Exploring the Role of Mechanical Force on Tendon Development in vivo Model: a Scoping Review

Abstract

The tendon transmits the skeletal muscle-generated forces to bones and drives the joint motion. While extensive research has indicated the fundamental role of mechanical forces in tendon development during embryonic and postnatal phases, a comprehensive summary regarding the mechanical forces applied to tendons is lacking. This scoping review aimed to summarize the current knowledge regarding the effect of mechanical forces on tendon development. The electronic database search using PubMed was performed in October 2021 and yielded 586 articles, of which 16 articles met the prespecified inclusion criteria. We defined the mechanical force in terms of muscle existence, muscle contraction, or weight-bearing locomotion and identified methodological heterogeneity in applying mechanical force to tendons. Despite the lack of consensus regarding standard intervention methods, the majority of the mechanical interventions were used to regulate the expression or synthesis of well-known growth factors required for tendon development, especially the Scx expression. Our results provide a novel point of view to build future research about mechanobiology in tendon development.

Full text

Page 1/19
Exploring the Role of Mechanical Force on Tendon
Development in vivo Model: a Scoping Review
Yuna Usami
Graduate School of Saitama Prefectural University https://orcid.org/0000-0002-3757-8362
Hirotaka Iijima
Nagoya University
Takanori Kokubun (  kokubun-takanori@spu.ac.jp )
Saitama Prefectural University https://orcid.org/0000-0001-6676-2356
Systematic Review
Keywords: Tendon development, mechanobiology, Scleraxis, animal model, movement, muscle contraction, scoping review
Posted Date: May 23rd, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1681832/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

Page 2/19
Abstract
The tendon transmits the skeletal muscle-generated forces to bones and drives the joint motion. While extensive research has
indicated the fundamental role of mechanical forces in tendon development during embryonic and postnatal phases, a
comprehensive summary regarding the mechanical forces applied to tendons is lacking. This scoping review aimed to
summarize the current knowledge regarding the effect of mechanical forces on tendon development. The electronic database
search using PubMed was performed in October 2021 and yielded 586 articles, of which 16 articles met the prespecied
inclusion criteria. We dened the mechanical force in terms of muscle existence, muscle contraction, or weight-bearing
locomotion and identied methodological heterogeneity in applying mechanical force to tendons. Despite the lack of
consensus regarding standard intervention methods, the majority of the mechanical interventions were used to regulate the
expression or synthesis of well-known growth factors required for tendon development, especially the
Scx
expression. Our
results provide a novel point of view to build future research about mechanobiology in tendon development.
Introduction
Tendons are connective tissue that transmits the forces generated by skeletal muscles to bones and drive joint movement.
They are repeatedly exposed to strong mechanical forces, such as the tensional forces generated due to muscle contraction
and endure mechanical forces throughout development. However, the current knowledge is not integrated into how
mechanical forces modulate tendon development from embryonic to early postnatal development phase.
In tendon development, general biological processes; formation, differentiation, migration, and pattern formation have
promoted understanding. Some transcriptional factors were identied in this process, but
Scleraxis
(
Scx
) is the most
representative marker for tendon maturation.
Scx
is a basic helix-loop-helix (bHLH) transcription factor, Schweitzer et al.
reported that
Scx
expression marks tendons maturation from the early progenitor stage to the formation of mature tendons
(Schweitzer et al., 2001).
Scx
is used as a detective target to evaluate the tendon maturation during the early stage of tendon
development. By observing this expression, we can gauge the progress of the tendon development. Throughout the early
stage of development,
Scx
was detected as a highly specic marker for tendons and ligaments in chicks, mice, and zebrash
(J. W. Chen and J. L. Galloway, 2014; R Schweitzer et al., 2001).
In recent years, ndings of biological processes put the spotlight on mechanical forces that can regulate and control key
cellular processes and induce molecular responses during the development process. Many researchers reported the novel
knowledge of tendon development using some species as a tendon development model. The chicks paralyzed by injecting d-
tubocurarine did not show any changes in the tendon cells, collagen, and immature elastic bers (C Beckham et al., 1977).
Transgenic mice model (e.g., mdg or spd mice) showed undeveloped tendons, which were detected using ScxGFP (Huang et
al., 2015). Similarly, paralyzed chick embryos showed reduced
Scx
expression, elastic modulus, and
lysyl oxidase
(
LOX
)
activity (Havis et al., 2016; X S Pan et al., 2018). However, these research on tendon development were confounded by some
factors such as species, age, intervention type, or tendon detection methods. Therefore, while the requirement of mechanical
forces for tendon development has been researched extensively, consensus regarding the amount or type of mechanical
forces to be applied to embryonic or postnatal animal tendons is lacking. Lack of consensus about the methods to modulate
the mechanical stress and interpretation of these research in tendon development induces confusion about the role and
effect of mechanical force on the molecular reactions in tendon development. A strict description of the mechanical force is
required to provide an overview or map of the evidence in this eld. Summarizing the available evidence is the logical rst
step toward establishing a consensus regarding the mechanical force used in the eld of tendon mechanobiology.
This scoping review aimed to (1) summarize the characteristics of the current animal models used for determining the
inuence of mechanical force on tendons and (2) dene the mechanical force in current literature. Furthermore, we assessed
(3) the effect of mechanical force by determining how some types of mechanical forces affect tendon development. This
review focuses on
in vivo
models because of the critical relationship between tendons and muscles or bones that is required
for utilizing mechanical force.

Page 3/19
Results
Manual database search yielded 586 articles (Figure 1). After the rst screening stage of titles and abstracts, 84 articles were
selected for full-text review. After the full-text screening, 68 articles were excluded, leaving 16 articles that met the inclusion
criteria. They were excluded because they failed to assess papers, were not animal models (e.g., human sample), did not
include interventions (e.g., observation of normal tendon development over time and gene modication model of the tendon),
and did not have any
in vivo
model (e.g.,
in vitro
and
ex vivo
), were not related to natural development (e.g., healing,
engineering research), did not match the age (within four weeks from the day of birth), and the outcome did not include
tendons (e.g., only muscle-tendon junction or enthesis) (Figure 1).
Experimental animal models were categorized into four species
The intervention methods and tendon development process vary with animal species, so we categorized by animal species.
Table 1 shows the characteristics of the experimental model in the included studies. Four different species were used; chicks
(n=13), mice (n=5), zebrash (n=3), and rats (n=2). The age of the chick models was between stage (st)18-19; it was
converted into the embryonic day E2-E3 (2 days from intervention at stage 4 and 5 (Hamburger and Hamilton, 1951)). Mice
aged E12.5 to P0 and rats aged P0 to P10 were used as models. Zebrash from 48 h post-fertilization (hpf) to 98 hpf were
used as models. Most studies used embryos, and only three studies used postnatal animals.
This scoping review identied interventions that decrease and increase the mechanical force to understand the role of
mechanobiology in tendon development. The interventions that reduced mechanical force involved 14 approaches; in
contrast, only three ways were used to increase mechanical force. The intervention type for reducing mechanical force was
categorized into ve groups for various animal intervention methods: transgenic, surgery, drug treatment, locomotion, and
added stimulation (Table 2). Five studies used multiple models. Three studies combined interventions that changed the
mechanical force and techniques that inhibited specic signaling.
In situ
hybridization (section or whole mount) was
frequently used to analyze tendons. Electronic microscopy, immunohistochemistry, transmission electron microscopy, and
mechanical testing were used for assessing the tendons. The other methods are shown in Table S1. Some biological markers
tested the effect of mechanical force. The most frequent marker was
Scx
, which is common in many studies.
Scx
mutant
mice showed a dramatic defect in tendon differentiation.
Scx
is recognized as required for tendon differentiation and
formation(Murchison et al., 2007). So, downregulated
Scx
expression in the intervention model means inhibiting tendon
development. GFP uorescence,
in situ
hybridization (ISH), and polymerase chain reaction (PCR) can also detect
Scx
expression.
The lack of consensus regarding standard intervention methods
We categorized types of intervention because that improved to catch the sight of dening mechanical force. Types of
intervention were identied as ones that decreased or increased the mechanical force. The intervention type of decreased
mechanical force was divided intofour groups. The transgenicgroup included several muscle-decient factor mutant models
that included
Myf5–/– Myod1–/–
double mutants(Brent et al., 2005; J. W. Chen and J. L. Galloway, 2014),
Pax3
(splotch or
splotch delayed) mutants (Schweitzer et al., 2001), muscular dysgenesis (
mdg
) mutant(Huang
et al.
, 2015), and voltage-
dependent L-type calcium channel subtype bata-1 (
cacnb1)
mutant(A. Subramanian et al., 2018). Full-length mRNA
encoding codon-optimized a-bungarotoxin (aBtx) was injectedinto the muscle(A. Subramanian
et al.
, 2018). Most studies
used the mouse model, except for the
cacnb1
mutant and aBtx mRNA intervention, which were in zebrash.
Myf5–/–
Myod1–/– 
double mutants do not contain any muscle progenitors, resulting in a muscle-less limb(Kassar-Duchossoy et al.,
2004).
Pax3
is required for the establishment of muscle progenitor cells in the limb, and mutations in
Pax3
(Splotch) and
Pax3
(Splotch-delayed) cause a severe defect in limb muscle formation(E Bober et al., 1994).
mdg
is an autosomal recessive
lethal mutation that results in the contraction of skeletal muscles(B A Adams and Beam, 1990). Full-length mRNA encoding
codon-optimized aBtx was injected in zebrash to prevent skeletal muscle contractions(Swinburne et al., 2015). The limb
was paralyzed in the
cacnb1
homozygous mutant(Zhou et al., 2006).

Page 4/19
Surgical procedures can remove muscle or neural tube in a region-specic manner. The surgery group experimented with
coelomic wing graft(F. Edom-Vovard et al., 2002; Kardon, 1998), neural tube ablation(F. Edom-Vovard
et al.
, 2002), and
dermomyotome removal(Ava E Brent et al., 2003)in chicks. Coelomic wing graft was performed in the lateral plate areas
corresponding to the future wing buds, or migratory myogenic cells were isolated from chick embryos. This weakened the
wing muscle. Neural tube ablation was performed on embryos before the exit of ventral root bers to produce complete
aneural wings. Dermomyotome removal is a method of surgically removing the dermomyotome before myotome formation,
making it muscle-less.
In addition, interventions using four types of drugs were also performed. They were decamethonium bromide (DMB)(Havis
et
al.
, 2016; J A Germiller et al., 1998; X S Pan
et al.
, 2018), pancuronium bromide (PB)(Havis
et al.
, 2016; X S Pan
et al.
, 2018),
d-tubocurarine(C Beckham
et al.
), and botulinum toxin(W.G.Hopkins, 1984). Botulinum toxin was used in mice, while the
other interventions were used for chicks. DMB prevented the effects of acetylcholine at the neuromuscular junction and
depolarized the end-plate region, inducing rigid paralysis(Paton and Zaimis, 1951). PB blocked the response to acetylcholine
and is characteristic of blocking drugs of the non-depolarizing type. Thus, chicks treated with doses of PB were paralyzed
and accid(W R Buckett et al., 1968). D-tubocurarine is an acetylcholine receptor antagonist that inhibits neuromuscular
activity(L T Landmesser and Szente, 1986; Paton and Zaimis, 1949). Botulinum toxin, an acetylcholine receptor antagonist,
decreased the amount of acetylcholine interacting with receptors, thereby reducing muscle contraction and motility(Pittman
and Oppenheim, 1978); PB, d-tubocurarine, and botulinum toxin induced accid paralysis.
In contrast, mechanical force was increased by injecting a group of drugs, which included only 4-aminopyridine (4-AP)(X S
Pan
et al.
), a neuromuscular blocking drug that blocks the potassium channels in neurons. When applied to chicks, it
stimulated the release of the neurotransmitter, acetylcholine (ACh) and enhanced its availability at the synaptic
cleft(Heywood et al., 2005). Therefore, the 4-AP model induces high-frequency movement and hypermobility(X S Pan
et al.
).
The locomotion group

focused on rodent locomotion (spinal cord(S. K.Theodossiou et al., 2021)) in the postnatal phase of
rats. The developing locomotor behavior during the postnatal period was believed to increase mechanical loading for limbs.
Rats with spinal cord transection did not show complete weight-bearing locomotion; hence, they were used as model systems
to disrupt locomotor development in neonates. The postnatal rats showed changes in spontaneous posture and locomotion
during the early postnatal week(H. E. Swann and M. R. Brumley, 2019). This locomotion development affects the limb
mechanical loading(S. K. Theodossiou et al., 2019).
The last group includes the electrically stimulated model(A. Subramanian
et al.
) that induces muscle contractions in
zebrash. This intervention was applied to both normal and paralyzed muscles to increase mechanical force.
Lack of mechanical force inhibited tendon maturation
We summarized the model to investigate the role of mechanical force in tendon development throughout
Scx
expression.
Tendon development of individual models is described below and summarized inTable 3 and Table 4.The most standard
method to control the mechanical force of the tendon is to arrange the force depending on the muscles. Many researchers
reported the way to modulate muscle function using a variable animal model. The dened mechanical force is roughly
divided into three types. One of the methods is the Muscle-less model using a surgical extraction,
Pax3
mutant mice model.
The others are muscle paralysis models that operate muscle contraction, such as DMD injection. The third is weight-bearing.
This section showed how each type of mechanical force affects the expression of
Scx
.
At rst, we show the relationship between muscle-less and
Scx
expression.
Myod1 Myf5
-decient zebrash expressed
Scx
53-
58 hpf, but not at 72 hpf(J. W. Chen and J. L. Galloway, 2014).
Myf5–/– ; Myod1–/–
double mutant muscle-less mouse
embryos survived
Scx
expression in the limbs but not in the epaxial region at E13.5(Brent
et al.
, 2005). In
Pax3
mutant
embryos, which were muscle-less similar to the
Myf5–/– Myod1–/–
double mutant,
Scx
expression in limbs was not affected
at E12.5(R Schweitzer
et al.
, 2001). In the E16.5
Pax3
mutant, Scx-GFP was not detected in the zeugopod tendon. However, it
persisted in the autopod tendon segments at E18.5(Huang
et al.
, 2015). At E16.5, Scx GFP was observed in the zeugopod of

Page 5/19
the
mdg
mutant. Moreover, muscle-lessness can also be induced by surgical manipulation. Induction of
Scx
was not
observed after surgically removing the dermomyotome before myotome formation(Ava E Brent
et al.
, 2003). Coelomic wing
graft surgery indicated that tenascin reduced the proximal and intermediate tendon but rescued the distal limb(Kardon,
1998). Another study on coelomic wing graft surgery involving chick embryos at E2 showed
Scx
expression at the forearm at
E6, but not at E10(F. Edom-Vovard
et al.
, 2002).
The tested model the effect of muscle contraction, in chick E8 and E9, the neural tube ablated embryos showed
downregulation of
tenascin
and
Scx
in tendons. Although
Scx
expression in the forearm at E10 (or E9) was limited in both
models, the digit expressed
Scx
at E10 (F. Edom-Vovard
et al.
, 2002). The expression of factors related to tendon
development, such as
Scx
and the
transforming growth factor-beta-2
(
Tgf-β2
), decreased at E6.5 in chicks paralyzed rigidly
using DMB, while
Tenomodulin
(
Tnmd
) expression decreased at E7.5(Havis
et al.
, 2016). Through E18, the tendons became
uniformly smaller and correlated with reduced chick movement due to paralysis(J A Germiller
et al.
, 1998). In chicks with
accid paralysis induced by PB,
Scx
expression decreased at E6.5 (Havis
et al.
, 2016) and
LOX
activity was reduced at HH43
(E17) (X S Pan
et al.
, 2018). The length of the muscles reduced in botulinum toxin-injected mouse, and the tendons were
longer than the muscles(W.G.Hopkins, 1984). The brocartilaginous area and elastic vinculum were not formed in chick
injected with d-tubocurarine. However, the tendon cells and collagen in immature elastic tendon bers did not change(C
Beckham
et al.
, 1977). The increased mechanical force in the 4-AP model increased the elastic modulus. Zebrash
stimulated by electronic stimuli to restore mechanical force reduced by aBtx-injection showed increase in axial tenocyte
projection length compared to that observed in aBtx-injected only animals. Several factors, such as
Thrombospondin 4b
(
Tsp4b
),
TGFb-induced protein
(
Tgfbip
), and
connective tissue growth factor a
(
Ctgfa2
), were upregulated to control levels by
electronic stimulation compared to that observed in aBtx-injected animals(A. Subramanian
et al.
, 2018).
The last is weight-bearing model. Theodossiou et al. reportedthe structural properties and cross-sectional area of the weight-
bearing Achilles tendon at P10 were higher than those of the non-weight-bearing phases P1 and P5 in the rat model(S. K.
Theodossiou
et al.
, 2019). But there is no research measuring
Scx
expression using the weight-bearing model.
Intervention for detecting molecular mechanism
In the above section, we focused on
Scx
expression. Moreover, we summarize several markers regulated by mechanical force
along with
Scx
. One study conrmed the relationship between expression and molecular mechanism by reimplanting a
source of
broblast growth factor 4
(
Fgf4
) in the aneural chick limbs and muscle-less chick wing(Havis
et al.
, 2016).
Fgf4
was not expressed in aneural and muscle-less wings; hence, reimplanting was performed to analyze the possible effects of
Fgf4
removal from aneural muscles on tendon markers.
Scx
and tenascin were downregulated in the aneural limbs and
muscle-less wings. Consistent with this, both models showed that grafts of
Fgf4
cells rescued the expression of the tendon-
associated molecules,
Scx
and
tenascin
. Another study tested whether Fgf rescued tendon gene expression without muscle
contraction in DMD-injected chick. mFgf4/ RCAS-producing cells were grafted into forelimb buds. While immobilization
following DMD induced a drastic decrease in
Scx
,
Tnmd
, and
Thrombospondin 2
(
Thbs2
) expression, the mFgf4-paralyzed-
limbs signicantly upregulated
Scx
,
ETS translocation variant 4
(
Etv4
) also known as
polyoma enhancer activator 3
(
Pea3
),
and
sprout 2
(
Spry2
). The relative mRNA levels of
Tnmd
,
Tgf2
,
and Tgfβ3
did not change under this condition. These results
suggested that the downregulation of several genes may be a molecular effect induced by the muscle-less state or inhibition
of contraction. The muscle may require both mechanical and molecular aspects.
Discussion
This scoping review summarized the effects of mechanical stimulation on tendon development in animal models. We
dened mechanical force in terms of muscle contraction, muscle existence, and weight-bearing (Fig.2). However, we
observed methodological heterogeneity in the application of mechanical force to tendons. Hence, the establishment of a
consensus for mechanical intervention is required. We showed to categorize the “mechanical force” will contribute to an
appropriate understanding of the effect of mechanical force based on muscle contraction or body movement
in vivo
to the

Page 6/19
tendon development. This knowledge of the detailed role and effect of mechanical force helps to reveal the mechanism of
tendon developmental mechanobiology.
What types of mechanical forces are dened and used in tendon
development?
Mechanical force is dened generally in terms of muscle existence or muscle contraction. Muscle loss or muscle paralysis is
considered a reduction of mechanical force. Few studies have shown that postnatal locomotion works as a mechanical force
in rodent limbs. This means weight-bearing associated with the locomotion increases the mechanical force on the limbs
compared to non-weight-bearing limbs. In contrast, the spinal cord transection was used as a model that couldn’t locomote
with weight-bearing for limbs.
These eld studies mostly used chick models, in addition to mouse, zebrash, and rat models. The chick embryo is amenable
to drug and surgical treatment(X S Pan et al., 2018). As chick embryos are present in eggs, they are easy to manipulate. The
mouse model is an established mammalian model organism. In addition, the mouse has been developed as a genetic model,
and it is a widely-used tool for targeting specic genes(Huang et al., 2015). The zebrash is also widely used as a genetically
modied model(A. Subramanian et al., 2018). The rat model can be used to observe limb movements after birth; their bodies
are bigger than those of mice and are hence easy to operate(S. K.Theodossiou
et al.
, 2021). Each animal has each strong
point to identify the role of mechanical force as the experiment model. Although these features vary the way of intervention
to operating mechanical force, each method has each limitation. In the genetic and drug model, muscle function can be
controlled easily, although the side effects of the loss of gene or injected drug on the tendon cannot be excluded. The weight-
bearing model cannot distinguish between loss of loading and only loss of muscle contraction due to spinal cord injury.
In this review, we targeted animals from the embryonic to postnatal stages. Many studies that we collected, targeted
embryos; conversely, studies on postnatal stage were few. Further studies are required to consider the effect of the
environment of each embryo on movement. For example, mice embryos exist with multiple litters in the womb, while the chick
grows in a rigid eggshell. These may limit body movements depending on the spatial limit. On the contrary, postnatal animals
can move without environmental limitations. We showed that muscle contraction and existence might play a key role as
force generators to the tendon, and that future studies on the function of mechanical force in the postnatal phase will be
important. Another important discovery is that mechanical force can be increased in three ways, that are muscle existence,
contraction, on load. Decreased mechanical force command a majority in the reports. In future studies, the model of
increased mechanical force should be developed. To compare decreased with increased mechanical force model, more
supported the idea that tendon needs mechanical force for development. We can judge the role of mechanical force
comprehensively by comparing the effects of enhanced and reduced intervention on tendon development.
What types of mechanical forces affect tendon development?
Many studies using muscle-less and inhibition of the muscle contraction models showed that the effect on tendon
development was subtle immediately after inducing tendon cells, but that the levels of factors contributing to tendon
development, such as
Scx
and tenascin, decreased or were absent in the late embryonic period. The role of other factors
varies with studies, and it is too early to arrive at conclusions. The methods used for evaluating tendon development are
confounded. This lack of common indicators has hindered advancement in the eld. Tenascin is one of the most classical
factors for detecting tendons; however, it is not a specic tendon marker. Identication of
Scx
as a tendon progenitor marker
maked a signicant breakthrough in research on tendon development. In the current study,
Scx
has been used to test the
effect of mechanical force on tendon. No one showed upregulating
Scx
expression in the animal model of reduced
mechanical force in early embryos (mice;~E12.5, chick; ~E6),
Scx
expression was not affected by the reduction in mechanical
force. Few studies reported that loss of muscle progenitors inhibited
Scx
expression (Ava E Brent et al., 2003; Brent et al.,
2005). Embryonic movement begins on E12.5 in mice (Shinoda, 1999), and at developmental day 2−6 in chick embryos (Wu
et al., 2001). After the beginning of limb movement, most studies showed decrease in
Scx
expression, with few exceptions.

Page 7/19
We suggest that investigating common
Scx
expression in multiple studies will be valuable, as no controlled denition of
“tendon development” is lacking.
We obtained not only trends but also contradictory results. While muscle-less
Pax3
mice did not express Scx-GFP at E16.5,
Scx-GFP expression persisted in
mdg
limbs, suggesting that the existence of muscles generated mechanical force as
connections from muscle to bone; in addition, muscle contraction exerts a tensile force on tendons. However, the results
obtained with the chick model differed. While at E6 ~ 6.5, DMD and PB paralysis models showed reduced
Scx
expression
(Havis et al., 2016), wing graft surgery, a muscle loss condition, resulted in
Scx
expression (F. Edom-Vovard et al., 2002).
These two studies used ISH to identify
Scx
expression. Although the intervention of these studies was the similar, the side
effects or intensity of the changed mechanical force may differ. We found that the current literature in this eld contains
many conicting reports. Future studies must consider the side effects of each intervention. For example, some transgenic
models almost lack of myogenic determination factor such as Pax3. However, it is not clear how contribute Pax3 oneself
tendon cell maturation. To resolve this problem, to use the same changing force in multiple intervention methods (muscle
contraction, muscle existence, or loading) within a single study is needed. Bias due to detection methods can be excluded if
the tendon reacts to mechanical force tested in a single publication. Although the molecular aspect may differ with the
intervention methods, the mechanical force does not change between models. In this study, we included ve studies involving
models with different types of mechanical forces; however, the differences in the effects of mechanical stress on the tendon
between models are less well understood. Furthermore, heterogeneity in species and intervention methods, such as genetics
and effect of drugs, render intertrial comparison dicult.
Moreover, the sampled parts of tendon varied with the body part, such as trunk or limb, forearm or digit. Kardon et al. (Kardon,
1998)and Edom et al. (F. Edom-Vovard et al., 2002) reported that the proximal tendon in chicks degenerated when the
muscles were induced less in the initial stage; subsequently, the distal tendons degenerated. Havis et al. (Havis et al., 2016)
showed that the decrease in
Scx
expression in chick embryos injected with DMD and PB was more evident in the
stylopod/zeugopod regions than in the digits. These results were observed not only in the forelimbs, but also in the
hindlimbs. Huang et al. (Huang et al., 2015) identied tendons within the zeugopod of Spd mice at E16.5 with diculty,
whereas the autopod tendons were identied easily. These results showed that initiated parts of degeneration are common,
that is a proximal part. These results indicate impairment of mechanical force due to muscle contraction or elongation of an
existing bone. This might be because the tendon bears tensile force from both muscle and bone. However, the elastic
modulus of the muscle-tendon complex should be considered while interpreting these results. Thus, if the tendon loads
tensile force via bone elongation, muscles with lower elastic modulus will stretch more than the tendon. The time scale on
development or sensitivity for force in each part might differ between proximal and distal tendons. However, why the results
vary with the body part remain unclear.
Several studies have tested the biological effects of changing the mechanical force using an
Scx
expression. In addition, one
study investigated the effects of
thrombospondin2
(
Thbs2
), an extracellular matrix protein that modulates collagen
brillogenesis and angiogenesis (Bornstein et al., 2000) on tendon mechanobiology. In connective tissue development, as
assessed using
Tnmd
expression (Qianman et al.), angiogenesis may potentially be involved in tissue maturation (Docheva
et al., 2005; Sato et al., 2014). The tail tendon degenerated in TSP2-null mice and provided an impression of increased laxity
in the tail (Kyriakides et al., 1998). There is no clear relationship between
Thbs2
and tendon development, although
Thbs2
may be involved in tendon maturation. Although some studies have combined the models to manipulate mechanical force by
adding or removing several factors, whether the intervention effect is due to mechanical force or solution effect remains
unclear. Therefore, the models introduced in this review induce both uctuating mechanical force and biochemical effects, for
example, because of the liquidity factor of the muscles. A model animal useful for elucidating the mechanism of
mechanotransduction for tendon development will be required in the future.
Limitations and future studies

Page 8/19
This review has some limitations. This review didn’t consider any risk of bias assessment due to the nature of scoping
reviews. It provides denitions of mechanical force used in tendon mechanobiology and knowledge regarding the
contribution of mechanical force to tendon development in the context of compelling evidence. However, to inspect the
function of mechanical force, we must conduct systematic reviews using studies on specic types of intervention and
compare different models; however, currently, consensus regarding the denition of mechanical force dened in this review is
lacking.
Conclusions
This scoping review provides insights regarding tendon development promoted by mechanical loading
in vivo
, as well as the
lack of consensus regarding the effect of mechanical force applied on tendons. We dened mechanical force in terms of
muscle existence, muscle contraction, or weight-bearing locomotion. Our results provide a novel point of view to build future
research about mechanobiology in tendon development.
Declarations
ACKNOWLEDGMENTS
AUTHOR CONTRIBUTIONS
Y.U. designed and performed the majority of the data collection and analysis and wrote the manuscript. H.I. designed the
experiments and reviewed & editing the manuscript. T.K. supervised and aided in the experimental design, carried out data
analysis, and reviewed & editing the manuscript.
DECLARATION OF COMPETING INTERESTS
The authors declare no competing interests.
References
1. A. Subramanian, Kanzaki, L.F., Galloway, J.L., and Schilling, T.F. (2018). Mechanical force regulates tendon extracellular
matrix organization and tenocyte morphogenesis through TGFbeta signaling. Elife
7
. 10.7554/eLife.38069.
2. Arksey, H., and O'Malley, L. (2005). Scoping studies: towards a methodological framework. International Journal of
Social Research Methodology
8
, 19–32. 10.1080/1364557032000119616.
3. Ava E Brent, Ronen Schweitzer, and JTabin, C. (2003). A Somitic Compartment of Tendon Progenitors. Cell
113
, 235–
248. 10.1016/s0092-8674(03)00268-x.
4. B A Adams, and Beam, K.G. (1990). Muscular dysgenesis in mice: a model system for studying excitation-contraction
coupling. FASEB J
4
, 2809–2816. 10.1096/fasebj.4.10.2165014.
5. Bornstein, P., Kyriakides, T.R., Yang, Z., Armstrong, L.C., and Birk, D.E. (2000). Thrombospondin 2 modulates collagen
brillogenesis and angiogenesis. J Investig Dermatol Symp Proc
5
, 61–66. 10.1046/j.1087 – 0024.2000.00005.x.
. Brent, A.E., Braun, T., and Tabin, C.J. (2005). Genetic analysis of interactions between the somitic muscle, cartilage and
tendon cell lineages during mouse development. Development
132
, 515–528. 10.1242/dev.01605.
7. C Beckham, R Dimond, and Jr, T.K.G. (1977). The Role of Movement in the Development of a Digital Flexor Tendon.
American Journal of Anatomy
150
, 443–460.
. Docheva, D., Hunziker, E.B., Fassler, R., and Brandau, O. (2005). Tenomodulin is necessary for tenocyte proliferation and
tendon maturation. Mol Cell Biol
25
, 699–705. 10.1128/MCB.25.2.699-705.2005.
9. E Bober, T Franz, H H Arnold, P Gruss, and Tremblay, P. (1994). Pax-3 is required for the development of limb muscles: a
possible role for the migration of dermomyotomal muscle progenitor cells. Development, 603–612.

Page 9/19
10. F. Edom-Vovard, Schuler, B., Bonnin, M.A., Teillet, M.A., and Duprez, D. (2002). Fgf4 positively regulates scleraxis and
tenascin expression in chick limb tendons. Dev Biol
247
, 351–366. 10.1006/dbio.2002.0707.
11. H. E. Swann, and M. R. Brumley (2019). Locomotion and posture development in immature male and female rats (Rattus
norvegicus): Comparison of sensory-enriched versus sensory-deprived testing environments. J Comp Psychol
133
, 183–
196. 10.1037/com0000147.
12. Hamburger, V., and Hamilton, H.L. (1951). A series of normal stages in the development of the chick embryo. J Morphol.
88
, 49–92.
13. Havis, E., Bonnin, M.A., Esteves de Lima, J., Charvet, B., Milet, C., and Duprez, D. (2016). TGFbeta and FGF promote
tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb
development. Development
143
, 3839–3851. 10.1242/dev.136242.
14. Heinemeier, K.M., Olesen, J.L., Haddad, F., Langberg, H., Kjaer, M., Baldwin, K.M., and Schjerling, P. (2007). Expression of
collagen and related growth factors in rat tendon and skeletal muscle in response to specic contraction types. J Physiol
582
, 1303–1316. 10.1113/jphysiol.2007.127639.
15. Heywood, J.L., McEntee, G.M., and Stickland, N.C. (2005). In ovo neuromuscular stimulation alters the skeletal muscle
phenotype of the chick. J Muscle Res Cell Motil
26
, 49–56. 10.1007/s10974-005-9007-8.
1. Huang, A.H., Riordan, T.J., Pryce, B., Weibel, J.L., Watson, S.S., Long, F., Lefebvre, V., Harfe, B.D., Stadler, H.S., Akiyama, H.,
et al. (2015). Musculoskeletal integration at the wrist underlies the modular development of limb tendons. Development
142
, 2431–2441. 10.1242/dev.122374.
17. J A Germiller, A L Lerner, R J Pacico, R T Loder, and Hensinger, R.N. (1998). Muscle and Tendon Size Relationships in a
Paralyzed Chick Embryo Model of Clubfoot. J Pediatr Orthop.
18 (3
), 314–318.
1. J. W. Chen, and J. L. Galloway (2014). The development of zebrash tendon and ligament progenitors. Development
141
, 2035–2045. 10.1242/dev.104067.
19. Kardon, G. (1998). Muscle and tendon morphogenesis in the avian hind limb. Development
125
, 4019–4032.
20. Kassar-Duchossoy, L., Gayraud-Morel, B., Gomes, D., Rocancourt, D., Buckingham, M., Shinin, V., and Tajbakhsh, S.
(2004). Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature
431
, 466–471.
10.1038/nature02876.
21. Kyriakides, T.R., Zhu, Y.H., Smith, L.T., Bain, S.D., Yang, Z., Lin, M.T., Danielson, K.G., Iozzo, R.V., LaMarca, M., McKinney,
C.E., et al. (1998). Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with
disordered collagen brillogenesis, an increased vascular density, and a bleeding diathesis. Journal of Cell Biology
140
,
419–430.
22. L T Landmesser, and Szente, M. (1986). Activation patterns of embryonic chick hind-limb muscles following blockade of
activity and motoneurone cell death. The Journal of Physiology
380
, 157–174.
23. Miake-Lye, I.M., Hempel, S., Shanman, R., and Shekelle, P.G. (2016). What is an evidence map? A systematic review of
published evidence maps and their denitions, methods, and products. Syst Rev
5
, 28. 10.1186/s13643-016-0204-x.
24. Micah D J Peters, C.M.G., Hanan Khalil, Patricia McInerney, Deborah Parker, Cassia Baldini Soares (2015). Guidance for
conducting systematic scoping reviews. Int J Evid Based Healthc.
13
, 141–146. 10.1097/XEB.0000000000000050.
25. Murchison, N.D., Price, B.A., Conner, D.A., Keene, D.R., Olson, E.N., Tabin, C.J., and Schweitzer, R. (2007). Regulation of
tendon differentiation by scleraxis distinguishes force-transmitting tendons from muscle-anchoring tendons.
Development
134
, 2697–2708. 10.1242/dev.001933.
2. Paton, W.D.M., and Zaimis, E.J. (1949). The pharmacological actions of polymethylene bistrimethyl-ammonium salts.
British Journal of Pharmacology and Chemotherapy,
4
, 381–400. 10.1111/j.1476-5381.1949.tb00565.x.
27. Paton, W.D.M., and Zaimis, E.J. (1951). The action of d-tubocurarine and of decamethonium on respiratory and other
muscles in the cat. J Physiol. 10.1113/jphysiol.1951.sp004531.

Page 10/19
2. Pittman, R.H., and Oppenheim, R.W. (1978). Neuromuscular blockade increases motoneurone survival during normal cell
death in the chick embryo. Nature
271
, 364–366.
29. Qianman, B., Jialihasi, A., Asilehan, B., Kubai, A., Aibek, R., Wupuer, A., Tangkejie, W., Maimaitiaili, A., Shawutali, N.,
Badelhan, A., et al. (2017). Active exercise promotes Achilles tendon healing and is accompanied by the upregulation of
collapsin response mediator protein2 in rats. Mol Med Rep
16
, 2355–2360. 10.3892/mmr.2017.6889.
30. R Schweitzer, Jay H. Chyung, Lewis C. Murtaugh, Ava E. Brent, Vicki Rosen, Eric N. Olson, Lassar, A., and Tabin, a.C.J.
(2001). Analysis of the tendon cell fate using Scleraxis, a specic marker for tendons and ligaments. Development
128
,
3855–3866.
31. S. K. Theodossiou, Bozeman, A.L., Burgett, N., Brumley, M.R., Swann, H.E., Raveling, A.R., Becker, J.J., and Schiele, N.R.
(2019). Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons. J Biomech
96
, 109354. 10.1016/j.jbiomech.2019.109354.
32. S. K.Theodossiou, Pancheri, N.M., Martes, A.C., Bozeman, A.L., Brumley, M.R., Raveling, A.R., Courtright, J.M., and Schiele,
N.R. (2021). Neonatal Spinal Cord Transection Decreases Hindlimb Weight-Bearing and Affects Formation of Achilles
and Tail Tendons. J Biomech Eng
143
. 10.1115/1.4050031.
33. Sato, I., Miwa, Y., Hara, S., Fukuyama, Y., and Sunohara, M. (2014). Tenomodulin regulated the compartments of
embryonic and early postnatal mouse masseter muscle. Ann Anat
196
, 410–415. 10.1016/j.aanat.2014.07.001.
34. Shinoda, T.S.a.Y. (1999). Highly reproducible spatiotemporal patterns of mammalian embryonic movements at the
developmental stage of the earliest spontaneous motility. European Journal of Neuroscience
11
, 2697–2710.
35. Swinburne, I.A., Mosaliganti, K.R., Green, A.A., and Megason, S.G. (2015). Improved Long-Term Imaging of Embryos with
Genetically Encoded α-Bungarotoxin. Plos One
10
. 10.1371/journal.pone.0134005.
3. Tricco, A.C., Lillie, E., Zarin, W., O'Brien, K.K., Colquhoun, H., Levac, D., Moher, D., Peters, M.D.J., Horsley, T., Weeks, L., et al.
(2018). PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med
169
, 467–
473. 10.7326/M18-0850.
37. W R Buckett, C E Marjoribanks, F A Marwick, and Morton, M.B. (1968). The pharmacology of pancuronium bromide
(Org.NA97), a new potent steroidal neuromuscular blocking agent.
3. W.G.Hopkins (1984). Axon growth in tendon organs of botulinum-paralyzed neonatal mouse muscles. Experimental
Neurology
86
, 405–409.
39. Wu, K.C., Streicher, J., Lee, M.L., Hall, B.K., and Muller, G.B. (2001). Role of motility in embryonic development I: Embryo
movements and amnion contractions in the chick and the inuence of illumination. J Exp Zool
291
, 186–194.
10.1002/jez.1068.
40. X S Pan, Li, J., Brown, E.B., and Kuo, C.K. (2018). Embryo movements regulate tendon mechanical property development.
Philos Trans R Soc Lond B Biol Sci
373
. 10.1098/rstb.2017.0325.
41. Zhou, W., Saint-Amant, L., Hirata, H., Cui, W.W., Sprague, S.M., and Kuwada, J.Y. (2006). Non-sense mutations in the
dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrash relaxed mutants. Cell Calcium
39
, 227–236.
10.1016/j.ceca.2005.10.015.
Methods
RESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be fullled by the lead contact,
Takanori Koubun (kokubun-takanori@spu.ac.jp).
Materials Availability

Page 11/19
This study did not generate new unique materials.
Data and Code Availability
This paper analyzes existing, publicly available data. These accession numbers for the datasets of the resource publications
are listed in the table. This paper does not report the code. Any additional information required to reanalyze the data reported
in this paper is available from the lead contact upon request.
METHOD DETAILS
The review followed the elaborate methodological framework according to the Preferred Reporting Items for Systematic
Reviews and Meta-Analyses extension for scoping reviews (PRISMA-ScR) guidelines(Tricco et al., 2018)and the ve-stage
framework outlined in Arksey and O’Malley(Arksey and O'Malley, 2005)(Supplemental Appendix S1 and S2). The detailed
protocol for this scoping review was not published or registered.
The central research question was: “What is known in the existing literature about the role of mechanical forces in tendon
development?” The specic research questions were as follows;
(1) What types of mechanical forces are dened and used in tendon development?
(2) What types of roles do mechanical forces in tendon development perform?
Inclusion criteria, literature search, and study selection
Inclusion criteria was set according to participants, concept, and context domains (PCC)(Micah D J Peters, 2015). The review
considered studies that included (1) embryo and postnatal animals (within four weeks from the day of birth) (participants
domain), (2) intervention to increase or decrease mechanical forces for the tendon (concept domain), and (3) tendon
development as an outcome (context domain).
PubMed databases were used for electronic database search from the time of database inception to October 2021. The
PubMed searches used the following search phrases: (((("Animals" [MeSH]) AND ("Tendons" [MeSH] OR tendon[Title])) AND
((((((("embryology"[MeSH]) OR ("Morphogenesis" [MeSH])) OR ("Gene Expression Regulation, Developmental"[MeSH])) OR
("infant, newborn"[MeSH])) OR (embryo)) OR (postnatal)) OR (neonatal))) AND (((((((Biomechanical Phenomena[MeSH
Terms]) OR (Movement[MeSH Terms])) OR (Stress, Mechanical[MeSH Terms])) OR (Paralysis[MeSH Terms])) OR
(Locomotion[MeSH Terms])) OR (tendons/metabolism[MeSH Terms])) OR (Weight-Bearing[MeSH]))) NOT (review[Publication
Type]). The last search for new manuscripts was performed in October 2021. References of the selected studies were
screened for further relevant studies.
The inclusion criteria were as follows: (1) full-length original research article, (2) the target was in embryonic or postnatal
stage (within four weeks from the day of birth), (3) induced uctuation of mechanical force in the tendon, and (4)
in vivo
model. Studies written in languages other than English were excluded.According to the prespecied inclusion criteria, two
reviewers (Y.U. and T.K.) independently screened titles, abstracts, and full texts of selected studies. Disagreements between
reviewers were resolved via discussion till a consensus was reached. The reference list of the reviewed articles was also
searched.
QUANTIFICATION AND STATISTICAL ANALYSIS
Data extraction
Basic study information regarding the author(s), year of publication, and the title was extracted. To answer the above
research questions, we summarized the following information: animal species, sample age, type of mechanical intervention,
comparator, duration of intervention, and biological ndings (molecular biology data, histological ndings, morphological

Page 12/19
analyses, and mechanics testing). The number of studies was considered the total number. When multiple models were used
in one study, the number of each intervention model, and not the number of papers, was considered. The biological data that
the authors specied in sentences or gures were extracted. Small variations did not write clearly but showed only in gures
were excluded.
Data synthesis
Data were synthesized in three sections, and we created an evidence map with a visual representation of outcome measures
to identify knowledge gaps (Miake-Lye et al., 2016). We also identied methodology gaps in the literature during the process
of collating and reporting the results using characteristics described in the data charting section. Given the focus on reporting
methods and inclination of data rather than data of concrete gures or statistically signicant, we didn’t execute the
statistical analysis.
Tables
Table 1. Summary of Studies
To answer the research questions, we summarized the following information: animal species, sample age, type of
mechanical intervention, comparator, duration of intervention, and biological ndings (molecular biology data, histological
ndings, morphological analyses, and mechanics testing).

Page 13/19
 Study
(Authors &
Year)
Animal
Species Age Tendon
Sample
Region
Intervention Comparator
 Force- Force
#1 Beckham C
et al.(1977) chick E18 limb d-tubocurarine − Ringer
solution, no
injection
#2 Hopkins WG
et al. (1984) CD-1
mouse P9 limb (gluteal
muscles
tendon)
botulinum toxin − PBS+Starved,
no intervention
#3 Kardon G et
al. (1998) chick st 28-
32 limb Coelomic graft
surgery − Normal
#4 Germiller JA
et al. (1998) chick E18 limb (achilles
tendon) decamethonium
bromide (DMB) − Saline
#5 Schweitzer R
et al. (2001) mouse E10 limb splotch mutant − WT
#6 Edom-Vovard
F et al.
(2002)
chick E6-
E10
E7-
E11
limb(forearm,
digit) Ceolomic Wing
Graft Surgery
Neural Tube
Ablation
− normal (no-
intervension)
#7 Brent AE et
al. (2003) chick stage
4 and
5+
2days
sclerotome surgically removed
the dermomyotome
(prior to myotome
formation)
− No
intervention
#8 Brent AE et
al. (2005) mouse At
E10.5-
11.0
trunk and
limb
Myf5
–/–;
Myod1
–/– − littermate
controls
(
Myf5
+/–;
Myod1
+/–)
#9 Eloy-Trinquet
S et al.
(2009)
mouse E12.5 limb
Pax3
mutant − WT
#10 Chen JW et
al. (2014) zebrash 53-58
hpf,
72hpf
the
craniofacial,
n, and
myosepta
Myod1
and
Myf5
Knockdown − WT
#11 Huang AH et
al. (2015) C57BL/6
mouse E16.5-
E18.5 limb (the
zeugopod
and
autopod)
Spd,mdg − WT
#12 Havis E et al.
(2016) chick E5.5
(24
h),
E6.5
(48
h),
and
E7.5
(72 h)
limb
(forelimb and
digits)
Decamethonium
bromide (DMB)
pancuronium
bromide (PB)
− Hank’s
solution
#13 Pan XS et al.
(2018) Chick HH45
(E19) limb (Achilles
Tandon) DMB
PB 4-AP
(hypermotility) saline
#14 Subramanian
A et al.
(2018)
zebrash 48
hpf ,
98hpf
axial Full-length mRNA
encoding codon-
optimized a-
bungarotoxin (aBtx)
injected

cacnb1
mutant
Electrical
stimulation WT

Page 14/19
#15 Theodossiou
SK et al.
(2019)
Sprague-
Dawley
rats
P1, 5,
and
10
limb (achilles
tendon, tail
tendon)
− locomotion
and weight-
bearing(P10)
non-weight-
bearing(P1,P5)
#16 Theodossiou
SK et al.
(2021)
Sprague-
Dawley
rats
P10 limb (achilles
tendon, tail
tendon)
spinal cord-
transected − sham

Table 2. Types of Intervention
The intervention type for reducing mechanical force was categorized into ve groups for various animal intervention
methods: transgenic, surgery, drug treatment, locomotion, and added stimulation.
Intervention methods Effect Force Animal Studies
Transgenic
Myod1
–/–
Myf5
–/– muscle-less ↓mouse #8
Myod1
and
Myf5
Knockdown muscle-less ↓zebrash #10
Pax3
-/- muscle-less ↓mouse #9
Pax3
(Splotch) muscle-less ↓mouse #5
Pax3
(Splotch delayed) muscle-less ↓mouse #11
Muscular dysgenesis (mdg) prevent muscle contraction ↓mouse #11
cacbn1
mutant prevent muscle contraction ↓zebrash #14
a-bungarotoxin (aBtx) mRNA prevent muscle contraction ↓zebrash #14
Surgery
Ceolomic wing graft muscle-less ↓chick #3, #6,
Neural tube ablation prevent muscle contraction ↓chick #6
Dermomyotome removals muscle-less ↓chick #7
Drug treatment
decamethonium bromide (DMB) rigid paralysis ↓chick #4,#12,#13
pancuronium bromide (PB) accid paralysis ↓chick #12, #13
d-tubocurarine accid paralysis ↓chick #1
botulinum toxin accid paralysis ↓mouse #2
4-aminopyridine (4-AP) hypermotility ↑chick #13
Locomotion    
spinal cord reduce weight-bearing ↓rat #16
locomotor development increase weight-bearing locomotor behavior ↑rat #15
Added stimulation    
Electrical stimulation increased muscle contraction ↑zebrash #14
Increased or decreased mechanical force ware shown in upward arrows or downward arrows.

Page 15/19
Table 3. Types of Mechanical Forces
We dened the mechanical force in terms of muscle existence, muscle contraction, or weight-bearing locomotion and
identied methodological heterogeneity in applying mechanical force to tendons.
Species Muscle Contraction Muscle Exist On Load
mouse/rat ↓Muscular dysgenesis (mdg) ↓
Myod1
–/–
Myf5
–/– ↓spinal cord
↓a-bungarotoxin (aBtx) mRNA ↓
Pax3
-/- ↑locomotor development
↓botulinum toxin ↓
Pax3
(Splotch)
↓
Pax3
(Splotch delayed)
chick ↓Neural tube ablation ↓Ceolomic wing graft 
↓decamethonium bromide (DMB) ↓Dermomyotome removals
↓pancuronium bromide (PB)
↓d-tubocurarine
↑4-aminopyridine (4-AP)
zebrash ↓
cacbn1
mutant ↓
Myod1
and
Myf5
Knockdown 
↑Electrical stimulation
Increased or decreased mechanical force were shown in upward arrows or downward arrows.
Table 4. Effects of Intervention
We summarized the model to investigate the role of mechanical force in tendon development throughout
Scx
expression.

Page 16/19
Effect Intervention Animal Duration Outcomes Study
(Authors &
Year)
decreased mechanical force
Transegenic
muscle-less
Myod1
–/–
Myf5
–/– mouse E0~ At
E10.5-11.0 E10.5: The limb/branchial arch Scx→
The somites Scx -
The apical ectodermal
ridge(Heinemeier et al.)Fgf4→
The anterior and posterior sclerotome
Pea3→
E13.5: The epaxial region Scx –
The limb Scx→
The axial tendin - 
Brent AE et
al. (2005)
Myod1
and
Myf5
Knockdown mouse E0~ 72hpf 53-58 hpf : the craniofacial and n
scxa+, xirp2a-
The myosepta scxa-, xirp2a-
72hpf: the head and n scxa-, xirp2a-
Chen JW et
al. (2014)
Pax3
-/- mouse E0 ~
E12.5 E12.5 Scx+ Eloy-Trinquet
S et al.
(2009)
Pax3
(Splotch) mouse E0~E10 Scx expression→Schweitzer R
et al. (2001)

Pax3
(Splotch
delayed) mouse E0~E16.5,
E18.5 E16.5 The zeugopod : ScxGFP-
     The autopod : Scx GFP+
The exor tendons (near the wrist):
Scx GFP-,
The extenser tendons: Scx GFP+,
several tendons were fused
E18.5 The autopod: ScxGFP+, an
aligned collagen matrix→, tendon size↓
Huang AH et
al. (2015)
prevent
muscle
contraction
Muscular
dysgenesis
(mdg)
mouse E0~E16.5 E16.5 The autopod: Scx GFP+, tendon
size↓
The zeugopod: Scx GFP+, The extenser
tendon fusion only near the wrist
Huang AH et
al. (2015)
cacbn1
mutant zebrash E0 to
98hpf 98hpf: fail to compact and elongate Subramanian
A et al.
(2018)
a-bungarotoxin
(aBtx) mRNA zebrash E0 to
48hpf 48hpf: axial tenocyte projection
length↓density↓
pSMAD3 signaling↓
tsp4b -, tgfbip -, ctgfa2 -, scxa→
Subramanian
A et al.
(2018)
Surgery
muscle-less Ceolomic wing
graft chick st16-st27-
35 st 28: tenascin →
st 29-30: (dorsal proximal and
intermediate) tenascin↓; do not
individuate, but instead degenerate
 (the distal tendon) tenascin→
st31-32: (the distal tendon) tenascin↓;
began to degenerate
Kardon G et
al. (1998)
 chick 17–25
somites The forearm: Scx E6 less segregated,
E8↓E10- Edom-Vovard
F et al.

Page 17/19
(E2)~E12 The digitScx
E10↓
[■mFgf4-Expressing Cells ]
Scx E10↑, tenascin E10→
*compare to only Wing Graft Surgery
(2002)
 Dermomyotome
removals chick stage 4
and 5+
2days
stage 4 and 5+ 2days: Scx- Brent AE et
al. (2003)
prevent
muscle
contraction
Neural tube
ablation chick 15–23
somites
(E2)~E11
(forearm/digit)
E7.  Fgf4→/→, Scx→/→, tenascin→/
→,   fgf8 n/d
E7.5 Fgf4↓/,   Scx↓/,    tenascin
n.d./,   fgf8↓/
E8 Fgf4↓/↓,  Scx↓/↓,   tenascin↓/↓,
    fgf8↓/
E9 Fgf4-/- ,   Scx↓/↓,  tenascin/↓,  
   fgf8-/-
E11  Fgf4-/-,   Scx -/↓,  tenascin
n.d./n.d., fgf8 n.d./n.d.
[■+mFgf4/RCAS-expressing cells]
The forearm: Scx E10↑, tenascin E10↑ ,
Fgf8 E10→
*compare to only Neural tube ablation
Edom-Vovard
F et al.
(2002)
Drug Treatment
rigid
paralysis decamethonium
bromide (DMB) chick E6~E18 CSA↓Germiller JA
et al. (1998)
chick E4.5~E7.5 forelimbs(stylopod and
zeugopod/digits *hole forelimbs;
SMAD7)
E5.5(24h) : Scx→
E6.5 (48h) : Scx↓/↓, COL1A2↓/↓,
ETV4↓/→, SPRY2↓/↓, FGF4↓/,
FGF8↓/, SMAD7↓, TGFB2↓/→
E7.5 (72h) : Scx↓/↓, COL1A2↓/
→,ETV4↓/↓, SPRY2↓/→, FGF4↓/,
FGF8↓/, SMAD7↓, TGFB2↓/→,TNMD-/,
THBS2-/,
The decrease of SCX expression was
more obvious in stylopod/zeugopod
regions compared with digits in ISH.
Hindlimbs(stylopod and
zeugopod/digits)
E6.5(48h) : Scx↓/→, ETV4↓/→,
SPRY2↓/→
E7.5(72h) : Scx↓/↓, ETV4↓/↓, SPRY2↓/
→
[■+mFgf4/ RCAS- producing cells vs.
DMB(forelimbs)]
E7.5(72h) SCX↑, ETV4↑, SPRY2↑
         COL1A2→, TNMD→,
THBS2→, FGF8→, TGFB2→,
TGFB3→,SMAD7→
*indicated by / showed that expression
was difference for each part.
Havis E et al.
(2016)
  chick HH43
chick
embryosto
- after 48h
Elastic modules↓, LOX activity ↓,
collagen content→
Pan XS et al.
(2018)
accid
paralysis pancuronium
bromide (PB) chick E8-E18 the proximal area : synovial cavity,
fuzzy layer not apperded.
the distal area : although a pulley did
Beckham C
et al.(1977)

Page 18/19
not appear to be as well developed as
that in the normal chicks.
the vinculum like the normal chicks
was not present.
several small blood vessels were
present. 
the tendon and their products
(collagen and immature elastic bers)
were not changed
chick E4.5~E7.5 E6.5(48h): Scx↓
E7.5(72h) Scx ↓
SCX expression was downregulated in
stylopod/zeugopod regions of
forelimbs and hindlimbs compared to
control limbs.
Havis E et al.
(2016)
chick HH43
chick
embryosto
- after 48h
LOX activity ↓Pan XS et al.
(2018)
d-tubocurarine chick E8-E18 the proximal area : synovial cavity,
fuzzy layer −
the distal area : pulley−, the vinculum−
several small blood vessels +
the tendon cells, collagen and
immature elastic bers →
Beckham C
et al.(1977)
botulinum toxin mouse newborn-
P9 length↑Hopkins WG
et al. (1984)
Locomotion
reduce
weight-
bearing
spinal cord rat P1~P10 Achilles tendons
linear region elastic modulus↑, cross-
sectional↓
Maximum force, displacement at
maximum force, linear region stiffness,
toe region elastic modulus, maximum
stress, strain at maximum stress, and
transition strain→
SHG images→
Tail tendons
linear region stiffness↑ cross-sectional
area↑
Maximum force , displacement at
maximum force, toe and linear region
elastic modulus, maximum stress,
strain at maximum stress, and
transition strain→
SHG images→
Theodossiou
SK et al.
(2011)
Increased Mechanical Force
Drug Treatment
hypermotility 4-aminopyridine
(4-AP) chick HH43
chick
embryosto
- after 48h
Elastic modules↑, LOX activity → ,
collagen content →
■+BAPN(inhibit LOX activity) vs 4-AP
  elastic module ↓
Pan XS et al.
(2018)
Locomotion
increase
weight-
bearing
locomotor
behavior
locomotor
development rat P1,5,10 P10: Achilles tendon: maximum force↑,
displacement at maximum force↑,
stiffness↑, cross-sectional
area↑(conpared to non-weight P1,P5)
elastic modulus→, maximum stress→,
Theodossiou
SK et al.
(2019)

Page 19/19
strain at maximum stress→(conpared
to non-weight P1,P5)
Added Stimulation
increased
muscle
contraction
Electrical
stimulation zebrash E0 to
48hpf 48hpf: axial tenocyte projection length,
density→
[■+EMS vs aBtx]
 axial tenocyte projection length
↑(conpared to αBtx-injected)
pSMAD3↑, scxa→, tsp4b↑, tgfbip↑,
ctgfa2↑ (conpared to αBtx-injected)
Subramanian
A et al.
(2018)
Increased or decreased value were shown in upward arrows or downward arrows. Loss of expression was shown minus.
Figures
Figure 1
Flow Diagram Showing Selection of Articles Used in The Study
Manual database search yielded 586 articles. After the rst screening stage of titles and abstracts, 84 articles were
selected for full-text review.
Figure 2
Types of Intervention Methods
We dened mechanical force in terms of muscle contraction, muscle existence, and weight-bearing for each animal
species.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
SupplementalAppendixS1.pdf
SupplementalAppendixS2.pdf
SupplementalTableS1.pdf