Abstract
Modern orthodontics since 1899 has failed to solve rapid and persistent
dentition maintenance after tooth movement till now. Traditional
post‐orthodontic retention using single mechanical fixation cannot
actuate periodontal tissue remodel, causing lifelong dependence, huge
time costs and discomforts. Current attempts by combining hormone drug
with mechanical fixation for improvement are limited by unsatisfactory
outcomes and global side effects. Targeting at this 120‐year dilemma, a
flexible magnetoelectric orthodontic retainer (FMOR) is presented using
customized additive manufacturing, serving as periodontal remodeler for
high‐performance post‐orthodontic retention. Wireless magnetoelectric
cues display considerable effects in periodontal microenvironment
remodel, switching periodontal ligament cell (PDLC) paracrine manner to
promote M2 macrophage formation, osteoclastic/osteoblastic balance and
collagen metabolism. Using rat and rabbit orthodontic model,
application of FMOR is confirmed to significantly strengthen the
maintaining of tooth movement outcomes. Mechanistically,
magnetoelectric treatment significantly upregulate PDLC genes related
to immune response, extracellular matrix organization and osteogenic
regulation. This study proposes a non‐destructive physical therapeutic
strategy for fast activation of periodontal reconstruction after
orthodontic treatment and inhibition of tooth relapse for the first
time, shedding light on development of next‐generation orthodontic
retention system for optimized solution of orthodontic relapse.
Keywords: additive manufacturing, periodontal remodel, post‐orthodontic
retention, wireless magnetoelectric
__________________________________________________________________
Orthodontic relapse persists as a 120‐year challenge, as traditional
retainers fail to remodel tissue, causing lifelong dependence. This
study demonstrates a flexible magnetoelectric retainer (FMOR) enabling
wireless periodontal remodeling. FMOR cues shift periodontal ligament
cell (PDLC) paracrine signaling, promoting M2 macrophages, osteogenic
balance, and collagen metabolism. Rat/rabbit models confirm FMOR
significantly strengthens tooth position maintenance, proposing a
non‐destructive physical strategy against relapse and enabling
next‐generation retention systems.
graphic file with name ADVS-12-e05020-g003.jpg
1. Introduction
Malocclusion serves as a common abnormality of dentition and
maxillofacial bone that causes great discomfort and secondary disease
risks to patients.^[ [42]^1 ^] Modern orthodontic treatment has been
developed to rectify malocclusion using biomechanical induction over a
century.^[ [43]^2 ^] Nevertheless, how to maintain the achieved
orthodontic effect still lies as an unsolved difficulty.^[ [44]^3 ,
[45]^4 ^] It should be noted that teeth are guided to a desired
position after finishing an orthodontic course. In this case,
periodontal tissues have not been transformed from aseptic inflammatory
status to pro‐reconstructing manner, which is mainly explained by
restored stress within the periodontal ligament (PDL) against opposite
orthodontic force.^[ [46]^5 , [47]^6 ^] To rebuild the periodontal
tissues around the orthodontic teeth, traditional mechanical retainer
is advocated to patients for permanent passive retention.^[ [48]^7 ,
[49]^8 ^] However, this is a huge burden to post‐treatment patients
owing to its significant demand of time and discomfort.^[ [50]^9 ^]
Once abandoning to persist in mechanical retention, the periodontal
tissues fail to recover, leading to rapid reverse of
orthodontic‐modified dentition due to instable periodontal status.^[
[51]^10 , [52]^11 ^] Therefore, key for this dilemma lies in the
disability of active regulation on periodontal microenvironment.
Post‐orthodontic periodontal tissues fail to get guided to perform
repair and reconstruction, making it especially susceptible to relapse
in response to external stimulus.^[ [53]^12 , [54]^13 , [55]^14 ^] In
summary, rapid periodontal microenvironment remodel for orthodontic
dentition is of great importance.
To solve this problem, orthodontists have explored collaboration of
mechanical fixation and drug delivery to enhance post‐orthodontic
retention.^[ [56]^15 , [57]^16 , [58]^17 ^] Supplementation of hormone
drugs, such as parathyroid hormone and atorvastatin, has been
identified to promote the stability of post‐orthodontic dentition by
enhancing periodontal remodel, including the counts of osteoclasts and
osteoblasts, as well as expression of bone metabolic proteins.^[
[59]^15 , [60]^16 ^] However, global administration of drugs poses some
unnecessary side effects, and deficiency in targeting to periodontal
tissues would lead to inadequate accumulation of effective constituents
in the local area, further compromising its clinical expectations.
Local application thus acts a proper alternative, but surface dressing
is difficult to penetrate into periodontal ligaments.^[ [61]^17 ^]
Repetitive injections would bring about sufferings and inconvenience to
patients, and this invasive operation is also harmful to periodontal
remodel.^[ [62]^18 , [63]^19 ^] In consequence, current studies still
failed to provide an optimal strategy for active and stable remodel of
periodontal tissues and tooth positions.
Biophysical electric cues have been identified to play an indispensable
role in tissue development, homeostasis and regeneration.^[ [64]^20 ,
[65]^21 , [66]^22 , [67]^23 ^] Endogenous bioelectric fields regulate
key regenerative processes including osteogenesis, angiogenesis, and
immunomodulation through voltage‐gated ion channels and
electrophysiological signaling pathways.^[ [68]^24 , [69]^25 ^]
Although mild local irritation and thermal effects may occur at high
intensities, clinical applications of exogenous bioelectric stimulation
demonstrate accelerated tissue repair with minimal side effects
compared to pharmacological interventions, owing to its non‐thermal,
non‐invasive nature.^[ [70]^26 , [71]^27 ^] In particular, we
previously confirmed that exogenous supplementation of electric signals
would benefit repair of nerve, skin and especially maxillofacial bone
tissues.^[ [72]^28 , [73]^29 , [74]^30 ^] Therefore, it is speculated
that bio‐electric signals might promote remodel of periodontal tissues.
Meanwhile, electric stimulation could be a non‐destructive method with
high‐penetration for biological intervention, which is suitable for
daily intervention post orthodontic treatment.^[ [75]^31 , [76]^32 ^]
Nevertheless, in orthodontic retention situation, the whole setup
(electrodes, wires and power sources) is rigid, unable to form superior
adaptation with soft tissues.^[ [77]^33 , [78]^34 ^] Limited by
definite intraoral space, integration of inner battery, wires and
electrodes into mechanical retainers remains largely impractical, and
might cause potential inconvenience and risks.^[ [79]^35 , [80]^36 ^]
Hence, development of novel wearable orthodontic retainers with
built‐in electrodes activated by external power exhibits huge value.
Flexible powering method based on Faraday's law of electromagnetic
transformation might be a potential solution for relapse inhibition,
whose advantages include wireless energy output, adequate current
generation and precise control in intensity.^[ [81]^37 , [82]^38 ^]
Besides, taking advantage of the combination of additive manufacturing
and dental CT scanning, customized flexible magnetoelectric device can
be designed and fabricated on basis of personal difference in anatomic
morphology.^[ [83]^39 , [84]^40 ^]
Here, we demonstrated a novel flexible magnetoelectric orthodontic
retainer (FMOR) that could maintain more than 60% tooth movement
outcomes, which reached almost twofold compared to pure mechanical
retention group, while the dentition lost almost the whole orthodontic
effect in the without retention 10 days after treatment. The FMOR
consists of a soft retainer backbone, liquid metal (LM) coils that
enables electric output in response to external alternating magnetic
field and bio‐interfaces for electric transmission into periodontal
tissues (Figure [85]1A–C). We identified that magnetoelectric output
from FMOR can remodel the orthodontic force‐induced pro‐inflammatory
periodontal microenvironment. In detail, magnetoelectric cues dominate
the transformation of periodontal ligament cell (PDLC) paracrine manner
from stress‐induced status, thus triggering M2 macrophage polarization,
re‐balancing osteoclastic and osteoblastic activity and facilitating
collagen deposition (Figure [86]1D). Mechanistic exploration delineates
that magnetoelectric retention treatment significantly upregulated the
genes related to immune response, extracellular matrix organization,
and osteogenesis regulation in PDLC. Application of wireless
magnetoelectric intervention achieved significant effect in inhibition
of post‐orthodontic relapses and reconstruction of periodontal tissues
in rat and rabbit models, evidenced by augmented alveolar bone
formation and collagen deposition.
Figure 1.
Figure 1
[87]Open in a new tab
Schematic illustration of the FMOR system and the post‐orthodontic
tissue reconstruction. A) The flexible magnetoelectric orthodontic
retainer and its components. B) The wearable magnetoelectric
transmitter and its disassembled electronic structure. C) The
activation of magnetoelectric retention therapy at the end of
orthodontic treatment. D) The magnetoelectric cues promote immune
repair and tissue reconstruction in periodontal tissues to inhibit
orthodontic relapse.
2. Results and Discussion
2.1. Design and Fabrication of FMOR System
The receiving and transmitting units of the FMOR system were designed
and constructed as shown in Figure [88]2A. Briefly, a 3D scanner was
used to capture the individual teeth and craniomaxillofacial recordings
into a digital model. The personalized FMOR and wearable transmitter
were designed by simulation design software, and the molded parts were
finally printed and encapsulated with LM coils by a light‐curing 3D
printing equipment. The retainers molded from polydimethylsiloxane
(PDMS) are biocompatible, ensuring that the FMOR, as the receiving end,
is harmless during application.^[ [89]^41 ^] The rationale behind the
choice of this flexible material is due to its elasticity and
toughness, which permit the retainer to have a certain degree of
physical retention based on improving the comfort of the patient's
wear, as well as its capacity to stably and safely encapsulate the
flexible LM coils and circuit structure inside (Figure [90]2B). The
overall dimensions of the FMOR are ≈6.2 × 5.6 × 4.3 mm^3. And FMOR
adopts a unified monolithic design with three integrated elements
(Figure [91]S1A, Supporting Information):
1. PDMS substrate: Encapsulates components while serving as fixation;
2. Embedded copper coil: Positioned within PDMS at gingival interface;
3. Surface‐mounted electrode: Adhered to PDMS and wire‐connected to
coil.
Figure 2.
Figure 2
[92]Open in a new tab
Fabrication and characterization of the FMOR system. A) Schematic
diagram of the fabrication process and the components of FMOR and the
wearable magnetoelectric transmitter. B) An optical image of FMOR and
the wearable magnetoelectric transmitter for rat experiments. C)
Magnetoelectric retention therapy for rats in vivo: i) Moving the rat
first molar to the target position after the OTM phase. ii) Wearing
personalized FMORs. iii) Initiating magnetoelectric retention therapy
with the wearable magnetoelectric transmitter. D) Voltage waveforms at
FMOR receiver and wearable magnetoelectric transmitter. E) Electrical
output of different input voltages between the receiver and
transmitter. F) Current output at different angels (θ) between FMOR
receiving coil and the transmitting coil.
This optimized design minimizes the intraoral footprint, which directly
addresses the spatial constraints mentioned in the introduction, while
ensuring mechanical robustness through complete component
encapsulation. Concurrently, in order to guarantee the precise delivery
of wireless magnetoelectric cues to the periodontal, the electrodes
were positioned on the inner side of the FMOR and securely attached to
the gums, while the remainder of the internal wiring was insulated from
the oral cavity (Figure [93]S1B, Supporting Information). Enhanced
cross‐sectional schematics and images of FMOR worn on the rat maxillary
specimens captured with a magnifying lens head demonstrated the contact
interface between the FMOR electrodes and the periodontal tissue
(Figure [94]S1C, Supporting Information, Figure [95]2C). Results of
Young's modulus showed that FMOR integrated with LM coil can meet the
strength requirements for intra‐oral wearing (Figure [96]S4, Supporting
Information). Then, we further compared the measured Young's modulus
with that of currently used medical‐grade materials (Table [97]S2,
Supporting Information). The results indicate that the PDMS used in
this work falls within the typical range for flexible transparent
materials. The most commonly used material for medical retainers is
PETG,^[ [98]^42 ^] which has a Young's modulus of up to 2.2 GPa. In
comparison, our PDMS exhibits significantly better flexibility, making
it more suitable for wearable and biomedical applications.
The wearable transmitter was designed by craniomaxillofacial 3D
scanning data. The core component of the wearable magnetoelectric
transmitter was the integrated circuit and the LM transmitter coil. The
integrated circuit can monitor and regulate current output parameters
of the system in real time (Figure [99]2B, Figure [100]S2, Supporting
Information). Similarly, the aforementioned process was utilized to
develop the personalized FMOR system that was subsequently applied to
rabbits (Figure [101]S3, Supporting Information). In summary, the
personalized FMOR was paired with the wearable magnetoelectric
transmitter for subsequent magnetoelectric retention therapy
(Figure [102]2C).
2.2. Wireless Magnetoelectric Output Performance of FMOR
FMOR is based on Faraday's law of electromagnetic transformation. The
alternating current created an alternating magnetic field around the LM
coil at the transmitter end, and the magnetic induction at any point in
space can be approximated by the Biot‐Saval law:^[ [103]^43 ^]
[MATH: dB⃗=μ04πId<
mover accent="true">l⃗×r^r2<
/mn> :MATH]
(1)
As the receiving end, the receiving coil in the FMOR that was under the
influence of an alternating magnetic field generates an induced
electromotive force within it (Figure [104]2D):
[MATH: ε=−∮A
dB⃗×dA⃗dt :MATH]
(2)
Subsequently, considering the spatial position of the coil is not fixed
in practical use, we conducted mutual inductance experiments to
regulate and evaluate the wireless magnetoelectric output performance
of the FMOR. We considered that the relative position and transmitted
power between the coils can significantly influence the magnetoelectric
efficiency.^[ [105]^44 , [106]^45 , [107]^46 ^] To more accurately
assess potential operational issues of the FMOR, we conducted
performance tests on the mutual inductance system. By adjusting the
voltage at the transmitter side, the voltage across the FMOR changes
synchronously (Figure [108]2E, Video [109]S1, Supporting Information),
which is crucial for maintaining a stable effective output current.
When the FMOR current deviates from the desired therapeutic range, it
can be effectively regulated by tuning the transmitter voltage. During
actual fabrication and wearing processes, it is difficult to ensure
consistent alignment between the FMOR coil and the transmitting coil.
Within the coverage area of the transmitting coil, variations in
horizontal offset, radial distance, and angular misalignment may occur.
Therefore, a series of experiments were carried out with the
transmitting voltage fixed at ≈4 V. The results show that the FMOR
receiver coil maintains a physiologically effective output current of
≈20 mA at distances up to 5 mm (Figure [110]S5, Supporting
Information). Moreover, an effective output current is still achieved
with angular deviations of up to 40° (Figure [111]2F). The series of
experiments demonstrated that while alterations in relative position
resulted in notable alterations in the magnetoelectric output, the
constructed parameters exhibited the physiological requirements. A
comparison with existing studies indicates that the magnetic field
strength and current applied in our experiments are within the
recognized safety limits.^[ [112]^47 , [113]^48 , [114]^49 ^]
Concurrently, real‐time temperature monitoring demonstrated that the
temperature fluctuations of the transmitting and receiving LM coils
during magnetoelectric retention therapy remained within the range of
37 °C, re‐ensuring the safety of FMOR system during its wireless
magnetoelectric output state (Figure [115]S6, Supporting Information).
In conclusion, preliminary in vitro experiments have demonstrated that
FMOR can achieve stable physiological‐grade wireless magnetoelectric
output and safe delivery to periodontal tissues through the
magnetoelectric induction of a wearable magnetoelectric transmitter.
2.3. FMOR Inhibits Orthodontic Tooth Relapse In Vivo
The schema in Figure [116]3A shows the experimental design in the rat
relapse model, including orthodontic force application and relapse
phase.^[ [117]^50 ^] The maxillary first molar (M1) of the rat was
rapidly moved by the 40 g of optimal orthodontic force provided by a
tension spring, resulting in the creation of a gap between it and the
second molar (M2). This stage is referred to as the Orthodontic
Movement (OTM) phase.^[ [118]^51 ^] Once the orthodontic force was
removed, the first molar entered the relapse phase under the
compressing and stretching forces of the periodontal ligament. Previous
studies have verified that the type of OTM in rats is tipping due to
the force was loaded on the neck of the tooth.^[ [119]^51 , [120]^52 ^]
By analyzing the stress distribution of the mesial root (MR) in
Figure [121]3B, it was shown that the periodontal tissues in the apical
distal region (A‐D region) were the major area subjected to orthodontic
pressure during the OTM phase, resulting in significant bone
resorption. While the cervical distal region (C‐D region) was the
tension side during the OTM phase, leading to bone deposition. In
contrast, orthodontic relapse resulted in a completely opposite
movement trajectory compared to the OTM phase. Additionally, it
produced completely opposite stress distributions and tissue behaviors.
In the relapse phase, C‐D region was under bone resorption in response
to the compressive stress, whereas A‐D region is the tension area with
bone deposition.
Figure 3.
Figure 3
[122]Open in a new tab
FMOR inhibits orthodontic tooth relapse in vivo. A) Schematic
illustration of the orthodontic and relapse rat model. Created by
BioRender.com. B) Schematic diagram of H&E staining showing regional
division of the first molar during orthodontic and relapse processes.
Labels: M1 the first molar, MR mesial root, AB alveolar bone, A‐D the
apical distal region, C‐D the cervical distal region, P tooth pulp. The
blue area indicates tension sides, the orange area indicates pressure
sides. C) Representative micro‐CT images of rats’ dentition after 5 and
10 days of orthodontic relapse. The width of the red area is the
distance between the first and second molars. Scale bar = 1 mm. D,E)
Quantitative analysis of the distance between first and second molars
and the relapse rate. Relapse rate = relapse distance / relapse
distance at 0 d. F,G) Micro–computed tomography images and quantitative
analysis of percent bone volume (BV/TV) of alveolar bone in the A‐D and
C‐D regions. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Sample size (n = 3).
To explore the effect of magnetoelectric retention therapy on
orthodontic relapse, during the relapse phase, rats were fitted with
FMOR for 12 h daily and the wearable magnetoelectric transmitter device
were adjusted to set varying magnetoelectric output gradients. Rats in
the control group did not wear retainers during the relapse phase,
while the passive retainer (PR) indicated that FMOR was equipped
without initiating the magnetoelectric retention therapy. The FMOR
group was classified into three subgroups based on current amplitude
(10, 20, and 30 µA). After 5 and 10 days of relapse, Micro‐CT 3D
remodeling and sagittal images of rat maxillary molars was performed
and the distance between M1 and M2 was measured. The results showed
that FMOR with magnetoelectric output significantly reduced the M1
relapse distance, with the most pronounced effect in the 20 µA current
group (Figure [123]3C,D and Figure [124]S7A, Supporting Information).
The M1 relapse rate was calculated by dividing the relapse distance by
the OTM distance. Results showed that the 20 µA‐FMOR therapy during the
10‐day relapse phase significantly reduced the relapse rate from 100%
in control group to about 50% (Figure [125]3E).
In order to analyze the dynamic variation of alveolar bone density in
the A‐D and C‐D regions of the MR distal region described above, we
visualized the tomographic images of the alveolar bone using Micro‐CT
data, which represented the level of bone density in terms of color.
Results of the tomographic images and bone volume fraction (BV/TV)
showed that the alveolar bone treated with magnetoelectric retention
therapy achieved higher bone density, especially in the 20 µA group
(Figure [126]3F,G). A comparison of the control and PR groups through
stress distribution analysis revealed that, as the deposition side, A‐D
area exhibited a more rapid osteogenesis rate during the relapse phase
under 20 µA‐FMOR therapy. Conversely, C‐D area demonstrated a notable
bone resorption inhibition effect as the bone resorption side under
20 µA‐FMOR therapy.
With the strong support in the distal region of the MR, and considering
that the biological changes occurred throughout the alveolar bone, we
further analyzed the regions of the distal roots as well. In previous
studies,^[ [127]^51 ^] the pressure in the distal root were mainly
distributed in the cervical mesial region (C‐M region, Figure [128]S7B,
Supporting Information). Based on this, we now performed a
comprehensive analysis of the C‐M region of the distal root. Micro‐CT
reconstruction and quantification of bone volume fraction (BV/TV)
showed that the alveolar bone density was significantly higher after
magnetoelectric retention treatment, especially in the 20 µA subgroup
(Figure [129]S7C, Supporting Information). The results of the distal
roots further reinforced the therapeutic effect of magnetoelectric
retention therapy.
Finally, it is imperative to undertake a critical evaluation to
ascertain the potential contribution of the magnetic field alone. We
established a dedicated magnetic field control group (MF Group) where
rats wore passive retainers (PR) without liquid metal coils and was
received identical 12‐h daily magnetic field exposure (20 µA‐equivalent
intensity). After 5 and 10 days of relapse, Micro‐CT measurements
showed no statistically significant differences in relapse distance or
relapse rate between MF and PR groups (Figure [130]S8A–D, Supporting
Information). Sagittal reconstructions and BV/TV quantification in
three critical regions (A‐D, C‐D, C‐M regions) revealed comparable
alveolar bone density in MF and PR groups, and both groups are
significantly lower than FMOR groups (Figure [131]S8E,F, Supporting
Information). These comprehensive analyses confirm that the actuating
magnetic field alone exerts no measurable effect on periodontal
remodeling or relapse inhibition, validating that the observed
therapeutic effect of FMOR requires magnetoelectric stimulation.
Hence, FMOR effectively inhibited orthodontic tooth relapse by
20 µA‐FMOR therapy, which may be related to magnetoelectric cues
modulating periodontal microenvironment during the relapse phase.
2.4. Magnetoelectric Output Dominate the Remodel of Periodontal
Microenvironment During the Relapse Phase
To further investigate the role of magnetoelectric retention therapy
during the relapse phase, we performed tissue staining on rat maxillary
specimens after 5 and 10 days of retention. TRAP and Cathepsin K (CTSK)
are relevant indicators that respond to osteoclastic activity and bone
resorption as previously mentioned.^[ [132]^51 ^] The results of the
TRAP staining and osteoclast density analysis demonstrated that
20 µA‐FMOR therapy markedly suppressed osteoblastic differentiation on
the bone resorption side (C‐D region) in comparison to the control and
PR groups (Figure [133]4A,B). Concurrently, on the bone deposition side
(A‐D region), magnetoelectric retention therapy also notably expedited
the clearance of TRAP^+ osteoclasts generated during the OTM phase
(Figure [134]S9, Supporting Information). Immunofluorescence staining
and intensity quantification of CTSK in C‐D and A‐D regions reconfirmed
the inhibition of osteoclastic activity, which was consistent with the
TRAP results (Figure [135]S10, Supporting Information).
Figure 4.
Figure 4
[136]Open in a new tab
Magnetoelectric output remodels periodontal microenvironment in
orthodontic relapse. A) TRAP staining on histological sections of
alveolar bone resorption in C‐D region. Black triangles indicate
TRAP‐positive cells. B) Quantitative analysis of the number of
osteoclasts. C,D) Representative immunofluorescence images and
quantitative analysis of CD206 in histological sections of alveolar
bone resorption in A‐D region. E,F) Representative immunofluorescence
images and quantitative analysis of RUNX2 in histological sections of
alveolar bone generation in A‐D region. G,H) Representative
immunofluorescence images and quantitative analysis of MMP‐1 in
histological sections of periodontal membrane resorption in C‐D region.
*p < 0.05, **p < 0.01, ***p < 0.001 and ****p <0.0001. Sample size (n =
3).
To further assess the role of magnetoelectric retention therapy on the
reconstruction of the tissue microenvironment, the number of M2
macrophages were counted and analyzed.^[ [137]^53 ^] As mechano‐induced
aseptic inflammation in orthodontics, shifts in macrophage M1/M2
polarization within the periodontal ligament (PDL) critically regulate
inflammatory and regenerative processes.^[ [138]^14 ^] During early
OTM, macrophages are recruited to inflammatory sites where cytokines
drive predominant M1 polarization, which is a phenotype associated with
sustained tissue destruction and root resorption. Conversely, M2
macrophages emerge significantly only during late OTM phase and after
force removal, which orchestrate bone resorption cessation and initiate
tissue repair. Crucially, prior studies confirm that elevating M2
proportions accelerates post‐inflammatory periodontal regeneration,^[
[139]^54 ^] while prolonged M1 dominance exacerbates damage.^[ [140]^53
^] Immunofluorescence staining for CD206 showed that 20 µA‐FMOR therapy
promoted sustained M2 macrophage polarization in both C‐D and A‐D
regions, achieving inhibition of osteoclastic behavior in the bone
resorption areas as well as promoting osteogenesis and tissue repair in
the bone deposition areas (Figure [141]4C,D and Figure [142]S11,
Supporting Information).
Furthermore, to understand the role of FMOR on bone regeneration,
runt‐related transcription factor 2 (RUNX2) were investigated.^[
[143]^55 ^] Immunofluorescence staining images showed that RUNX2 was
abundantly expressed in the alveolar bone in A‐D region under
20 µA‐FMOR therapy, suggesting that magnetoelectric cues have a
facilitating effect on osteogenesis (Figure [144]4E,F). Meanwhile,
compared with the control and PR groups, 20 µA‐FMOR therapy similarly
promoted an increased number of RUNX2^+ cells in the bone resorption
zone (Figure [145]S12, Supporting Information). Considering the
simultaneous inhibition of osteoclastic behavior in the C‐D region by
magnetoelectric retention therapy, this suggests that magnetoelectric
cues help to rebalance osteoblast and osteoclast activity, thereby
promoting local bone repair and counteracting pressure‐induced bone
resorption behavior. Finally, to explore the effect of magnetoelectric
retention therapy on periodontal fiber remodeling, matrix
metalloproteinase‐1 (MMP‐1) was labelled as an important protease for
extracellular matrix and collagen fiber resorption.^[ [146]^56 ^] The
results showed that 20 µA‐FMOR therapy significantly reduced the
intensity of MMP1 expression in the periodontal membranes of C‐D and
A‐D regions, which effectively protected the periodontal fibers on the
pressure side from resorption and promoted matrix fiber restoration on
the tension side (Figure [147]4G,H and Figure [148]S13, Supporting
Information).
In summary, due to aseptic inflammation caused by orthodontic force
during OTM phase and pressure‐induced resorption of the unbalanced
periodontal tissues during the relapse phase, self‐reconstruction of
periodontal tissues is slowly initiated. By promoting matrix collagen
metabolism and deposition, M2 macrophage polarization, rebalancing of
osteogenesis and osteoclasis, magnetoelectric retention therapy thus
dominate the reversal of periodontal destruction during the relapse
phase.
2.5. Magnetoelectric Output Mediates Immune Remodel and Initiates Restorative
Activity After Removal of Orthodontic Pressure In Vitro
To clarify the intrinsic connection between magnetoelectric retention
therapy and periodontal remodel, cell co‐culture system was used to
simulate the periodontal microenvironment in vitro (Figure [149]5A).
During orthodontic treatment, periodontal membrane cells (PDLC)
function as the primary sensors in response to mechanical signals that
not only regulate periodontal soft tissue remodeling but also rely on
the paracrine signaling network to control the local immune
microenvironment and alveolar bone remodeling.^[ [150]^57 ^] Based on
this, primary human PDLCs were seeded in Transwell chambers and applied
a pressure of 2 g cm^−2 for 24 h to mimic the in vivo orthodontic
pressure during OTM phase as previously described.^[ [151]^50 ^]
Subsequently, following magnetoelectric retention therapy for PDLC, the
Transwell chambers were transferred to culture plates inoculated with
macrophages, osteoblasts or osteoclasts, respectively, for cell
co‐culture. Quantification of CD206^+ macrophages by flow cytometry
showed a massive increase in CD206^+ macrophages from 33.48% to 74.55%
in 20 µA‐FMOR therapy group compared with the control group
(Figure [152]5B,C). Meanwhile, the results of immunofluorescence
staining of macrophages demonstrated that magnetoelectric retention
therapy markedly reduced the expression of the M1 macrophage marker
CD86 while concurrently elevating the expression of the M2 macrophage
marker CD206(Figure [153]5D,G,H). In addition, real‐time quantitative
PCR (RT‐qPCR) results showed that the expression of IL12, a gene
associated with M1 polarization, was significantly decreased in
macrophages in 20 µA‐FMOR therapy group, whereas the expression of the
genes CD163, VEGFα, and TGFβ, associated with M2 polarization, was
significantly increased (Figure [154]5J).
Figure 5.
Figure 5
[155]Open in a new tab
Magnetoelectric output mediates immune remodeling and promotes alveolar
bone osteogenesis in vitro. A) Schematic diagram of the Transwell
co‐culture system of PDLCs with other cells under wireless
magnetoelectric output. Created by BioRender.com. B,C) Flow cytometry
and quantitative analysis of the cell surface marker CD206 in
macrophages co‐cultured with PDLCs under wireless magnetoelectric
output. D,G,H) Representative immunofluorescence images and
quantitative analysis of CD206 (green), CD86 (red) and cell nuclei
(DAPI, blue) in macrophages co‐cultured with PDLCs under wireless
magnetoelectric output for 48 h. E,I) Representative immunofluorescence
images and quantitative analysis of RUNX2 (green), F‐actin (red) and
cell nuclei (DAPI, blue) in h‐FOBs co‐cultured with PDLCs under
wireless magnetoelectric output for 5 days. F) Representative images of
ALP staining and Alizarin Red staining of h‐FOBs co‐cultured with PDLCs
under wireless magnetoelectric output for 5,10, and 21 days. J) mRNA
expression levels of representative cytokine genes in macrophages. K)
mRNA expression levels of classic osteogenic gene markers in h‐FOBs for
5 and 10 days. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Sample size (n = 3).
To explore the balance of bone deposition resorption with
magnetoelectric retention therapy, osteogenesis and osteoclasis was
evaluated similarly in vitro. Immunofluorescence of human osteoblasts
(hFOBs) showed that RUNX2 (Figure [156]5E,I) and COL1A1 (Figure
[157]S14, Supporting Information) exhibited the highest expression
intensity with 20 µA‐FMOR therapy. Alkaline phosphatase (ALP) staining
at 5 and 10 days was conducted to demonstrate the osteogenesis process
of hFOBs in a cell co‐culture system. Additionally, Alizarin Red
staining was employed to label the extracellular calcified nodules
after 21 days. The results indicated that the 20 µA‐FMOR therapy
exhibited the highest osteogenic and calcification efficiencies
(Figure [158]5F). Moreover, the expression of osteogenic‐related marker
genes RUNX2, OPN, COL1A1 and OCN were analyzed by RT‐qPCR after 5‐ and
10‐day culture, suggesting 20 µA‐FMOR therapy provided the highest
osteogenic efficiency (Figure [159]5K). On the other hand,
immunofluorescence staining of human osteoclasts showed that 20 µA‐FMOR
therapy significantly downregulated CTSK expression (Figure [160]6A,C).
TRAP staining and quantitative measurement confirmed that 20 µA‐FMOR
therapy significantly downregulated TRAP activity by more than half
compared to the control group. (Figure [161]6B,D).
Figure 6.
Figure 6
[162]Open in a new tab
Magnetoelectric output inhibits osteoclastic behavior and promotes
periodontal remodeling in vitro. A,C) Representative immunofluorescence
images and quantitative analysis of CTSK (red) and cell nuclei (DAPI,
blue) in OCs co‐cultured with PDLCs under wireless magnetoelectric
output for 10 days. B,D) Representative images of TRAP staining and the
TRAP activity of OCs co‐cultured with PDLCs under wireless
magnetoelectric output for 10 days. E,F,G,H) Representative
immunofluorescence images and quantitative analysis of MMP‐1 (green),
POSTN (green) and cell nuclei (DAPI, blue) of PDLCs under wireless
magnetoelectric output for 10 days. I) mRNA expression levels of
representative genes associated with fiber and extracellular matrix
disruption in PDLCs for 10 days. J) mRNA expression levels of
representative genes associated with fiber and extracellular matrix
reconstruction in PDLCs for 10 days. *p < 0.05, **p < 0.01, ***p <
0.001 and ****p < 0.0001. Sample size (n = 3).
Last, the effect of magnetoelectric retention therapy on periodontal
fibre metabolism was assessed. Immunofluorescence staining of PDLCs
showed that MMP‐1 associated with collagen fiber resorption was
significantly down‐regulated with 20 µA‐FMOR therapy
(Figure [163]6E,G). Whereas the periostin (POSTN), which is associated
with PDLCs proliferation as well as periodontal fiber secretion and
reconstruction,^[ [164]^58 ^] was significantly up‐regulated
(Figure [165]6F,H). Gene expression levels related to extracellular
matrix and collagen metabolism were examined by RT‐qPCR. Results showed
that MMP‐1 and MMP‐2 were significantly down‐regulated
(Figure [166]6I), while POSTN and ACTA2 was increased in PDLCs with
20 µA‐FMOR therapy (Figure [167]6J), suggesting deposition and
remodeling of extracellular matrix and fibrillar collagen.
2.6. Magnetoelectric Output Remodels the Periodontal Microenvironment via
Modulating the Immune Signaling and Metabolic Pattern of PDLCs
To further elucidate the response of the periodontal microenvironment
to the magnetoelectric retention therapy from transcriptomic
perspective, we performed RNA sequencing on PDLCs after 7‐day
magnetoelectric retention therapy in vitro. Principal component
analysis (PCA) demonstrated a notable divergence in global gene
expression in 20 µA‐FMOR group (Figure [168]7A). Differential
Expression Gene (DEG) counts and Wayne plots among groups showed that,
compared with the other two group, the number of DEGs up‐regulated in
the 20 µA‐FMOR group was 1434 and 1107, respectively, of which 910 DEGs
were overlapped (Figure [169]7B,C). These results provided genetic
validation of the previous results, indicating that a significant
effect of magnetoelectric retention therapy need the magnetoelectric
output to reached 20 µA.
Figure 7.
Figure 7
[170]Open in a new tab
Magnetoelectric output modulates the immune signaling and metabolic
patterns of PDLCs. A) Principal Component Analysis (PCA) of the RNA
sequencing profile of PDLCs after 7 days of culture. B) Differentially
expressed genes from RNA‐seq analysis of PDLCs after 7 days of culture.
C) The heatmap of differentially expressed genes from RNA sequencing.
D) Gene Ontology (GO) enrichment analysis of the upregulated genes in
PDLCs cultured after 7 days. E) Kyoto Encyclopedia of Genes and Genomes
(KEGG) analysis of upregulated genes in PDLCs cultured after 7 days. F)
Representative immunofluorescence images of Flou‐4 (green) signals in
PDLCs cultured for 7 days. G,H) Western blot images and quantitative
analysis of the phosphorylation levels of PI3K and AKT in PDLCs in
different groups. I,J) Flow cytometry and quantitative analysis of the
cell surface marker CD206 in macrophages co‐cultured with PDLCs in
different groups. K) Representative images of TRAP staining of OCs
co‐cultured with PDLCs in different groups for 10 days. L)
Representative images of ALP staining of h‐FOBs co‐cultured with PDLCs
in different groups for 5 days. M) Representative immunofluorescence
images POSTN (green) and cell nuclei (DAPI, blue) of PDLCs in different
groups for 10 days. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p <
0.0001. Sample size (n = 3).
Further, GO enrichment analysis revealed that the up‐regulated DEGs are
mainly related to macrophage activation, type 2 immune response,
epithelial cell proliferation, extracellular matrix metabolism, and
skeletal system development in 20 µA‐FMOR group (Figure [171]7D).
Furthermore, KEGG pathway enrichment analysis indicated that the
upregulation of PI3K‐Akt signaling pathway, Calcium signaling pathway,
cAMP signaling pathway, MAPK signaling pathway and etc. was correlated
with 20 µA‐FMOR therapy (Figure [172]7E). Previous studies have
investigated that activated PI3K‐Akt signaling pathway demonstrates
superiority in inducing PDLCs to immune repair, collagen formation and
promote alveolar bone mineralization by up‐regulating TGFβ and M2‐type
macrophage polarization,^[ [173]^59 , [174]^60 ^]which potentially
linked to orthodontic relapse inhibition.^[ [175]^61 ^] Meanwhile,
Established evidence positions calcium ions (Ca^2⁺) as critical
secondary messengers in intracellular signaling, with growing
literature emphasizing electro‐activated Ca^2⁺ influx in triggering
downstream pathways.^[ [176]^62 ^]
Based on this, immunohistochemical staining of key marker proteins
(p‐PI3K, PI3K, p‐AKT, AKT) in PI3K‐AKT signaling pathway was performed
in rat periodontal specimens (Figure [177]S15, Supporting Information).
The results of the phosphorylation ratios preliminarily confirmed the
effect of magnetoelectric retention therapy on the activation of
PI3K‐AKT signaling pathway in vivo. Subsequently, in vitro rescue
experiments were designed to further investigate the signaling between
the pathways. To validate the roles of calcium signaling and PI3K‐AKT
pathways, we introduced gadolinium chloride (GdCl₃) to inhibit calcium
channels in FMOR group (FMOR+GdCl₃ group) and LY294002 (FMOR+LY group)
to suppress PI3K‐AKT activation. Fluo‐4 AM fluorescence imaging
revealed significantly elevated intracellular Ca^2⁺ levels in FMOR
group versus PR control, while FMOR+GdCl₃ group showed no significant
difference from PR levels (Figure [178]7F). This result confirmed that
magnetoelectric retention therapy activated calcium signaling to
enhance intracellular Ca^2⁺ concentration. Western blot analysis
further demonstrated that calcium channel activation initiates
downstream PI3K/AKT signaling. Phosphorylation levels of PI3K/AKT
pathway proteins were markedly higher in FMOR group than PR control,
consistent with the RNA sequencing data (Figure [179]7G,H). Notley,
both GdCl₃ and LY294002 reduced phosphorylation to PR baseline levels.
Subsequently, in vitro functional assays including macrophage
polarization (flow cytometry of CD206 positive macrophages in
Figure [180]7I,J), osteogenic/osteoclastic activity (ALP staining of
h‐FOBs in Figure [181]7L and TRAP staining of osteoclasts in
Figure [182]7K), and collagen formation (immunofluorescence images of
POSTN of PDLCs in Figure [183]7M) revealed that GdCl₃ treatment
abolished FMOR therapeutic effects, comparable to that of the FMOR+LY
group.
Collectively, these results suggested a coherent mechanistic framework
wherein magnetoelectric retention therapy may involve Ca^2⁺ influx
through PDLC calcium channels. In this model, Ca^2⁺ functions as a
secondary messenger, potentially activating PI3K/AKT signaling during
magnetoelectric retention therapy. This cascade could initiate
paracrine signaling networks that contribute to periodontal
reconstruction and relapse inhibition. Notably, considering that ion
signaling is also associated with mechanotransduction channels, such as
Piezo1, the above mechanisms require additional validation beyond KEGG
analysis.
2.7. Application of FMOR Greatly Inhibits Post‐Orthodontic Relapses in a
Rabbit Model
Considering the constrained jaw dimensions, a 3‐week OTM phase, and an
applied force of 40 grams in the rat model, it is slightly less
appropriate for assessing magnetoelectric retention therapy under
clinically relevant orthodontic forces (i.e., ≥50 grams) or extended
OTM distances. In contrast, the rabbit model exhibited a higher
tolerance for orthodontic forces (100 g) and a greater OTM distance
(six times the distance observed in the rat model).^[ [184]^63 ^] This
greater capacity enables a better simulation of clinical situations of
periodontal remodeling subsequent to tooth movement (Figure [185]8A).
As described previously, the mandibular first molars of the rabbit
entered the relapse phase after rapidly moved by a constant orthodontic
force of 100 g.^[ [186]^64 ^] During the relapse phase, rabbits were
treated with magnetoelectric retention therapy for 12 h
(Figure [187]8B). After 10 and 21 days of relapse, micro‐CT imaging
demonstrated that the 20 µA‐FMOR therapy exhibited the smallest relapse
distance (Figure [188]8C,E), while effectively reducing the 100%
relapse rate observed in the control and PR groups to ≈40% for 21 days
(Figure [189]8F). Representative tomographic reconstruction images and
bone volume fraction results showed that 20 µA‐FMOR therapy was
effective in promoting bone repair and increasing alveolar bone density
in relapse phase (Figure [190]8D,G). The number of TRAP‐positive
multinucleated cells reduced significantly in the 20 µA‐FMOR therapy
group, indicating an effective inhibition in osteoclast differentiation
during relapse phase (Figure [191]8H,J). Furthermore,
immunofluorescence staining of alveolar bone showed a decrease of
CD86^+ and an increase of CD206^+ macrophages with 20 µA‐FMOR therapy
(Figure [192]8I,K,L), confirming the magnetoelectric retention therapy
remodeled periodontal microenvironment by promoting M2‐type macrophage
polarization in relapse phase.
Figure 8.
Figure 8
[193]Open in a new tab
FMOR magnetoelectric retention therapy greatly improved orthodontic
retention efficiency in vivo. A) Schematic illustration of the
orthodontic and relapse rabbit model. Created by BioRender.com. B)
Magnetoelectric retention therapy for rabbits in vivo: i) Moving the
first molar using springs with a force of 100 g in the OTM phase. ii)
Wearing personalized FMORs for each rabbit. iii) Initiating
magnetoelectric retention therapy with the wearable magnetoelectric
transmitter. C) Representative micro‐CT images of rabbit dentition
after 10 and 21 days of orthodontic relapse. The width of the red area
indicates the distance between the first and second molars. Scale bar =
1 mm D) Micro–computed tomography images of alveolar bone between M1
and M2 in rabbits. E,F) Quantitative analysis of the distance between
first and second molars and the relapse rate. Relapse rate = relapse
distance / relapse distance at 0 d. G) Quantitative analysis of percent
bone volume (BV/TV) of alveolar bone between M1 and M2 in rabbits. H,J)
TRAP staining and quantitative analysis of the number of osteoclasts on
histological sections of alveolar bone resorption regions. Black
triangles indicate TRAP‐positive cells. I,K,L) Representative
immunofluorescence images and quantitative analysis of CD206 (green)
and CD86 (red) in histological sections of alveolar bone resorption
regions. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Sample
size (n = 3).
Therefore, compared with the control and PR groups in the rabbit model,
we further identified the efficacy and clinical prospects of FMOR. Due
to the lack of physiological active regulation, the retention effect of
the clinical passive retainer was limited for its restricted wearing
time per day. However, FMOR achieves a significant retention efficiency
for its periodontal immune environment reversal, osteogenesis and
osteoclastic balance and periodontal fiber reconstruction. In
conclusion, the development of FMOR with active regulation is of
clinical significance and provides insights for optimizing orthodontic
relapse treatment.
3. Conclusion
In conclusion, the magnetoelectric retention therapy has been
successfully developed for efficient periodontal restoration and
reconstruction after orthodontics. The therapy consists of an
additively fabricated flexible magnetoelectric orthodontic retainer
(FMOR) configured with a receiving liquid metal (LM) coil and a
wearable magnetoelectric transmitter configured with a transmitting LM
coil. Following orthodontic treatment, the patient wears the FMOR,
which generates a self‐powered magnetoelectric output through wireless
remote modulation of the wearable magnetoelectric transmitter. Thus,
magnetoelectric cues reversed pressure‐induced aseptic inflammation and
promoted reconstruction of the periodontal environment, which triggers
M2 macrophage polarization, rebalances osteoclastic and osteoblastic
activity and facilitates collagen deposition. The mechanistic
exploration revealed that 20 µA‐FMOR therapy significantly upregulated
the genes related to immune response, extracellular matrix
organization, and osteogenesis regulation in PDLC. Our study provides a
high‐performance strategy for optimizing orthodontic retention therapy,
highlighting the promise of additively fabricated flexible
magnetoelectric devices for clinical tissue repair and reconstruction.
4. Experimental Section
Fabrication of the FMOR system
1) Preparation and encapsulation of FMOR
The maxilla of the rats and mandible of the rabbits were recorded into
digital models using a 3D scanner (Creality 3D CR‐Scan Otter), which
preserves various detailed features of teeth. The results of the
scanning were designed into braces that fit the teeth using model
design software, and the corresponding detachable negative and positive
molds were designed. The mold parts were then printed and shaped using
a commercial light‐cured printing device (Formlabs Form3+). In order to
allow FMOR to be smoothly peeled off from the molds, the residual
photosensitive resin on the surface of the molds was removed here using
a plasma cleaner (PD150).
The base polymer of polydimethylsiloxane (PDMS) was mixed with the
cross‐linking agent in a mass ratio of 10:1, stirred thoroughly and
vacuumed to make the precursor liquid. The prepared precursor liquid
was injected into the mold in layers, and a prefabricated copper coil
and its output electrode with a layered spatial structure were placed.
The FMOR was crosslinked at a temperature of 60 °C for 2 h. FMOR was
prepared by removing the PDMS from the mold after curing.
2) Preparation and encapsulation of the wearable magnetoelectric
transmitter
Head structures of rats and rabbits were recorded as digital models
using a 3D scanner. Model design software was used to create a model of
a headgear universally suitable for rats or rabbits with a circular
slot in the cheek area directly opposite the teeth. The headgear was
printed using a light‐curing printing device. Electronic components
such as the control chip, voltage regulator module, rectifier module,
and communication module were integrated into the main board and placed
in the circular slot together with the emitting copper coil. The
wearable magnetoelectric transmitting coil is powered by a dry battery
and outputs magnetoelectric signals to the FMOR.
General Characterization of the FMOR System
1) Structural characterization
To evaluate the mechanical behavior of the PDMS, we prepared the
precursor solution using a consistent mixing ratio. Two types of 1 cm^3
PDMS solid blocks — one with embedded coils and one without — were
fabricated using a mold. Stress‐strain data were obtained under
uniaxial compression with a displacement of 1 mm. Young's modulus was
calculated using the formula:
[MATH: E=σε=−F
L0AL−L0 :MATH]
(3)
The FMOR was placed 5 cm away from the emitting coil and the emitting
coil was kept at an output current of 50 µA. An infrared camera was
used to measure the temperature change of the FMOR.
2) Electrical characterization and signal acquisition
In order not to interfere with the normal activities of the
experimental subjects, the orthodontic retention system was designed as
two parts, the transmitting end and the receiving end. The receiving
end is fixed to the rat's teeth by FMOR, and two electrode sheets are
affixed to the area to be electrically stimulated. The transmitting end
converts the direct current from the dry battery into an alternating
current that changes periodically, and the alternating magnetic field
formed supplies energy to the receiving end through electromagnetic
induction. The electrical stimulation waveform of the electrodes at the
receiving end can be changed by modulating the waveform characteristics
at the transmitting end.
In order to investigate the ability of the retainer to cope with
various states during use, the experiment was conducted by modulating
the relevant variables to simulate different scenarios. In actual use,
the coils at the transmitter and receiver ends do not guarantee an
absolute positional relationship. In the experiment, the horizontal
height distance, axial central distance, relative angle θ and input
voltage of the two coils are adjusted to investigate the transformation
of the stimulus current at the output end under different states.
Animals
All animals were purchased from Beijing Vital River Laboratory Animal
Technology Company. All animal experiments were authorized by the
Animal Ethics Committee of Tongji Medical College (Wuhan, China) and
approved by the Institutional Animal Care and Use Committee of Tongji
Medical College ([2022] IACUC Number: 3977).
Male 6‐week‐old Sprague‐Dawley (SD) rats were randomly divided into
five groups. The rats were housed under specific pathogen free (SPF)
conditions with standard rodent chow and provided free access to water
during a 12‐h light‐dark cycle. After 2 weeks of acclimatization, the
rats were anaesthetized with an intraperitoneal injection of sodium
pentobarbital (40 mg kg^−1 body weight) and a 0.2 mm nickel‐titanium
helical spring, adjusted to a tensile force of ≈40 g, was attached
between the maxillary first molar and the maxillary incisor. In order
to minimize the effects of mesial tooth displacement, the orthodontic
appliance was checked daily and also reactivated weekly. After 3 weeks
of loading and the M1 reaches an OTM distance of 500 µm, the
orthodontic appliance was removed and the rats entered the relapse
phase. The rats were treated with magnetoelectric retention therapy
during the relapse phase. Specifically, rats were fitted with FMOR for
a period of 12 h each day. The wearable magnetoelectric transmitter
device was adjusted to set varying magnetoelectric output gradients
(10, 20, and 30 µA). After 5 and 10 days of relapse, Micro‐CT 3D
remodeling and sagittal images of rat maxillary molars was performed
and the distance between M1 and M2 was measured.
Similarly, male 2.5 kg New Zealand rabbits were randomized into 3
groups. After 2 weeks of adaptation to the environment, the rabbits
were anaesthetized with an intravenous injection of 2% sodium
pentobarbital saline solution (1 mL kg^−1 body weight) at the ear
margin, and a 0.2‐mm nickel‐titanium coil spring, adjusted to a tension
of ≈100 g, was attached between the mandibular first molar and the
maxillary incisor. Meanwhile, the orthodontic appliance was checked
daily while it was reactivated weekly. After loading for 4 weeks and
the M1 reaches an OTM distance of 3 mm, the orthodontic device was
removed and the rabbits entered the relapse phase. The rabbits were
treated with magnetoelectric retention therapy during the relapse
phase. Specifically, the rabbits were treated with magnetoelectric
retention therapy by wearing FMOR for 12 h per day. The wearable
magnetoelectric transmitters were adjusted to set magnetoelectric
output intensities of 20 µA. FMOR group was compared with PR group
(without initiating magnetoelectric retention treatment) and Control
group (without wearing FMOR). After 10 and 21 days of relapse, Micro‐CT
3D remodeling was conducted and specimens were harvested for
histological analysis.
Micro‐CT Scanning Evaluation
Animal intraoral measurements were obtained through standardized
micro‐CT scanning (SkyScan 1176, Bruker, Belgium) followed by 3D
reconstruction and quantitative analysis. Specifically, animals were
scanned at 70 kV, 350 µA, 180° rotation, 0.3° rotation step and 9 µm
resolution after survival anesthesia (OTM phase) or terminal anesthesia
(relapse phase). Raw datasets were reconstructed using NRecon
(v1.6.10.4, Bruker) then analyzed in DataViewer (v1.5.2.4, Bruker) and
CTAn (v1.15.4.0, Bruker) with triple measurements per parameter.
With reference to other relevant studies,^[ [194]^65 ^] the OTM
distance was defined as the distance between the nearest contact points
of the first molar (M1) and the second molar (M2) at the end of the OTM
phase. Relapse distance was the OTM distance minus the distance between
the nearest contact points of M1 and M2 after relapse. The relapse rate
is the ratio of the relapse distance to the OTM distance. Each result
was measured three times.
Hematoxylin Eosin Staining
Maxillary specimens were decalcified with 10%
ethylenediaminetetraacetic acid (EDTA) decalcification solution after
micro‐CT scanning. When the bone tissue was softened, the samples were
observed morphologically following a routine Hematoxylin Eosin (H&E)
staining procedure. Briefly, the samples were dehydrated, embedded in
paraffin and cut into 4 µm thick sections. After deparaffinization and
rehydration, the sections were stained with hematoxylin and
differentiated with acidic alcohol. Subsequently, the specimen sections
were stained with eosin solution, then dehydrated and cleared.
Histological images were taken with an optical microscope.
TRAP Staining
The paraffin sections were prepared as described above. TRAP staining
was performed according to the instructions of the TRAP kit (Solarbio,
China) to label osteoclasts. Briefly, TRAP incubation solution was
prepared by mixing AS‐BI buffer, GBC solution and TRAP buffer in the
ratio of 10:1:90. After deparaffinization and rehydration, the sections
were rinsed with TRAP Incubation Solution for 45–60 min at 37 °C and
then washed with ultrapure water. Sections were then restained with
hematoxylin solution for 5 min. Relative images were obtained using an
optical microscope.
Immunofluorescence for Tissue Staining
Paraffin‐embedded maxillary and mandibular tissue sections were blocked
with 5% bovine serum albumin (BSA) for 1 h to avoid non‐specific
staining. Subsequently, the sections were incubated with primary
antibodies CD206, CD86, RUNX2, MMP‐1 and CTSK at 4 °C overnight in a
lucifugal chamber. Primary antibody dilutions were 1:200. The slides
were then washed with PBS for three times, followed by incubation with
secondary antibody (1:200) for 1 h in the dark. Cell nuclei were
stained with 4′,6‐Diamidino‐2‐phenylin‐dole (DAPI, Sigma, USA) for 5
min. The fluorescent images were captured with a confocal microscope
(Nikon A1‐Si, Japan). The antibodies used in immunofluorescence are
described in Table [195]S2, Supporting Information.
Cell Culture
Human periodontal cells (PDLCs) were obtained from orthodontically
extracted premolar teeth. Briefly, the periodontal ligaments of
premolar teeth were collected with a sterile blade, followed by
digestion of the periodontal ligaments with collagenase I (C8140,
Solarbio) at 37 °C for 1 h. PDLCs were cultured in T25 flasks with
α‐MEM medium containing 10% fetal bovine serum (FBS, Gibco, USA) and 1%
penicillin‐streptomycin (HyClone, USA), which was incubated in a
humidified incubator containing 5% CO[2] at 37 °C. PDLCs were passaged
every 5 days and were used in this research were in passages 4–6.
The human monocytic leukemia cell line (THP‐1) was obtained from the
American Type Culture Collection (ATCC). THP‐1 cells were maintained in
RPMI 1640 medium (HyClone, USA) supplemented with 10% FBS and 1%
penicillin–streptomycin in an incubator at 37 °C with 5% CO[2]. THP‐1
cells were differentiated in different ways to obtain macrophages or
osteoclasts. With 100 ng mL^−1 phorbol 12‐myristate 13‐acetate (PMA,
P8139‐1MG, Sigma, USA) for 48 h, THP‐1 cells were differentiated into
macrophages to probe the direction of macrophage polarization. To probe
osteoclastic behavior, THP‐1 cells were differentiated into osteoblasts
with 20 ng mL^−1 receptor activator of nuclear factor κ‑B ligand
(RANKL, Novoprotein, China), 20 ng mL^−1 macrophage colony‑stimulating
factor (M‐CSF, Novoprotein, China) and 100 ng mL^−1 PMA for at least 10
days.
The human fetal osteoblast cell line (hFOB 1.19) was obtained from
ATCC. The cells were cultured in DMEM/F12 medium (HyClone, USA)
supplementing 0.3 mg mL^−1 G418 (Biosharp, China), 10% FBS and 1%
penicillin‐streptomycin. To evaluate osteogenic behavior, hFOB cells
were activated osteogenic expression with 10 mmol L^−1
β‐glycerophosphate (Sigma, USA), 10 nmol L^−1 dexamethasone and
50 µg mL^−1 ascorbic acid for 7 days.
Cell Co‐Culture System Simulating the Periodontal Microenvironment In Vitro
PDLCs were plated at a density of 1 × 10^5 cells per well in Transwell
chambers of 6‐well plates. When the cells were cultured to a density of
70% – 80%, they were continuously compressed according to the uniform
compression method described previously.^[ [196]^50 ^] Briefly, the
plates were placed in the Bionic Pressure Cell Culturator (NK‐P40,
Naturethink, China) and the instrument parameters were adjusted to
achieve a compressive force of 2 g cm^−2 for 24 h in 5% CO[2] at 37 °C.
Next, the PDLC in the Transwell chambers were magnetoelectrically
stimulated using a receiver coil, and the stimulation intensity was
controlled by a transmitter coil. Finally, Transwell chambers were
transferred to culture plates seeded with macrophages, osteoblasts or
osteoclasts to mimic the state of the periodontal microenvironment
during the relapse phase after orthodontic treatment.
Flow Cytometry Analysis
Expression of the M2 macrophage surface marker CD206 was detected using
flow cytometry. After 48 h of culture, cells were scraped, fixed with
4% (w/v) paraformaldehyde, blocked with 1% (w/v) BSA/PBS, and then
incubated with FITC Anti‐Human CD206/MMR Antibody (E‐AB‐F1161C,
Elabscience, China). Cells were analysed using a BD FACS Canto II flow
cytometer.
Alkaline Phosphatase and Alizarin Red S staining
Alkaline phosphatase assay was performed using the BCIP/NBT Alkaline
Phosphatase Staining Kit (Beyotime, China). After 5 and 10 days of
osteogenic activation of hFOB in an in vitro simulated periodontal
microenvironmental cell co‐culture system, the medium was discarded and
the cells were rinsed with PBS, and BCIP/NBT staining solution was
added to each well (PBS, containing BCIP (3.3 µL mL^−1) and NBT
(6.5 µmol mL^−1). Images were taken with a microscope after 30 min of
incubation at room temperature.
Alizarin Red S Staining Kit (Beyotime, China) for displaying osteoblast
mineralized nodules in vitro. As described previously, after 21 days of
hFOB osteogenic activation, the culture medium was removed and fixed
with 4% paraformaldehyde. After 3 washes using PBS, Alizarin Red S
staining solution was added and stained for 30 min at room temperature.
After a final thorough washing using distilled water, images were
captured under a light microscope.
Quantitative Real‐Time PCR
Total RNA was extracted using TRIZOL (TaKaRa, Japan) according to the
manufacturer's instructions. Complementary DNA (cDNA) was reverse
transcribed from the RNA templates using HiScript III RT SuperMix for
qPCR (+gDNA wiper) (Vazyme, China). qRT‐PCR was performed using ChamQ
Universal SYBR qPCR Master Mix (Vazyme, China) on Step One Plus
real‐time PCR systems (Applied Biosystems, Thermo Fisher, USA).
Quantification of target gene expressions was analyzed by the 2−ΔΔCt
method, normalized to the expression of glyceraldehyde 3‐phosphate
dehydrogenase (GAPDH) gene, and was presented as mean ± SD of
replicates. The primer sequences used in qRT‐PCR are described in Table
[197]S3, Supporting Information.
Immunofluorescence for Cell Staining
Cells were fixed with 4% paraformaldehyde for 15 min and then
permeabilized by 0.5% Triton X‐100 for 15 min. Subsequently, cells were
blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature.
The cells were incubated overnight at 4 °C in a humid chamber using
primary antibody (1:200). Cells were washed three times the following
day and incubated for 1 h at room temperature with secondary antibody.
Cytoskeleton was stained with TRITC Phalloidin (YEASEN, China) for 30
min and cell nuclei were counterstained with DAPI for 5 min. Images
were captured using a laser‐scanning confocal microscope (Nikon A1‐Si,
Japan). The primary antibodies used in immunofluorescence are described
in Table [198]S2, Supporting Information.
RNA Sequencing
PDLCs were treated with different intensities of magnetoelectric
retention therapy for 7 days in the cell co‐culture system in vitro.
Cell lysates were then collected using TRIZOL (TaKaRa, Japan) according
to the manufacturer's instructions. Three independent replicate samples
from three groups were sent to Beijing Novogene Co, Ltd. for global
transcriptome analysis. Before transcriptome sequencing, quality
control tests were performed on the samples, including 1) RNA
concentration greater than 50 ng uL^−1 and total amount greater than
1.5 µg; 2) 260 nm/280 nm absorbance ratio close to the range of
1.8–2.1, and the samples were free of macromolecular contamination; and
3) the samples were kept intact and free of degradation.
In the differential expression analysis, DESeq2 was applied to identify
differentially expressed genes. Differentially expressed genes (DEGs)
were chosen as p‐value < 0.05 and |FoldChange| ≥ 1. For Gene Ontology
(GO) enrichment analysis of DEGs, clusterProfiler R package was
implemented. GO terms with corrected p‐value <0.05 were considered
significantly enriched by DEGs. The Kyoto Encyclopedia of Genes and
Genomes (KEGG) enrichment analysis of differentially‐expressed genes
was also performed by clusterProfiler R package.
Differentially‐expressed genes were significantly enriched for KEGG
pathways with a p‐value less than 0.05.
Statistical Analysis
All numerical data were presented as mean ± standard deviation (SD) and
statistically analyzed using GraphPad Prism 8.0 (GraphPad Software
Inc., USA) with three or more replicate values. Significant differences
were determined using Student's t‐test for comparison of two groups and
one‐way ANOVA with post‐hoc Tukey's test for multiple comparisons. The
statistical significance was declared when *p < 0.05, **p < 0.01, ***p
< 0.001 and ****p < 0.0001.
Ethics Approval and Consent to Participate
All animal experiments were performed in accordance with the Guidelines
for Care and Use of Laboratory Animals of Tongji medical college,
Huazhong University of Science and Technology and approved by the
Animal Ethics Committee of Tongji medical college, Wuhan ([2022] IACUC
Number: 3977).
Conflict of Interest
The authors declare no conflict of interest.
Author Contributions
H.L., J.S., and P.C. contributed equally to this work. H.L. contributed
to writing – review & editing, writing – original draft, visualization,
validation, methodology, investigation, formal analysis, data curation,
and conceptualization; J.S. to writing – original draft, validation,
investigation, funding acquisition, formal analysis, and
conceptualization; P.C. to writing – original draft, investigation,
formal analysis, and data curation; L.H. to investigation,
visualization, and software; X.Z. to visualization and methodology;
S.G. to investigation and software; Q.T. to methodology and software;
X.H. to data curation and investigation; L.C. to methodology,
validation, and investigation; and B.S. to project administration,
resources, supervision, conceptualization, and funding acquisition.
Supporting information
Supporting Information
[199]ADVS-12-e05020-s002.docx^ (11.6MB, docx)
Supplemental Video 1
[200]Download video file^ (14.1MB, mp4)
Acknowledgements