
described in Su Y. et al. [
56
]. The presence of these ions can lead to conversion, which can
be beneficial to the bone regeneration process as the pins are optimally integrated into the
bone matrix.
According to our study results regarding the area of previously installed pins, we
determined a regenerate containing calcium phosphate and, presumably, hydroxyapatite.
Additionally, some researchers also believe that the formation of more complex Mg
3
(PO
4
)
2
is possible [
27
,
29
]. However, such data are mainly described with the subcutaneous
injection of a rod based on magnesium alloys. When studying the chemical composition of
the interface on the element distribution map, we found Zn only 1 month after implantation.
Klima et al. also studied the chemical composition of the implant–bone interface using
SEM-EDS and found a thin and compact phosphate-based layer (3–5
µ
m) on the surface of
Zn-based implanted screws, regardless of the implantation period [
46
]. They found calcium
and zinc in this layer, which led them to suggest the formation of a complex or mixture of
Ca/Zn phosphates under
in vivo
conditions. They explained the formation of such a layer
by the interaction of Zn
2+
ions released upon dissolution of the experimental alloy with
body fluids containing Ca
2+
, HXPO4(3–x)–, and other ions. These formed phosphates are
subsequently deposited on the implant surface [
56
,
57
]. The formation and precipitation
of these phosphates is also affected by the presence of proteins that can complex zinc
and ions from solutions. These complex compounds subsequently enhanced phosphate
precipitation [
58
,
59
]. Kubasek J. et al. discovered that a phosphate-based layer is formed at
an early stage (up to several days) after implantation [
18
,
60
]. The formation of a phosphate
layer is beneficial for further bioresorption. According to Su et al., the surface layer of zinc
phosphate (ZnP) increases the cyto- and hemocompatibility of Zn-based materials [
56
].
They also reported increased antibacterial activity in ZnP-coated samples. Chou et al.
studied the effects of ZnP coatings on the behavior of organic bone regeneration (GBR)
membranes and described the antibacterial activity of zinc phosphate [
61
]. Thus, the
formation of a ZnP layer with predictable antibacterial activity, which was observed by
Klima et al., may be the reason why only a very limited inflammatory response has been
recorded [
46
]. In our study, the nail was in close contact with both the cancellous bone and
cortical layer. In the dynamics of our experiment, and especially at the post-six-months
stage, young bone-tissue formation in the osteotomy zone is visualized, which indicates
the complete biodegradation of the installed pins.
Furthermore, it is impossible to detect residual Mg by EDS surface scanning if it
diffused into the bone to a depth greater than a few microns, and we could not find residual
Mg on the specimen surface after 3 and 6 months of implantation.
Based on our results, we believe that the tested biomaterials can be used in a clinical
setting without causing side effects. However, it is necessary to further increase the number
of observations and conduct additional clinical studies using full-fledged structures for
osteosynthesis, as well as using additive technologies for 3D printing individual products.
Moreover, our plan is to investigate our hypothesis using new samples as follows:
Materials 2022,15, 7529 12 of 14
(a) Prepare specimens with implanted Mg-based pins at 1-month steps until 6 months;
(b) Cut, grind, and polish the cross-section of each specimen;
(c) Perform final delicate polishing with Ga-FIB in the SEM chamber to remove the
oxide layer;
(d) Reveal Mg bone diffusion by combining FIB-EDS and FIB-TOF-SIMS methods.
5. Conclusions
In our study, we successfully implanted Mg-Zn-Ga alloy pins in 12 Wistar rats, which
we followed up for 6 months with sampling at 1, 3, and 6 months. The alloy is completely
resorbed within a period of 1 to 6 months. We determined a spiral structure when analyzing
the “bone–implant” interface. This structure is presumably similar to hydroxyapatite
crystals. In the future, it will be necessary to conduct additional research on a larger
number of animals and the use of finished products. Our obtained results will make it
possible to create the most effective types of fixing structures consisting of bioneutral and
low-toxic elements made of bioresorbable metals for various branches of medicine, making
it possible to avoid repeated surgical intervention in the future.
Author Contributions:
Conceptualization, A.D. and A.K.; methodology, A.D. and E.S.S.; software,
A.M.; validation, Z.G., N.R. and I.S.; formal analysis, N.R.; investigation, I.S.; resources, I.R.; data
curation, N.R.; writing—original draft preparation, Z.G.; writing—review and editing, A.D., A.K.,
V.B. and E.S.S.; visualization, N.R.; supervision, A.D.; project administration, O.Y. and I.R.; funding
acquisition, A.D., O.Y. and I.R. All authors have read and agreed to the published version of the
manuscript.
Funding:
The authors are grateful to the Ministry of Science and Higher Education of the Russian
Federation for financial support (Megagrant No. 075-15-2022-1133).
Institutional Review Board Statement:
The animal study protocol was approved by the Intercolle-
giate Ethics Committee (protocol №04, 15.04.2021).
Informed Consent Statement: Not applicable.
Acknowledgments:
The authors express sincere gratitude to the staff of the Kuban State Medical
University (Krasnodar, Russia) for their help in conducting the experimental part of the study.
Conflicts of Interest: The authors declare no conflict of interest.
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