Introduction
Tendon injury is a common musculoskeletal disorder
that frequently causes pain, functional limitation and long-term
impairment in quality of life (1,2).
Because tendon tissue is relatively hypocellular and hypovascular,
its intrinsic regenerative capacity is limited, and healing often
culminates in fibrosis, inferior mechanical performance and an
increased risk of re-injury (3,4).
Although current therapeutic strategies, including surgical repair,
biomaterial-based approaches and cell-based interventions, may
improve structural restoration, complete functional regeneration
remains difficult to achieve (5-7).
Accordingly, there is a clear need to identify biological
strategies that can more effectively promote tendon repair.
The mechanical microenvironment is now recognized as
a major determinant of stem cell behavior and lineage commitment
(8,9). Mechanical cues regulate proliferation,
migration and differentiation through mechanotransduction pathways
involving integrins, cytoskeletal remodeling, Rho GTPases, and
transcriptional coactivators such as Yes-associated protein (YAP)
and transcriptional coactivator with PDZ-binding motif (TAZ)
(10-14).
In tendon biology, scleraxis (SCX) is a canonical transcription
factor required for tendon development and maintenance of the
tenogenic program (15,16). The YAP/TAZ-SCX axis therefore
represents a plausible molecular interface through which external
mechanical stimulation may influence tenogenesis-associated cell
fate decisions.
Low-intensity pulsed ultrasound (LIPUS) is a
noninvasive biophysical intervention with documented regenerative
and anti-inflammatory effects (17,18).
By delivering acoustic mechanical energy to cells and tissues,
LIPUS can modulate mechanosensitive signaling and alter cellular
behavior (19,20). Previous studies have suggested that
LIPUS may facilitate tendon and tendon-bone healing (21,22);
however, the molecular events linking LIPUS-derived mechanical
stimulation to TSC fate remain insufficiently defined, particularly
with respect to YAP/TAZ- and SCX-associated signaling.
In the present study, transcriptomic analysis was
combined with in vitro experiments to examine whether LIPUS
activates mechanotransduction-related pathways in rat TSCs and
thereby enhances YAP/TAZ- and SCX-associated responses. It was
hypothesized that LIPUS would induce a tenogenesis-associated
molecular program characterized by increased YAP/TAZ signaling,
upregulation of SCX, and enhanced expression of matrix-related
markers.
Materials and methods
Primary cell source
Two specific pathogen-free male Sprague-Dawley rats,
aged 3-4 weeks and weighing 80-100 g, were used exclusively for
primary tendon stem cell (TSC) isolation. The animals were
maintained under standard laboratory conditions, including a
controlled temperature of 20-26˚C, relative humidity of 40-70%, and
a 12/12-h light/dark cycle. Food and sterile drinking water were
provided ad libitum throughout the experimental period. All
subsequent experiments were performed using expanded in
vitro cell passages derived from these primary cultures and
were repeated independently. The animals were obtained from the
Experimental Animal Center of Shanxi Medical University. The
present study was approved by the Animal Welfare Ethics Committee
of the Second Hospital of Shanxi Medical University (approval no.
DW20230008; Taiyuan, China), and all procedures were conducted in
accordance with institutional and national guidelines for animal
care and use. At the end of tissue collection, rats were euthanized
by intraperitoneal injection of an overdose of sodium pentobarbital
(200 mg/kg). Death was confirmed by the absence of spontaneous
respiration, heartbeat, and pedal withdrawal reflex before tendon
harvest.
Main reagents and instruments
The major reagents used in the present study were
PBS (cat. no. P7209; LABLEAD), penicillin-streptomycin (cat. no.
15140122; Thermo Fisher Scientific, Inc.), serum-free
cryopreservation solution (cat. no. 12648010; Thermo Fisher
Scientific, Inc.), collagenase type I (cat. no. 17100017; Thermo
Fisher Scientific, Inc.), RIPA lysis buffer (cat. no. 89901; Thermo
Fisher Scientific, Inc.), protease inhibitor cocktail (cat. no.
78429; Thermo Fisher Scientific, Inc.), TRIzol reagent (cat. no.
15596026CN; Thermo Fisher Scientific, Inc.), reverse transcription
kit (cat. no. 4366596; Thermo Fisher Scientific, Inc.), SYBR Green
qPCR mix (cat. no. K0223; Thermo Fisher Scientific, Inc.), BCA
protein assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.),
0.25% trypsin-EDTA (cat. no. 25200056; Gibco; Thermo Fisher
Scientific, Inc.), α-MEM (cat. no. 12571063; Gibco; Thermo Fisher
Scientific, Inc.), fetal bovine serum (cat. no. 10099141C; Thermo
Fisher Scientific, Inc.), PVDF membranes (cat. no. IPVH00010) and
Verteporfin (VP; cat. no. HY-B0146; CL 318952; MedChemExpress).
Primary antibodies used for western blotting and
immunofluorescence were all purchased from Abcam, including
anti-YAP (cat. no. ab52771), anti-TAZ (cat. no. ab84927), anti-SCX
(cat. no. ab58655), anti-COL I (cat. no. ab34710) and anti-β-actin
(cat. no. ab8226). Antibodies used for flow cytometry were
purchased from Thermo Fisher Scientific, Inc., including CD90 (cat.
no. A15761), CD105 (cat. no. 12-1057-42), CD146 (cat. no.
11-1469-42), CD11b (cat. no. 12-0112-82), CD34 (cat. no.
48-0341-82) and CD45 (cat. no. MHCD4530). Corresponding horseradish
peroxidase-conjugated and fluorescent secondary antibodies (cat.
no. ab6721) were obtained from Abcam.
The principal instruments included a humidified
CO2 incubator (Thermo Fisher Scientific, Inc.), an
inverted fluorescence microscope (Leica Microsystems GmbH), a
real-time PCR system (Bio-Rad Laboratories, Inc.), a microplate
reader (BioTek; Agilent Technologies, Inc.), a biosafety cabinet
(Shanghai Lishen), a high-speed centrifuge (Eppendorf SE), a flow
cytometer (Beckman Coulter, Inc.), and a LIPUS device (Welld).
Isolation and culture of TSCs
Achilles tendons were harvested aseptically, minced
into fragments of ~1 mm, and digested with collagenase type I at
37˚C for 1 h with gentle agitation. After centrifugation at 300 x g
for 5 min at room temperature, the cell suspension was seeded into
culture dishes containing α-MEM supplemented with 10% fetal bovine
serum and 1% penicillin-streptomycin and maintained at 37˚C in a
humidified atmosphere of 5% CO2. During primary culture,
non-adherent cells were removed by repeated medium replacement,
which allowed enrichment of the adherent TSC population. The
adherent cells were expanded by routine passaging when cultures
reached 80-90% confluence. Cells were passaged at a ratio of 1:3,
and passage-3 TSCs were used for all subsequent experiments. All
experiments were independently repeated at least three times using
independently prepared cultures derived from the same primary TSC
isolation.
Flow cytometric identification of
TSCs
Passage-3 TSCs were dissociated with 0.25%
trypsin-EDTA, washed three times with PBS, and resuspended as
single-cell suspensions at a final concentration of
~1x106 cells/ml. Cells were incubated with
fluorophore-conjugated antibodies against CD90 (1:100), CD105
(1:100), CD146 (1:100), CD11b (1:100), CD34 (1:100) and CD45
(1:100) for 30 min at 4˚C in the dark, with matched isotype
controls included for each channel. After washing, the cells were
analyzed using a flow cytometer (FC500; Beckman Coulter, Inc.), and
the data were processed using the corresponding acquisition
software (FlowJo v10.8.1; FlowJo LLC). Cells positive for CD90,
CD105 and CD146 and negative for CD11b, CD34 and CD45 were
considered consistent with the TSC phenotype.
Experimental grouping and
treatment
Passage-3 TSCs were assigned to three groups:
Control, LIPUS and LIPUS + VP. Cells in the LIPUS group were
exposed to LIPUS at a frequency of 1.5 MHz, an intensity of 50
mW/cm2, a pulse repetition frequency of 1 kHz, a duty
cycle of 20%. Cells received two treatment sessions of 25 min each,
with a 24-h interval between sessions. Cells in the LIPUS + VP
group were pretreated with VP at a final concentration of 1 µmol/l
for 1 h before each LIPUS session. Control cells were cultured
under identical conditions without LIPUS or VP treatment. All
downstream assays were performed after the treatment period.
Although a VP-only group was not included, the
concentration of VP (1 µmol/l) and the 1-h pretreatment duration
were selected based on previously established protocols. The
Control group was maintained under identical culture conditions
without LIPUS or VP treatment.
Scratch-wound assay
TSCs were seeded in 6-well plates at
5x105 cells/well and cultured until a confluent
monolayer was formed. A linear scratch was created using a sterile
200-µl pipette tip, and detached cells were removed by washing with
PBS. Cells were then maintained in basal medium, and images of the
same wound area were acquired at 0, 24, and 48 h under an inverted
microscope. Scratch closure was quantified using ImageJ software
according to the following formula: wound closure (%)=[(initial
wound area-residual wound area)/initial wound area x100%].
Western blotting
TSCs were seeded in 6-well plates at
2x105 cells/well. After treatment, total cellular
protein was extracted using RIPA lysis buffer supplemented with
protease inhibitors. Protein concentration was determined using a
BCA assay, and equal amounts of protein (20 µg per lane) were
loaded per lane. Samples were separated on 10% SDS-PAGE gels and
transferred onto PVDF membranes. After blocking with 5% non-fat
milk for 2 h at room temperature, the membranes were incubated
overnight at 4˚C with primary antibodies against YAP (1:1,000), TAZ
(1:1,000), SCX (1:1,000), COL I (1:1,000) andβ-actin (1:5,000).
After washing with TBST (containing 0.1% Tween-20), membranes were
incubated with horseradish peroxidase-conjugated secondary
antibodies (1:5,000) for 1 h at room temperature. Immunoreactive
bands were visualized using enhanced chemiluminescence (cat. no.
P0018AS; Beyotime Institute of Biotechnology) and quantified using
ImageJ. Equal amounts of total protein were loaded in each lane,
and β-actin was used as the loading control for normalization. The
total β-actin signal showed no apparent difference among the
experimental groups under the present LIPUS treatment
conditions.
Reverse transcription-quantitative PCR
(RT-qPCR)
Total RNA was extracted from treated cells using
TRIzol reagent according to the manufacturer's protocol. The
thermocycling conditions were as follows: Initial denaturation at
95˚C for 30 sec, followed by 40 cycles of denaturation at 95˚C for
5 sec and annealing/extension at 60˚C for 30 sec; a melt-curve
analysis was performed to confirm amplification specificity.
Complementary DNA was synthesized using a reverse transcription
kit, and quantitative PCR was performed using SYBR Green chemistry
on a real-time PCR system. Reverse transcription was performed
using the kit according to the manufacturer's instructions. The
expression levels of target genes, including COL1A1 and TNMD were
normalized to GAPDH, and relative mRNA expression was calculated
using the 2-ΔΔCq method (23). Each assay was performed using at
least three independent biological replicates. The sequences of
primers used in the present study are as follows: TNMD forward,
5'-GTGAAGGTGGAGAAGACCCG-3' and reverse, 5'-TTGCCTCGACGGCAGTAAAT-3';
COL1A1 forward, 5'-GTTTGGAGAGAGCATGACCGA-3' and reverse,
5'-ACAAGCGTGCTGTAGGTGAA-3'; and GAPDH forward,
5'-TCACTGCCACCCAGAAGAC-3' and reverse,
5'-TGTAGGCCATGAGGTCCAC-3'.
Immunofluorescence staining
TSCs were seeded in 6-well plates at
2x105 cells/well and processed after treatment. Cells
were fixed with 4% paraformaldehyde for 30 min, washed with PBS,
permeabilized with 0.3% Triton X-100 for 5 min, and blocked with 5%
goat serum (cat. no. BL1097A; Labgic) for 30 min at room
temperature. The samples were then incubated overnight at 4˚C with
primary antibodies against SCX (1:1,000) and COL I (1:1,000),
followed by incubation with fluorescent secondary antibodies
(1:100) for 1 h at room temperature in the dark. Nuclei were
counterstained with DAPI (5 mg/ml; cat. no. C1002; Beyotime
Institute of Biotechnology). Fluorescence images were acquired
using identical exposure settings for all groups and quantified
using ImageJ software (version 2.14.0; National Institutes of
Health). For each sample, at least three representative fields were
analyzed.
Transcriptomic analysis
Transcriptomic data were obtained from Gene
Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) (GSE273466)
(24). Raw data underwent quality
control and normalization. Differentially expressed genes between
Control and LIPUS groups were identified using |log2
fold change|>1 and FDR <0.05. Gene Ontology (GO) enrichment
analysis was performed to identify biological processes (BP),
cellular components (CC) and molecular functions (MF) associated
with LIPUS exposure. Gene set enrichment analysis (GSEA) evaluated
YAP1- and SCX-associated signatures. Analyses were performed using
Python-based workflows, including statsmodels, bioinfokit and
gseapy.
Statistical analysis
Statistical analyses were performed using SPSS 21.0,
and graphs were generated using GraphPad Prism 9.0 (Dotmatics). All
experiments were repeated at least three times using independently
prepared cultures. Data distribution was assessed using the
Shapiro-Wilk test. For normally distributed data, comparisons
between two groups were performed Transcriptomic data were obtained
from GEO (GSE273466). Raw data underwent quality control and
normalization. Differentially expressed genes between Control and
LIPUS groups were identified using |log2 fold
change|>1 and FDR <0.05. GO enrichment analysis was performed
to identify BP, CC and MF associated with LIPUS exposure. GSEA
evaluated YAP1- and SCX-associated signatures. Analyses were
performed using Python-based workflows, including statsmodels
(https://www.statsmodels.org/),
bioinfokit (https://github.com/reneshbedre/bioinfokit) and gseapy
(https://gseapy.readthedocs.io/). For
normally distributed data, comparisons between two groups were
performed using the unpaired two-tailed Student's t-test. For data
that did not meet the assumptions of normality, the Mann-Whitney U
test was applied. Data are presented as the mean ± SD. Unless
otherwise stated, all experiments were repeated independently at
least three times. P<0.05 was considered to indicate a
statistically significant difference.
Results
LIPUS-induced transcriptomic
alterations are associated with mechanotransduction and tenogenic
differentiation
To investigate the molecular basis of the LIPUS
response, the transcriptomic dataset GSE273466 was first analyzed.
The heatmap and volcano plot revealed a distinct separation in gene
expression patterns between the Control and LIPUS groups,
indicating that LIPUS substantially altered the transcriptional
landscape of TSCs (Fig. 1A and
B). GO enrichment analysis further
showed that the differentially expressed genes were predominantly
associated with mechanotransduction-related processes, cytoskeletal
organization and structural adaptation (Fig. 1C).
Representative RNA-seq results further indicated
altered expression of selected mechano-sensing-related and
tendon-associated transcripts after LIPUS exposure (Fig. 1D and E). In parallel, GSEA demonstrated
significant enrichment of YAP1- and SCX-related signatures in the
LIPUS group (Fig. 1F and G). Taken together, these data support the
view that LIPUS induces a transcriptional program consistent with
enhanced mechanotransduction and activation of
tenogenesis-associated pathways.
Primary rat TSCs are successfully
identified, and LIPUS enhances their migratory capacity
Primary cells isolated from rat Achilles tendon
adhered readily to the culture surface and, after expansion,
displayed a uniform spindle-shaped, fibroblast-like morphology
characteristic of TSCs (Fig. 2A).
Flow cytometric analysis showed that the cultured cells were
strongly positive for CD90, CD105 and CD146. By contrast, only
small fractions of cells were positive for the
hematopoietic/inflammatory markers CD11b (5.65%) and CD45 (2.34%),
while CD34 expression was also low (Fig. 2B). Thus, the overwhelming majority
of cultured cells displayed the expected TSC-associated
immunophenotypic profile, supporting successful enrichment of a
stem/progenitor-like tendon-derived cell population with minimal
hematopoietic contamination.
 | Figure 2Characterization of primary rat TCSs,
and the pro-migratory effect of LIPUS. For the LIPUS-treated
samples in panels C-E, cells were exposed to LIPUS at 1.5 MHz and
50 mW/cm², with a pulse repetition frequency of 1 kHz and a 20%
duty cycle, for 25 min per session on two occasions separated by 24
h. (A) Representative microscopic images of primary rat TSCs at low
and high magnification, showing a spindle-shaped, fibroblast-like
morphology. (B) Flow cytometric analysis of surface markers in
cultured TSCs. Cells showed high expression of CD90, CD105 and
CD146, whereas only minor fractions were positive for CD11b (5.65%)
and CD45 (2.34%), supporting enrichment of a TSC-associated cell
population. (C) Scratch-wound assay showing that LIPUS accelerated
wound closure at 24 and 48 h compared with the Control group, with
corresponding quantitative analysis of closure percentage (D). (E)
Flow cytometric analysis of apoptosis using Annexin V-FITC/PI
staining after LIPUS treatment. Representative plots showing
apoptotic cell populations in the Control and LIPUS groups. Data
are presented as the mean ± SD. ***P<0.001 and
****P<0.0001 vs. Control. TCSs, tendon stem cells;
LIPUS, low-intensity pulsed ultrasound. |
It was then examined whether LIPUS affected TSC
migratory behavior using a scratch assay. Baseline wound widths
were comparable between groups. At both 24 and 48 h, the LIPUS
group showed a greater reduction in scratch area than the Control
group, and quantitative analysis confirmed significantly increased
scratch closure after LIPUS treatment (Fig. 2C and D). These findings indicate that LIPUS
enhances the migratory activity of TSCs in vitro. Cell
apoptosis was assessed by Annexin V-FITC/PI double staining and
flow cytometry. As shown in Fig.
2E, compared with the Control group, LIPUS treatment did not
increase apoptosis. The total apoptotic rate decreased from 14.3
and 11.7% to 10.9 and 8.5% at 6 and 30 h, respectively, indicating
that LIPUS treatment exhibited favorable cytocompatibility without
inducing obvious apoptosis.
LIPUS activates YAP/TAZ signaling and
promotes tenogenic differentiation of TSCs
To determine whether LIPUS induced
tenogenesis-associated molecular changes in TSCs, the expression of
tendon-related markers was assessed together with key
mechanotransduction mediators. Immunofluorescence staining showed
that LIPUS markedly increased SCX and COL I expression relative to
the Control group (Fig. 3A and
B), and quantitative analysis
confirmed significantly higher fluorescence intensities after LIPUS
treatment (Fig. 3C and D).
 | Figure 3LIPUS enhances YAP/TAZ-associated
signaling and increases tenogenesis-associated marker expression in
tendon stem cells. TSCs in the LIPUS group were treated with LIPUS
at 1.5 MHz and 50 mW/cm², with a pulse repetition frequency of 1
kHz and a 20% duty cycle, for two 25-min sessions separated by 24
h. (A and B) Representative immunofluorescence images showing
increased SCX and COL I expression in TSCs after LIPUS treatment.
(C and D) Quantitative analysis of immunofluorescence intensity
together with reverse transcription-quantitative PCR analysis of
tendon-associated markers, showing increased expression in the
LIPUS group. (E and F) Western blot analysis and densitometric
quantification demonstrating increased YAP, TAZ and SCX protein
levels after LIPUS stimulation. Immunofluorescence analysis of YAP
localization after LIPUS treatment. (G) Representative images of
YAP (red), F-actin (green) and nuclei (DAPI, blue). (H)
Quantification of nuclear YAP localization ratio. Data are
presented as the mean ± SD. *P<0.05,
**P<0.01 and ***P<0.001 vs. Control.
LIPUS, low-intensity pulsed ultrasound; TCSs, tendon stem cell;
SCX, scleraxis. |
At the transcriptional level, RT-qPCR demonstrated
significant upregulation of Col I and Tnmd in the LIPUS group,
supporting enhanced expression of matrix-related and
tendon-associated genes. Western blot analysis further showed that
LIPUS significantly increased the protein expression of YAP, TAZ
and SCX (Fig. 3E and F). Collectively, these findings indicate
that LIPUS activates YAP/TAZ-associated signaling and enhances a
tenogenesis-associated molecular phenotype in TSCs. YAP subcellular
localization was evaluated by immunofluorescence staining. Compared
with the Control group, LIPUS treatment significantly increased the
nuclear localization ratio of YAP, indicating enhanced YAP nuclear
translocation and activation of YAP signaling (Fig. 3G and H).
Inhibition of YAP/TAZ suppresses
LIPUS-induced tenogenic differentiation and matrix synthesis
To determine whether the effects of LIPUS were
associated with YAP/TAZ signaling, pharmacological inhibition
experiments were performed using VP. Immunofluorescence staining
showed that the LIPUS-induced increases in SCX and COL I were
markedly attenuated by VP treatment (Fig. 4A and B), and quantitative analysis confirmed a
significant reduction in fluorescence intensity in the LIPUS + VP
group (Fig. 4C and D).
 | Figure 4Pharmacological inhibition of YAP/TAZ
attenuates LIPUS-induced tenogenesis-associated responses and
matrix-related marker expression. TSCs in the LIPUS and LIPUS + VP
groups received LIPUS at 1.5 MHz and 50 mW/cm², with a pulse
repetition frequency of 1 kHz and a 20% duty cycle, for two 25-min
sessions separated by 24 h. In the LIPUS + VP group, cells were
pretreated with VP (1 µmol/l) for 1 h before each LIPUS session. (A
and B) Representative immunofluorescence images of SCX and COL I in
the Control, LIPUS and LIPUS + VP groups. (C and D) Quantitative
immunofluorescence and reverse transcription-quantitative PCR
analyses showing that VP reduced the LIPUS-induced increase in
tendon-associated marker expression. (E and F) Western blot
analysis and densitometric quantification demonstrating that VP
suppressed the LIPUS-mediated increase in YAP, TAZ, SCX and COL I
protein expression. (G) Proposed working model illustrating that
LIPUS enhances tenogenesis-associated responses in TSCs through
mechanotransduction-dependent activation of YAP/TAZ-associated
signaling and upregulation of SCX. Data are presented as the mean ±
SD. *P<0.05, **P<0.01 and
***P<0.001. LIPUS, low-intensity pulsed ultrasound;
TCSs, tendon stem cell; VP, Verteporfin. |
Consistent with these findings, RT-qPCR showed that
the LIPUS-induced upregulation of Col I and Tnmd was significantly
suppressed by VP. Western blot analysis further demonstrated that
VP reduced the expression of YAP, TAZ, SCX and COL I relative to
LIPUS treatment alone (Fig. 4E and
F). On the basis of these results,
a working model was presented, in which LIPUS-derived mechanical
cues activate YAP/TAZ-associated signaling, increase SCX
expression, and thereby enhance tenogenesis-associated responses
and matrix-related gene expression in TSCs (Fig. 4G).
Discussion
In the present study, LIPUS induced transcriptomic
and molecular changes in rat TSCs that were consistent with
activation of mechanotransduction and enhancement of a
tenogenesis-associated program. By integrating bioinformatic
analysis with in vitro experiments, it was found that LIPUS
enriched pathways related to mechanical signaling and cytoskeletal
remodeling, increased scratch closure, and upregulated YAP, TAZ,
SCX, COL I and TNMD. Moreover, pharmacological inhibition with VP
attenuated these effects, supporting the involvement of
YAP/TAZ-associated signaling in the cellular response to LIPUS.
A key observation in the present study is that LIPUS
reshaped the transcriptomic landscape of TSCs toward a
mechanosensitive state. The enriched BP were primarily related to
mechanotransduction and cytoskeletal organization, in line with the
concept that stem cells convert physical cues into transcriptional
outputs through membrane-associated mechano-sensors and
actin-dependent signaling networks (25-28).
This finding is mechanistically relevant because YAP/TAZ activity
is tightly coupled to cytoskeletal tension, cell shape and
cell-matrix interactions (13,14,29,30).
The present transcriptomic data therefore provide a plausible
upstream framework linking LIPUS-derived mechanical stimulation to
downstream changes in tendon-associated gene regulation. Finally,
the upstream mechanosensitive pathways linking LIPUS exposure to
YAP/TAZ activation were not directly examined. Thus, although the
present findings support the involvement of the YAP/TAZ/SCX axis,
they do not establish the precise upstream molecular sequence.
Further studies are required to define the roles of integrins,
Piezo channels, calcium signaling and cytoskeletal remodeling in
this process.
Because LIPUS may influence cytoskeletal signaling
and YAP/TAZ activity, the suitability of β-actin as a loading
control warrants consideration. Mechanical stimulation can alter
actin filament organization, polymerization, intracellular tension
and cell morphology; however, such changes do not necessarily
indicate a change in total β-actin protein abundance. In the
present study, β-actin bands showed no apparent differences among
the Control, LIPUS and LIPUS + VP groups under the applied
treatment conditions (50 mW/cm²; two 25-min sessions separated by
24 h). Therefore, β-actin was used to normalize protein loading in
the western blot analyses. This result should be interpreted as
stability of total β-actin abundance under the specific conditions
of the present study and does not exclude LIPUS-induced remodeling
of the actin cytoskeleton.
It should be noted that primary tendon-derived
cultures are intrinsically heterogeneous. Although CD11b and CD45
were used as negative markers for TSC characterization, low
proportions of CD11b-positive (5.65%) and CD45-positive (2.34%)
cells were detected in the present study. These minor populations
may represent residual non-TSC cells after primary isolation or
low-level background staining. Importantly, the predominant cell
population was positive for CD90, CD105 and CD146 and negative for
hematopoietic-associated markers overall. Therefore, TSC
identification was based on the combined marker profile rather than
on complete absence of each negative marker in every individual
cell.
SCX is a central regulator of tendon development and
maintenance of the tenogenic program (15,16,31,32).
In the present study, SCX was increased after LIPUS treatment at
the transcriptomic and/or targeted experimental levels, together
with increased expression of COL I and TNMD in targeted assays.
These findings support the interpretation that LIPUS enhances a
tendon-associated molecular phenotype rather than merely altering a
non-specific stress-response program. Because YAP/TAZ function as
mechanosensitive transcriptional coactivators that regulate
lineage-associated gene expression (33-39),
the concurrent increase in YAP/TAZ and SCX is consistent with the
involvement of a functional YAP/TAZ-SCX signaling axis in this
process. Notably, COL I and TNMD were validated primarily by
targeted assays rather than emphasized in the RNA-seq display,
which most likely reflects differences in transcript selection,
analytical thresholds and figure presentation rather than a true
biological inconsistency.
The inhibitor experiments using VP further
strengthened this interpretation. VP attenuated the LIPUS-induced
increases in YAP, TAZ, SCX, COL I, and TNMD, indicating that
YAP/TAZ activity contributes materially to the downstream molecular
response. This observation is biologically plausible because
YAP/TAZ signaling interacts with other developmental pathways,
including TGF-β and Wnt, to regulate cell fate decisions (37,38,40-45).
Although these pathways were not directly interrogated in the
present study, the current findings position YAP/TAZ as an
important signaling node through which mechanical stimulation may
be integrated with broader regulatory networks during tendon
repair.
Another notable finding is that LIPUS enhanced TSC
migratory behavior in vitro. Efficient cell migration is
relevant to tendon repair because progenitor cells must populate
the injury site before participating in tissue remodeling. In this
context, the increased scratch closure observed after LIPUS
exposure suggests improved migratory capacity rather than
accelerated tissue healing per se, and the wording should therefore
be interpreted accordingly.
Several limitations should be acknowledged. First,
the current data support enhancement of a tenogenesis-associated
molecular phenotype, but they do not by themselves establish
complete tenocyte maturation. Second, the study was performed
entirely in vitro and therefore cannot fully recapitulate
the biomechanical, inflammatory, vascular and extracellular matrix
microenvironment of injured tendon tissue in vivo. Thus, the
observed effects of LIPUS should not be directly extrapolated to
functional tendon healing outcomes. Future studies using rat or
mouse Achilles tendon injury models will be required to evaluate
histological repair, collagen organization, biomechanical strength
and in vivo activation of the YAP/TAZ/SCX axis. Third, the
transcriptomic analysis was based on an external LIPUS-related
dataset rather than the current TSC model, which may introduce
system-dependent differences. Therefore, these findings should be
considered supportive evidence and require further validation in
LIPUS-treated TSCs. Finally, additional lineage markers, functional
assays and in vivo validation will be needed to strengthen
causal inference and clarify the hierarchical relationship between
YAP/TAZ, SCX, and other mechanosensitive pathways.
Furthermore, TSCs in the present study were derived
from 3-4-week-old male rats; thus, variability associated with
donor age, sex, individual donors and cell batches was not
systematically evaluated. Future studies should include multiple
donors of different ages and sexes to improve the generalizability
of the findings. In addition, neither a VP-only group nor a
vehicle-control group was included. Although no overt morphological
abnormalities or cytotoxicity were observed under the treatment
conditions, potential off-target effects of VP cannot be completely
excluded. Future investigations should therefore incorporate
appropriate pharmacological controls and complementary genetic
approaches, such as YAP/TAZ knockdown, to further confirm pathway
specificity.
Taken together, these findings indicate that LIPUS
is not merely a passive physical adjunct, but a biologically active
modulator of TSC behavior. By engaging mechanotransduction-related
signaling, particularly the YAP/TAZ/SCX axis, LIPUS may represent a
promising noninvasive approach for the enhancement of tendon
regeneration.
In conclusion, LIPUS enhances tenogenesis-associated
molecular responses and matrix-related marker expression in rat
TSCs. Mechanistically, LIPUS activates mechanotransduction-related
signaling, increases YAP/TAZ-associated activity, upregulates SCX,
and elevates the expression of COL I and TNMD. Pharmacological
inhibition with VP attenuates these effects, indicating that the
cellular response to LIPUS is closely associated with activation of
the YAP/TAZ/SCX axis. These findings provide a mechanistic
rationale for further evaluation of LIPUS in tendon
regeneration.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the Open Fund of the
Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy,
China Three Gorges University (grant no. 2024KZL010).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
SL and YL conceived and designed the study. SL and
YH contributed to methodology design and performed data analysis
using software. KZ, LC and DL conducted formal analysis. YL was
responsible for data curation and investigation. SL and DL wrote
the original draft. All authors reviewed and critically revised the
manuscript, approved the final version of the manuscript, and
agreed to be accountable for all aspects of the work. SL and YL
confirm the authenticity of all the raw data.
Ethics approval and consent to
participate
The present study was approved by the Animal Welfare
Ethics Committee of the Second Hospital of Shanxi Medical
University (approval no. DW20230008; Taiyuan, China), and all
procedures were conducted in accordance with institutional and
national guidelines for animal care and use.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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