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Mechanical Stimulation of Mesenchymal Stem Cells Biomechanical Stimulation and its Clinical Prospects 33
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Mesenchymal Cell Activation by Biomechanical Stimulation and its Clinical Prospects, 2016, 35-48 35
Nahum Rosenberg (Ed)
All rights reserved-© 2016 Bentham Science Publishers
CHAPTER 4
Bio-Mechanical Stimulation of Skin Fibroblasts
Yaron Har-Shai
1*
, Isaac Zilinsky
1
, Rei Ogawa
2
and Chenyu Huang
3
1
The Unit of Plastic Surgery, Carmel Medical Center, 7 Michal St. Haifa, 3436212, and the Bruce
Rappaport Faculty of Medicine, Technion- Israel Institute of Technology, Haifa, Israel;
2
Department of Plastic, Reconstructive and Aesthetic Surgery, Nippon Medical School, Tokyo,
Japan and
3
Department of Plastic, Reconstructive and Aesthetic Surgery, Beijing Tsinghua
Changgung Hospital; Medical Center, Tsinghua University, China
Abstract: The fibroblast is the most common cell of connective tissue. Fibroblast
synthesizes the extracellular matrix and has a crucial role in wound healing. Fibroblast
has different morphological characteristics according to their anatomical location and
according to their metabolic activities. Although this unremarkable from their shape,
when implanted ectopically these cells keep their original tissue characteristics at least
over a few generations. Fibroblasts synthesize GAG (glycosaminglycan), different types
of collagen, glycoprotein and other fibrous proteins. Additionally they secrete various
growth factors, including thymic stromal lymphopoietin (TSLP). Fibroblasts generate
the extracellular fibrous matrix as part of their metabolic secretion activity as part of the
repair process following tissue damage.
This chapter is divided into two sections i.e. the biomechanical, histomorphological and
structural properties of the skin and its behaviour following biomechanical stimulation
and the discussion on the cellular mechanotransduction, mechanosensores and
mechanosignaling pathways in the fibroblast.
Keywords: Fibroblast, molecular signaling, skin, strain/stress curve.
THE BIOMECHANICAL, HISTOMORPHOLOGICAL AND STRUCTURAL
PROPERTIES OF THE SKIN DURING STRETCHING
Overview
Normal skin can be described as a non-linear, anisotropic, viscoelastic material
with a special ability to preserve and restore homeostasis. When the skin is
stretched and relaxed within its elastic limits, the relaxation phase demonstrates a
strain/stress (force/gain) curve different from what observed during the stretching
phase. The area between the two curves (hysteresis) represents the energy
consumed. When the skin is repeatedly stretched and relaxed, additional
elongation of the collagen fiber network occurs, also termed stress relaxation. It
allows skin to stretch gradually and to exceed the boundaries of its natural
*Corresponding author Har-Shai Yaron: The Unit of Plastic Surgery, Carmel Medical Center, 7 Michal
St. Haifa, 3436212, and the Bruce Rappaport Faculty of Medicine, Technion- Israel Institute of Technology,
Haifa, Israel; Email: yaron07@yahoo.com
36 Biomechanical Stimulation and its Clinical Prospects Har-Shai et al.
extensibility [1-8]. The effect of stress relaxation is most evident clinically when
cyclic stretching of the skin is followed by short periods of relaxation, a technique
known as load cycling [3, 9]. An interesting clinical example for these phenomena
was demonstrated by Hirshowitz [2]. He indicated that there was a high
dissatisfaction rate amongst post - face-lift patients due to insufficient lift and
sagging back of the jowls. Despite surgical pulling of the skin to a tight position
and securing it to a fixed location, stress relaxation occurs within a couple of
hours to days. To overcome this phenomenon, a two-stage face-lift was proposed
with an interval of approximately four weeks. The reaction of the skin to
mechanical loading is time dependent, due to the resistance from the viscous
mucopolysacharide and glycoproteins (fibrous matrix) in the dermis. It was shown
that adipocytes, which are exclusively concentrate in the superficial musculo-
aponeurotic system of the face (SMAS) provide a certain degree of firmness to
this layer and support the longevity of face-lift procedures which includes SMAS
plication [8].
In several reports on skin expansion it was shown that intrinsic changes occur on
the microscopic level [1, 10, 11]. Normally, the collagen fibers in the dermis are
arranged in a convoluted and coiled structure and interwoven with elastic fibres.
With skin stretching and mechanical creep, the dermal collagen fibres are
straightened and converted into a compact and parallel configuration [6, 10, 11].
Subsequentially the mucopolysaccharide ground substance and the interstitial
fluid are displaced, and additional surface area is gained.
The extrusion of interstitial fluids from among the collagen fibres of the dermis
causes change in the proportion of viscous components in the fibre mat, with
subsequent mechanical stiffness of the skin. When the skin relaxes, renewed
energy sources enter the system through the circulation, enabling the tissues to
return to their normal state. Collagen does not have the ability to retract back to its
original position. Retraction is dependent on elastin fibres which are distributed
among collagen fibres and are interconnected by end to side bonds. Their role is
to pull back the stretched collagen to its original coiled position. At low loads, the
collagen fibres move against each other with relative ease, therefore the relaxed
skin can be readily stretched, while in a more stretched skin a resistance to further
extension rapidly increases, as the fibres become aligned and take up the strain.
This phenomenon was demonstrated in the standard strain/stress curve.
The collagen fibres contribute to the considerable tensile strength of skin and
generate resistance to its further extension and/or deformation when pulled.
Bio-Mechanical Stimulation of Skin Fibroblasts Biomechanical Stimulation and its Clinical Prospects 37
Attempt to stretch skin beyond its natural extensibility by using a mechanical unit
or two dimensional mechanical load might tear the collagen fibres with
subsequential formation of striae.
Other elements of the skin, such as blood vessels and nerves, are not affected by
skin stretching and tolerate it relatively well. It has been documented that in skin
edges subjected to stretching, the microcirculation rapidly adapts to tension. Most
likely, the blood vessels in the dermis demonstrate an initial inherent resistance to
the loading [4, 5], a resistance that yields with time. However, acute excessive
stretch may cause damage, possibly resulting in hematoma, oedema, ischemia and
pain.
In chronic skin wounds the composition of the mucopolysaccharide matrix is
altered and an immature scar tissue is formed in its place, impairing the normal
lubrication of the collagen fibres. In such conditions the time required for the
harnessing of the skin’s viscoelastic properties through stretching is extended.
Biological creep differs from mechanical creep. It involves slowly expanding
subcutaneous forces that come into play in conditions such as a growing tumor,
obesity, gravid uterus, or subcutaneous spherical tissue expanders applied over a
time period of weeks to months [9]. It is associated with significant stretching
involving metabolic activity and tissue growth. Although skin remains
homeostatic for long periods of time, repeated loading, resulting from normal
activity, actinic degradation, and biologic senescence eventually produce changes
which are similar to the associated with the term “fatigue” in non-living materials
and “aging” in living tissues. This process of attrition accelerates with age, as the
tissue becomes stiffer and less elastic, and the ability of potential elongation
diminishes [12].
Histological Changes
Research of histological changes during tissue expansion was related to the
clinical use of spherical devices for chronic expansion, which induce biological
rather than mechanical creep. In contrast, mechanical creep is gained with the
short-time acting linear devices. Nevertheless, it is clear that the gain of excessive
skin in chronic expansion is not generated purely due to biological changes, but
this is rather a combination of biological creep, some degree of mechanical creep,
and the recruitment of tissue from the neighbouring area. In epidermis the changes
seen during expansion include: hyperplasia, hyperkeratosis, parakeratosis, and
spongiosis, some inflammation, and increased melanocytic activity. The basilar
layer shows an increased rate of mitosis, significant rise in the number of labelled
38 Biomechanical Stimulation and its Clinical Prospects Har-Shai et al.
basal and suprabasal keratinocytes, and an increase in epidermal thickness [13].
The thickening is at the level of the stratum spinosum, while there is some
flattening of the rete ridges [14]. This finding suggests existence of a net gain of
tissue, not only by stretching the expanded area, but through the generation of
new tissue. In the studies using labelled thymidine a significant increase in
epidermal mitosis, indicated by an increased rate of thymidine uptake, following
each inflation of the expansion prosthesis, and a decline in the rate of epidermal
mitosis with deflation of the prosthesis, were demonstrated [15-17]. On electron
microscopic imaging the cells of the malpighian layer revealed abundancy with
groups of tonofilaments that form tonofibrils with decreased intercellular space in
all layers of the epidermis and greater undulation of the basal lamina and laminar
surfaces [14].
In the dermis more substantial changes were observed. During the first three
weeks of skin expansion noticeable changes, including reduction in the thickness
of the dermis, fibroplasia, and collagen rearrangement with increased volume,
were observed. The collagen became more compact with more myofibroblasts
[15]. This observation was confirmed by detecting higher levels of
hydroxyproline in expanded skin [18]. The thickened bundles of collagen are
located mainly at the reticular dermis and oriented parallel to the tension vector
[6, 7, 8, 14]. There was also thickening of elastic fibres and some loosening of
their normal structure that showed fragmentation and disintegration. The result
was a decrease in the ability of the expanded skin to retract [14].
Constant strain on the dermal extracellular matrix, like that occurring in
pregnancy, may remodel the elastic fibre network and be clinically evident by
generation of the striae distensae.
The mean fraction of elastin and collagen fibres in the facial skin is in the range of
6.1% ± 1.8% (SE ± 0. 04%); the fraction of collagen fibres in the skin is in the
range of 48.5% ± 7.0% (SE ± 0.2%). Therefore there is a significant difference in
the content of the collagen and elastin groups (p < 0.0001). No gender or age
differences were found in the content of elastin and collagen fibres [8]. In the
adnexa, i.e. hair follicles, sweat glands, peripheral nerves, receptors, and blood
vessels, usually there is no changes after expansion. For this reason patients do
not notice hair loss or excessive skin dryness at the expansion area of skin. During
expansion, there may be some temporary neuropathy, which returns to normal
after several months [14]. Subcutaneous fat is the most sensitive tissue to pressure
and stretch. It reacts to pressure by inflammation and necrosis. This may result in
Bio-Mechanical Stimulation of Skin Fibroblasts Biomechanical Stimulation and its Clinical Prospects 39
a deformation such as a depression or dent. In some cases fibrous tissue replaces
the fat lobules. Biopsies from expanded skin have revealed thinning of the
subcutaneous tissue. The mean thickness of the subcutaneous fat in expanded skin
found to be 869µ comparing to 2076µ in unexpanded tissue [14]. Muscle is
tolerant to stretch and pressure. During expansion, the muscle becomes flat and
compact with abnormal sarcomeres and myofibrils and increased mitochondria,
but usually without a decrease of its function. Muscle mass resumes its former
volume after extraction of the expander device. In some cases, muscle, like fat,
undergoes atrophy and is totally replaced by fibrous tissue [14, 19]. In the long-
term histopathological evaluation, two years after the expander was removed,
there were no substantial differences in the microscopic appearance of epidermis
in the expanded compared to the non- expanded skin. However in the dermis of
the expanded skin there was some elastosis and fragmentation of elastic fibres
[14, 20]. After two years all the histopathological changes in the epidermis, the
dermis and in the blood vessels returned to pre-expansion baseline appearance and
the previously observed capsule was not detectable any more [14].
MECHANOTRANSDUCTION, CELULAR MECHANOSENSORS AND
MECHANOSIGNALING PATHWAYS DURING MECHANICAL
STIMULATION OF FIBROBLASTS
Overview
Various complex biomechanical forces are gained to skin and cutaneous wounds,
including tension [21], shear force [22], scratch [23], compression [22], negative
pressure [24], hydrostatic [25] and osmotic [26] pressures are finally perceived by
cellular mechanosensors (mechanoreceptors) [22-29]. This process is called
mechanotransduction when physical forces are sensed and converted into
biochemical signals and then result in cellular responses.
Mechanoreceptors in cells include the mechanosensitive (MS) ion channels (e.g.,
Ca
2+
, K
+
, Na
2+
, and Mg
2+
) [30-33], cytoskeleton (actin filaments) [34] and cell
adhesion molecules (CAMs) such as integrins [35]. Fibroblasts are attached to the
extracellular matrix (ECM) via CAMs, and the cytoskeleton is connected to MS
ion channels and CAMs [30]. When the ECM is damaged by biomechanical
forces, such as skin tension, the cytoskeleton is altered and MS ion channels are
activated [30]. In contrast, extracellular fluid (ECF) pressure cannot activate MS
ion channels through cytoskeletal alteration, since hydrostatic pressure affects ion
inflow without altering cell shape [36]. Mechanical stimuli to cells are converted
into electrical signals through mechanoreceptors, which accelerate cellular
40 Biomechanical Stimulation and its Clinical Prospects Har-Shai et al.
activities, e.g. proliferation, angiogenesis, and epithelialization. These effects are
transmitted via different mechanotransduction and mechanosignaling pathways
[37] involving TGF/Smad, integrin, MAPK/G protein, TNF-a/NF-B and
interleukin, Wnt/-catenin, calcium ion pathways and have been the subject of
extensive research in fibroblasts.
TGF-β/Smad Signalling
TGF- is a multifunctional growth factor which is a potent modulator of
connective tissue remodelling and gene expression for synthesis of ECM [38]. In
mammalian cells three isoforms have been identified, namely TGF-1, TGF-2,
and TGF-3. The biochemical signal initiated by TGF- is propagated
downstream through Smads [39]. TGF- is intimately involved in scar tissue
reaction to mechanical forces.
Wang et al. found that in comparison to normal skin fibroblasts, keloid-derived
fibroblasts, when exposed to equibiaxial strain, produce high levels of TGF-1,
TGF- 2 and collagen1α at both the mRNA and protein levels [40]. They also
generate more focal adhesion complexes and show increased activation of focal
adhesion kinase (FAK). Moreover, TGF-1 up regulates myofibroblast
contractility, which is associated with pathological contractures, by inducing the
formation of important structural elements, namely stress fibres, vinculin-
containing fibronexus adhesion complexes and fibronectin fibrils [41]. TGF-1
also up regulates the fibroblast contractile marker a-smooth muscle actin (aSMA)
in myofibroblasts in a dose-dependent manner [41]. With regard to the other TGF
isoforms, TGF-3 has been shown to be a promising target for reducing dermal
scarring [42]. In addition, when skin fibroblasts are cultured in stressed lattices,
they produce the mRNAs of both TGF-1 and 3 [43]. Moreover, analysis of
dermal fibroblasts in a three-dimensional (3D)-collagen culture model revealed
that TGF-1 and 3 have similar effects on the contractile forces generated by the
cells [44]. The TGF- signalling pathway crosstalks actively with other pathways,
including the integrin a21 pathway. The latter pathway plays a central role in the
TGF-1 induced regulation of a-SMA and in the maintenance of the intracellular
tension [45]. Integrin-linked kinase is required for the TGF-1 mediated induction
of dermal myofibroblast differentiation [46]. In addition, the TGF- signalling
pathway works is combined with Wnt/-catenin signalling. This interaction may
participate in the pathogenesis of hypertrophic scars and keloids [47]. TGF- can
activate also the extracellular signal-related kinase (ERK), c-Jun N-terminal
kinase (JNK) and p38 MAPK pathways [48].
Bio-Mechanical Stimulation of Skin Fibroblasts Biomechanical Stimulation and its Clinical Prospects 41
Integrin Signalling
The integrins act as cellular mechanosensors by spanning the cell membrane and
being connected at one end to the cytoskeleton and at the other end to the ECM
[49]. They are composed of α and subunits that make up at least 24
heterodimers. There are three main integrin types: those that involved in fibroblast
proliferation [50], those that are part of collagen contraction [51] and those that
are part of myofibroblast differentiation [52], i.e. a11, a21 and a111 types.
The a21 is the primary integrin that is involved in the fibroblast binding to the
3D collagen lattices, while wound remodelling [53, 54] and induces collagenase
synthesis [55].
Integrins play a role in several different cellular pathways. Propagation of tensile
force through integrins induces RhoA activation at force-transfer sites in
fibroblasts, thereby mediating the assembly of actin through the Rho - ROCK-
LIMK cofilin pathway [56]. The activation of p38 in dermal fibroblasts, after
exposure to cyclic strain, is dependent on integrin 1, since pre-incubation of the
fibroblasts with integrin 1 antibody before mechanical stimulation can reduce the
p38 activation [57]. Integrin 1 also participates in the mechano-dependent
ERK1/2 activation in human keratinocytes [58], and the cyclic stretch-induced
activation of FAK and Rho in dermal fibroblasts can be abrogated by pre-
incubation with anti-integrin 137. Furthermore the activation of integrin by
mechanical strain can be mediated by the PI3K (an initiator of a cellular pathway
regulating the cell cycle) followed by increased integrin binding to the ECM
proteins [59].
MAPK and G Protein Signalling
There are three main subgroups of MAPK pathways that are involving ERK1/2,
JNK or p38 proteins. A typical signalling cascade is mediated by the activation of
an upstream mitogen-activated protein kinase kinase kinase (MAP3K) followed
by the MAP kinase (MAPK) cascade, where there is sequential phosphorylation
of the constituent MAP kinase kinase (MAP2K) and the MAPK [60]. With regard
to the putative mechanobiological role of MAPK in scarring, ERK and p38
MAPK can be activated in fibroblasts during collagen matrix contraction under
isometric tension [61]. Moreover, in dermal fibroblasts, p38MAPK and ERK1/2
are significantly activated by high-frequency repetitive stretching, but not by
intermittent stretching; apparently the frequency value of the stretching also
affects this signal activation profile [62].
42 Biomechanical Stimulation and its Clinical Prospects Har-Shai et al.
ERK is believed to participate in the very first steps of the healing process,
whereas p38MAPK mostly participates in the stimulation of genes that control the
intermediate and late healing phases and significantly influence cell migration and
proliferation [63].
G protein signalling is closely interlinked with MAPK signalling. G protein
consists of α, and γ subunits, and the α-subunits are further classified the into
the Gs, Gi, Gq and G12 groups [64]. Two small G proteins, RhoA and Rac-1,
have been shown to participate in the sensitivity of human fibroblasts to
mechanical strain and to modulate ERK pathway signalling [65].
TNF-α/NF-κB and Interleukin Signalling
Although TNF- α/NF-κ [66] and interleukins (e.g.,IL-6[67], IL-1[68], and IL-
13[69]) actively participate in the pathophysiology of scars or keloids, there is
only limited evidence that they participate in mechanobiology. Thus, their roles in
scar development remain unclear. Hypertrophic scars (HS) have higher basal
levels of both IL-1 and TNF- α than normal scars, and the compression of HSs
can elevate their IL-1 secretion and diminish their TNF-a production [70].
Sustained epidermal loading was also shown to increase the release of IL-1 α and
TNF-α by commercially available epidermal equivalents [71]. Cyclic strain can
also induce IL-1 mRNA expression in keratinocytes [72].
Wnt/β-catenin Signalling
The Wnt/-catenin pathway directly interconnects extracellular signals, gene
transcription, and cell cycle control [73]. Canonical Wnt signals are transduced
through Frizzled family receptors [74] and LRP5/LRP6 [75]. In the presence of
Wnt, Frizzled-1 inactivates GSK3, stabilizes -catenin (which will otherwise be
destabilized through GSK3 and APC [76] in complex with Dishevelled proteins),
and leads to the accumulation of -catenin and its translocation to the nucleus
[77]. Subsequently, the accumulated nuclear -catenin binds T-cell
factor/lymphoid enhancer binding factor (TCF/LEF) (transcription factors) and
activates the transcription of target genes [78]. Several studies have shown that
the levels of the effector molecule of this pathway, -catenin, are elevated during
fibroproliferation [78] in HSs or keloids. Fibroblasts also exhibit increased
proliferation, motility and invasiveness when they express stabilized -catenin,
and the overexpression of stabilized -catenin leads to excessive scar formation
[78]. Wnt/-catenin signalling can act in a combination with the TGF- signalling
pathway, when TGF- activates -catenin-mediated transcription in human
dermal fibroblasts via the Smad3 and p38 MAPK pathways. It was suggested that
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