Received: 28 October 2017 | Accepted: 30 March 2018

DOI: 10.1002/jcp.26637

MINI-REVIEW

Magnetotherapy: The quest for tendon regeneration

Tamagno Pesqueira1,2 | Raquel Costa-Almeida1,2 | Manuela E. Gomes1,2,3

1 3B's Research Group − Biomaterials,

Biodegradables and Biomimetics, University of Tendons are mechanosensitive tissues that connect and transmit the forces generated
Minho, Headquarters of the European
by muscles to bones by allowing the conversion of mechanical input into biochemical
Institute of Excellence on Tissue Engineering
and Regenerative Medicine, Zona Industrial da signals. These physical forces perform the fundamental work of preserving tendon
Gandra, Barco, Guimarães, Portugal
homeostasis assuring body movements. However, overloading causes tissue injuries,
2 ICVS/3B's − PT Government Associate
Laboratory, Guimarães, Portugal
which leads us to the field of tendon regeneration. Recently published reviews have
3 The Discoveries Centre for Regenerative and broadly shown the use of biomaterials and different strategies to attain tendon
Precision Medicine, Headquarters at regeneration. In this review, our focus is the use of magnetic fields as an alternative
University of Minho, Barco, Guimarães,
Portugal therapy, which has demonstrated clinical relevance in tendon medicine because of
their ability to modulate cell fate. Yet the underlying cellular and molecular mechanisms
Correspondence
Manuela E. Gomes, 3B's Research Group − still need to be elucidated. While providing a brief outlook about specific signalling
Biomaterials, Biodegradables and Biomimetics,
pathways and intracellular messengers as framework in play by tendon cells,
University of Minho, Headquarters of the
European Institute of Excellence on Tissue application of magnetic fields as a subcategory of physical forces is explored, opening
Engineering and Regenerative Medicine,
up a compelling avenue to enhance tendon regeneration. We outline here useful
Avepark − Parque de Ciência e Tecnologia,
Zona Industrial da Gandra, 4805–017 Barco, insights on the effects of magnetic fields both at in vitro and in vivo levels, particularly
Guimarães, Portugal.
on the expression of tendon genes and inflammatory cytokines, ultimately involved in
Email: megomes@dep.uminho.pt
tendon regeneration. Subsequently, the potential of using magnetically responsive
Funding information biomaterials in tendon tissue engineering is highlighted and future directions in
Fundação para a Ciência e a Tecnologia,
Grant numbers: IF/00593/2015, SFRH/BD/ magnetotherapy are discussed.
96593/2013
KEYWORDS
contact-free technology, electromagnetic field, magnetic biomaterials, mechano-responsive
tissue, mechanotransduction, tendon

Abbreviations: AC, alternating current; AR, adenosine receptors; BMP, bone morphoge- 1 | SNAPSHOT ON TENDON PHYSIOL OGY
netic proteins; C3H10T1/2, mouse mesenchymal stem cell line; CaM, calmodulin; CMF,
combined magnetic field; COL1A1, Type I Collagen alpha 1; COL1A2, Type I Collagen
alpha 2; COL3A1, Type III Collagen alpha 1; DC, direct current; DCN, decorin; ECM,
Tendons, as well as ligaments, together with bone, cartilage, and
extracellular matrix; EGR, early growth response; FMOD, fibromodulin; hFOB1.19, human muscle, are all components of the musculoskeletal system. Among
fetal osteoblastic cell line; IBSP, bone sialoprotein; IL, interleukins; KLF4, krueppel-like
these, tendons act as a mechanically active bridge, being responsible
factor 4; MNP, magnetic nanoparticles; MKX, mohawk; MSCs, mesenchymal stromal cells;
NO, nitric oxide; Oct4, octomer-binding transcription factor 4; PCL, Poly(ε-caprolactone); for connecting and transmitting forces between muscles or from
PEMF, pulsed electromagnetic field; PMF, pulsed magnetic field; PDGF, platelet-derived
muscles to bones, generating motion and allowing movement of body
growth factor; PGE2, prostaglandin E synthase 2; SCX, scleraxis; SPP1, osteopontin; TCPs,
tendon stem/progenitor cells; T/C-28a2, human chondrocyte cell line; TIMP, tissue parts. Morphologically, these mechanoresponsive connective tissues
inhibitor of metalloproteinases; TGF, transforming growth factor; TNF, tumor necrosis are composed of uniaxially oriented fibres following a specific
factor; TNC, tenascin-C; TNMD, tenomodulin; VEGF, vascular endothelial growth factor;
VGCC, voltage-gated calcium channel. hierarchy, from collagen fibrils (50–500 nm in diameter) to fibre

J Cell Physiol. 2018;1–11. wileyonlinelibrary.com/journal/jcp © 2018 Wiley Periodicals, Inc. | 1

2 |
bundles (1–10 mm in diameter) wrapped by an anti-friction sheath to
allow tendon gliding.
Tendon tissue is constituted by a mixed population of resident
cells that includes mature tenocytes and tendon stem/progenitor cells
(TSPCs) embedded in a protein-rich niche (Bi et al., 2007). In this
tendon niche, extracellular matrix (ECM) contains primarily fibrillar
collagens (e.g., collagen type I, collagen type III), as well as
proteoglycans (e.g., decorin, biglycan, fibromodulin) and glycoproteins
(e.g., elastin, tenascin C, tenomodulin, cartilage oligomeric matrix
protein), and cells communicate with each other and with the
surrounding microenvironment by specific cellular membrane proteins
responsible for regulating cell-cell (e.g., Connexin-32, Connexin-43,
Cadherin-11) and cell-ECM (e.g., collagen-binding integrins α1β1,
α2β1, α10β1, α11β1) interactions (Costa-Almeida et al., 2015).
Moreover, tendon exhibits a graded morphology at both ends: on
one side, the tendon-to-bone insertion (enthesis) and on the other
extremity the tendon-to-muscle (myotendinous) junction. Thus, the
singular architecture of tendons together with a harmonious interplay
of mechanical forces are translated into an intriguing biological
complexity and a delicately powerful functional role in the musculo-
skeletal system. Altogether, such tissues are constantly exposed to
mechanical loading, starting by posture control (gravity) and including
forces associated with daily activities. Therefore, understanding the
influence of mechanical stimuli on tendon repair and regeneration is of
paramount importance toward developing more effective clinical
strategies to improve tissue healing and functional recovery.
Tendon injuries are of utmost concern worldwide with more than
102.5 million patients in US alone suffering from injuries annually
(Lomas et al., 2015). These injuries frequently take place in the Achilles
tendon, rotator cuff, and flexor tendon, with partial or total rupture
occuring within the mid-substance or enthesis (Egger & Berkowitz,
2017; Jarvinen et al., 2001; Myer & Fowler, 2016; Thorsness & Romeo,
2016). Upon injury, tendon undergoes a process of repair implying the
formation of scars, an acellular tissue which establishes adhesions with
surrouding tissues limiting tendon gliding and motion amplitude
(Kannus, 2000), instead of a process of regeneration, which would lead
to full recovery of tissue functionality without scar formation.
Nonetheless, unsatisfatory outcomes of classical treatments implying
oral administration of anti-inflammatory drugs and/or physical
rehabilitation, and surgical interventions lay the foundation for
ingenious approches with ameneable translation into the clinics.
This attractive approaches range from the development of natural/
synthetic substitutes (Lomas et al., 2015), instructive bioactive
molecules (Docheva, Müller, Majewski, & Evans, 2015), cell- (Gaspar,
Spanoudes, Holladay, Pandit, & Zeugolis, 2015) and gene- (Tang, Zhou,
Wu, Liu, & Wang, 2016) based therapies to contact-free instructive
signals of magnetic- or electromagnetic-based fields, herein called
magnetotherapy.
In this review, useful insights to attain tendon regeneration are
discussed, starting off by exploring the importance of mechanical
stimulation to maintain tissue homeostasis, emphasising the signalling
pathways and intracellular messengers involved in mechanotransduc-
tion. Then, the underlying cellular and molecular mechanisms in play
PESQUEIRA ET AL.
both in vitro and in vivo upon magnetic stimulation, subcategory of
physical forces, are elucidated particularly as toolkit with immuno-
modulatory ability and to govern gene expression. Lastly, instructive
magnetically responsive biomaterials as a novel strategy toward
bioengineering tendon constructs, as well as future steps into the field
of magnetotherapy and tendon tissue engineering are discussed.
2 | HO W PH Y SIC AL FO RCES REGU LAT E
TEN DO N H O M E O S TAS I S
The delicate equilibrium between mechanical forces to which tendon
cells are exposed mediates tissue homeostasis and development of
pathologies. Tendon homeostasis is underpinned by physiological
levels of mechanical loading (Popov et al., 2015) while overloading
leads to tendinopathies (Spiesz et al., 2015). In a healthy state,
exposing tendons to stress ≤ 25 MPa and strain ≤ 2.5%, the so-called
“physiological levels of mechanical loading,” maintains their homeo-
stasis during daily activities (Maganaris & Paul, 1999) However,
repetitive movements (wear and tear), as well as acute trauma, or
chronic diseases may culminate in tendon injury. Thus, to attain
successful tendon regeneration it is pivotal to understand the in vivo
microenvironment housing tissue homeostasis and healing, together
with the basic cellular and molecular mechanisms occurring in such
mechanoresponsive tissues. In this section, we briefly highlight the role
of intracellular calcium (Ca2+) in response to mechanical stimulation
and transforming growth factor (TGF)-β signalling pathway along with
two transcriptional factors mohawk (MKX) and early growth response
1 (EGR1) as major players of mechanotransduction in tendon cells,
particularly at dictating gene expression.
2.1 | Insights into tendon mechanotransduction and
molecular effectors
Cells sense their surroundings by a process called mechanotransduc-
tion, a route by which they translate mechanical forces into
biochemical signals, generating a biological response (Jaalouk &
Lammerding, 2009). Tendon cells respond to physical forces via
activation of mechanosensitive complexes involving focal adhesion
complex, ion channels, and second messengers interacting in an
autocrine and paracrine manner (Ackermann & Hart, 2016). Calcium,
an important intracellular messenger involved in numerous cellular
processes (Berridge, Lipp, & Bootman, 2000) has a transient response
when cells are mechanically stimulated (Wall & Banes, 2005). Once
generated, the Ca2+ wave propagates to the neighbouring cells via gap
junctions (Wall & Banes, 2005). As reported in an earlier study, the
concentration of intracellular Ca2+ [Ca2+]ic increased from 100 nM
(basal levels) to approximately 1,500 nM when avian tendon cells were
stimulated with mechanical indentation (1 Hz for 8 hr daily during 3
days) (Banes et al., 1999). A similar increase is likely to occur in a
combinatory stimulation composed of cyclic tensile strain (4%) and
shear stress (1,666 μm/s) (Maeda, Hagiwara, Wang, & Ohashi, 2013).
Upon mechanical stimulation, the mechanosensing L-type voltage-
PESQUEIRA ET AL.
| 3

gated calcium channels (VGCC) have been found in tenocytes and an
2+
increment in [Ca ]ic levels was detected upon repetitive mechanical
stretching (1.0 Hz) in vitro in a time and magnitude-dependent manner,
pointed as a major risk factor leading to cytoskeleton damage by
disrupting actin filaments (Chen, Deng, Zhang, & Tang, 2015). Despite
2+
a transient response in [Ca ]ic, mechanical stimulation also leads to
alterations in Adenosine 5′-triphosphate (ATP) and changes in gene
and protein expression (Figure 1) (Lavagnino et al., 2015). Tendon cells
express purinoceptors P2Y in the cell membrane which makes them
sensitive to extracellular ATP (Tsuzaki, Bynum, Almekinders, Faber, &
Banes, 2005) and, once stimulated, such receptors are responsible for
increasing [Ca2+]ic due to enhanced influx of ATP (Yamazaki et al.,
2003). For example, within physiological levels of mechanical loading,
the concentration of ATP released in human tendon cells increases
from 0.5–1 nM (basal levels) to 14–17 nM, which returns to basal
levels within 30 min via ecto-ATPase (Tsuzaki et al., 2005); however,
elevated levels of ATP temporarily block gap junctions signalling and
desensitise tendon cells to mechanical stimulation (Yamazaki et al.,
2003). In this manner, depending on the intracellular machinery that is
activated, tendon tissue perceives distinct stimuli differently.
At the gene level, mechanical stimulation has been demonstrated
to influence the expression of several tendon-associated genes, highly
dictating the physiology of tendon cells. When stimulated, the signal
propagates from the cell membrane all the way to the nucleus via a
harmonious nexus of signalling pathways. In tendon cells, these
pathways include the TGF-β signalling (Maeda et al., 2011) and
guanosine triphosphate Rho and Rho-associated protein kinase
(ROCK) (Rho/ROCK) proteins (Xu et al., 2012), ultimately involved in
the commitment of stem cells toward the tenogenic lineage (Li et al.,
2015; Xu et al., 2012). When the biochemical signals reach the nucleus,
the transcription factors MKX, activated by transcription factor II–I
repeat domain-containing protein 1 (gtf2ird1), and zinc finger EGR1
have been identified as molecular effectors in tendon cells (Gaut et al.,
2016; Kayama et al., 2016). Emerging evidence from an in vivo study
using the mouse model C57BL6/N demonstrated that animals
mechanically stimulated (treadmill) for 1 hr per day 5 days a week
during 4 weeks, increased the expression of Mkx in comparison with
unexposed controls. On top of increased Mkx expression, an
upregulation of tendon-associated genes Tenomodulin (Tnmd), colla-
gen type I alpha 1 (Col1a1), collagen type I alpha 2 (Col1a2) and
fibromodulin (Fmod) was also detected; however, the expression of
Scleraxis (Scx) remained similar to controls (Kayama et al., 2016). On
the other side, Scx increased in Mkx−/− mouse but no changes in
comparison with controls were seen (Kayama et al., 2016), which
suggests that upon mechanical stimulation, Mkx is pivotal to modulate
the expression of vital tendon-associated genes independently of the
expression of Scx. Importantly, Mkx is required for proper tendon fibres
formation with a compelling role in regulating collagen type I
production (Ito et al., 2010). From a regenerative perspective, Mkx
highly accelerates tendon stem cell differentiation while preventing

FIGURE 1 Molecular interplay of mechano-magnetic stimulated tendon-derived cells on the dynamic of Ca2+ and ATP. When cells are
mechanically stimulated, due to membrane depolarization, calcium channels enhance the intracellular concentration of calcium. Part of
intracellular calcium migrates to the endoplasmic reticulum but also calcium stores within the endoplasmic reticulum release Ca2+ into the
cytoplasm. Increased cytoplasmic calcium concentration stimulates mitochondrial oxidative metabolism, which either directly via ATP synthase
activity or indirectly via dehydrogenase activity triggers the synthesis and release of ATP. The calcium/ATP dynamics is involved in
cytoskeletal organization, protein expression profiles and balance of matrix metalloproteinases
4 |
phenotypic drift toward chondrogenic/osteogenic lineages. (Suzuki
et al., 2016).
Early growth response 1 is likely important for the maintenance of
proper tendon phenotype via expression of tendon-associated genes
Scx, Col1a1, and Col1a2, and to keep suitable mechanical properties of
the tissue as demonstrated in vivo (Guerquin et al., 2013). As Egr1
positively affects tendon repair and commits the differentiation of
mesenchymal stem cells into the tenogenic lineage in vitro (Guerquin
et al., 2013), it was demonstrated that murine pluripotent stem cells
(C3H10T1/2) transfected to overexpress Egr1 exhibited enhanced
expression of Scx, Tnmd, Col1a1, Col1a2 when encapsulated within a
3D fibrin-based system under tension (Gaut et al., 2016). In the same
manner as Mkx, Egr1 expression is sensitive to mechanical stimulation
(tension) but strikingly, its overexpression was able to rescue the
expression of these tendon-associated genes even under reduced
loading conditions (Gaut et al., 2016). Importantly to note is that Scx,
Mkx, and Egr1 are all three transcription factors in play during tendon
development and they are involved in the tenogenic commitment of
stem cells improving tendon repair upon injury (Milet & Duprez, 2015).
Importantly, upregulation of Scx, Tnmd, Col1a1, and Col1a2 upon injury
improved tendon healing in vivo in the presence of Egr1 over-
expressing MSCs or TSPCs, demonstrating their commitment toward
tenogenesis (Guerquin et al., 2013; Tao, Liu, Chen, Zhou, & Tang,
2015). Interestingly, the healing mechanism is likely to follow particular
signalling branches of TGF-β pathway, partially due to active SMAD2/
3 signalling in a rat model of injured Achilles tendon (EGR-1
overexpressing MSCs) (Guerquin et al., 2013), and BMP12/Smad1/
5/8 signalling (EGR-1 overexpressing TSPCs) in a rabbit model of
injured rotator cuff tendon (Tao et al., 2015).
Altogether, these findings highlight the interplay between
numerous molecular effectors and mechanical forces, which may
have a role in directing tendon regeneration. Further studies at the
cellular and molecular level could uncover potential mechanisms
directing tenogenic differentiation of stem cells and improving cell-
based therapies for tendon tissue engineering and regenerative
medicine.
3 | B E Y O N D R E P A I R : SH O O T I N G FO R
REGENE RATION
Magnetic and electromagnetic field therapy, herein called as magneto-
therapy, is currently used in the clinics for the treatment of several
health conditions (e.g., diabetes, multiple sclerosis) in distinct medical
fields, given its safety and non-invasiveness characteristics (Afshari
et al., 2016; Sun, Kwan, Zheng, & Cheing, 2016). As a US Food and
Drug Administration (FDA)-approved therapy, pulsed electromagnetic
field (PEMF) is the most commonly used magnetotherapy modality
(Markov, 2007). In orthopaedics, for example, PEMF positively
modulated inflammation and reduced pain in rats suffering from
tendinitis and in patients recovering from arthroscopic reconstruction
of anterior cruciate ligament (Benazzo et al., 2008; Lee, Maffulli, Li, &
Chan, 1997). Indeed, patients with persisted rotator cuff tendinitis
PESQUEIRA ET AL.
exposed to an external PEMF (75 Hz) during 5–9 hr daily over 4 weeks
showed significant improvement in the range of motion of the shoulder
and a decrease in inflammation (Binder, Parr, & Hazleman, 1984).
However, a recent study suggests that PEMF was useful only as a
short-term (about 5 months) modulator of inflammation including
reduced local inflammation, postoperative joint swelling and pain in
patients undergoing rotator cuff repair as no differences were
observed after 2 years follow-up (Osti, Buono, & Maffulli, 2015).
Nonetheless, how magnetotherapy modalities affect tendon resident
cells still remains elusive.
Recently, magnetic stimulation of tendon cells has re-emerged as a
trend in investigating novel tissue engineering and regenerative
medicine (TERM) approaches aiming at clinical translation toward
tendon regeneration. Figure 2 schematically depicts main strategies in
TERM that take advantages of magnetic field application. In this
regard, magnetic tissue engineering approaches involve the in vitro
generation of mature tendon tissue constructs through the use of
magnetically responsive biomaterials. Moreover, the translation of
magnetotherapy into the clinics may involve (i) the application of an
external magnetic field directly at the site of injury; (ii) the implantation
of a tissue-engineered construct together with magnetic actuation; or
(iii) the implantation of cell-free magnetically responsive biomaterials
in combination with magnetic actuation. In this section, we explain
how magnetic stimulation directly affects the physiology of cells during
an inflammatory and regenerative state, and the potential of
magnetically responsive biomaterials as an instructive toolbox to
guide tendon regeneration.
3.1 | Insights of magnetic actuation dictating tendon
cell behaviour
In a regenerative medicine scenario, the basic understanding of how an
external stimulus modulates cell physiology is one of the imposed
challenges. At the cellular level, electromagnetic field affects cell
communication, governs the cytoskeletal organisation, and structural
components of the plasma membrane and changes the dynamics of
[Ca2+]ic, as reviewed elsewhere (Pesce, Patruno, Speranza, & Reale,
2013). In particular, electromagnetic stimulation has been shown to
increase the release of the anti-inflammatory cytokine interleukin (IL)-
10, simultaneously leading to a decrease of the release of pro-
inflammatory cytokines, such as IL-6 and tumor necrosis factor (TNF-
α), both in macrophages and lymphocytes (Vergallo et al., 2013), as well
as in tendon cells (de Girolamo et al., 2013, 2014), particularly, in a time
and field orientation (Milovanovich et al., 2016; Ross & Harrison,
2013), as well as field intensity and exposure period dependent
manner (de Girolamo et al., 2014). The anti-inflammatory effect of
PEMF may somewhat rely on the fact that it is an agonist for adenosine
receptors (ARs), particularly increasing the density and functionality of
A2A and A3 ARs seen by enhanced release of IL-10 (anti-inflammatory
cytokine) and diminished release of IL-6, TNF-α (pro-inflammatory
cytokines) in musculoskeletal tissue related cell lines (human T/C-28a2
chondrocytes and hFOB 1.19 osteoblasts) (Vincenzi et al., 2013).
Additionally, PEMF (5 Hz, 4 μT) for 90 min restored the plasma
PESQUEIRA ET AL.
| 5

FIGURE 2 Schematic illustration of magnetic tissue engineering approach involving the development of magnetically responsive
bioengineered tendon constructs via generation of mature tendon tissue in vitro. Upon clinical translation, as a contact-free therapy, the
magnetic field can be applied directly at the site of injury alone, in combination with cell-free magnetic biomaterial or bioengineered magnetic
tendon construct further modulating cell response while stimulating the tissue mechanically, therefore aiding tendon regeneration

membrane Ca2+ ATPase activity and [Ca2+]ic, subsequently inhibiting
the synthesis of prostaglandin E2 (PGE2), a principal mediator of
inflammation (Park, Pillinger, & Abramson, 2006), in blood lympho-
cytes in rats suffering from rheumatoid arthritis (Selvam et al., 2007).
Because the cell membrane is considered as the main target of
electromagnetic fields (Markov, 2007), stimulation strongly influences
the influx of Ca2+ via volatge-gated calcium channel (VGCCs), such as
the L-type calcium channel (Pall, 2013). Enhanced intracellular calcium
levels together with exposure to electromagnetic field modulate the
activation rate of calmodulin (CaM), which, in turn, by forming the
complex Ca/CaM are involved in the dynamics of nitric oxide (NO) at
the target tissue, an essential molecule involved in the progression of
tendon healing cascade (Bokhari & Murrell, 2012). For example,
electromagnetic stimulation directly modulating CaM signals increased
the production of NO (2-fold in comparison with unexposed cells) in
2+
human fibroblasts when high levels of [Ca ]ic were observed (Pilla,
2012). However, these mechanisms of action are not completely
understood in tendon cells. Taking a step back on the anti-
inflammatory effect of electromagnetic field by intensifying the
release of IL-10 in tendon cells, they also release growth (e.g., TGF-
β) and angiogenic factors (e.g., vascular endothelial growth factor
(VEGF)) (de Girolamo et al., 2013, 2014). Moreover, stimulated cells
proliferated and migrated faster than unexposed controls, even when
two different magnetic stimuli settings were used (magnetic flow (e.g.,
0.25 μT up to 0.4 mT) and frequency (e.g., 7.8 Hz up to 50 Hz))
regardless the time of exposure (e.g., 30 min versus 7 days of
continuous stimulation) (Denaro et al., 2011; Seeliger, Falldorf,
Sachtleben, & van Griensven, 2014). In this case, the same intracellular
machinery activated upon stimulation could somehow explain the
similarities in cell proliferation and migration in face of distinct
anatomic locations from where tenocytes were harvested; however
further studies are needed to elucidate this correlation fully.
Nonetheless, the release of TGF-β and VEGF together with increased
migration highlight the regenerative potential of electromagnetic field
stimulation of tendon tissue. Cell cycle and total collagen accumula-
tion, on the other side, seem not to be influenced upon stimulation in
vitro neither culturing human tenocytes harvested from supraspinatus
and quadriceps tendons (Denaro et al., 2011; Liu et al., 2016).
Surprisingly, TSPCs electromagnetically stimulated (30 Hz,
1.5 mT) for 60 min presented a similar migratory profile compared to
unexposed cells (Randelli et al., 2016). In this study, authors also
reported that electromagnetic stimulation is likely to maintain TSPCs in
an undifferentiated state by regulating stem cell reprogramming
factors octamer-binding transcription factor 4 (Oct4), Kruppel-like
factor 4 (KLF4) and Nanog, up to 48 hr following stimulation (Randelli
et al., 2016). In a different study, tendon-derived cells stimulated with
 6
|

TABLE 1 The influence of electromagnetic field stimulation on human tendon-derived cells in vitro
Stimulation
In vitro
PEMF (frequencies between 10 and 30 Hz
and magnetic flow between 0.5–1.5 mT)
PEMF (frequency 75–Hz and magnetic
flow of 1.5 mT)
PEMF (frequency 75 Hz and magnetic
flow of 1.5 mT or 3.0 mT)
PEMF (frequency 7.8 Hz or 33 Hz, and
magnetic flow of 0.25 µT or 3.16 µT)
PEMF (frequency 50 Hz and magnetic
flow of 0.4 mT)
PEMF (Physio-Stim® PEMF system,
Orthofix Inc.)
Treatment Cell type Outcome Ref.
Single treatment for 1 hr up to 48 hr Tendon stem/progenitor cells Significant upregulation of Oct4, KLF4, and Nanog. Randelli
cell culture from supraspinatus tendon No effects on the expression of tendon markers et al.
COL1A1 and TNC. (2016)
Single treatment for 4–hr, 8 hr, and Tendon-derived cells from 8 hr treatment significantly enhances the de Girolamo
12 hr up to 48 hr cell culture semitendinous and gracilis expression of SCX (58%) and COL1A1 (49%). Release of et al.
tendons IL-6 (360%) and IL-10 (133%), (2013)
TGF-β (11-fold) and VEGF-A (41%)
Single and repeated treatments for Tendon-derived cells from Repeated treatment enhances the de Girolamo
8 hr and 12 hr up to 48 hr cell semitendinous and gracilis release of IL-10 (70%) as compared with unexposed et al.
culture tendons cells (2014)
Combined treatment for 30 min Tendon-derived cells from Enhances cell migration and proliferation Seeliger
(10 min of 33 Hz plus 20 min of patellar tendon et al.
7.8 Hz) (2014)
Continuous exposure for 7 days Tendon-derived cells from Enhances cell migration Denaro et al.
supraspinatus and quadriceps (2011)
tendon
Single treatment for 3 hr daily up to Tendon-derived cells from Increases the expression of COL1A1 (2.9-fold), Liu et al.
2 weeks supraspinatus tendon TGF-β1 (3.5-fold), PDGF (6.3-fold), BMP-12 (3.0-fold), (2016)
TIMP-4 (5.1-fold) at 2 weeks under inflammatory
conditions (10 ng/ml of IL-1α)

BMP, Bone Morphogenetic Protein; COL1A1, Collagen type I; PDGF, Platelet Derived Growth Factor; SCX, Scleraxis; TIMP, Tissue Inhibitor of Metalloproteinase; TNC, Tenascin C.
PESQUEIRA
ET AL.
PESQUEIRA ET AL.
| 7

low-frequency static magnetic field (2 Hz, 350 mT) had enhanced

Hu, & Lu, 2014

Robotti, Zimbler,

Lee et al. (1997)

Xu, Zhang, Qu,

expression of COL1A1, TNC, DCN, and SCX after a single exposure for

Strauch et al.

Grossman,
Greenough

Kenna, &
8 hr (Pesqueira, Costa-Almeida, & Gomes, 2017). Subsequently, the

(1996)

(2006)

1999
expression of COL1A1, COL3A1, and TNC increased in culture upon
Ref.

magnetic stimulation for 8 hr every 24 hr up to 14 days (Pesqueira

et al., 2017). Despite the emerging evidence, it is challenging to claim
an optimal window of magnetotherapy for tendon regeneration, given
diverse cell responses observed and magnetic settings used. Major

± 1.41 MPa), energy to failure (0.87 ± 0.17 J), and a number of

Higher load to failure (311.0 ± 59.4 N), ultimate strength (8.46
outcomes of in vitro and in vivo studies with magnetic stimulation are

PMF 17 Hz showed reduced inflammation, better collagen

summarised in Tables 1 and 2, respectively. The distinct behavior may
No differences were observed compared with control

No differences were observed compared with control

Increased in tensile strength (69%) of treated tendon

be exploited by the impact that intrinsic magnetic settings such as the

magnetic field and magnetic field gradient have on intracellular forces

alignment, and fibroblasts were more mature

proteoglycans (36%) than the control group.

(Zabloskii et al., 2016) Overall, this raises an increasing attention
toward more detailed and concise studies both at the cellular and
molecular levels. As a contact-free technology and simultaneously
acting both as a mechanical stimulation and modulation of inflamma-
tory response, of particular importance for tendon regeneration,
modulate cell fate, and ultimately, direct tenogenic differentiation,
magnetic field can represent an interesting candidate to remotely
guide tissue regeneration.
Outcome

3.2 | Magnetically responsive biomaterials in

musculoskeletal tissues: The first steps in tendon
the forepaw of New Zealand

tissue engineering
Flexor digitorum profundus of

hind leg of Sprague-Dawley

Achilles tendon of Sprague-

Flexor profundus tendon of
Achilles tendon of the right

the central toe in White

Zealand White rabbits

Three-dimensional (3D) biomaterials are an essential toolbox to

Patellar tendon of New

provide physical cues, therefore orchestrating vital cell responses

Leghorn chickens

AC, alternating current; DC, direct current; CMF, combined magnetic field; PMF, pulsed magnetic field.
White Rabbits

which culminate in tissue development (Furth, Atala, & Van Dyke,

Dawley rats

2007). Among the classes of biomaterials, magnetically responsive

Cell type
The influence of electromagnetic field stimulation on tendon tissue in vivo

ones combined with on-demand magnetic stimulation are becoming an

rats

attractive strategy to regulate cell fate (Santos, Reis, & Gomes, 2015).
Such biomaterials can be fabricated through the incorporation of
Single treatment for 15 min daily for
Single treatment for 8 hr daily for 3

magnetic nanoparticles (e.g., ferromagnetic, superparamagnetic) into a

Double treatment for 30 min daily

daily up to 16 weeks following

Combined treatment for 30 min

polymeric network by simply mixing both solutions (blending method),

between Day 6 and Day 21
Single treatment for 6 hr daily

promoting in situ precipitation of magnetic precursors (in situ

third postoperative day

precipitation method) or chemically modifying the surface of nano-

particles enabling them to act as cross-linking agents (grafting-onto
for 3 weeks

method) (Li et al., 2013). Upon fabrication, the bulk properties are
Treatment

4 weeks

hooked on the production method adopted and type of nanoparticles

weeks

used (Dai & Nelson, 2010). In particular, superparamagnetic nano-

particles are promising candidates within tissue engineering strategies
given that (i) they do not retain magnetic fluctuations after removal of
± 200 mG; amphimagnetic field = ± 600 mG)
PEMF 15 Hz with 19 quasi-rectangular pulses

PMF with quasi-sinusoidal rectified waveform

the applied external magnetic field; (ii) there is reduced interparticle

magnetic interaction; and (iii) they are stable under physiological
frequency = 76.6 Hz; DC: strength =
Radio frequency PEMF of 27.12 MHz

conditions (Dai & Nelson, 2010). Bringing this knowledge together,

(17/50 Hz) PEMF (15/46 Hz)

magnetically responsive biomaterials have been reported to exert a

CMF (AC: strength = 400 mG,

positive role in bone regeneration (Singh et al., 2014). For instance,

PEMF (frequency 15 Hz)

culturing rat bone marrow MSCs on a magnetic nanofibrous scaffold

sinusoidal waves

(15% (w/v) MNPs within a polycaprolactone (PCL) mesh) resulted in an

upregulation of osteogenic genes COL1A1, osteopontin (SPP1) and
Stimulation
TABLE 2

bone sialoprotein (IBSP) (Singh et al., 2014). Additionally, in vivo

In vivo

implantation of a magnetic nanocomposite scaffold (5–10% w/w

MNPs in PCL) combined with exposure to a static magnetic field
8 |
enhanced bone formation 6 weeks after surgery in a critical-sized
defect created in mice calvaria (Yun et al., 2016). This class of
biomaterials has also been reported for cartilage (Zhang, Lock, Sallee, &
Liu, 2015) and muscle regeneration (Cezar et al., 2016). For example,
the implantation of biphasic hydrogels (7% iron oxide in alginate
covalently modified with RGD peptide) magnetically stimulated (1 Hz
for 5 min every 12 hr) resulted in muscle regeneration with reduced
fibrous capsule formation, fibrosis, and inflammation following 2 weeks
post-implantation in mouse defect model of tibialis anterior muscle
(Cezar et al., 2016). Also, magnetically responsive biomaterials have
been proposed for tendon tissue engineering (Gonçalves et al., 2016;
Santos et al., 2016; Silva et al., 2017). Indeed, protein expression of
tendon-related ECM proteins (collagen type I and tenascin C) was
found to be increased under magnetically stimulated (2 Hz, 350 mT)
adipose-derived stem cells cultured on aligned fibrous magnetic
scaffold (0.018:1 w/w MNPs in PCL-based blend), therefore suggest-
ing the tenogenic commitment of this cell population (Gonçalves et al.,
2016). Furthermore, subcutaneous implantation of the same bioma-
terial formulation of PCL-based blend magnetically responsive
membrane (0.018:1 w/w MNPs) in a rat model has shown its ability
to moderate inflammation (Santos et al., 2016). The presence of M2
macrophages (anti-inflammatory phenotype) in the fibrous tissue was
reduced and the magnetically responsive membrane also prevented
mast cell infiltration and fibrous tissue thickening over time (9 weeks)
(Santos et al., 2016). This study is an important step to understand the
immunomodulatory ability of magnetically responsive biomaterials to
attain tissue regeneration, particularly because healing generally
involves numerous cell types including inflammatory cells. The
modulation of inflammation in early stages of tissue healing may
lead to improved healing as a result of the presence of M2
macrophages in acute inflammation frequently being favorable for
immunosuppression, scar resolution, and remodeling (Thomopoulos,
Parks, Rifkin, & Derwin, 2015).
Overall, besides acting as a physical support for cells, such
biomaterials will provide the necessary mechanical stimuli through
structural deformations by the application of an external magnetic
field, which enables the creation of a dynamic microenvironment
triggering distinct cell responses, as discussed above (Silva et al., 2017).
4 | C ONC LU SI ON S
Tendon physiology is intrinsically dependent on mechanical stimuli
from the moment of development onwards, highly dictating the
adaptation and survival of tendon cells. Increasing body of research has
uncovered how mechanical actuation modulates cell behavior by
pointing out mechanosensing molecules (Egr1, Mkx), intracellular
messengers (Ca2+, ATP) together with signaling pathways (TGF-β,
MAPK) responsible for keeping tendon cells either in the right way or
to steer the commitment of stem cells toward differentiated cells.
Magnetotherapy is becoming an attractive strategy in modulating cell
physiology with promising, although junior outcomes in tendon
healing; partially due to lack of standardized magnetic settings (e.g.,
PESQUEIRA ET AL.
frequency, magnetic flow, and magnetic gradient) and poor knowledge
of their effects on intracellular forces and machinery involved. By
knowing the underlying molecular mechanisms in command upon
stimulation, magnetotherapy could be an important adjuvant in tendon
regeneration, potentially enabling to substitute nonsteroidal anti-
inflammatory drugs, for example, highly used by patients with
inflammation. In this sense, future research is needed to explore (i)
how undifferentiated and differentiated cells recognize different
magnetotherapy-based settings; (ii) which signalling pathways are
responsible for transducing magnetic stimuli; (iii) to what extent
magnetic stimulation can modulate tendon cell behavior in inflamma-
tory and non-inflammatory environment; (iv) and whether the
expression of tendon-associated genes and proteins is altered. To
conclude, we expect that contact-free magnetically stimulated 3D
environment based approaches will advance the treatment of tendon
disorders and establish a novel paradigm for regenerative medicine
broadly.
ACKNOWLEDGMENTS
The authors acknowledge the financial support from FCT (Fundação
para a Ciência e Tecnologia) in the framework of FCT-POPH-FSE for
R.C.-A. PhD grant SFRH/BD/96593/2013, and M.E.G. FCT grant IF/
00593/2015. The authors related works were financed by the project
“Accelerating tissue engineering and personalized medicine discover-
ies by the integration of key enabling nanotechnologies, marine-
derived biomaterials and stem cells,” supported by Norte Portugal
Regional Operational Programme (NORTE 2020), under the
PORTUGAL 2020 Partnership Agreement, through the European
Regional Development Fund (ERDF).
AUTHOR S’ C ONT R I B UT I ON
TP and RC-A have contributed equally to literature review and
manuscript preparation. All authors have read and approved the final
version of the manuscript.
CONFLICTS OF INTEREST
The authors have no competing interests.
ORCID
Raquel Costa-Almeida http://orcid.org/0000-0001-7188-645X
Manuela E. Gomes http://orcid.org/0000-0002-2036-6291
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How to cite this article: Pesqueira T, Costa-Almeida R,
Gomes ME. Magnetotherapy: The quest for tendon
regeneration. J Cell Physiol. 2018;1–11.
https://doi.org/10.1002/jcp.26637