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Development of a novel multiphysical approach for the characterization of mechanical properties of musculotendinous tissues

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At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.
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Development of a novel
multiphysical approach for the
characterization of mechanical
properties of musculotendinous
tissues
Malek Kammoun1, Redouane Terni1, Vincent Dupres2, Philippe Pouletaut1, Sandra Même3,
William Même3, Frederic Szeremeta3, Jessem Landoulsi
4, Jean-Marc Constans5,
Frank Lafont2, Malayannan Subramaniam6, John R. Hawse6 & Sabine F. Bensamoun1
At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical
properties of muscle and tendon tissues. Further, there are no reports regarding characterization of
mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting
the ability to monitor changes in these tissues during disease progression or response to therapy.
Therefore, we sought to develop novel protocols for the characterization of mechanical properties in
musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given
that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of
skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using
TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of
glue or chemical agents for muscle and tendon ber immobilization during acquisition of transversal
cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live
animals, we have also developed an ultrasound elastography protocol using a new linear transducer,
SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo.
This protocol allows for the identication of changes in muscle and tendon elasticities. Such innovative
technological approaches have no equivalent to date, promise to accelerate our understanding of
musculotendinous mechanical properties and have numerous research and clinical applications.
Musculotendinous tissue is complex and consists of well-organized hierarchical structures that inuence its func-
tion and mechanical properties. At present, the mechanical properties of muscle tissue are characterized in vitro
using stretching tests (at dierent velocities) or contractile tests (using calcium) to measure the passive and active
properties of the muscle13. However, few studies have performed transversal tests, such as indentation, to ana-
lyze the transversal elasticity of the tissue4,5. Atomic Force Microscopy (AFM) is a perfectly suited technique for
carrying out such tests. Indeed, in the last two decades, AFM has emerged as a ubiquitous technique utilized in
biological research under near-physiological conditions from single molecules to living cells610. In addition to
its outstanding imaging capabilities, force-distance curve based modes are widely used to probe the mechanical
properties of biological samples1113. Using AFM, the viscoelastic behavior of skeletal muscle cells (myoblasts)14
and dystrophic muscle15 have been characterized using the Young’s modulus to quantify the functional benet
1Alliance Sorbonne Universités, Université de Technologie de Compiègne, Biomechanics and Bioengineering
Laboratory, UMR CNRS 7338, Compiègne, France. 2Université Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille,
U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France. 3Centre de Biophysique
Moléculaire, CNRS UPR4301, Orléans, France. 4Sorbonne Université, Laboratoire de Réactivité de Surface UMR
7197, Paris, France. 5Institut Faire Faces, EA Chimère 7516 UPJV, CHU Amiens Imagerie Médicale, Amiens, France.
6Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Correspondence and
requests for materials should be addressed to S.F.B. (email: sabine.bensamoun@utc.fr)
Received: 17 September 2018
Accepted: 3 May 2019
Published: xx xx xxxx
OPEN
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aer gene therapy. At the level of muscle bers, AFM has been used on xed tissue samples from Mdx, or Col6a1
KO mice to analyze the mechanical properties of the sarcolemma16. However, it is dicult to compare the elastic
values measured with AFM to other studies due to the experimental conditions such as preparation and xation
of the samples, type of cantilever employed and mathematical models used to extract the Young’s modulus from
the force-distance curves which are specic to each protocol17. For the rst time, we have devised a novel AFM
protocol with the ability to be performed on intact living muscle and tendon bers that can be used for numerous
research applications across the eld. We have also applied the most recent AFM data acquisition modes, more
specially the Quantitative Imaging mode (QImode), in this new protocol allowing for the generation of high res-
olution elasticity maps. is technique highlights the distribution of the mechanical properties along the bers
and reveals the striated structure of the tissue.
However, AFM has limitations in that it is unsuitable for application on live animals. us, non-invasive
imaging techniques such as elastography (magnetic resonance imaging (MRI) or ultrasound (US)) have begun
to be used for in vivo estimation of the mechanical properties of mouse or rat muscle18,19. Elastography is based
on the propagation of shear waves using an external driver placed on the muscle. Previous studies have imple-
mented needle drivers implanted in the hindlimb of mice to acquire magnetic resonance elastography (MRE)
based acquisitions for assessment of the mechanical anisotropic ratio of the shear storage moduli18. Recently,
Nelissen et al.20 have developed a MRE-based approach to analyze skeletal muscle damage in rats. With regard to
US elastography, a recent study19 has used the Aixplorer machine to show correlations between elastic properties
and pathological characteristics of spinal cord injuries in rats. For ultrasound elastography, the driver is a probe
placed on the tissue which generates localized acoustic radiation force to produce waves that propagate transver-
sally and provide a two-dimensional real-time quantitative map of the elasticity of muscle. In the present report,
we have utilized these technologies to devise a non-invasive US elastography protocol that allows for the charac-
terization ofmuscle and tendon tissues mechanical properties in live animals. Ultrasound elastography is a more
practical and available technique compared to MRE which requires the use of a MRI machine. Additionally, we
have developed this protocol using a novel clinical probe that could allow for the use of this technique for clinical
applications.
Typically, elastography and AFM protocols for assessment of skeletal muscle and tendon tissues are developed
independently and therefore have substantial dierences between them regarding machine settings, data analysis
protocols etc. One goal of the present report was to develop a protocol that was identical for analysis of both skel-
etal muscle and tendon and that could therefore be universally applied to both tissues for mice.
In addition, we have also validated and correlated the elastic results with other analyses including MRI tex-
ture analysis (MRI-TA) and Transmission Electron Microscopy (TEM). For all of our studies, we have utilized
wild-type (WT) and TIEG1 KO mice as our model system given their well-characterized biological and structural
defects throughout the musculotendinous system. Such innovative technological approaches have no equivalent
to date and promise to accelerate our understanding of musculotendinous mechanical properties.
Results
Atomic force microscopy (AFM) analysis of the elastic properties of musculotendinous tis-
sues. AFM experiments were performed on skeletal muscle and tendon bers to generate elasticity maps. e
sarcomeric structure of muscle bers was observed with this technique demonstrating its capability to measure
elasticity of single muscle bers (Fig.1A–D). e Young’s modulus was signicantly decreased for TIEG1 KO
muscle bers isolated from both the soleus and the EDL muscles compared to WT controls. is decrease in the
Young’s modulus is represented by less intense color mapping for the TIEG1 KO bers (Fig.1). ese analyses
also revealed that slow twitch bers isolated from WT soleus muscle have greater elasticity than fast twitch bers
isolated from WT EDL muscle (Fig.2A). Conversely, TIEG1 KO tendon bers exhibited a signicant increase
in the Young’s modulus compared to WT tendon bers (Figs1E,F and 2B). e Young’s modulus of the WT ten-
don (53.2 kPa) and WT EDL (46.5 kPa) were nearly identical indicating a similar range of the transversal elastic
modulus for these two tissues. Finally, these results indicate that the impact of TIEG1 expression on elasticity is
tissue specic.
Transmission electron microscopy (TEM) analysis. In order to further understand the basis for the
defects in the elastic properties of skeletal muscle tissues derived from TIEG1 KO mice via AFM, we performed
TEM studies. Longitudinal sections of WT and TIEG1 KO soleus and EDL muscles revealed disorganization of
the muscle structure in the absence of TIEG1 expression (Fig.3). Specically, smaller sarcomere lengths (1.5 µm
for KO and 2.5 µm for WT) and the near complete disappearance of the I band were observed.
Evaluation of the musculotendinous elastic and textural properties using ultrasound elastogra-
phy. Given that AFM and TEM evaluation of tissues is performed on ex vivo tissues, we sought to evaluate the
utility of ultrasound elastography of skeletal muscle and tendon tissues in live animals. From the B-mode image
(Figs4A and 5A), no signicant dierence of echo intensity mean or coecient of variation were found between
WT and TIEG1 KO tissues. Similar results were found for the contrast measured within the muscle region of
interest (ROI) (TIEG1 KO: 54.5 ± 18.2% vs WT: 50.6 ± 14.1%) and the tendon ROI (TIEG1 KO: 40.9 ± 14.0% vs
WT: 36.0 ± 9.7%). However, the TIEG1 KO and WT ROIs were well discriminated by texture analysis as high-
lighted in the PCA images (Fig.6A,B).
From the elastography images, no signicant dierence in mean stiness, coecient of variation or con-
trast was observed. More specically, no signicant dierence in the stiness was detected between TIEG1
KO (36.1 ± 10.3 kPa) and WT (44.4 ± 13.4 kPa) muscle nor between TIEG1 KO (157.3 ± 56.3 kPa) and WT
(161.1 ± 54.1 kPa) tendon (Fig.6C,D). However, we noted signicant stiness dierences between WT muscle
(44.4 ± 13.4 kPa) and WT tendon (161.1 ± 54.1 kPa) with higher values observed in the tendon tissue.
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Hierarchical ascending classication (HAC) of skeletal muscles. Comparison of the Hierarchical
Ascending Classications (HAC) for all muscles (tibialis anterior, plantaris, gastrocnemius lateralis, gastrocne-
mius medialis, soleus and extensor digitorum longus) analyzed via MRI revealed two distinct groups of texture
proles which clustered as a function of mouse genotype (Fig.7). Dierences between Class I (CI) and Class
II (CII) demonstrate a genotype eect on the dierent muscles. ese results indicate dierent texture proles
between WT and TIEG1 KO muscles. e global values for the tibialis anterior, plantaris, gastrocnemius lateralis,
gastrocnemius medialis, soleus, and EDL were found to be 73% (WT vs. TIEG1), 62.5% (WT vs. TIEG1), 81.2%
(WT vs. TIEG1), 59% (WT vs. TIEG1), 76% (WT vs. TIEG1) and 70% (WT vs. TIEG1), respectively.
Discussion
Muscle bers are composed of dierent compartments (myobril, extra sarcomeric cytoskeleton and sarco-
lemma) which ideally would be characterized in longitudinal and transverse directions in order to comprehen-
sively characterize the entire ber. However, with the use of existing technology and protocols, measurements in
the transversal direction are oen neglected despite the fact that important information could be obtained from
Figure 1. Elasticity maps (15 µm× 15 µm) obtained from the AFM protocol for slow (A,B soleus: Sol) and fast
(C,D EDL) twitch muscle bers and tendon bers (E,F) of wild-type (WT) and TIEG1 knockout (KO) mice.
e muscle and tendon cartographies are represented by dierent colors which are representative of lower
(blue/green) and higher (red/orange) elasticities.
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such analyses for structural proteins located perpendicular to the ber axis21. For instance, desminopathy is a
muscle disease related to the defect of an intermediate lament (called desmin), which is aligned in a transverse
manner through the Z-disks, known to play a critical role in the maintenance of structural integrity and mechan-
ical properties22.
e AFM technique, which is primarily used to image biomolecular and cellular systems, is currently poorly
utilized for analysis of mechanical properties in brous tissues. Past studies have employed AFM on tissue cry-
osections4 or following tissue xation5 at resolutions of the whole tendon (diameter units: millimeter) or at the
tendon bril level (diameter units: nanometer). For the rst time, we report the development of a novel protocol
for the use of AFM on living tendon tissues at the resolution of single tendon bers (diameter units: micrometer).
From an experimental standpoint, a challenge with such analyses is to immobilize the muscle ber during
the indentation test without using glue or chemical agents16,23 which could alter the mechanical properties of the
tissue. e acquisition of the force curve during the indentation is also a key point of this technique. Indeed, the
curve of the force, which is recorded for each pixel (128 × 128) of the cartography, must be validated according to
the artifact of ber movement leading to incorrect interpretation of the Young’s modulus. Another novel aspect
Figure 2. Quantication of Young’s modulus obtained via AFM for wild-type (WT) and TIEG1 knockout (KO)
soleus (SOL) and EDL muscle bers (A) and tendon (B) bers. ***p < 0.001 between indicated groups.
Figure 3. Transmission Electronic Microscopy (TEM) acquisition of soleus (Sol) and extensor digitorum
longus (EDL) muscles isolated from wild-type (WT) and TIEG1 knockout (KO) mice. Longitudinal sections
revealed a disorganized ultrastructure in both TIEG1 KO Sol and EDL muscles (BD) compared to WT
littermates (AC).
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of this study was to generate high resolution elasticity maps of single muscle and tendon bers through the use of
the QI mode of AFM. is allows for fast data acquisitions that are required to generate high resolution maps that
highlight the spatial distribution of the musculotendinous mechanical properties.
e maps generated from these studies have revealed the regular periodic structure of the muscle ber, with
smaller sarcomeres (1.5 µm) observed in the TIEG1 KO muscle, and the collagenous parallel laments within
the tendon bers. ese structural observations are in agreement with TEM acquisitions performed on WT and
TIEG1 KO muscle which revealed a disorganization of the muscle structure in both TIEG1 KO soleus and EDL
muscles compared to WT littermates. Specically, smaller sarcomere lengths (1.5 µm for KO and 2.5 µm for WT)
and disappearance of the I band where observed in both slow and fast twitch TIEG1 KO skeletal muscles. ese
results demonstrate that the eciency of the AFM protocols (experimental and acquisition) are representative of
the main structures of muscle and tendon bers with regard to the elastic cartography allowing for the avoidance
of topographic acquisition that is currently performed with AFM.
Regardless of mouse genotype, slow skeletal muscle bers (soleus) exhibited higher elasticity compared to fast
bers (EDL). ese results most likely relate to the oxidative and glycolytic ber composition of these specic
skeletal muscles. In addition, the lower elasticity as detected by AFM in the TIEG1 knockout soleus and EDL
bers relative to the WT bers may be reective of the disorganized structure. e disorganization of TIEG1 KO
skeletal muscle bers likely leads to a decreased resistance under the tip of the AFM probe. e control (WT)
values are of interest for numerical modelling, which need referent data to simulate the true mechanical behavior
of biological samples. We also observed higher elasticity for the Z-disk region of the muscle structure compared
to the M-band and this observation was in agreement with Ognevas study21 for rat muscle bers.
In comparison with muscle bers, the transversal elasticity of WT tendon bers is within the same range as
those of the WT muscle bers. Interestingly, deletion of the TIEG1 gene increased the transversal elasticity of the
tendon bers compared to WT control bers. Previous TEM studies on tendon tissue along the longitudinal axis of
the tendon ber did not reveal any structural changes between the dark and light bands24. However, Gumez et al.25
reported dierent X-ray diraction patterns between WT and TIEG1 KO tendon bers demonstrating that lack
of TIEG1 expression results in a disorganization of the collagenous structure and a signicant distortion of the
triple helix network26.
It is interesting to note that deletion of TIEG1 has opposite eects on the elasticity of muscle vs. tendon bers.
Given the intimate association of these two tissues with one another, it is possible that these dierences are due
Figure 4. Representative B-mode image of the mouse hindlimb (A) and the corresponding elastogram (B)
showing the elasticity of the dierent tissues present within the hindlimb. e region of interest (ROI) analyzed
for quantication of the Young’s modulus (E)is indicated (B).
Figure 5. Representative B-mode image of the mouse hindlimb (A) and the corresponding elastogram (B)
showing the elasticity of the dierent tissues present within the hindlimb. e region of interest (ROI) for
measurement of the Young’s modulus (E)of the Achilles tendon is indicated (B).
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to compensation of one tissue for another. However, it is also possible that these eects are due to tissue specic
dierences exerted by this transcription factor. TIEG1 is known to mediate the down-stream eects of many
cytokines and hormones including TGFβ, BMP, EGF, and estrogen among others2730. It is therefore possible that
Figure 6. Principal component analysis of texture parameters from the B-mode acquisitions of muscle (A)
and tendon (B) according to mouse genotype (1: TIEG1 knockout (KO) vs 2: wild-type (WT)). Signicant
dierences for both tissues between the two genotypes are apparent. Quantication of muscle stiness (C) and
tendon stiness (D) according to mouse genotype.
Figure 7. Hierarchical ascending classication (HAC) of the six muscles as a function of mouse genotype
(wild-type (WT) vs TIEG1 knockout (KO)). Dierences between Class I (CI) and Class II (CII) demonstrate a
genotype eect on the dierent muscles.
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loss of TIEG1 expression dierentially impacts these signaling pathways in these two tissues. It is also possible
that other transcriptional co-regulators and/or transcription factors that are essential for muscle and tendon
development/function are dierentially impacted by loss of TIEG1 expression. Additional studies are needed to
identify the precise mechanisms by which loss of TIEG1 expression results in opposite eects with regard to the
elasticity of muscle and tendon bers.
To overcome the necessity to sacrice mice, recent elastography imaging techniques (MRI, US) have enabled
the measurement of mechanical properties in live animals. While magnetic resonance elastography has been
used on muscles of mdx (Duchene) mice18, US elastography has never been performed on mouse muscle or
tendon. In the present study, US elastography was performed with the Aixplorer machine which is clinically used
for human tissues including muscle31 and sciatic nerve32 among others. Here, we have applied this technique to
characterize the elastic properties of the musculotendinous system in a non-invasive manner. We have utilized
a high frequency probe (SLH20-6) that has been recently adopted in clinical practice to image the functionality
of supercial musculotendinous tissues. It should be noted that ultrasound imaging of mouse tissue is usually
performed with homemade constructor probes. Using the SLH20-6 probe, muscle ber structure and Achilles
tendon were accurately analyzed with the use of the B-mode image (resolution 38 µm). Moreover, texture anal-
ysis revealed signicant dierences as a function of mouse genotype for both tissues, demonstrating that US
elastography (Aixplorer) has the capability of identifying structural defects in muscle and tendon tissues. We
have validated these ndings with MRI texture analysis (MRI-TA) which also indicated signicant structural
changes between TIEG1 KO and WT muscle bers. In comparison with the US technique, MRI has the ability
to analyze the structure of all individual muscles within the hindlimb, while US can only characterize groups
of muscles3335. Interestingly, the hierarchical ascending classication (HAC) revealed that deletion of TIEG1
aected all muscle-texture proles regardless of muscle type (oxidative: soleus, glycolytic: EDL, mix: gastrocne-
mius, plantaris, tibialis).
While US was able to detect structural changes as a function of genotype, the elastography acquisitions were
not able to dierentiate between WT and TIEG1 KO mice with regard to the elastic properties. is result indi-
cates that US elastography, based on the analysis of shear waves, is not suciently sensitive to detect dierences in
elasticity. e development of higher frequency probes could allow for better spatial resolution and may allow for
better resolution for detection of dierences in elastic properties. erefore, US elastography performed on the
musculotendinous system cannot currently replace in vitro tests. However, this technique was able to dierentiate
the elasticity between tendon and muscle tissues regardless of mouse genotype. is dierentiation was most
likely related to the increased stiness of the Achilles tendon compared to skeletal muscle.
It should be noted that the protocols developed in the present study have some limitations. Concerning
AFM experiments, it remains dicult to compare the elastic modulus values with the ones obtained from sim-
ilar experiments. is is primarily due to the numerous experimental conditions (type of AFM tip, calibration
method, maximal indentation force, acquisition mode, mathematical models used to extract the Young’s modulus
from the force-distance curves, etc ) which inuence the measurement of the elastic properties. Nevertheless,
this problem is being increasingly recognized, and some improvements have recently been proposed with the
introduction of the Standardized Nanomechanical AFM Procedure (SNAP)17. We are perfectly aware of these
limitations and for this reason we apply a series of precautions to ensure that our results are comparable from one
experiment to another. is implies keeping the same parameters for the whole study, not changing the laser posi-
tion and using the same type of cantilever (and where possible, the same cantilever) and the same mathematical
model for the analysis.
As an extension of the results presented here, future studies will focus on developing the AFM protocol for
analysis of muscle bers under active conditions by altering calcium concentrations during the mechanical test.
Furthermore, it is of interest to use this technique on tissues undergoing various degrees of stretching to provide
a more dynamic measure of their functional properties. As for the US elastography protocol, the clinical probe
utilized in this study did not allow for dierentiating the elastic properties as a function of mouse genotype. To
improve upon this limitation, it will be necessary to design a specic US probe with higher frequency that is
compatible with the Aixplorer machine allowing for a better characterization of the mechanical properties of
rodent so tissues (muscle and tendon). In the future, it will be also of interest to adapt the present elastography
protocols for other tissues.
In conclusion, we have developed a novel multiphysical approach that enables a better understanding of the
structural and mechanical properties of so living tissues including muscle and tendon. ese novel applications
will be useful in both research and clinical applications for the evaluation of normal and diseased states. Finally,
through the use of these mouse models, we have further implicated important roles for TIEG1 in the musculo-
tendinous system and have demonstrated that loss of TIEG1 expression reduces the elastic properties of muscle
bers while improving the transversal elastic properties of tendon bers.
Methods
Mice and study design. e generation of TIEG1 KO mice has been previously described36 and initial char-
acterization of TIEG1 expression and function in skeletal muscle and tendon were previously reported24,25,3741.
For the studies presented here, 3 month old littermate female animals from heterozygous breeding pairs were
used. All mice were maintained in a temperature controlled room (22 ± 2 °C) with light/dark cycle of 12 hours.
Animals had free access to water and were fed with standard laboratory chow ad libitum. is study was carried
out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of
the National Institutes of Health. e protocol was approved by the French ministry of higher education, research
and innovation (Permit Number: DUO-4776) and the Mayo Clinic Institutional Animal Care and Use Committee
(Permit Number: A9615).
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Atomic force microscopy (AFM) measurements. All experiments were performed on a commer-
cial AFM (NanoWizard 3, JPK Instruments, Berlin, Germany) combined with an inverted optical microscope
(AxioObserver.Z1, Zeiss) driven by JPK NanoWizard soware 6.0 and Zen Blue 2012. Data were acquired in
Quantitative Imaging mode (QImode) using PFQNM-LC cantilevers with a nominal spring constant of 0.08 N/m
and a 3.5 µm high conical tip with 8 nm tip radius (Bruker, Santa Barbara, CA, USA). Cantilever spring constant
and sensitivity were calibrated before each experiment using the thermal uctuation method of the JPK soware,
yielding spring constant values ranging from 0.019 to 0.025 N/m.
Control (NWT_Tendon = 7, NWT_Soleus = 7, NWT_EDL = 7) and TIEG1 KO (NKO_Tendon = 7, NKO_Soleus = 7,
NKO_EDL = 7) bers were extracted from slow (soleus) and fast (EDL) muscles39 as well as tail tendon24. Perfect
immobilization of the sample is a prerequisite during each AFM experiment. is is particularly the case in exper-
iments conducted on bers where the slightest movement (vertical or lateral) is likely to generate artifacts during
the acquisition of force curves, which will result in making the elasticity maps unusable. e absence of move-
ment during transverse indentations is therefore a fundamental element of our experimental protocol. Fibers
were immobilized on plexiglass and submersed in a drop of PBS. Bright eld imaging was used to identify the
area of interest on bers. A visual control of the immobility of the ber was performed using the optical micro-
scope coupled with the AFM instrument. is ensured that only Quantitative Imaging (QI) les were recorded
in a static condition. Subsequently, the AFM tip was engaged in the central portion of the ber (to ensure a near
plane contact surface to avoid curvature eects leading to artifacts) to scan an area of 15 µm × 15 µm. e AFM
tip was scanned on three areas along each muscle and tendon ber by applying a force trigger of 2 nN with a 3 µm
Z ramp (i.e. the range between the two extreme positions of the AFM tip), at 80 µm/s. Compared to conventional
Force-Volume (FV) mode, QI mode uses a specic movement algorithm that enables precise control of the verti-
cal force at every pixel of the image without applying any lateral force that could damage so biological samples.
Combined with the new generation of short cantilevers, QI mode allows fast tip displacements, thus reducing the
time required to capture elasticity maps. Hence, we were able to perform 128 × 128 pixel force mapping scans in
less than 15 minutes while more than 5 hours would be needed for the same resolution with FV.
Data analysis. e AFM data analysis was performed using in-house pyAF (python Atomic Force) soware,
version 1.5.1. An algorithm based on the tting of the baseline was used to detect the point of contact on the
force curve. A linear t was made using the baseline of the approach curve. A noise threshold is used to shi the
t along the force axis. e noise parameter can be manually adjusted by the user to optimize the detection of
the point of contact. e intersection between this shied t and the force curve denes the position of the point
of contact. Subsequently, the elasticity was deduced from the Young’s modulus which was determined from the
indentation portion of the curve using the elastic contact model for conical indenters (named Sneddon model42):
π
α
δ=
F
E
v
2tan
1
22
where F is the measured force, E the elastic modulus, α the half opening angle of the tip, δ the indentation, and ν
the sample’s Poisson’s ratio, that was set to 0.5.
Elasticity maps. Arrays of force curves in the x and y planes (typically 128 curves × 128 curves) were recorded on
areas of a given size (15 µm × 15 µm). From these force mapping scans, the Young’s modulus was then estimated for
each force curve and displayed as colored pixels that reect the magnitude of ber stiness (elasticity maps).
Box plot. Box plots were generated by pooling all of the data used to create elasticity maps for each ber
type (tendon, muscle) (elastic modulus deduced from each individual force curve) as a function of mouse geno-
type. Unpaired t-test with Welch’s correction (which assume no equal SDs) was performed to compare elasticity
between mouse genotypes for muscle (soleus, EDL) and tendon bers (***p-value < 0.0001). Bottom and top of
the boxes represent the rst and third quartiles. e line within the box represents the second quartile, i.e. the
median. e end of the whiskers represents the 9th and the 91st percentiles.
Transmission electron microscopy (TEM). Prior to processing for TEM, the soleus (N = 7) and the EDL
(N = 7) muscles were dissected from WT and TIEG1 KO mice with both extremities pinned to avoid muscle
contraction and to maintain original length. Muscles were then immediately placed in xative [1% (vol/vol)
glutaraldehyde and 4% (vol/vol) paraformaldehyde in 0.1 M phosphate buer, pH 7.2]43 and incubated over-
night at 4 °C. Subsequently, two pieces (1 mm × 2 mm) of muscle were dissected from the middle of the tissue
and rinsed for 30 min in three changes of 0.1 M phosphate buer, pH 7.2, followed by a 1 h secondary xation in
phosphate-buered 1% OsO4 and 30 min in 1% uranyl acetate at room temperature. Following dehydration in a
series of ethanol washes, tissue was embedded in EMbed 812 resin (EMS, Hateld PA) and polymerized at 60 °C
for 18h44. Ultrathin (90 nm) sections were cut using a Leica UC7 ultramicrotome (Bualo Grove, IL) and placed on
200 mesh copper grids and stained with lead citrate. Five micrographs of each specimen were randomly captured
across the muscle tissue using a JEOL 1400Plus TEM (Peabody, MA), operating at 80 kV with a magnication
of 42000×. ImageJ 1.46/Java 8 soware (National Institute of Health, Bethesda, MD, United States)45 was used to
manually quantify the sarcomere length.
Shear wave elastography imaging. Mice (NWT = 7, NKO = 7) were anesthetized with 1.5% isourane
and a mixture of O2/air (1:1) at an output of 0.7 L/min and placed in a prone position. Landmarks were dened
to orient the mice in a reproducible manner with the paw bent at a xed angle (Fig.8). Tendon and muscle
in the right hindlimb were imaged by the same operator with an ultrasound machine (Aixplorer MultiwaveTM
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System, Supersonic Imagine, Aix-en-Provence, France) using the SLH 20-6 linear transducer probe having the
specic characteristics: resolution: 38 µm, 2.38 cm footprint, 192 composite elements, eective bandwidth from
6 to 20 MHz.
For muscle imaging, the probe was placed on the surface of the hindlimb, which was shaved, parallel to
the Achilles tendon. A thin layer of water-soluble transmission gel was applied for optimal acoustic coupling.
To ensure that the probe was reproducibly placed, a visual control was performed using the B-mode image
where the bone and the heel, considered as referent tissues, were identied. Subsequently, the same box size
(10 mm× 10 mm) was dened for all acquisitions according to the length of the muscle and bone.
For tendon imaging, the probe was shied to clearly visualize the Achilles tendon on the B-mode image. Bone
and heel were also used as referent tissues and a box (10 mm× 10 mm) was placed in the same area for each
mouse. e Aixplorer MultiwaveTM System generates two types of waves that propagate within the tissue: a com-
pression wave that creates a high-quality B-mode image showing the anatomical structure within the hindlimb
(skin, muscle, tendon, bone), and a shear wave that provides a quantitative color-coded map of tissue elasticity
(Figs7 and 8). e setting parameters for B-mode image were:
==
μ
sp
ulse duration 600
Frequency
number _half_cycle
2
, gain
50%, pulse repetition frequency: 20 kHz and spatial resolution: 38 µm. To obtain mapping of elasticity with the
shear wave elastography (SWE) sequence the setting parameters were: musculoskeletal preset, resolution mode
enabled, tissue tuner at 1540 m/s, gain at 50%, dynamic range at 60 dB and the spatial resolution is 0.9 mm. e
SWE mode is based on the generation of localized acoustic radiation force by the probe in the tissue; the locally
excited tissue then produces waves which propagate transversally. In addition, the range of elasticity (Young’s
modulus) was set between 0 kPa and 180 kPa, which corresponds to a shear wave velocity range of 0–7.7 m/s.
Image analysis. In the B-mode image for US analysis (anatomical), a region of interest (ROI) was manually
drawn within the hindlimb muscle group (lateralis and medialis gastrocnemius, soleus, EDL: Extensor Digitorum
Longus, plantaris and tibialis) and within the surface Achilles tendon. ImageJ soware (NIH, Bethesda, USA) was
used to create the ROI. e ROI was then superimposed on B-mode images and elasticity images. Subsequently,
the ROI Manager Tool of ImageJ was used to measure the following parameters: mean and standard deviation
for echo intensity and stiness, contrast (ratio of the dierence (maximum - minimum) to the sum (maxi-
mum + minimum)) and coecient of variation (ratio of standard deviation to mean).
e ROI of B-mode images were also analyzed for texture analysis using Mazda 4.6 soware45, which includes
several texture-analysis methods. In this study, gray-level histogram parameters and co-occurrence parameters
were used. Principal Component Analyses (PCA) were performed to identify the most signicant texture param-
eters that were able to discriminate KO muscle/tendon from WT muscle/tendon.
Statistical analysis. e SystatTM V11 soware package (Systat Soware Inc., CA, USA) was used to per-
form all statistical analyses. Non parametric two-sample Kolmogorov-Smirnov tests were performed in order to
compare the elasticity values and the B-mode values between WT and TIEG1 KO mice. e statistical analysis
was considered signicant for P < 0.05.
Figure 8. Model indicating the orientation and placement of the ultrasound probe on the mouse hindlimb.
is image was drawn by Frank M. Corl from the department of Biomedical and Scientic Visualization, Mayo
Clinic. Used with permission of Mayo Foundation for Medical Education and Research, all rights reserved.
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Magnetic resonance imaging (MRI). Selection of skeletal muscles. MRI experiments were performed on
10 TIEG1 KO and 10 WT mice. For each mouse, muscles known to have dierent ber types46,47 (slow, mixed,
fast) were selected, namely tibialis anterior, plantaris, gastrocnemius lateralis, gastrocnemius medialis, soleus,
and EDL muscles.
MRI acquisition. Prior to MRI analysis, mice were anesthetized with 1.5% isourane and a mixture of O2/air
(1:1) at an output of 0.7 L/min. Respiration was monitored during the entire experiment, and body tempera-
ture was maintained at 37 °C using a warm-water circulation system. MRI was performed on a 9.4T horizon-
tal ultra-shielded superconducting magnet dedicated to small-animal imaging (94/20 USR Bruker Biospec,
Wissembourg, France) and was equipped with a 950 mT/m gradient set. A loop gap coil (10 mm inner diameter)
was used for both proton transmission and reception. Axial images of the TIEG1 KO and WT hindlimb muscles
were obtained using a gradient echo (Flash) sequence with the following parameters: TE/TR = 6 ms/252 ms, ip
angle = 20°, FOV size = 2 × 2 cm, matrix size = 256×256, bandwidth = 50 kHz, slice thickness = 570μm, to dis-
play 78 µm× 78 μm in plane resolution for a duration of 1 min (one accumulation).
Texture analyses. Acquired MR images were transferred to an external computer for data processing. ROIs
were manually drawn as large as possible within the selected muscles and subsequently analyzed using Mazda
software48 (Mazda 4.6, MRI analysis software, ©1998–2007 by P.M.Szczypinski), which includes several
texture-analyses methods. In this study, the greylevel histograms, composed of various parameters including
mean, standard deviation, skewness, kurtosis and dierent percentiles were used. In addition, a co-occurrence
matrix based on the following parameters: contrast, correlation, entropy, homogeneity, energy and run length
matrix with run length and grey level non uniformity, long and short run emphasis, wasapplied.
Data analyses. PCA (principalcomponent analyse) were performed to identify the most signicant texture
parameters able to discriminate TIEG1 KO muscles from the WT muscles with a condence level of 0.95. A
two-class (WT/TIEG1 KO) Hierarchical Ascending Classication (HAC) was performed with the most relevant
texture parameters. is was performed for all muscles: tibialis anterior, plantaris, gastrocnemius lateralis, gas-
trocnemius medialis, soleus and extensor digitarum longus. e global value (true positive + true negative/total
number of mice) was calculated for each HAC. is parameter represents the number of mice that are grouped
into WT or TIEG1 KO by means of their texture proles. A value superior to 60% is considered good.
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Acknowledgements
is work was carried out and funded in the framework of the Labex MS2T. It was supported by the French
Government, through the program “Investments for the future” managed by the National Agency for Research
(Reference ANR-11-IDEX-0004-02). is work was also supported by the National Institutes of Health (R01
DE14036 to JRH and MS). We would also like to thank Dr. omas Spelsberg for his excellent mentorship.
We thank the Institut Pasteur Core for experimental and technical support (ANR 10-EQPX-04-01 & FEDER
12,001,407 to FL). We acknowledge Frank Corl as the artist, who has created the illustration (Figure 8), as a work-
for-hire for Mayo Foundation for Medical Education and Research.
Author Contributions
S.F.B., M.K., J.R.H. and M.S. conceived the project. V.D., M.K., R.T., S.F.B., M.S., J.R.H., S.M., W.M., F.S., J.L.
and P.P. designed and performed the experiments. V.D., M.K., R.T., S.F.B., M.S., J.R.H., S.M., W.M., J.L. and P.P.
analyzed and interpreted data. V.D., M.K., R.T., S.F.B., M.S., J.R.H., S.M., W.M., F.L., J.M.C., J.L. and P.P. wrote and
revised the manuscript. All authors have read and approved the nal manuscript.
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... The stiffness of organisms-which is resistance to deformation in response to an applied force-varies by several orders of magnitude depending on the type of tissue, such as neurons (Young's modulus E ~ 10 2 Pa), muscle (~10 4 Pa), and bone (~10 9 Pa) [1][2][3]. Even inside the same tissue, the microscopic spatial distribution of stiffness is heterogeneous, with spatial scales ranging from 10 to 1000 μm [4][5][6][7]. For example, in pituitary gland tissue, soft and stiff areas are heterogeneously distributed at the cell scale (2-20 μm), and the spatial gradient of Young's modulus reaches ~10 kPa/μm [8]. ...
... These results imply that cells sense the general spatial modulation of stiffness and regulate directional cell movement. The soft and stiff domains in living tissues vary in size, ranging from single-cell size (10-100 μm) to multicellular size (100-1000 μm) [4][5][6][7]. Thus, as the next step, we focused on the dependency of cell shaping and migration on the cell-scale heterogeneity of the matrix stiffness [39]. ...
... For example, in the simulation, the peak of the probability distribution of the MSC position shifts slightly to the stiff region (Fig. 5 (e)). To reproduce the peak of the probability distribution at the boundary between the soft and stiff regions (Fig. 4 (d)), another term is required in Eq. (6). c E µ ∇ causes uniaxial elongation of the cell body along stripes. ...
Article
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In living tissues where cells migrate, the spatial distribution of mechanical properties, especially matrix stiffness, is generally heterogeneous, with cell scales ranging from 10 to 1000 μm. Since cell migration in the body plays a critical role in morphogenesis, wound healing, and cancer metastasis, it is essential to understand the migratory dynamics on the matrix with cell-scale stiffness heterogeneity. In general, cell migration is driven by the extension and contraction of the cell body owing to the force from actin polymerization and myosin motors in the actomyosin cytoskeleton. When a cell is placed on a matrix with a simple stiffness gradient, directional migration called durotaxis emerges because of the asymmetric extension and contraction of the pseudopodia, which is accompanied by the asymmetric distribution of focal adhesions. Similarly, to determine cell migration on a matrix with cell-scale stiffness heterogeneity, the interaction between cell-scale stiffness heterogeneity and cellular responses, such as the dynamics of the cell-matrix adhesion site, intracellular prestress, and cell shape, should play a key role. In this review, we summarize systematic studies on the dynamics of cell migration, shaping, and traction force on a matrix with cell-scale stiffness heterogeneity using micro-elastically patterned hydrogels. We also outline the cell migration model based on cell-shaping dynamics that explains the general durotaxis induced by cell-scale stiffness heterogeneity. This review article is an extended version of the Japanese article, Dynamics of Cell Shaping and Migration on the Matrix with Cell-scale Stiffness-heterogeneity, published in SEIBUTSU BUTSURI Vol. 61, p.152-156 (2021). Fullsize Image
... This suggests a convenient way to tune vegetable stiffness by controlling autoclaving time. 1.5 hours of autoclaving is sufficient for Chinese chives (Young's modulus reduced from average 772.5 kPa to 40.7 kPa, Fig. 5d) to gain a muscle-mimicking stiffness (10-40 kPa) 35 , and 20 min for mushroom (Young's modulus reduced from 34.5 kPa to 17.2 kPa, Fig. 5k). 20 min of autoclaving softened loofah from 3.5 kPa to 0.7 kPa ( Supplementary Fig. 5d), to match the mechanical range of fat tissue (0.6-2 kPa) 36 . ...
Article
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Cultured meat needs edible bio-scaffolds that provide not only a growth milieu for muscle and adipose cells, but also biomimetic stiffness and tissue-sculpting topography. Current meat-engineering technologies struggle to achieve scalable cell production, efficient cell differentiation, and tissue maturation in one single culture system. Here we propose an autoclaving strategy to transform common vegetables into muscle- and adipose-engineering scaffolds, without undergoing conventional plant decellularization. We selected vegetables with natural anisotropic and isotropic topology mimicking muscle and adipose microstructures respectively. We further adjusted vegetable stiffness by autoclaving, to emulate the mechanical properties of animal tissues. Autoclaved vegetables preserve rich cell-affinitive moieties, yielding a good cell culture effect with simplified processing. Autoclaved Chinese chive and Shiitake mushroom with anisotropic micro-patterns support the scalable expansion of muscle cells, improved cell alignment and myogenesis. Autoclaved isotropic loofah encourages adipocyte proliferation and lipid accumulation. Our engineered muscle- and fat-on-vegetables can further construct meat stuffing or layered meat chips. Autoclaved vegetables possess tissue-mimicking stiffness and topology, and bring biochemical benefits, operational ease, cost reduction and bioreactor compatibility. Without needing decellularization, these natural biomaterials may see scale-up applications in meat analog bio-fabrication.
... To generate elasticity maps of the isolated skeletal muscle independent of any neurohormonal activation, AFM measurements were performed as described previously [41]. Briefly, the experiments were performed via AFM (Nanowizard 4, JPK Instruments, Berlin, Germany) combined with an inverted optical microscope (AxioObserver ZI, Zeiss), driven by JPK Nanowizard software 6.0. ...
Article
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Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14 weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in the Gastrocnemius and soleus muscles were analyzed by LC/MS–MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius muscle were decreased after 14 weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5–2fold in the gastrocnemius muscle after 14 weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7 nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4 nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting.
... Recently, non-invasive imaging techniques such as elastography (Kammoun et al., 2019;Qin et al., 2013) make it possible to characterize such mechanical properties in vivo allowing for longitudinal assessment and ultimately non-invasive monitoring of pathological progression in the case of specific muscle diseases. In a previous study (Ternifi et al., 2020), ultrasound shear wave (SWE) elastography was performed on Klf10 KO mice and the passive mechanical behaviors of a group of muscles were measured as it was not possible to independently analyze slow and fast twitch muscles. ...
... In vitro testing of different muscle components is commonly performed with mechanical devices enabling the longitudinal stretching of an entire muscle [3], single fibers [4] or myofibrils [5][6][7]. Other experimental techniques, such as atomic force microscopy [8,9] and elastography [10] are used to provide the transverse local elastic modulus and longitudinal stiffness, respectively. ...
... Cell stiffness is associated with changes in function, increasing as myocytes differentiate to myotubes [26]. Although previous studies utilizing AFMbased indentation to quantify skeletal muscle stiffness have reported variable stiffness values for skeletal muscle, absolute stiffness values measured by AFM indentation protocols are strongly dependent on the experimental parameters, such as the model and the method used to analyze the results [28][29][30][31]. For example, cell fixation with PFA increases the measured stiffness [32]. ...
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Background Skeletal muscle development and regeneration depend on cellular fusion of myogenic progenitors to generate multinucleated myofibers. These progenitors utilize two muscle-specific fusogens, Myomaker and Myomerger, which function by remodeling cell membranes to fuse to each other or to existing myofibers. Myomaker and Myomerger expression is restricted to differentiating progenitor cells as they are not detected in adult myofibers. However, Myomaker remains expressed in myofibers from mice with muscular dystrophy. Ablation of Myomaker from dystrophic myofibers results in reduced membrane damage, leading to a model where persistent fusogen expression in myofibers, in contrast to myoblasts, is harmful. Methods Dox-inducible transgenic mice were developed to ectopically express Myomaker or Myomerger in the myofiber compartment of skeletal muscle. We quantified indices of myofiber membrane damage, such as serum creatine kinase and IgM+ myofibers, and assessed general muscle histology, including central nucleation, myofiber size, and fibrosis. Results Myomaker or Myomerger expression in myofibers independently caused membrane damage at acute time points. This damage led to muscle pathology, manifesting with centrally nucleated myofibers and muscle atrophy. Dual expression of both Myomaker and Myomerger in myofibers exacerbated several aspects of muscle pathology compared to expression of either fusogen by itself. Conclusions These data reveal that while myofibers can tolerate some level of Myomaker and Myomerger, expression of a single fusogen above a threshold or co-expression of both fusogens is damaging to myofibers. These results explain the paradigm that their expression in myofibers can have deleterious consequences in muscle pathologies and highlight the need for their highly restricted expression during myogenesis and fusion.
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Cultured meat needs edible bio-scaffolds that provide not only an appropriate growth milieu for muscle and adipose cells, but also biomimetic microstructures to sculpt tissue architecture. Current meat-engineering technologies struggle to achieve scalable cell production, efficient cell differentiation, and tissue maturation in one single culture system. Here we propose a simple strategy to transform common vegetables into muscle- and adipose-engineering scaffolds, without undergoing conventional plant decellularization. We selected vegetables with natural anisotropic and isotropic topology mimicking muscle and adipose tissue microstructures respectively. We further adjusted vegetable stiffness by autoclaving to emulate the mechanical characteristics of animal tissues. Our selected anisotropic Chinese chive and Shiitake mushroom stem supported the scalable expansion of murine myoblasts and porcine myosatellite cells, and improved cell alignment and subsequent myogenesis. We also chose loofah with isotropic topology to construct lipid-containing fat tissues. Our cultured muscle and fat tissues can be assembled into meat stuffing or layered meat chips. Our results highlight naturally micro-patterned vegetables, without needing decellularization, as promising meat-engineering scaffolds. These edible biomaterials are simple to fabricate, low-cost, and possess tissue-mimicking topology and stiffness. Vegetable scaffolds may see broad applications in the bio-fabrication of meat analogs, muscle-powered robots, and transplantable tissue patches.
Article
Volumetric muscle loss (VML), which refers to a composite skeletal muscle defect, most commonly heals by scarring and minimal muscle regeneration but substantial fibrosis. Current surgical interventions and physical therapy techniques are limited in restoring muscle function following VML. Novel tissue engineering strategies may offer an option to promote functional muscle recovery. The present study evaluates a colloidal scaffold with hierarchical porosity and controlled mechanical properties for the treatment of VML. In addition, as VML results in an acute decrease in insulin-like growth factor 1 (IGF-1), a myogenic factor, the scaffold was designed to slowly release IGF-1 following implantation. The foam-like scaffold is directly crosslinked onto remnant muscle without the need for suturing. In situ 3D printing of IGF-1-releasing porous muscle scaffold onto VML injuries resulted in robust tissue ingrowth, improved muscle repair, and increased muscle strength in a murine VML model. Histological analysis confirmed regeneration of new muscle in the engineered scaffolds. In addition, the scaffolds significantly reduced fibrosis and increased the expression of neuromuscular junctions in the newly regenerated tissue. Exercise training, when combined with the engineered scaffolds, augmented the treatment outcome in a synergistic fashion. These data suggest highly porous scaffolds and exercise therapy, in combination, may be a treatment option following VML.
Article
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Extracellular matrix stiffness is a major regulator of cellular states. Stiffness‐sensing investigations are typically performed using cells that have acquired “mechanical memory” through prolonged conditioning in rigid environments, e.g., tissue culture plastic (TCP). This potentially masks the full extent of the matrix stiffness‐driven mechanosensing programs. Here, a biomaterial composed of 2D mechanovariant silicone substrates with simplified and scalable surface biofunctionalization chemistry is developed to facilitate large‐scale cell culture expansion processes. Using RNA sequencing, stiffness‐mediated mechano‐responses of human tendon‐derived stromal cells are broadly mapped. Matrix elasticity (E.) approximating tendon microscale stiffness range (E. ≈ 35 kPa) distinctly favors transcriptional programs related to chromatin remodeling and Hippo signaling; whereas compliant stiffnesses (E. ≈ 2 kPa) are enriched in processes related to cell stemness, synapse assembly, and angiogenesis. While tendon stromal cells undergo dramatic phenotypic drift on conventional TCP, mechanovariant substrates abrogate this activation with tenogenic stiffnesses inducing a transcriptional program that strongly correlates with established tendon tissue‐specific expression signature. Computational inference predicts that AKT1 and ERK1/2 are major stiffness‐sensing signaling hubs. Together, these findings highlight how matrix biophysical cues may dictate the transcriptional identity of tendon cells, and how matrix mechano‐reciprocity regulates diverse sets of previously underappreciated mechanosensitive processes in tendon fibroblasts.
Article
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It is a long held belief that maximal joint range of motion (ROM) is restricted by muscle tension. However, it exists indirect evidence suggesting that this assumption may not hold true for some joint configurations where non-muscular structures, such as the peripheral nerves, are stretched. Direct evidences are lacking. This study aimed to determine whether a static stretching aiming to load the sciatic nerve without stretch within plantar flexors is effective to: (i) alter nerve stiffness; and (ii) increase the ankle's maximal ROM. Passive maximal ankle ROM in dorsiflexion was assessed with the hip flexed at 90° (HIP-flexed) or neutral (HIP-neutral, 0°). Sciatic nerve stiffness was estimated using shear wave elastography. Sciatic nerve stretching induced both a 13.3 ± 7.9% (P < 0.001) decrease in the nerve stiffness and a 6.4 ± 2.6° increase in the maximal dorsiflexion ROM assessed in HIP-flexed. In addition, the decrease in sciatic nerve stiffness was significantly correlated with the change in maximal ROM in dorsiflexion (r = -0.571, P = 0.026). These effects occurred in the absence of any change in gastrocnemius medialis and biceps femoris stiffness, and ankle passive torque. These results demonstrate that maximal dorsiflexion ROM can be acutely increased by stretching the sciatic nerve, without altering the muscle stiffness.
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There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.
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Purpose While several studies demonstrated the occurrence of intermuscular mechanical interactions, the physiological significance of these interactions remains a matter of debate. The purpose of this study was to quantify the localized changes in the shear modulus of the gastrocnemius lateralis (GL), monoarticular dorsi- and plantar-flexor muscles induced by a change in knee angle. Method Participants underwent slow passive ankle rotations at the following two knee positions: knee flexed at 90° and knee fully extended. Ultrasound shear wave elastography was used to assess the muscle shear modulus of the GL, soleus [both proximally (SOL proximal) and distally (SOL distal)], peroneus longus (PERL), and tibialis anterior (TA). This was performed during two experimental sessions (experiment I: n = 11; experiment II: n = 10). The shear modulus of each muscle was compared between the two knee positions. Results The shear modulus was significantly higher when the knee was fully extended than when the knee was flexed (P < 0.001) for the GL (averaged increase on the whole range of motion: + 5.8 ± 1.3 kPa), SOL distal (+ 4.5 ± 1.5 kPa), PERL (+ 1.1 ± 0.7 kPa), and TA (+ 1.6 ± 1.0 kPa). In contrast, a lower SOL proximal shear modulus (P < 0.001, − 5.9 ± 1.0 kPa) was observed. Conclusion As the muscle shear modulus is linearly related to passive muscle force, these results provide evidence of a non-negligible intermuscular mechanical interaction between the human lower leg muscles during passive ankle rotations. The role of these interactions in the production of coordinated movements requires further investigation.
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The goal of the present study were (1) to investigate the pathological characteristics of gastrocnemius muscle (GM) and quantitatively assess GM tissue stiffness in rat models with spinal cord injury (SCI) and (2) to explore the correlation between pathological characteristics changes and Young’s modulus value of GM. 24 Sprague Dawley male rats were allocated into normal control groups and SCI model subgroups, respectively. GM stiffness was assessed with shear wave sonoelastography technology. All GMs were further analyzed by pathological examinations. GM weights were decreased, the ratio of type I fibers was decreased, and the ratio of type II fibers was increased in the GM in the model group. MyHC-I was decreased, while MyHC-II was increased according to the electrophoretic analysis in model subgroups. The elastic modulus value of GM was increased in the model group. A significant negative correlation was found between Young’s modulus value of GM and the ratio of type I fibers of GM in model subgroup. Our studies showed that the stiffness of GM is correlated with pathological characteristics during the initial stages of SCI in rats. We also identified shear wave sonoelastography technology as a useful tool to assess GM stiffness in SCI rat models.
Article
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We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.
Article
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Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.
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Deformation of skeletal muscle in the proximity of bony structures may lead to deep tissue injury category of pressure ulcers. Changes in mechanical properties have been proposed as a risk factor in the development of deep tissue injury and may be useful as a diagnostic tool for early detection. MRE allows for the estimation of mechanical properties of soft tissue through analysis of shear wave data. The shear waves originate from vibrations induced by an external actuator placed on the tissue surface. In this study a combined Magnetic Resonance (MR) compatible indentation and MR Elastography (MRE) setup is presented to study mechanical properties associated with deep tissue injury in rats. The proposed setup allows for MRE investigations combined with damage-inducing large strain indentation of the Tibialis Anterior muscle in the rat hind leg inside a small animal MR scanner. An alginate cast allowed proper fixation of the animal leg with anatomical perfect fit, provided boundary condition information for FEA and provided good susceptibility matching. MR Elastography data could be recorded for the Tibialis Anterior muscle prior to, during, and after indentation. A decaying shear wave with an average amplitude of approximately 2 μm propagated in the whole muscle. MRE elastograms representing local tissue shear storage modulus Gd showed significant increased mean values due to damage-inducing indentation (from 4.2 ± 0.1 kPa before to 5.1 ± 0.6 kPa after, p<0.05). The proposed setup enables controlled deformation under MRI-guidance, monitoring of the wound development by MRI, and quantification of tissue mechanical properties by MRE. We expect that improved knowledge of changes in soft tissue mechanical properties due to deep tissue injury, will provide new insights in the etiology of deep tissue injuries, skeletal muscle damage and other related muscle pathologies.
Article
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As transforming growth factor (TGF)-β inducible early gene-1 is highly expressed in skeletal muscle, the effect of TIEG1 gene deletion on the passive mechanical properties of slow and fast twitch muscle fibers was analyzed. Twenty five muscle fibers were harvested from soleus (Sol) and extensor digitorum longus (EDL) muscles from TIEG1⁻/⁻ (N = 5) and control (N = 5) mice. Mechanical tests were performed on fibers and the dynamic and static stresses were measured. A viscoelastic Hill model of 3rd order was used to fit the experimental relaxation test data. In parallel, immunohistochemical analyses were performed on three serial transverse sections to detect the myosin isoforms within the slow and fast muscles. The percentage and the mean cross sectional area of each fiber type were calculated. These tests revealed a significant increase in the mechanical stress properties for the TIEG1⁻/⁻ Sol fibers while a significant decrease appeared for the TIEG1⁻/⁻ EDL fibers. Hill model tracked the shape of the experimental relaxation curve for both genotypes and both fiber types. Immunohistochemical results showed hypertrophy of all fiber types for TIEG1⁻/⁻ muscles with an increase in the percentage of glycolytic fibers (IIX, and IIB) and a decrease of oxidative fibers (I, and IIA). This study has provided new insights into the role of TIEG1, known as KLF10, in the functional (SoltypeI: more resistant, EDLtypeIIB: less resistant) and morphological (glycolytic hypertrophy) properties of fast and slow twitch skeletal muscles. Further investigation at the cellular level will better reveal the role of the TIEG1 gene in skeletal muscle tissue.
Article
Introduction: TIEG1 is a transcription factor that is highly expressed in skeletal muscle. The purpose of this study was to characterize the structural properties of both fast- (EDL) and slow- (soleus) twitch muscles in the hind-limb of TIEG1 deficient (TIEG1(-) /(-) ) mice. Methods: Ten slow and 10 fast muscles were analyzed from TIEG1(-) /(-) and WT mice using MRI texture (MRI-TA) and histological analyses. Results: MRI-TA could discriminate between WT slow and fast muscles. Deletion of the TIEG1 gene led to changes in the texture profile within both muscle types. Specifically, muscle isolated from TIEG1(-) /(-) mice displayed hypertrophy, hyperplasia, and a modification of fiber area distribution. Discussion: We demonstrate that TIEG1 plays an important role in the structural properties of skeletal muscle. This study further implicates important roles for TIEG1 in the development of skeletal muscle and suggests that defects in TIEG1 expression and/or function may be associated with muscle disease. This article is protected by copyright. All rights reserved.