Cellular and Molecular Responses to Mechanical Expansion of Tissue

Abstract

The increased use of tissue expander in the past decades and its potential market values in near future give enough reasons to sum up the consequences of tissue expansion. Furthermore, the patients have the right to know underlying mechanisms of adaptation of inserted biomimetic, its bioinspired materials and probable complications. The mechanical strains during tissue expansion are related to several biological phenomena. Tissue remodelling during the expansion is highly regulated and depends on the signal transduction. Any alteration may lead to tumour formation, necrosis and/or apoptosis. In this review, stretch induced cell proliferation, apoptosis, the roles of growth factors, stretch induced ion channels, and roles of second messengers are organized. It is expected that readers from any background can understand and make a decision about tissue expansion.

Full text

REVIEW
published: 15 November 2016
doi: 10.3389/fphys.2016.00540
Frontiers in Physiology | www.frontiersin.org 1November 2016 | Volume 7 | Article 540
Edited by:
Mauricio Antonio Retamal,
Universidad del Desarrollo, Chile
Reviewed by:
Roberta Tasso,
Ospedale San Martino (IRCCS), Italy
Xinhua Qu,
Shanghai Ninth People’s Hospital,
China
*Correspondence:
Mohammad T. Rahman
m.tariqur.rahaman@gmail.com
Specialty section:
This article was submitted to
Membrane Physiology and Membrane
Biophysics,
a section of the journal
Frontiers in Physiology
Received: 26 July 2016
Accepted: 27 October 2016
Published: 15 November 2016
Citation:
Razzak MA, Hossain MS, Radzi ZB,
Yahya NAB, Czernuszka J and
Rahman MT (2016) Cellular and
Molecular Responses to Mechanical
Expansion of Tissue.
Front. Physiol. 7:540.
doi: 10.3389/fphys.2016.00540
Cellular and Molecular Responses to
Mechanical Expansion of Tissue
Muhammad Abdur Razzak 1, Md. Sanower Hossain 1, Zamri Bin Radzi 1,
Noor Azlin B. Yahya1, Jan Czernuszka 2and Mohammad T. Rahman1*
1Department of Children’s Dentistry and Orthodontics, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia,
2Department of Materials, University of Oxford, Oxford, UK
The increased use of tissue expander in the past decades and its potential market values
in near future give enough reasons to sum up the consequences of tissue expansion.
Furthermore, the patients have the right to know underlying mechanisms of adaptation
of inserted biomimetic, its bioinspired materials and probable complications. The
mechanical strains during tissue expansion are related to several biological phenomena.
Tissue remodeling during the expansion is highly regulated and depends on the signal
transduction. Any alteration may lead to tumor formation, necrosis and/or apoptosis.
In this review, stretch induced cell proliferation, apoptosis, the roles of growth factors,
stretch induced ion channels, and roles of second messengers are organized. It is
expected that readers from any background can understand and make a decision about
tissue expansion.
Keywords: tissue expansion, growth factors, focal adhesion complex, apoptosis, ion channels, secondary
messengers
INTRODUCTION
Since the first utilization in 1957 (Neumann, 1957), the use of tissue expansions have become
widespread in maxillary and craniofacial surgery (Kobus, 2007), burn scar excision (Hafezi et al.,
2009), breast reconstruction following mastectomy (Lohsiriwat et al., 2013), ophthalmology (Hou
et al., 2012), management of omphalocele (Clifton et al., 2011), nasal reconstruction (Kheradmand
et al., 2011), scalp alopecia (Guzey et al., 2015) and other deformities in plastic reconstructive
surgery (Motamed et al., 2008; Laurence et al., 2012; Santiago et al., 2012). Tissue expander
generates new tissues, by exploiting the viscoelastic properties of the skin and adjusted histological
changes which follows the principle of the controlled mechanical skin overstretch (Argenta, 1984;
Pamplona et al., 2014). It involves the insertion of a biomimetic and bioinspired material (i.e.,
hydrogel tissue expander) adjacent to a wound or defect that needs to be resurfaced (Motamed et al.,
2008; Swan et al., 2012). The expanded tissue can then be used to resurface a defect or incorporate
permanent prostheses (Kasper et al., 2012; Swan et al., 2012).
Nevertheless, tissue expansion for the reconstructive surgery are also associated with a variety of
complications (Adler et al., 2009; Huang et al., 2011). Swan et al. (2012) observed mucoperiosteal
ulceration while using uncoated self-inflating anisotropic hydrogel tissue expander in the porcine
hard palate. Minor side effects on skin histology and circulation resulted in skin stretching with
staples or hypodermic needles, thus proving the Pavletic device to be non-feasible in primary
wound closure (Tsioli et al., 2015). Incidence of infection, being the most common complication
(Huang et al., 2011), has witnessed a total of 16 cases out of 215 children who underwent
reconstruction with tissue expanders (Adler et al., 2009). However, the pivotal concern is to ensure
normal tissue patterning and prevent tumor or scar formation (Huang and Ingber, 1999; Aarabi
et al., 2007).

Razzak et al. Cellular Response to Mechanical Stress
Recent studies revealed that rapid changes in extension,
alignment, and collagen adapt to mechanical expansion (i.e.,
stretch or strain). Both elastin and collagen realign in a parallel
fashion in response to stretch and/or expansion (Verhaegen
et al., 2012; Tsioli et al., 2015), and the elongation occurs to the
direction of stretching (Figure 1). Mechanical stretch on tissue
is related to several physiological phenomena such as cellular
growth enhancement and/or expansion with a significantly
higher vascularity of expanded tissue (Yano et al., 2004). Strain
beyond physiological limit may lead to alteration of cell function
such as tumor formation, necrosis and/or apoptosis (Chen et al.,
1997; Huang and Ingber, 1999; Wernig et al., 2003; Knies et al.,
2006). Hence lies the clinical implications of tissue expansion
(Swenson, 2014; Kwon et al., 2016).
In physiological condition, tissue development and
remodeling are highly regulated. A number of studies have
focused on the cellular and molecular mechanisms (such
as integrated network of cascades, implicating growth factors,
cytoskeleton, protein kinase family, synthesis of DNA, expression
of gene) leading to the increase of skin surface area (Plenz et al.,
1998; Takei et al., 1998; Skutek et al., 2003; Knies et al.,
2006; Jaalouk and Lammerding, 2009; Wong et al., 2011; Wu
et al., 2015). Under mechanical stress, the cell phenotype and
FIGURE 1 | (A) Effects of tissue expansion on surrounding tissues. (B) Tissue expander before implantation and implanted in rat. Pictures taken from ongoing
research in author’s lab.
the nature of the physical stimuli determine which signal
transduction pathways are activated during tissue expansion
(Hsieh and Nguyen, 2005). This review, will focus the reports of
molecular events of skin-derived cells in response to mechanical
strain. The response of cells to mechanical stretch, the roles of
growth factors, effects on extracellular matrix, cell membrane,
and stretch induced ion channels, roles of second messengers,
and cellular interactions will be organized from the extracellular
to intracellular pathways with future perspectives in the
conclusion.
RESPONSE OF CELLS TO MECHANICAL
STRETCH
The viscoelastic properties of skin to increase surface area in
response to forces are the basic biology of tissue expansion
(Bascom and Wax, 2002). The external forces are transmitted
through the multi-layered skin which consists of epidermis
connected to the dermis and the underlying subcutaneous
tissues (Schwartz and DeSimone, 2008). The morphological
and physiological consequences of tissue expansion on various
layers of skin and other cellular and muscular components are
summarized in Table 1.
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Razzak et al. Cellular Response to Mechanical Stress
TABLE 1 | Responses of tissues to expansion.
Tissues Effects observed References
Epidermis • Increased density and thickness of epidermis up to 40% instead of normal state (10%) in expanded skin
• Reduced intercellular spaces in all layers of the epidermis
• Remarkably increased the mitotic activity of epidermis; resulting increased DNA synthesis and therefore
cellular proliferation
• Maintained phenotypical characteristic of epidermis
Austad et al., 1982; Vander Kolk
et al., 1987; van Rappard et al.,
1988; Silver et al., 1992.
Dermis • Thinned dermal thickness rapidly with an average of 20% and thickness may return to normal within 2
years following expansion
• Decreased the density of hair follicles in the expanded skin but quantitatively and functionally remain
unchanged
• Increased collagen synthesis in the dermis during tissue expansion
• Observed temporary hyperpigmentation in expanded tissue upon up-regulation of melanin expression
during tissue expansion
Austad et al., 1982; Pasyk et al.,
1988; Johnson et al., 1993.
Fat • Lost subcutaneous fat permanently
• Decreased the thickness of adipose tissue and markedly decreased the number of fat cells by as much as
30 to 50%
• May flattened or disappeared adipocytes altogether during the expansion process
• Occurred a varying amount of fat necrosis during tissue expansion process, the degree of which is related
to the rate of expansion
Leighton et al., 1988; Pasyk et al.,
1988; Takei et al., 1998.
Muscle • Sensitive to tissue expansion and changed ultra-structural
• Thinned muscle in expanded skin without changing the number of cells
• Increased number and size of mitochondria, number of vesicles and amount of sarcoplasm
• Undergo atrophy and weakness after expansion resulting in the so-called bath-tub depression, but
permanent sequelae are rare
Pasyk et al., 1982; Sasaki and
Pang, 1984; Stark et al., 1987;
Johnson et al., 1993.
Capsule • Developed a dense fibrous capsule around the expander after few days of implantation
• Elongated fibroblasts, which stimulates the synthesis of collagen
• Developed double-layered capsule within 7 days of expander implantation
• Increased the thickness of capsule after 2 to 2.5 months of expansion
Austad et al., 1982; Johnson et al.,
1993.
Blood vessels • Observed rapid angiogenesis and distention of capillaries during expansion
• Increased the number of arterioles and venules within few days of expansion
• Elongated veins and arteries rapidly with no loss of diameter or intimal integrity
Sasaki and Pang, 1984; Stark et al.,
1987; Saxby, 1988.
Nerve • Nerve tissue is tolerant to tissue expansion and no demyelination or necrosis of nerve tissue
• Lengthen the peripheral nerve without significant damage
• No neurologic change in response to expansion during tissue expansion (Intraluminal pressure more than
44 mm Hg may cause reduction of axon potential)
Swenson, 2014.
Bone • Tissue expansion causes significant but reversible cranial and long bone changes
• Reduced bone thickness and volume during tissue expansion
• Noticed erosion beneath the expander without changing bone density
• Nothing changed in the inner table of the skull or stigmata
Antonyshyn et al., 1988; Moelleken
et al., 1990; Johnson et al., 1993.
Vascular plexus • Enhanced angiogenesis in expanded tissues might be caused of increased gene expression and VEGF level
• Raised more vascularized flaps in expanded tissue and survived to a greater length, averaging 117% over
control flaps
Saxby, 1988; Nikkhah et al., 2015.
Numerous researchers have linked the mechanisms that lead
to an increased length with skin’s elasticity (Kenedi et al., 1975;
Bader and Bowker, 1983; Larrabee Jr and Sutton, 1986). Gibson
et al. (1965) associated the increase in skin length with the
interstitial displacement of fluids and skin’s creep behavior.
Austad et al. (1982) reported that the increased length was as
a result of cellular proliferation. Siegert et al. (1993) simplified
these findings relating the strain, time and mechanism of skin
expansion as shown in Figure 2. Because of its elasticity, the
skin expands practically without temporal delay after expansion
pressure is exerted. Interstitial displacement of fluids can be
seen (in oedema) after skin expansion. Larrabee Jr et al. (1986),
Gibson et al. (1965) and Wilhelmi et al. (1998) suggested that
the biological creep (i.e., the generation of new tissue) is due to
the chronic stretching forces. It is also most likely that similar
events such as interstitial fluid displacement and elasticity beyond
the tolerance limit of the tissue might induce necrosis and/or
apoptosis of the tissue (Linder-Ganz and Gefen, 2004).
Cell stretching, in some contexts causes apoptosis, and in
others promotes cell proliferation (Takei et al., 1998; Skutek
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Razzak et al. Cellular Response to Mechanical Stress
FIGURE 2 | Physiological and cellular response of skin to mechanical
stress. It is most likely that mechanical stress beyond the limit of tolerance of
elasticity might induce necrosis and/or apoptosis at the cellular level (Modified
from Siegert et al., 1993).
et al., 2003). Similarly, apoptosis and proliferation pathways
share many common elements, and they converge and influence
each other at different levels (Wernig et al., 2003). Application
of mechanical stretch (stimulus) activates mechanosensitive
ion channels, G-protein coupled receptors, protein kinases,
integrin-matrix interactions and other membrane-associated
signal-transduction molecules to convert physical cues to
biologic responses (Schwartz and DeSimone, 2008; Jaalouk and
Lammerding, 2009) (Figure 3).
Stretch Induced Proliferation
In response to mechanical stretch, cells of the cutaneous tissues,
such as fibroblasts, receive the signals and prepare to proliferate
(Silver et al., 2003). The extracellular matrix (ECM) plays a
central role in strain-induced cell proliferation (Hynes, 2002).
The extracellular forces transmitted through the ECM leading
to the deformation of the matrix, followed by alteration of
plasma membrane and adhesion complexes (Chien, 2007).
The transmembrane protein integrin communicate with both
extracellular matrix and cytoplasmic proteins such as talin,
paxilin, and vinculin. Integrins also sense the physical properties
of the ECM and organize the cytoskeleton accordingly (Zamir
and Geiger, 2001). Binding of talin to the integrin cytoplasmic
tail induce a conformational change from an inactivated to an
activated state with an increase affinity for the ECM (Tadokoro
et al., 2003). Upon the activation of integrins, the βsubunit
complexes with numerous structural and signaling proteins to
form a focal adhesion complex (FAC) to provide both the
physical link between integrin-adhesion receptors and the actin
cytoskeleton, as well as sites of signal transduction into the cell
interior (Carragher and Frame, 2004; Wozniak et al., 2004). The
activated FAC then activate signal transduction pathways that co-
ordinate cell proliferation (Figure 4). Hence it is well evident that
a number of growth factors in ECM regulate cell proliferation
(Singh et al., 2009; Bush and Pins, 2010).
Recently, Jiang et al. (2016) demonstrated that, static stretch
conditions can increase collagen I levels but decrease fibronectin
levels compared to a cyclic stretch conditions where collagen I
is significantly reduced but fibronectin is markedly increased.
Thus, cyclic stretch suppressed human fibroblast proliferation
compared to that with static stretch. Again, nuclear envelope
proteins such as emerin or lamin A/C were shown to play
critical roles in suppressing vascular smooth muscle cells
hyperproliferation induced by hyperstretch (Qi et al., 2016).
Stretch Induced Apoptosis
A balanced cell proliferation/growth and apoptosis is a pre-
requisite for normal development and for adaptation to a
changing environment (Jacobson et al., 1997). Too little apoptosis
can promote cancer and autoimmune diseases; whereas, too
much apoptosis can augment ischaemic conditions and drive
neurodegeneration (Czabotar et al., 2014). Apoptosis can be
triggered either by external receptor-dependent stimuli (ligation
of death receptors with their cognate ligands, such as FasL,
TRAIL or TNF) or internal mitochondria-mediated signaling
(Adams, 2003; Özören and El-Deiry, 2003).
Different stimuli such as intracellular damage, cytotoxic
compounds and developmental activates the mitochondrial
(intrinsic) pathway of apoptosis (Liao et al., 2004, 2005). In
this pathway, stretch activates pro-apoptotic effectors Bax and
Bak, which then disrupt the mitochondrial outer membrane
resulting in the release of cytochrome c (Figure 3). Cytochrome
c then leads to the formation of the apoptosome with the help
of apoptotic protease-activating factor 1 (apaf-1) that promotes
caspase 9 activation (Li et al., 1997; Luo et al., 1998; Zou et al.,
1999). In the death receptor-mediated pathways (extrinsic) of
apoptosis, certain death receptor ligands of the tumor necrosis
factor (TNF) family (such as Fas ligand and TNF) bind with
their cognate death receptors (FAS and TNFR1, respectively) on
the plasma membrane, leading to caspase 8 activation via the
Fas-associated death domain protein (FADD) and the TNFR-
associated death domain protein (TRADD) in a cytosolic death-
inducing signaling complex (DISC) also known as complex II
(Wang et al., 2008; He et al., 2009). These two pathways converge
at activation of the effector caspases (caspase 3, caspase 7, and
caspase 6) (Adams, 2003).
Necrosis, known as a catastrophic form of death, is typically
not associated with caspases activation and mediates cells’ demise
in response to severe injuries or in case of a pathological evet
(Vanden Berghe et al., 2004). Although, apoptosis and necrosis
may occur simultaneously in response to specific stimuli, the
morphological characteristics of cell undergoing necrosis are
distinct from those seen in cells undergoing apoptosis (Kroemer
and Levine, 2008). However, mechanisms of necrosis due to
tissue expansion are not fully understood.
MAJOR ROLES OF GROWTH FACTORS IN
TISSUE EXPANSION
The cellular growth, tissue integrity and eventually the
reestablishment of the barrier function of the skin is executed
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Razzak et al. Cellular Response to Mechanical Stress
FIGURE 3 | Possible signaling pathways activated in response to mechanical stretch. Upon application of forces, extracellular matrix deformed and the
plasma membrane altered resulting activation of the ion channel and activate the integrins, G-protein coupled receptors, tyrosine kinase receptors and others
membrane bound signaling pathways.
and regulated by the coordinated efforts of several cell types
(keratinocytes, fibroblasts, macrophages, platelets etc.) and
numerous growth factors (biologically active polypeptides)
(Werner et al., 2007; Gurtner et al., 2008). The epidermal
growth factor (EGF) family, transforming growth factor beta
(TGF-β) family, fibroblast growth factor (FGF) family, vascular
endothelial growth factor (VEGF), platelet derived growth factor
(PDGF), connective tissue growth factor (CTGF), interleukin
(IL) family are all important in stress (either mechanical or
physiological) induced cell growth (Werner et al., 1994; Shimo
et al., 1999; Steiling and Werner, 2003; Shirakata et al., 2005;
Secker et al., 2008). The functions of growth factors depend
on source and binding with specific receptors and can act
by paracrine, autocrine, juxtacrine, and endocrine mechanisms
(Barrientos et al., 2008). Earlier studies showed that, EGF, FGF-2,
TGF-β, PDGF, and VEGF levels are increased in early after injury
and decreased at chronic states and IL-1 and 6, and TNF-αlevels
increased both in early and chronic states (Brown et al., 1986;
Frank et al., 1995). The functions of various growth factors are
summarized in Table 2.
Among the growth factors families, the EGF family and the
TGF-βfamily are thought to play central roles (Hashimoto,
2000) and they provide dual-mode regulation of keratinocyte
growth via the proliferation-stimulating effect of EGF and the
proliferation-inhibiting effect of TGF-β(Amendt et al., 2002;
Secker et al., 2008). Although, these growth factors appear to
share several downstream pathways of cell membrane molecules,
the direct effects of mechanical stress on TGF and EGF are yet to
be investigated (Takei et al., 1998). Although, human epidermal
keratinocytes express ErbB1, ErbB2, and ErbB3, they do not
express ErbB4 (Hashimoto, 2000). Similarly, signals originating
from ErbB1 play crucial roles in mediating the pro-survival
and proliferative programs of keratinocytes (Shirakata et al.,
2010). The expression of cadherins, integrins, and various other
ECM components that contribute to the maturation of new
blood vessels are regulated by FGF2 (Cross and Claesson-Welsh,
2001). HB-EGF shows a starring role in the reepithelialisation
and granulation tissue formation (Marikovsky et al., 1996). The
strongest autocrine stimulation to cell growth is provided by
amphiregulin (Piepkorn et al., 1994).
ION CHANNEL RELATED TO MECHANICAL
STRAIN
Mechanical stress to the cell surface activates the
mechanosensitive ion channels along with other membrane-
associated signal-transduction molecules (De Filippo and Atala,
2002; Wang et al., 2009). The precise mechanism of activation
and modulation of ion channels by mechanical forces that
results in biologically meaningful signals are subjects of intensive
research (Martinac, 2014). Sachs (1991) reported that, in order
to make conformational changes of a channel, external forces
must do work on the channel and be dominated by the distance
the force move. Howard and Hudspeth (1988) estimated that
the stress activated channels change their dimensions by 4 nm
between the closed and open states. These stretch-induced ion
channels are mainly cation (Ca2+, K+, and Na+) channels and a
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Razzak et al. Cellular Response to Mechanical Stress
FIGURE 4 | Signaling pathways activated by mechanical stretch leading to either cell proliferation or apoptosis. The integrins organize the cytoskeleton
according the physical properties of the extracellular matrix (ECM). The membrane bound ion channels, G-protein, tyrosine kinase receptor and other molecules
activate specific pathways to proliferation. In case of apoptosis, receptor-like molecules such as integrins, focal adhesion proteins become activated and these
molecules in turn activate a limited number of protein kinase pathways (p38 MAPK, PI3K/Akt, JNK etc.), which amplify the signal and activate enzymes (caspases)
that promote apoptosis. Activation of death receptors (Fas and/or TNFR) leads to the formation of a death-inducing signaling complex (DISC), resulting in the cleavage
of procaspase-8 to its active form. Caspase-8 in turn activates downstream proteins that lead to apoptosis. Bax, induces the release of cytochrome c from the
mitochondria and promotes apoptosis. Moreover, cytochrome c complexes with apaf-1 and procaspase-9 to form an apoptosome. This leads to the activation of
caspase-9, which in turn activates effector caspases (3, 6, and 7) and subsequent apoptosis. Among the stretch-activated ion channels, rapid influx of Ca2+activate
several pathways including signal transduction cascades leading to cell proliferation, apoptosis, cell contraction, activation of potassium channel. Potassium channels
play roles in maintaining optimal membrane potentials. Mechanical forces and calcium influx also open chloride channels which act as apoptotic agents through a
delineated mechanism.
few anion (Cl−) channels (Jackson, 2000; Nilius and Droogmans,
2001).
The vast majority of channels open because of the changes
in lipid bilayer, membrane fluidity or tension and are regulated
by voltage, extracellular ligands, phosphorylation, influx of Ca2+
and direct (physical interactions between G-protein subunits
and the channel protein) or indirect (via second messengers
and protein kinases) interaction with activated G proteins
(Christensen, 1987; Maroto et al., 2005; Lumpkin and Caterina,
2007; Hahn and Schwartz, 2009). The mechanosensitive activities
of ion channels are cell dependent and vary from cell to cell
(Hsieh and Nguyen, 2005). The elevated intracellular Ca2+levels
are cytotoxic and provide the apoptotic stimulus in multiple cell
types. The studies of past decades indicated the involvement of
different ions in stretch induced response and cytoskeleton are
also associated (Jackson, 2000; Wang et al., 2001). However, the
precise ion channels related mechanisms for tissue expansion are
yet to be studied.
SECOND MESSENGERS SYSTEM IN
STRAIN-INDUCED RESPONSES
The exact role of second messengers system in response to
tissue expansion (i.e., epithelial cell proliferation) is not clearly
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Razzak et al. Cellular Response to Mechanical Stress
TABLE 2 | Growth factors in response to mechanical or physical stress on different tissues.
Growth factor Native cells Experimental condition
(expansion or stress)
Effect on
growth factor
Major observations References
Epidermal growth
factor (EGF)
Macrophages, Fibroblasts Burn injuries ↑Keratinocyte proliferation and
migration
Grayson et al.,
1993
2 mm incisional wounds on the
PU.1 null mouse
↑Reepithelialisation Martin et al.,
2003
Heparin-binding
epidermal growth
factor (HB-EGF)
Macrophages Keratinocyte-specific
HB-EGF-deficient mice
↓Wound closure was markedly
impaired
Shirakata et al.,
2005
Cells treated with tetracycline
(TET)
↑↑ Overexpression of HB-EGF inhibits
proliferation
Stoll et al., 2012
Fibroblast growth
factor 1, 2, and 4 (FGF
1, 2, and 4)
Fibroblasts,
Macrophages, Endothelial
cells, Smooth muscle
cells, Chondrocytes, Mast
cells
Cultured fibroblasts stimulated
with IL-1α
↑Fibroblast proliferation
Angiogenesis
Maas-
Szabowski and
Fusenig, 1996
Transforming growth
factor-α(TGF-α)
Macrophages,
Keratinocytes
Macrophages isolated from a
wound site
↑Keratinocyte migration and
reepithelialisation
Rappolee et al.,
1988
Transforming growth
factor-β1-3 (TGF-β1-3)
Macrophages,
Fibroblasts, Keratinocytes,
Neutrophils
Adult and fetal wounds II
↑
Reepithelialisation of skin
Epidermal differentiation
Cowin et al.,
2001a
Fetal and adult sheep incisional
skin wounding
↑TGF-β3 is anti-scarring Scheid et al.,
2002
Amphiregulin (AR) Keratinocytes Serum free cultured human
keratinocytes
↑Keratinocyte proliferation Piepkorn et al.,
1994
Keratinocyte growth
factor (KGF or FGF7)
Fibroblasts Wounded mice skin ↓Delayed re-epithelialization due to
reduced proliferation rate of
epidermal keratinocytes
Werner et al.,
1994
Platelet derived growth
factor (PDGF)
Macrophages, Endothelial
cells
Acute incisional wounds in an
aging mouse colony
↓The low levels of PDGF in the old
cause initial delay in fibroblasts and
inflammatory cell infiltration and
proliferation within the wounds
Ashcroft et al.,
1997
Hepatocyte growth
factor (HGF)
Mesenchymal cells,
Hepatocytes, Adipocytes,
Keratinocytes
Adult rat excisional wounds ↑Keratinocyte migration, and
proliferation Angiogenesis
Cowin et al.,
2001b
Vascular endothelial
growth factor (VEGF)
Neutrophils,
Macrophages, Endothelial
cells, Fibroblasts,
Immobilized VEGF in porous
collagen scaffold
↑Endothelial cell proliferation,
migration, and angiogenesis
Shen et al.,
2008
Connective tissue
growth factor (CTGF)
Fibroblasts, Endothelia Scratched human corneal
epithelial cells
↑CTGF is strongly induced and
caused pathophysiology in tissues
by inducing matrix deposition,
conversion of fibroblasts into
contractile myofibroblasts
Secker et al.,
2008
Insulin-like growth
factor-I (IGF-I)
Fibroblasts, neutrophils,
macrophages,
hepatocytes and skeletal
muscle
Estrogen-deprived mice ↑Keratinocyte and fibroblast
proliferation and migration
Collagen synthesis and
re-epithelialization
Emmerson
et al., 2012
Rat surgical incision ↑Re-epithelization Todorovic et al.,
2008
(Continued)
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Razzak et al. Cellular Response to Mechanical Stress
TABLE 2 | Continued
Growth factor Native cells Experimental condition
(expansion or stress)
Effect on
growth factor
Major observations References
Interleukin-I αand β
(IL-I αand β)
Neutrophils, Monocytes,
Macrophages,
Keratinocytes
Irradiated fibroblasts ↑Keratinocyte activation, migration
and proliferation
Induce KGF expression and
fibroblasts creation
Maas-
Szabowski
et al., 2000
Endothelin-I (ET-I) Keratinocytes, Fibroblasts,
Endothelial cells
Cyclic stretch of cultured rat
aortic smooth muscle cells
(raSMC) and porcine aortic
endothelial cells (PAEC)
↑(PAEC)
↓(raSMC)
Reveal central role for the
endothelin system in
stretch-induced apoptosis of the
smooth muscle cells.
ET-1 binding to the ETBreceptor
subtype results in apoptosis rather
than proliferation
Cattaruzza
et al., 2000,
2001.
Activin Keratinocytes, Fibroblasts,
Inflammatory cells,
Macrophages
Normal and wounded skin ↑Stimulates keratinocyte migration,
fibroplasia, and matrix production
Hübner et al.,
1996
↑, increased in response to mechanical strain; ↓, decreased in response to mechanical strain; ↑↑, overexpression in response to mechanical strain; II, unchanged in response to
mechanical strain.
TABLE 3 | Effects of mechanical strain on major second messengers.
Second messenger Experimental condition (expansion or
stress)
Effects on second
messenger
Major observation References
Cyclic adenosine
monophosphate (cAMP)
Cyclical elongation and relaxation of smooth
muscle cells grown on elastic membrane
↑Collagen production inhibited by
raised cAMP.
Kollros et al., 1987
Round tissue expanders were placed dorsally ↓Protein production increased in
expanded tissue
Johnson et al., 1988
Constant and cyclic strain (150 mmHg for 5
days) of human keratinocytes
↓Protein production significantly
increased
Takei et al., 1997
Prostaglandin E2 (PGE2) Cyclical elongation and relaxation of smooth
muscle cells grown on elastic membrane
↑Collagen production inhibited by
increased PGE2.
Kollros et al., 1987
Constant and cyclic strain (150 mmHg for 5
days) of human keratinocytes
↓Protein production significantly
increased
Takei et al., 1997
Phosphodiesterase IV (PDE IV) Constant and cyclic strain (150 mmHg for 5
days) of human keratinocytes
↑Controll cAMP levels in human
keratinocytes
Takei et al., 1997
↑, increased in response to mechanical strain; ↓, decreased in response to mechanical strain.
elucidated (De Filippo and Atala, 2002). Several investigations in
last decades of the past century reported that, cyclic adenosine
monophosphate (cAMP) plays an important role to influence
cell growth, differentiation, proliferation and protein synthesis
depending on the source of cells and experimental conditions
(Bang et al., 1992; Florin-Christensen et al., 1993; Zhang et al.,
2016). Takei et al. (1997) found significant increase of protein
production in keratinocytes subjected to cyclic strain. Moreover,
net collagen amount decreases when the levels of cAMP in skin
fibroblasts is increased. Study of Acute and chronic cyclic strain
reduces adenylate cyclase activity in cultured coronary vascular
smooth muscle cells that could promote strain-induced cell
contraction (Wiersbitzky et al., 1994). The findings of previous
researches on second messengers are listed in the Table 3.
Inositol phosphate (IP), c-fos, and phospholipids (PL) are
thought to mediate extracellular signals to the nucleus but the
precise mechanisms need further reaserch (Takei et al., 1998).
Moreover, Molinari (2015), proposed hydrogen ion (H+) as a
second messenger to mediate Ca2+mobilization especially in
IP3/Ca2+signaling pathway. At the beginning of 21st century,
Buscà et al. (2000) reported that the BRAF gene (which
mediates growth signaling at a level just below RAS) can be
activated by cAMP in melanocytes. Extracellular signals (growth
factors) that activate G-protein-couples receptor can result in
the activation of adenylate cyclase to upregulate cAMP leading
to the activation of RAS and further activation of BRAF and
the downstream cascades (Simonds, 1999; Davies et al., 2002;
Pollock and Meltzer, 2002). Likewise the studies on second
messengers have been done on different cell lines, this study
was also performed with cultured cell lines derived from human
tumors, so further investigations are needed to be executed with
expanded tissue and acutely stretched skins to determine the
Frontiers in Physiology | www.frontiersin.org 8November 2016 | Volume 7 | Article 540

Razzak et al. Cellular Response to Mechanical Stress
precise roles of the ubiquitous and archetypal intracellular second
messengers.
CONCLUSION AND FUTURE
PERSPECTIVES
In this article, recent advances in tissue expansion in the
field of plastic and reconstructive surgery were described with
a special focus on the biological response and the activated
pathways leading to either proliferation or apoptosis. Emphasis
was given on the roles of membrane bound molecules such
as integrins, G-protein, growth factors, stretch-activated ion
channels, and secondary messengers. Although, studies of past
decades demonstrated that, mechanical stimulation is capable
to activate highly integrated signaling cascades resulting in
the new skin production, questions remain on how different
types of stimulation works on, different cells following different
signal transduction pathways. For example, studies on the
cells from the kidney differ significantly compared to the
cells of skin which are subjected to constant expansion
or mechanical forces. Moreover, studies using cultured cells
rather than intact tissue (skin) cannot clarify the exact effects
of tissue expansion. Similarly, stimulus such as shearing,
heat, and shock cannot provide natural microenvironment
to better understand how cell adapt to changes during
tissue expansion. Furthermore, the signaling pathways activated
by different biochemical factors were investigated in linear
methods such as single pathway analysis, which is insufficient
to describe multiple signaling pathways involved in cell
proliferation and/or apoptosis. Therefore, in depth comparative
proteomic and genomic analysis with expanded tissue or
acutely stretched skin would reveal the pathways and molecules
responsible for cell proliferation and/or apoptosis ultimately skin
regeneration.
AUTHOR CONTRIBUTIONS
Concept development: MTR. Writing the manuscript: MAR,
MTR, MSH, ZR, NY, and JC. Literature review for data collection:
MAR, MTR, MSH, and ZR. Figure and Tables: MAR, MTR, MSH.
ACKNOWLEDGMENTS
This work was made possible by grant
(UM.C/625/1/HIR/MOHE/DENT/21) from Ministry of
Higher Education, Malaysia, to Associate Professor ZR Faculty
of Dentistry, University of Malaya. Authors wish to acknowledge
MAR for language editing.
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Conflict of Interest Statement: The authors declare that the research was
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