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European Journal of Trauma and Emergency Surgery (2020) 46:231–244
https://doi.org/10.1007/s00068-020-01324-1
REVIEW ARTICLE
Electrical stimulation inbone tissue engineering treatments
LiudmilaLeppik1 · KarlaMychellyneCostaOliveira1 · MitBalvantrayBhavsar1 · JohnHowardBarker1
Received: 10 December 2019 / Accepted: 4 February 2020 / Published online: 20 February 2020
© The Author(s) 2020
Abstract
Electrical stimulation (EStim) has been shown to promote bone healing and regeneration both in animal experiments and
clinical treatments. Therefore, incorporating EStim into promising new bone tissue engineering (BTE) therapies is a logi-
cal next step. The goal of current BTE research is to develop combinations of cells, scaffolds, and chemical and physical
stimuli that optimize treatment outcomes. Recent studies demonstrating EStim’s positive osteogenic effects at the cellular
and molecular level provide intriguing clues to the underlying mechanisms by which it promotes bone healing. In this review,
we discuss results of recent invitro and invivo research focused on using EStim to promote bone healing and regeneration
and consider possible strategies for its application to improve outcomes in BTE treatments. Technical aspects of exposing
cells and tissues to EStim in invitro and invivo model systems are also discussed.
Keywords Electrical stimulation· Bone regeneration· Bone tissue engineering· In vitro· In vivo
Introduction
Bone is one of the few tissues in mammals, that when frac-
tured “regenerates” on its own. However, in cases where
large volumes of bone are missing, like in severe injury,
surgical extirpation of large amounts of infected bone or
tumors, and congenital skeletal abnormalities, these regen-
erative capabilities are overwhelmed and complex and costly
treatments must be employed to close the defect. Among
conventional treatment options, bone autografts are consid-
ered to be the gold standard. However, in spite of the suc-
cess they enjoy, autografts are associated with drawbacks,
like donor site morbidity, limited availability in overly large
defects, and the risk of infection (reviewed in [1]), which
continue to fuel the search for better, alternative treatments.
Bone tissue engineering (BTE) has recently been introduced
as an alternative to conventional treatments, for large non-
healing bone defects, and holds great promise for promoting
bone healing and regeneration without the associated draw-
backs [2]. BTE approaches, in many ways, simulate bone
autografts, in that they fill the defect with bone-forming
stem/progenitor cells, scaffolds that restore missing bone
volume, and growth factors that control cell–cell and
cell–scaffold interactions [3]. Success of BTE approaches, in
clinical settings, depends largely on the choice of cells, scaf-
fold material, and signaling stimuli added to the cell–scaf-
fold mix, and/or present in the microenvironment of the
healing defect. While pre-clinical and clinical BTE treat-
ments have demonstrated encouraging early outcomes [4,
5], the logistics associated with harvesting, isolating and
amplifying the cells, and the time required to do so, are not
optimal and continue to stimulate the search for strategies to
manipulate/fine-tune the type, quantity, and composition of
stem cells, scaffolds, and stimuli (reviewed in [3]).
For decades, electrical stimulation (EStim) has been stud-
ied and used successfully in clinical practice to stimulate
bone healing (reviewed in [6]). While the detailed mecha-
nisms by which EStim promotes healing are poorly under-
stood, several recently published invitro studies suggest
that EStim’s pro-healing effect is due to its influence on the
behavior and/or function of bone-forming stem cells, such
as migration [7, 8], proliferation [9], differentiation [10, 11],
mineralization [12], extracellular matrix deposition [13], and
attachment to scaffold materials [14]. Importantly, all these
cell behaviors/functions that are central to healing could
potentially be used to optimize outcomes in BTE treatments.
In this review, we provide an overview of different methods
and results using EStim to treat cells, scaffolds, and tissues
* Liudmila Leppik
Liudmila.Leppik@kgu.de
1 Frankfurt Initiative forRegenerative Medicine, Experimental
Orthopedics andTrauma Surgery, J.W. Goethe University,
Frankfurt/Main, Germany
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232 L.Leppik et al.
1 3
in invitro and invivo model systems, with an eye toward
its potential use in BTE treatments. We discuss mechanisms
by which EStim acts at cellular and molecular levels and
discuss limitations and technical aspects of delivering EStim
both in experimental and clinical settings. This knowledge
could assist in the development of future clinical strategies
for combining EStim and BTE treatments.
Applying EStim inbone tissue engineering
treatments
EStim could potentially be added in clinical BTE treatments
either exvivo, when the cell–scaffold construct is prepared,
or invivo, after the cell–scaffold construct is delivered into
the bone defect.
EStim’s eects oncell function
Previous invitro experiments that exposed cells and/or
scaffolds to EStim, demonstrated its ability to influence cell
functions associated with enhanced bone healing [7–14].
These experiments were conducted on a variety of differ-
ent cell types; bone marrow-derived mesenchymal stem
cells (BM-MSC), from human and animal origin [10, 11,
15–25]; adipose-derived mesenchymal stem cells (AT-MSC)
[11, 20, 26–30], mouse osteoblast-like cells [31–33] and
more recently, human dental pulp stem cells (DPSC) [34].
The above cell types are commonly studied for use in BTE
applications. Based on these findings, one can speculate that
treating cell–scaffold constructs with EStim exvivo, prior
to placing the mix in a bone defect, would greatly improve
outcomes in BTE in treatments. How DC EStim affects these
cells is summarized in the following paragraphs and Fig.1.
Cell proliferation andapoptosis
The number of stem/progenitor cells that can be obtained
for use in BTE construct preparation is often limited by
the amount of donor material that can be harvested, usu-
ally from bone marrow or adipose tissue. While possible,
invitro expansion of donor cells, to reach adequate numbers
for therapeutic doses, is not an optimal solution, as it is time
consuming and can negatively impact stem cell “quality”
[35]. Although EStim has mainly been shown to increase the
rate of cell proliferation, contrary findings exist, that show
EStim can also decrease, or have no effect on cell prolifera-
tion (for general review refer to [36]). Our own experience
showed that daily application of 50–150mV/mm DC EStim
Fig. 1 Cellular mechanisms and functions activated by EStim
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233Electrical stimulation inbone tissue engineering treatments
1 3
has no effect on rat BM-MSC and AT-MSC proliferation,
when cultured in 2D or 3D (with scaffolds) [11, 29, 37]. That
said, others have shown that longer application of DC EStim
enhanced rat BM-MSC [22] and human BM-MSC prolifera-
tion. In addition, using EStim, in the form of a degenerating
sine wave, which deteriorates over time (degenerate wave—
DW), Griffin etal. were able to show an even greater effect
on cell proliferation [16]. When treating osteoblasts in static
medium conditions, Kumar etal. describes DC EStim as
having no or negative effects on cell proliferation [38]. Oth-
ers have shown positive effects of DC EStim on prolifera-
tion in 3D invitro studies with fetal human or neonatal rat
osteoblasts [39–41].
The effect of EStim on cell apoptosis, which may accom-
pany enhanced cell proliferation [42] is unclear, as some
studies have reported enhanced effect, while others describe
a decrease or no effect at all (for more details see [36]). In
summary, EStim’s effect on cell proliferation and apoptosis
appears to be heavily dependent on the type and origin of
the cells, the stimulation regimen and culture conditions [16,
43].
Cell dierentiation
EStim has been shown to enhance MSC osteogenic dif-
ferentiation in a number of studies. We and others have
demonstrated that DC EStim stimulates osteogenic differ-
entiation in rat BM-MSC and AT-MSC, cultured in osteo-
genic medium in both 2D and 3D (with scaffold) culture
conditions [10, 11, 16, 29, 37, 44, 45]. Interestingly, recent
studies showed that applying EStim, in the early stages of
MSC osteogenic differentiation (first 7 days), is sufficient to
induce a strong, sustained, and long-lasting pro-osteogenic
effect [10, 45]. As it relates to BTE treatments, this approach
would benefit the logistics of treatment, making it possi-
ble to pretreat cells + scaffold with EStim, exvivo, prior to
placing them in a bone defect. In theory, by triggering this
sustained pro-osteogenic effect, EStim-pretreatment, would
promote healing in the defect long after discontinuing its
delivery. We are currently testing this hypothesis in ongoing
invitro and invivo experiments in our laboratories.
Cell alignment
Cell alignment plays a critical role in embryonic develop-
ment, growth, and regeneration [46], as it provides specific
hierarchy of cells’ physical and mechanical properties and
biological functions at the tissue level (reviewed in details
in [46]. As it relates to BTE treatments, cell alignment is
critical in cell–cell and cell–scaffold interactions during
osteogenic differentiation and mineralization. DC EStim
has been shown to significantly affect cell alignment. Sev-
eral invitro 2D-culture studies report DC EStim causing
MSC and osteoblasts to undergo retraction and elongation,
ultimately resulting in the realignment of the long cellular
axis perpendicular to the electric field [30, 44, 47, 48]. In
invitro 3D‐culture studies, Yang etal. describes EStim as
“promoting synergy” between cells and scaffold material
[48]. Finally, it was shown that not only cell alignment, but
also cell division plane, could be controlled by externally
applied EStim [49, 50], theoretically making it possible to
control the direction of cellular expansion.
Cell migration
Cell migration is a behavior that is essential in embryonic
development, and in tissue growth and repair, and in BTE
applications can play an important role in cell infiltration
into scaffolds and integration with host tissues. When
exposed to externally applied EStim, similar in magnitude
to endogenous electrical fields, many types of cells migrate
in specific direction, and the speed and direction of cell
migration are voltage dependent (reviewed in details [51,
52]). The movement of cells along an electric field gradi-
ent, or electrotaxis, appears to be dependent on species and
cell subtype differences. For example, cells of osteosarcoma
cell line, SAOS, migrate in the opposite direction as rat cal-
varia osteoblasts [53, 54]. Interestingly, similar cells from
different origin were shown to have different electro-kinetic
properties. For example, AT-MSC display different traveling
wave velocity and rotational speed compared to BM-MSC
[55, 56].
Cell attachment/adhesion
Cell attachment/adhesion is known to affect cell behavior
and function. For example, osteogenic stem cell differen-
tiation was shown to be positively influenced by strong
adhesion to surfaces with rough microtopographies [57].
3D scaffold material, upon which cells are seeded in tissue
engineering applications, provides anchorage for cells and
are said to create a microenvironment which promote cell
differentiation, metabolic activity, and cell–cell signaling
[58]. The positive effect of EStim on cell–scaffold attach-
ment has been demonstrated and described by several groups
in several different invitro experimental protocols [39, 59,
60]. In the rapidly growing field of smart biomaterials, scaf-
folds, made of electrically active biomaterials, are specifi-
cally designed to deliver EStim to cells, to promote tissue
formation (reviewed in details in [61]).
In summary, at this point, the incorporation of EStim
into BTE treatments, is mostly at the invitro stage of devel-
opment, and can potentially make it possible to fine-tune
cell alignment, cell division, differentiation, migration, and
attachment to scaffolds. These invitro studies are laying the
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234 L.Leppik et al.
1 3
groundwork for subsequent invivo studies that can be used
to optimize outcomes in future BTE clinical studies.
EStim‑induced cell response—mechanisms
Exposing cells to exogenous EStim generates a response
called electrocoupling, caused by high resistance of the
plasma membrane, which prevents the penetration of elec-
tric stimuli, independent of the cytoplasm conductive capac-
ity [62]. One of the possible electrocoupling mechanisms
involves asymmetric redistribution/diffusion of electrically
charged cell membrane receptors in response to electric
fields, which further activates numerous downstream sign-
aling cascades. Another possible mode of action is related
to the cell membrane depolarization due to direct activation
of voltage-gated Ca2+ channels, which leads to increase in
intracellular calcium ion concentration, a cellular response
consistently reported after electric stimuli. These and other
mechanisms are discussed with more details below.
Signal transduction pathways Electrical signals are
sensed and converted into biochemical cues by multi-
ple pathways within cells, resulting in various biological
responses. The activation of the MAPK (mitogen-activated
protein kinase) cascades represents a major signal transduc-
tion pathway, which regulates specific mRNAs transcription
as consequence of external stimuli [63]. This leads to the
activation of extracellular signal-regulated kinase ERK1/2
and 5, JNK and p38MAPK, that consecutively intervenes
in important cell activities, such as proliferation, differen-
tiation, apoptosis and others, depending on the type of cell
and stimuli [64, 65]. Fast and sustained phosphorylation of
extracellular signal-regulated kinase (ERK), p38 mitogen-
activated kinase (MAPK), Src and Akt, was demonstrated by
Zhao etal. in cells migrating under the influence of electrical
fields [66, 67]. EStim was shown to induce direction and
movement of adult stromal cells through the activation of
PI3K and ROCK signaling pathways [59].
Ca2+transients Increase in intracellular Ca2+ is one of
the prompt effects of EStim on cellular response. Calcium
ions are important cellular mediators, which play a role in
many important vital activities such as proliferation, differ-
entiation, and apoptosis [68]. Intracellular Ca2+ could be
increased via two essential events; by the passage of extra-
cellular Ca2+ into intracellular space through plasma mem-
brane ion channels, or by activation of specialized receptor/
channels on the surface of the endoplasmic reticulum (ER),
which release Ca2+ from internal stores in the ER [69]. Cal-
cium oscillations can increase the efficiency and specificity
of gene expression, which guides the direction of cell dif-
ferentiation [70, 71]. EStim was shown to facilitate differ-
entiation of hMSC by changing Ca2+ oscillation patterns to
patterns similar to those seen in osteoblasts [72]. Of note,
EStim exposure can directly stimulate L-type voltage-gated
Ca2+ channels (VGCCs) in the plasma membrane [73] that
can elicit many regulatory responses through the enzymatic
action of the Ca2+/calmodulin-dependent nitric oxide syn-
thases [74].
Mechanotransduction—cytoskeletal reorganization and
actin distribution Mechanotransduction is the conversion
of external mechanical stimuli into intracellular electri-
cal or chemical signals [75]. The inverse effect of mecha-
notransduction is the transformation of electrical stimuli into
mechanical activity that causes tension in the cytoskeleton
due to reorganization of the cytoskeletal filaments and actin
redistribution. Changes in the actin structure could occur
as consequence of interactions between plasma membrane
and electrical stimuli [44]. Hereof, EStim has been shown to
cause either direct effects on the cytoskeleton, or intervene
on cellular processes regulated by the cytoskeleton [76].
Surface receptor redistribution Most likely, DC EStim is
restrained by cellular plasma membrane, which holds high
electrical resistance, and events take place at the cell surface
rather than penetrating inside the cell. As a result, most of
the biochemical signal transduction cascades in response to
EStim, arise due to the redistribution of charged cell surface
receptors (CSRs) at the external space of cell membrane
[77]. It is reported that the exposure to 100–3000mV/mm of
external EStim results in redistribution membrane proteins
and lipids on external site of the cell due to induction of rela-
tive electrophoretic movement these components on the cell
exterior [78]. Specifically, epidermal growth factor receptor
(EGFR) was shown to be up-regulated by the application of
low levels of EStim, which also induces EGFR redistribu-
tion and accumulation at the cathode side of the cell [79]. In
addition to promoting an asymmetric distribution of EGFR,
colocalization of membrane lipids and second-messenger
signaling molecule ERK ½ could also occur due to influence
of small amounts of EStim, resulting on the triggering of
MAPK signaling cascade [49, 79].
ATP synthesis Direct current EStim, ranging from 10
to 1000 µA, is known to stimulate membrane-bound ATP
synthesis [80]. This is thought to be due to EStim guiding
migrating protons to reach the mitochondrial membrane-
bound H1-ATPases, to generate ATP. This is supported by
the observation of high levels of released ATP measured in
electrically stimulated cells [81]. The relationship between
ATP synthesis and actin cytoskeleton is one of the intrigu-
ing mechanisms to explain how cells sense electrical fields.
It has been well documented that intracellular ATP is con-
sumed for the conversion of monomeric G-actin to poly-
meric F-actin [82] and that EStim-induced ATP depletion
is implicated in the reorganization of actin cytoskeleton in
electrically stimulated hMSC [76].
Heat shock proteins It has been generally hypothesized
that cells’ response to EStim (especially at levels higher
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235Electrical stimulation inbone tissue engineering treatments
1 3
that those occurring naturally) could follow physiological
stress response and function through activation of stress
heat shock proteins [83]. The involvement of heat shock
proteins (hsp 27 and hsp 70) in the upregulation of some
of the transcription factors was previously reported in
hMSC osteogenic differentiation [84].
Reactive oxygen species Participating in crucial signal-
ing pathways, reactive oxygen species (ROS) is consid-
ered another important mechanism involved in stem cell
response to EStim [85]. Controlled induction of ROS at
physiologic levels can benefit interactions of other sign-
aling molecules influencing differentiation. Numerous
studies have demonstrated that MAPK pathways and the
subsequent signaling cascades of ERK1,2, JHK, and p38
are activated by moderate levels of ROS [86]. Prolifera-
tion and differentiation of MSC were shown to be medi-
ated by a mild rise in hypoxia-induced ROS [87–89].
Lipid rafts Recent research has shown that in addition
to proteins, lipids in the cellular membrane also partici-
pate in the response to externally applied EStim. It was
shown that due to externally applied EStim, plasma mem-
brane glycolipids could redistribute and congregate into
nanodomain structures, known as lipid rafts [90]. Acting
as the initial sensor of electric fields, these nanodomain
structures polarize, coalesce, and segregate membrane
proteins, which in consequence trigger intracellular sign-
aling events to guide cell migration [91].
All these cellular mechanisms are involved in a com-
plex and finely orchestrated network of signaling and
responses. Biological processes are programmed as a
chain reaction starting from a cellular activity, which nor-
mally leads to implications in the tissues they compose.
Therefore, it follows that external interferences in the cell
response, promoted by the application of EStim, influence
not only cells but also tissues as well.
Eects ofEStim onbone healing
Bone healing is a complex and well-orchestrated process,
both in time and space, requiring coordinated function
of different cell types and systems [92]. EStim’s ability
to promote bone healing has been demonstrated both in
animal experiments and clinical settings (reviewed in [6]).
When EStim is applied to a bone defect, alone or in com-
bination with BTE constructs, its effect on all the resident
cells needs to be considered. In the following lines we
discuss how EStim, when added to BTE treatment might
influence key bone healing parameters, like osteogenesis,
vascularization, and inflammation.
Osteogenesis
The positive effect of EStim on osteogenesis is well doc-
umented in numerous invitro and invivo studies, and in
clinical applications (reviewed in [6]). In our own invivo
studies, we recently showed that DC EStim, when applied in
combination with BTE treatment [29] resulted in significant
new bone formation, by stimulating MSC proliferation and
differentiation. The underlying mechanism suggested for this
effect is that EStim enhances bone healing by stimulating
the calcium–calmodulin pathway secondary to the upregula-
tion of bone morphogenetic proteins, transforming growth
factor-β and other cytokines (reviewed in [93]; [29, 94]).
Chondrogenesis
Endochondral ossification plays a pivotal role in bone heal-
ing. During this process, progenitor cells differentiate into
chondrocytes which later undergo maturation and miner-
alization, finally resulting in new bone tissue (reviewed
in [95]). Whereas the positive effects of EStim on endo-
chondral ossification have been shown in previous studies
[29, 96, 97], there few studies focused on analyzing the
effects EStim has on endochondral ossification and MSC
chondrogenic differentiation (reviewed in [98]). In our own
invitro studies, we were not able to detect a positive effect
of direct current EStim on rat MSC chondrogenic differen-
tiation (unpublished data). However, others have reported
invitro studies using pulsed EStim that showed the oppo-
site (reviewed in [99]. One study showed that EStim alone
stimulated MSC chondrogenic differentiation [23], and oth-
ers showed that only applying an electrical field together
with a chemical inducer (transforming growth factor-β3)
induced MSC chondrogenic differentiation [100, 101]. A
new interesting approach that applies nanosecond pulse
EStim treatment to potentiate MSC chondrogenic activity
was recently reported and showed encouraging preliminary
results both invitro and invivo [99, 102]. Although these
recent examples suggest that EStim has a positive effect on
chondrogenesis, additional studies are needed to confirm this
effect and to sort out the underlying mechanisms.
Vascularization
New vessel formation plays a pivotal role in all forms of
healing and regeneration and BTE is no exception [103]. In
the case of defects that require large volumes of cells and
scaffold material, once the construct is placed in the defect,
its innermost part does not receive adequate vascularization
causing ischemia and cell death in the graft. A number of
studies have been performed that test different methods of
stimulating new vessel formation into the tissue engineered
constructs (reviewed in [104]). Several studies, in dermal
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236 L.Leppik et al.
1 3
wounds, have demonstrated EStim’s ability to stimulate new
vessel ingrowth from pre-existing blood vessels in adjacent
tissues into ischemic wounds [105–109]. DC EStim was
shown to promote important angiogenic responses of vas-
cular endothelial cells and selectively regulate production
of growth factors and cytokines important in angiogenesis
through a feedback loop mediated by VEGF receptors [110,
111]. In our own studies, in a rat femur large defect model,
adding EStim to BTE-treated bone defects caused a signifi-
cant increase in new vessel formation into the defect [29].
Inammation
It is well known that bidirectional cross talk between
immune cells and bone cells is crucial for bone remodeling
and repair [112]. The close interaction between the immune
system and bone healing is well documented. In the emerg-
ing research field of osteoimmunology, the early inflam-
matory phase of healing is a promising target for immu-
nomodulatory approaches to enhance bone healing [113].
Even though the role of immune cells and cytokines in bone
healing has been recognized for 20years now, and EStim
has been used to treat bone fractures even longer, little is
known about the effects of EStim on immune cells during
bone healing.
The immune system plays a crucial role as the host’s first
responder following injury, in which case macrophages are
rapidly recruited to the site of injury initiating the inflam-
matory response [114]. Whereas early studies showed that
DC EStim does not alter macrophage phenotype [115],
more recent studies describe EStim causing macrophages
and monocytes to migrate away from the stimuli. Moreo-
ver, EStim was shown to significantly enhance macrophage
phagocytic uptake and to selectively modulate cytokine
production [116]. EStim’s effect of upregulating osteogenic
gene (Spp2 and Bmp2) transcription in macrophages could
help explain its role in stimulating osteogenesis [89]. Invivo,
low-voltage EStim was shown to modify macrophage
response by changing the M1 to M2 macrophage ratio [97].
Overall, these findings suggest that EStim can exert a signifi-
cant effect on these macrophage sub-populations. The goal
of ongoing studies is to use EStim to fine-tune the response
of macrophages, and other immune cells from pro-inflam-
mation to pro-regenerative in BTE treatments.
EStim inbone tissue engineering
treatments—current status andlimitations
There are a growing number of studies in the literature that
focus on combining EStim and BTE treatments [117]. In
addition to studying how best to use EStim to manipulate
cell behavior, it is also important to consider technical
aspects of delivering EStim to cell + scaffold constructs and/
or tissues in clinical treatments. EStim devices for use in
treating exvivo cells prior to transplantation will have to be
developed, while commercially available DC bone growth
stimulators (OsteoGen® from Biomet EBI and Zimmer
direct current bone growth stimulator, reviewed in [118,
119]) could be adapted for treating transplanted BTE con-
structs in clinical settings.
Applying EStim invitro
In most invitro applications, cells grown in 2-D or 3-D
culture can be treated with specific regimens of EStim in
purpose-built chambers. As these chambers are not commer-
cially available, different laboratories have developed their
own to satisfy their specific needs. Below, we describe a few
of the most commonly used setups (Fig.2).
Metallic electrode EStim chamber
Perhaps the simplest in design and to use, are chambers
in which EStim is delivered directly to cells in culture by
means of metallic electrodes. Different types of metals are
used for the electrodes, including stainless steel [120], cop-
per [121], platinum [122, 123], silver/silver chloride [124],
iridium oxide, and titanium nitride [125]. Although platinum
is inferior in stiffness to other metals and is the most expen-
sive, it is nevertheless preferred over other metals since it
is less prone to corrosion [126]. Generally, one end of the
electrodes is bent to fit into a cell culture well where they are
submerged into culture medium containing the cells, and the
other end of the electrodes is connected to a power supply.
Standard 6-, 12- or 24-well cell culture plates are used,
and the electrodes are attached to removable lid(s) or
inserted directly into the well(s) [37]. This type of setup has
the advantage that multiple samples (wells) can be stimu-
lated simultaneous, thus increasing reproducibility. The volt-
age range deliverable with this setup is generally from tens
to hundreds of mV/mm. For example, in one of our studies
using this setup, we exposed rat MSC to 100mV/mm of
DC EStim for 1h/day, for 7–21days, and demonstrated that
this EStim regimen improved mineralization and expression
of osteogenic marker genes [10, 11, 37]. In another study,
Wang etal. showed that 200mV/mm of DC EStim for 4h
enhanced migration, proliferation, and differentiation of rat
BM-MSC [22].
The main advantage of this type of EStim chamber
is that it is a simple design that does not require special
equipment/knowledge to build and use. A detailed video
demonstration of how to build and use this type of EStim
chamber is available at [122]. A drawback associated with
this type of setup is the possible generation of cytotoxic
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237Electrical stimulation inbone tissue engineering treatments
1 3
faradic products on the electrode surface, which limits the
duration and intensity of EStim that can be applied to the
cells [37, 122]. The addition of a peristaltic pump to the
culture plate(s) that regularly exchanges culture medium
would avoid these limitations [38].
Salt bridge EStim chamber
Another commonly used EStim chamber delivers EStim
to cells through salt bridges submerged in the culture
media. The salt bridges separate the culture medium,
and cells from the metallic electrodes thus preventing
them from being exposed to cytotoxic electrochemical
byproducts and pH changes [127].Salt bridges contain
a saturated solution of inert salt, usually NaClO4, KCl,
or KNO3. These act as electrochemical cells that work
like batteries, transferring electric current to ionic cur-
rent through the salt bridges via redox reactions [128].
The voltage required using salt bridge EStim chambers is
relatively large, around 70V, to overcome the resistance
of the bridges [124].
While the salt bridge setup/method has been widely
used to study the effects of EStim on cultured cells, it has
a number of limitations: (1) small working area limiting
the number of cells that can be studied in a single set-
ting; (2) limited EStim exposure time due to the concen-
tration and heat differences between the bridge contents
and the media; (3) technically complicated to set up and
run experiments, making sterility and reproducibility a
challenge; (4) setup differs significantly from metallic
electrode stimulators used invivo and in clinical settings;
therefore, the correlation and consequently interpretation
between invitro and invivo study results are problematic.
Microuidic chip EStim chambers
In the EStim chamber designs described above, the cells are
often exposed to toxic electrolysis products and the elec-
trical field generated is not homogenous. These limitations
overcame in microfluidic chip EStim chambers [129–132].
Microfluidic EStim chambers consist of (1) an inlet for
loading cells, (2) a main fluidic channel, (3) a constriction
microchannel/microchip, (4) a pair of stimulation electrodes
for applying electrical stimulation and reference electrodes
for measuring extracellular field potential simultaneously,
and (5) an outlet reservoir for collection of cells after EStim
[133]. To use this chamber, cells are first loaded through the
inlet, then, by controlling the driving pressure of the flow
and using a constriction channel, cells are trapped on the
surface of measurement electrodes, where they are exposed
to EStim. After stimulation, cells are driven to an outlet
where continuous measurements are performed. The small
cross section of the chamber limits the amount of electrical
current applied and reduces the cytotoxic products that can
harm the cells. Some limitations of microfluidic chip EStim
chambers are that their small size requires that they be spe-
cially manufactured, the setup procedure prior to running an
experiment is complicated and their small size results in low
cell yield and poor cell product recovery [132].
While the EStim chambers described above are relatively
well suited to the needs of researchers for conducting invitro
experiments to study the effects of EStim on different cell/
scaffold combinations, their use for preparing large biomi-
metic BTE constructs for transplantation invivo or exvivo
in a clinical setting is not adequate. For this, special EStim
chambers will have to be developed/adapted to accommo-
date clinical requirements such as scaling up (size adapta-
tion), on line monitoring, standardization, sterilization, and
Fig. 2 EStim setups commonly used to stimulate cells in vitro. a
Metallic electrode EStim chamber delivers EStim to cells via metal-
lic electrodes submerged directly in culture medium in standard cell
culture plates. b Salt bridge EStim chamber delivers EStim to cells
through salt bridges submerged in culture medium. c Microfluidic
EStim chamber, uses micropumps to move cells in and out of con-
stricted channels where they are trapped and exposed to EStim
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
238 L.Leppik et al.
1 3
cost considerations. To grow and cultivate engineered bone
constructs long term, biomimetic perfusion bioreactors are
under development that take into consideration flow of cul-
ture medium and fluid-shear stress, position specific oxygen
gradients, mechanical, and physical stimulations [134–136].
These adaptations could allow direct comparisons between
invitro studies, move the above mentioned exciting new lab-
oratory findings closer to invivo applications and closer to
the ultimate goal of clinical application in BTE treatments.
Clinical EStim bone treatment devices
There are a number of commercially available clinical
EStim devices that could be used/adapted to treat BTE
constructs with EStim, before they are loaded into bone
defects. Devices used for EStim bone treatment in clinical
settings, can be categorized into external and internal stimu-
lators, that deliver EStim via an external field or percutane-
ously, and via internal surgically implanted electrodes. The
external stimulators deliver capacitive coupling (CC) and
inductive coupling (Pulsed ElectroMagnetic Field—PEMF)
EStim, and the internal stimulators deliver direct current
(DC) EStim [137, 138] (Fig.3).
Capacitive coupling stimulators are small, lightweight
devices, which use an external power source. Despite the
obvious advantages of not having to be surgically implanted,
and ease of use, disadvantages include, patients must change
batteries daily, skin irritation, and patient non-compliance
are common problems [138]. There are only a few clinical
studies available that support the effectiveness of CC devices
(reviewed in [6]).
Inductive coupling or pulsed electromagnetic field
(PEMF): The electrodes of these devices can be placed
under casting material or used through a cast. These devices
create low-level electromagnetic signals, which after reach-
ing the fracture site, are converted into electric currents and
are said to mimic the body’s normal physiologic processes.
The primary advantage of PEMF bone stimulators is their
noninvasive application; however, drawbacks include, the
heavy weight of these devices, difficulty assessing treatment
dosage, and patient non-compliance [139].
DC electrical stimulators have the benefits of providing
constant and uniform current delivery, the EStim is focused
at the bone defect, and elimination of patient non-compli-
ance. Surgical implantation consists of placing a cathode
at the fracture site and an anode in the nearby subcutane-
ous tissue, that deliver electric current flow between them.
The electrodes are connected to a stimulator device, which
is implanted subdermally [140, 141]. The power source of
these devices typically last from 6 to 8months, at which time
the implanted device and electrodes must be removed in a
second procedure. Over the last 3 decades, numerous studies
support the clinical efficacy of these DC EStim stimulators
[138, 142, 143]. Recent clinical studies using implantable
DC EStim stimulators, alone [144], or in combination with
bone grafts [145] have reported increased bone healing rates.
Other physical stimulation techniques, like magnetic and
vibration stimulations, have been tried and found to pro-
mote bone healing [146], in general their administration in
patients in clinical settings have come up against serious
limitations. The wearable devices used for their administra-
tion are cumbersome, thus when used for prolonged periods
tends to interfere with patients’ daily activities leading to
decreased compliance [6]. In addition, these units have been
reported to give inconsistent results, and one of the reasons
for this has been attributed to the fact that the stimulation
energy they generate is not focused at the fracture site. In
contrast, when DC EStim is administered with surgically
implanted device compliance is not an issue and the electri-
cal energy is focused at the fracture site, which has led to the
reporting of more consistent treatment outcomes.
Despite EStim’s demonstrated effectiveness improving
bone healing, both in pre-clinical animal studies and in clini-
cal settings, few studies have investigated the effectiveness
of combining EStim with BTE treatments invivo [27, 29,
Fig. 3 Clinical EStim devices. External stimulators deliver capacitive coupling and inductive coupling (pulsed electromagnetic field) EStim, and
internal stimulators deliver direct current (DC) EStim via surgically implanted device
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
239Electrical stimulation inbone tissue engineering treatments
1 3
94, 147, 148]. In one of these studies, our group treated large
defects in rat femurs with AT-MSC + Scaffold + EStim. We
found that the rate and quality of bone healing at 8weeks,
in defects treated with AT-MSC + Scaffold + EStim, was
significantly better than controls [29]. Our own experience
from this study, together with reports in the literature [141,
149], suggests that the problems of combining EStim with
BTE in clinical settings would be similar to those experi-
enced in current clinical EStim bone treatments. Namely,
complications associated with the surgical procedures used
to implant and explant the EStim device, electrode breakage
or dislodgement, and infection.
Current developments
Summarizing, invitro experiments that expose cells and/or
scaffolds to EStim, generally show positive results, although,
a lack of standardization of cell types, models and protocols
make it difficult to draw definitive conclusions. Additional
studies are needed to develop strategies for transferring these
encouraging invitro findings into meaningful invivo BTE
applications. In addition, the logistics of combining EStim
and BTE treatments in practical, cost effective ways in clini-
cal settings must be considered.
Some exciting new developments that could be incorpo-
rated into these strategies include the use of electroactive
smart polymeric biomaterials that could potentially combine
scaffold and EStim into one. Recent advancements in poly-
mer science, using “smart” biomaterials, that enable built-
in stimulus/response behavior capabilities, have tremendous
potential [150]. Electroactive smart polymeric biomaterials
could be used to build scaffolds that offer precise control
over the amount, duration, and localization of the electrical
stimulus, thus obviating the need for bone stimulators. These
materials have already been tested invitro, where they have
demonstrated the ability to improve cell proliferation and
differentiation [151–153]. If in addition to promoting these
pro-osteogenic activities, these smart biomaterials can also
be designed to biodegrade after a given useful time period,
this would be yet another benefit [154].
Conclusions
EStim has the demonstrated ability to improve osteogenic
potential in various types of MSC and osteoblast-like cells
invitro, and to stimulate new tissue formation like, bone,
cartilage, and vessels invivo. It is important to recognize
that these encouraging early findings are strongly dependent
of many factors like type and origin of cells, EStim regi-
men, and area of the defect to be treated. The variability of
these results reported in the literature make it difficult to
compare and develop a single optimal EStim + BTE proto-
col. Accordingly, it is important that future invitro experi-
ments be planned and conducted with an eye toward apply-
ing the findings in invivo models and regimens that can be
transferred into to clinical protocols. Combining EStim and
BTE treatments has the potential to create synergies that
could result in outcomes that far exceed those achieved by
either treatment on its own.
Acknowledgements Open Access funding provided by Projekt DEAL.
Funding This study was supported by the Friedrichsheim Foundation
(Stiftung Friedrichsheim) based in Frankfurt/Main, Germany.
Conflict of interest The authors declare that they have no conflict of
interest.
Informed consent No informed consent is necessary.
Human and animal rights This study does not include any human par-
ticipants and/or animals.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.
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