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REVIEW
published: 26 May 2020
doi: 10.3389/fcell.2020.00360
Edited by:
Shiro Jimi,
Fukuoka University, Japan
Reviewed by:
Rajprasad Loganathan,
Johns Hopkins University,
United States
Lydia Masako Ferreira,
Federal University of São Paulo, Brazil
*Correspondence:
Susan Gibbs
s.gibbs@amsterdamumc.nl
Specialty section:
This article was submitted to
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Frontiers in Cell and Developmental
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Received: 02 December 2019
Accepted: 22 April 2020
Published: 26 May 2020
Citation:
Limandjaja GC, Niessen FB,
Scheper RJ and Gibbs S (2020) The
Keloid Disorder: Heterogeneity,
Histopathology, Mechanisms
and Models.
Front. Cell Dev. Biol. 8:360.
doi: 10.3389/fcell.2020.00360
The Keloid Disorder: Heterogeneity,
Histopathology, Mechanisms and
Models
Grace C. Limandjaja1, Frank B. Niessen2, Rik J. Scheper3and Susan Gibbs1,4*
1Department of Molecular Cell Biology and Immunology, Amsterdam University Medical Center (location VUmc), Vrije
Universiteit Amsterdam, Amsterdam, Netherlands, 2Department of Plastic Surgery, Amsterdam University Medical Center
(location VUmc), Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 3Department of Pathology, Amsterdam University
Medical Center (location VUmc), Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 4Department of Oral Cell Biology,
Academic Centre for Dentistry (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam, Netherlands
Keloids constitute an abnormal fibroproliferative wound healing response in which
raised scar tissue grows excessively and invasively beyond the original wound borders.
This review provides a comprehensive overview of several important themes in
keloid research: namely keloid histopathology, heterogeneity, pathogenesis, and model
systems. Although keloidal collagen versus nodules and α-SMA-immunoreactivity have
been considered pathognomonic for keloids versus hypertrophic scars, conflicting
results have been reported which will be discussed together with other histopathological
keloid characteristics. Importantly, histopathological keloid abnormalities are also
present in the keloid epidermis. Heterogeneity between and within keloids exists which is
often not considered when interpreting results and may explain discrepancies between
studies. At least two distinct keloid phenotypes exist, the superficial-spreading/flat
keloids and the bulging/raised keloids. Within keloids, the periphery is often seen as
the actively growing margin compared to the more quiescent center, although the
opposite has also been reported. Interestingly, the normal skin directly surrounding
keloids also shows partial keloid characteristics. Keloids are most likely to occur after an
inciting stimulus such as (minor and disproportionate) dermal injury or an inflammatory
process (environmental factors) at a keloid-prone anatomical site (topological factors)
in a genetically predisposed individual (patient-related factors). The specific cellular
abnormalities these various patient, topological and environmental factors generate
to ultimately result in keloid scar formation are discussed. Existing keloid models can
largely be divided into in vivo and in vitro systems including a number of subdivisions:
human/animal, explant/culture, homotypic/heterotypic culture, direct/indirect co-culture,
and 3D/monolayer culture. As skin physiology, immunology and wound healing is
markedly different in animals and since keloids are exclusive to humans, there is a
need for relevant human in vitro models. Of these, the direct co-culture systems that
generate full thickness keloid equivalents appear the most promising and will be key
to further advance keloid research on its pathogenesis and thereby ultimately advance
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Limandjaja et al. The Keloid Disorder
keloid treatment. Finally, the recent change in keloid nomenclature will be discussed,
which has moved away from identifying keloids solely as abnormal scars with a purely
cosmetic association toward understanding keloids for the fibroproliferative disorder that
they are.
Keywords: keloid, cicatrix, hypertrophic, keloid anatomy and histology, keloid etiology, keloid pathology, keloid
heterogeneity, keloid model, keloid phenotype
INTRODUCTION
As early as approximately 3000 B.C., the existence of keloid scars
has been acknowledged in the description of a “swelling on his
breast, large, spreading, and hard,” which felt like “touching a
ball of wrappings” in the Edwin Smith Papyrus, the first known
surgical treatise describing ancient Egyptian medical practice
(Breasted, 1930;Berstein and Roenigk, 1996). Keloids are not
mentioned in modern day literature until the early 19th century
when Jean Louis Alibert, the father of French Dermatology,
first described tumor-like scars which he initially referred to
as ‘les cancroïdes de la peau.’ When it became clear these
cicatricial tumors were in fact non-cancerous, Alibert changed
the name to ‘cheloïde’ or ‘keloïde’ in reference to the Greek word
‘χηλi’ (khçlçé) for crab’s claw and the suffix -oid meaning ‘like.’
Together this is meant to reflect not only the claw-like extension
of the keloids but also refers to their horizontal invasive growth
beyond the initial wound margins into the surrounding skin
(Alibert, 1825;Delpech, 1881;Berstein and Roenigk, 1996).
The keloid incidence rate varies greatly and is known to be
influenced by racial ethnicity. The risk of keloid development
significantly increases with increasing pigmentation (Burd and
Huang, 2005;Wolfram et al., 2009). In the Black and the Hispanic
general population, the incidence varies from 4.5–6.2 to 16%
(Cosman et al., 1961;Oluwasanmi, 1974;Rockwell et al., 1998);
while the incidence in the Taiwanese Chinese and Caucasians is
reported to be as low as <1% (Bloom, 1956;Seifert and Mrowietz,
2009;Sun et al., 2014). However, these numbers are largely based
on studies from several decades ago with the oldest dating back
to 1931. To our knowledge there are no new incidence numbers
of keloid scarring in the general population. More recent data
is available for very specific subpopulations: in head and neck
surgical patients (Young et al., 2014) as well as women after
caesarian section (Tulandi et al., 2011), the incidence of keloid
scar formation was significantly increased in African Americans
(0.8 and 7.1%, respectively) compared with the Caucasian (0.1
and 0.5%, respectively) and Asian or other (0.2 and 5.2%,
respectively) population. Interestingly, in Africans with albinism
the prevalence rate of 7.5% was not statistically different from
the overall prevalence rate of 8.3% in the general population or
the 8.5% observed in the normally pigmented African population
(Kiprono et al., 2015). It would therefore seem that increased
pigmentation in and of itself cannot solely explain the reported
ethnic differences in incidence rates (Bran et al., 2009).
In addition to the obvious cosmetic disfigurement, keloids
can also produce symptoms of itching and pain (Lee S. S. et al.,
2004;Bijlard et al., 2017). A study comparing the quality of life
in patients with keloids to that of psoriasis patients found that
patients with abnormal scars demonstrated the same reduced
quality of life levels as psoriasis patients when compared with
healthy controls (Balci et al., 2009). Similarly, a cross-sectional
survey on the burden of keloid disease (Bijlard et al., 2017)
showed that having keloids was associated with considerable
impairment of emotional wellbeing. In summary, keloids may
affect a very specific demographic for reasons we do not yet know,
but for those affected, these abnormal scars can have significant
consequences beyond cosmetics.
The mechanisms behind keloid scarring in particular are still
poorly understood (Slemp and Kirschner, 2006;Robles and Berg,
2007;Seifert and Mrowietz, 2009), and this is reflected in our
inability to satisfactorily manage this abnormal scar (Niessen
et al., 1999;Butler et al., 2008b). Known for its therapy-resistant
nature, excision alone has recurrence rates of 55–100% and can
even result in the development of a worse scar than before (Robles
and Berg, 2007;Butler et al., 2008b;Balci et al., 2009;Shih
et al., 2010). To further advance research on the pathogenesis
underlying keloid scar formation, there is an urgent need for
relevant, true-to-life keloid scar models that resemble the in vivo
keloid phenotype accurately. This review on keloid scars will
discuss histopathological characteristics, inter- and intralesional
heterogeneity, the pathogenetic mechanisms, as well as existing
scar model systems of keloids.
KELOID HISTOPATHOLOGY
Keloids are primarily a clinical diagnosis (Gulamhuseinwala
et al., 2008), and as such are not usually sent in for further
analysis by the pathologist. Although the histopathological
definition of a keloid scar was not further detailed in the
article, Gulamhuseinwala et al. (2008) found that retrospective
analysis of H&E stainings of 568 clinically diagnosed keloids
only proved accurate in 81% of the cases. Experienced plastic
surgeons diagnosed keloids based on the following clinical
criteria: the presence of a scar with a history of antecedent local
trauma and growth extending beyond its boundary. The non-
keloid diagnoses included acne keloidalis (11%), hypertrophic
(6%), and even normotrophic (2%) scars and a single pilonidal
abscess. Importantly though, no malignancies or dysplasias were
reported. Based on these findings, the authors suggested that
sending excised keloid tissue for histopathological examination
is not necessary if the clinician is an expert and there is a
strong clinical suspicion (Gulamhuseinwala et al., 2008). In
response to this study, however, Wong and Ogawa pointed
out that many clinicians would not be comfortable with the
incorrect diagnosis rate of 19% and therefore advocate for
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post-surgical histopathological confirmation (Wong and Lee,
2008;Ogawa et al., 2009).
The histopathological abnormalities of the full scarring
spectrum and normal skin have been summarized in
Supplementary Table S1, specific cellular abnormalities in
keloid scars are summarized in Supplementary Table S3 and will
be elaborated upon in the section ‘Keloid cellular abnormalities’.
The histopathological findings on keloid scars will be briefly
summarized in this section. The epidermal thickness in keloid
scars has been described as anything from atrophic (Koonin,
1964;Bakry et al., 2014) and normal (Moshref and Mufti, 2009;
Huang et al., 2014), to sometimes (Ehrlich et al., 1994;Materazzi
et al., 2007) or always increased (Bertheim and Hellström, 1994;
Chua et al., 2011;Syed et al., 2011;Sidgwick et al., 2013;Jumper
et al., 2015;Suttho et al., 2017;Shang et al., 2018). However, the
overwhelming majority supports the observation of increased
epidermal thickness in keloid scars, and what is more, this was
confirmed when thickness was measured in µm (Hellström et al.,
2014) as well as number of viable cell layers (Limandjaja et al.,
2017, 2019). Similarly, conflicting findings have been reported
with regards to rete ridge formation. Reports range from normal
rete ridge formation (Lee J. Y. Y.et al., 2004;Moshref and Mufti,
2009) to reduced (Koonin, 1964;Chong et al., 2015;Jumper
et al., 2015;Suttho et al., 2017;Shang et al., 2018) or complete
absence thereof (Ehrlich et al., 1994;Meenakshi et al., 2005;
Huang et al., 2014), although none have attempted to objectively
measure the extent of rete ridge formation. Overall, most
studies, including our own histopathological studies (Limandjaja
et al., 2017, 2019), appear to support the findings of a flattened
epidermis with increased thickness. Epidermal differentiation
appears mostly unaffected (Bloor et al., 2003;Ong et al., 2010;
Limandjaja et al., 2017, 2019). Although increased epidermal
activation/proliferation has been observed (Prathiba et al., 2001;
Bloor et al., 2003;Ong et al., 2010), this is in contrast with the
findings from our extensive immunohistochemical analysis
of the keloid epidermis (Limandjaja et al., 2017). We showed
normal levels of epidermal proliferation and differentiation in the
thickened keloid epidermis, save for the precocious expression
of terminal differentiation marker involucrin. We therefore
proposed that increased epidermal thickness was not the result of
epidermal hyperproliferation, but was associated with abnormal
differentiation instead. Interestingly, keloid keratinocytes
also showed increased expression of epithelial-mesenchymal
transition (EMT) markers (Chua et al., 2011;Ma et al., 2015;Yan
et al., 2015;Hahn et al., 2016;Kuwahara et al., 2016).
Histopathological studies of the keloid dermis showed that
fibroblasts were present in higher numbers (Ueda et al., 1999;
Tanaka et al., 2004;Meenakshi et al., 2009;Jiao et al., 2017).
Other dermal cell types residing in keloids include myofibroblasts
(Santucci et al., 2001;Kamath et al., 2002;Lee J. Y. Y.et al.,
2004; Lee et al., 2012;Moshref and Mufti, 2009;Shin et al., 2016)
and fibrocytes (Iqbal et al., 2012;Shin et al., 2016), both were
present in increased numbers. In our whole biopsy image analysis
of keloid tissue, CD34 expression was found to be absent from
the keloid dermis, but constitutively and abundantly present in
normal skin and normotrophic scars (Limandjaja et al., 2019).
Interestingly, within these CD34−dermal regions, we found
senescent (p16+) mesenchymal cells (vimentin+) as well as
myofibroblasts (α-SMA+).
Overall trends in the dermal ECM composition include
increased levels of collagen I and III (Naitoh et al., 2001;Syed
et al., 2011) with increased collagen bundle thickness (Verhaegen
et al., 2009); increased fibronectin (Kischer and Hendrix, 1983),
glycosaminoglycans (Carrino et al., 2012), chondroitin sulfate
(Ikeda et al., 2009), biglycan (Hunzelmann et al., 1996), versican
(Carrino et al., 2012), tenascin (Dalkowski et al., 1999), and
periostin (Zhou et al., 2010;Maeda et al., 2019); while levels
of elastin (Szulgit et al., 2002;Ikeda et al., 2009;Theoret et al.,
2013), and decorin (Carrino et al., 2012) were reduced. Dermal
hyaluronic acid expression showed variable results (Alaish et al.,
1995;Meyer et al., 2000;Ikeda et al., 2009;Yagi et al., 2013), but
unlike normal skin, its expression was equal in both the keloid
epidermis and dermis (Tan et al., 2011). Reports on vascularity
are also highly variable, both increased (Ehrlich et al., 1994;
Amadeu et al., 2003;Tanaka et al., 2004;Materazzi et al., 2007;
Ong et al., 2007b;Syed and Bayat, 2012;Bakry et al., 2014) and
decreased vascular density (Beer et al., 1998;Ueda et al., 2004;
Kurokawa et al., 2010;Theoret et al., 2013) has been observed
in keloids. Nerve fiber density appears to be increased in keloids
compared with normal skin (Hochman et al., 2008;Drummond
et al., 2017). Lastly, keloids also show increased immune cell
infiltration (Amadeu et al., 2003;Sharquie and Al-Dhalimi, 2003;
Tanaka et al., 2004;Shaker et al., 2011;Jiao et al., 2015;Luo et al.,
2017), with higher quantities of macrophages (Boyce et al., 2001;
Shaker et al., 2011;Jiao et al., 2015) and T-lymphocytes (Shaker
et al., 2011;Murao et al., 2014;Jiao et al., 2015) in particular. An
extensive review by Jumper et al. (2015) on the histopathology
of keloid scars has reiterated most of the aforementioned
findings, and further emphasizes the following as most frequently
occurring and therefore most discerning features of keloid scars:
a thickened, flattened epidermis; a tongue-like advancing edge
in the dermis; haphazard, thick, hyalinized collagen bundles as
the predominant dermal feature, with subsequent loss of the
papillary-reticular boundary; increased dermal cellularity; signs
of inflammation; and variable α-SMA expression.
Unlike their keloid counterparts, hypertrophic scars are
raised scars whose growth remains within the borders of the
original wound (Burd and Huang, 2005) and they can be
difficult to distinguish histopathologically. Keloidal collagen
(Cosman et al., 1961;Santucci et al., 2001;Ogawa et al.,
2009) vs. α-SMA and dermal nodules (Ehrlich et al., 1994;
Huang et al., 2014) have often been cited as pathognomonic
features for keloids or hypertrophic scars, respectively, but
conflicting reports abound (Muir, 1990;Ehrlich et al., 1994;
Santucci et al., 2001;Lee J. Y. Y.et al., 2004;Ogawa et al.,
2009;Ali et al., 2010;Bux and Madaree, 2010;Huang et al.,
2014). In a histopathological study (Limandjaja et al., 2019)
comparing both scar types, α-SMA and dermal nodules were
present in both scars and while keloidal collagen remained
a strong keloid marker, it was also observed in one of the
hypertrophic scars. Additionally, a thickened epidermis with
involucrin overexpression and a CD34−/α-SMA+/p16+dermal
cell population could be found in both scar types, although
α-SMA and p16 immunoreactivity were present in higher degrees
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in hypertrophic scars vs. keloids, respectively. In short, despite
the clearly defined clinical distinction between the two abnormal
scar types, the histological distinction between hypertrophic and
keloid scars remains a source of contention, especially in the early
stages (Burd and Huang, 2005).
INTER- AND INTRALESIONAL KELOID
HETEROGENEITY
Interlesional Heterogeneity
Several reports suggest that distinct keloid phenotypes may exist.
As early as 1960, Conway et al. (1960) discerned between nodular
raised keloids and flat keloids often observed on the sternum.
More recently, Bella et al. (2011) studied large multigenerational
pedigrees of patients with familial keloids in three rural African
tribes. The superficial spreading phenotype predominated in two
of the tribes, while the raised vertical phenotype predominated
in the remaining tribe (Figure 1). Superficial spreading keloids
show irregular subepidermal spread with irregular areas of hyper-
and hypopigmentation, they are mostly raised at the edges and
are characterized by a central flattened and quiescent area. This
central area is often regularly or hypopigmented, while the
margins show hyperpigmentation. In contrast, raised keloid scars
are prominently bulbous in shape with distinct borders and
may have limited areas of central quiescence. Another form of
interlesional keloid heterogeneity was proposed by Akaishi et al.
(2010), who distinguished between regular keloids with a round
shape and clear curving lines, and irregular keloids with irregular
shapes and lines. The authors found that the irregularly shaped
keloids showed a significant increase in infection and previous
surgery rates, and proposed that in contrast, the shape of regular
keloids was determined by skin tension alone. In predilection
body sites exposed to constant stretching (e.g., scapula, chest, and
shoulder), the butterfly, the crab’s claw or the dumbbell have also
been described as typical keloid shapes which are predominantly
determined by local mechanical factors (Huang et al., 2017). In
short, several distinct keloid phenotypes have been described,
the division of which appears to be based predominantly on the
keloid’s growth pattern and/or the resulting shape.
Intralesional Heterogeneity
It is also likely that heterogeneity exists within keloid scars
(see Supplementary Table S1). Based on clinical observations,
the most often described distinction is that of a red, raised
peripheral margin which actively invades the surrounding skin
and more depressed, lighter colored center showing clinical
regression (Louw et al., 1997;Lu et al., 2007a;Seifert et al., 2008).
This peripheral-central distinction matches the description of
the superficial spreading keloid phenotype (Bella et al., 2011).
Symptoms of strong itching (Lee S. S. et al., 2004) predominate
in the more pigmented keloid margin (Louw et al., 1997;
Le et al., 2004;Lu et al., 2007a;Bella et al., 2011), together
with hypercellularity (Appleton et al., 1996;Ladin et al., 1998;
Akasaka et al., 2001;Varmeh et al., 2011;Huang et al., 2014),
increased vascularity (Appleton et al., 1996;Le et al., 2004;Touchi
et al., 2016) and immune cell infiltration (Appleton et al., 1996;
FIGURE 1 | Spreading vs. bulging keloid phenotypes. Watercolor illustration.
Left figure shows a typical keloid of the ‘spreading’ phenotype located on the
anterior chest, with quiescent center and an actively growing peripheral
margin. Right figure shows a keloid of the ‘bulging’ phenotype, which are
bulbous in shape and can often be observed on the earlobe. Figure first
published in Limandjaja (2019), used with permission.
Le et al., 2004). Reduced apoptosis (Lu et al., 2007a;Seifert et al.,
2008), increased proliferation (Varmeh et al., 2011;Suttho et al.,
2017) and increased cellular activity (Louw et al., 1997), all
contribute to enlarging the pool of ECM-producing fibroblasts in
this region and support the hypothesized increased keloid activity
(see Supplementary Table S2A). In contrast, the central keloid
region shows either hypopigmentation or regular pigmentation
(Louw et al., 1997;Le et al., 2004;Lu et al., 2007a;Bella et al.,
2011) with pain as the main symptom (Lee S. S. et al., 2004), as
well as hypocellularity (Appleton et al., 1996;Ladin et al., 1998;
Akasaka et al., 2001;Varmeh et al., 2011;Huang et al., 2014)
and reduced vascularity (Appleton et al., 1996;Le et al., 2004;
Touchi et al., 2016) (see Supplementary Table S2A). Fibroblasts
derived from this central region generally show signs of inactivity
(Louw et al., 1997), as well as reduced proliferation (Varmeh et al.,
2011;Suttho et al., 2017), increased apoptosis (Lu et al., 2007a;
Seifert et al., 2008) and senescence (Varmeh et al., 2011). Taken
together with the increased expression of ECM-degrading genes
(Seifert et al., 2008), the central region appears to be the area of
relative quiescence.
Overall, the majority of studies support the concept of an
actively developing periphery and a more quiescent central
region, but the reverse has also been postulated with an active
role for the central region rather than the periphery. The
keloid center has been reported to show increased proliferation
(Giugliano et al., 2003;Tsujita-Kyutoku et al., 2005), the absence
of apoptosis (Appleton et al., 1996;Sayah et al., 1999;Akasaka
et al., 2001), increased expression of fibrosis-associated genes
(e.g., TGFβRI, SMAD 2, and SMAD3) (Tsujita-Kyutoku et al.,
2005) and certain wound healing mediators (IL-6 and VEGF)
(Giugliano et al., 2003), all of which support a more pro-active
role for this region. In line with these findings, our in vitro
reconstructed (Limandjaja et al., 2018b) different keloid regions
showed that differences existed between the regions in terms
of scar parameter expression. The central deep keloid region
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showed the more exaggerated keloid phenotype with respect to
increased contraction, increased epidermal thickness, reduced
HGF secretion and reduced collagen type IV α2 chain dermal
gene expression.
Comparison of the keloid heterogeneity findings remains
difficult due to the varying definitions of what constitutes the
periphery and the center within a keloid, as this may differ
significantly between studies. Additionally, we suspected that the
apparent dichotomy between results supporting an active keloid
periphery and those whose findings ascribe this active role to
the keloid center, may be explained by the existence of different
keloid phenotypes (Limandjaja et al., 2018b). Various keloid
phenotypes have been described in the previous paragraph, but
we would like to propose an even simpler classification based on
the work of Bella et al. (2011) and Supp et al. (2012b): namely that
of a more concave ‘superficial spreading’ and raised or ‘bulging’
keloids (see Figure 1) (Bella et al., 2011). Depending on the
phenotype, the actively expanding region could be located in
the periphery or the deeper central region (Supp et al., 2012b).
As all the keloids included in the study were of the ‘bulging’
phenotype, it follows that the central deep region would show the
most aggressive keloid phenotype.
Furthermore, heterogeneity has also been observed in regions
other than the periphery and the center, these are summarized
in Supplementary Table S2B. An often-used division is that
between different dermal layers (Russell et al., 1989, 1995;Luo
et al., 2001;Supp et al., 2012b;Chong et al., 2015, 2018;
Jiao et al., 2017), in which case the middle or deepest dermal
layers were usually found to act more aggressively compared
with the more superficial layers. The conflicting reports on the
peripheral and central keloid regions notwithstanding, it is clear
that differences exist between different lesional sites within a
keloid and these abnormal scars should therefore not simply
be considered or treated as a homogenous growth. Because of
this, we strongly advocate for the inclusion of a description of
the keloid phenotype/shape and the use of schematic drawings
to indicate from where within a keloid samples were taken
for experimentation.
Normal Skin Surrounding Keloids
The normal skin directly adjacent to and surrounding keloid scars
is also rarely included in keloid research. Although similarity to
normal skin has been reported (Jumper et al., 2017), the majority
of reported results suggest that the surrounding normal skin
behaves more like keloid tissue than normal skin. For example,
the surrounding normal skin often itches like the keloid periphery
(Lee S. S. et al., 2004) and shows increased blood flow compared
with unaffected normal skin (Liu et al., 2016). On a cellular level,
increased staining of the hematopoietic stem cell marker c-KIT
(Bakry et al., 2014) and heat shock protein 70 (Lee et al., 2013) has
been observed in both keloids and their surrounding normal skin,
while keratinocytes and fibroblasts from the surrounding normal
skin shared the abnormal expression of many of the same genes in
keloid-derived keratinocytes and fibroblasts (Hahn et al., 2013).
In the dermal compartment, the epidermal appendages lost from
keloid tissue reappear in the surrounding normal skin in reduced
capacity; and the dense and excessive collagen deposition of the
keloid can extend into the ECM of the surrounding normal skin,
which is otherwise loosely organized, with thin, wavy or even
fragmented collagen fibers (Lee et al., 2013; Lee W. J. et al., 2015;
Bakry et al., 2014;Jiao et al., 2017). Similarly, portions of nodules
from the adjacent keloid have also been found to extend into the
surrounding normal skin (Kischer and Pindur, 1990). The dermis
adjacent to keloids is more cellular but less crowded compared
with healthy skin, and shows significant lymphocyte infiltration
(Bakry et al., 2014;Lee W. J. et al., 2015;Jiao et al., 2017). The
skin surrounding keloids also differed from unaffected healthy
skin with respect to proliferation and apoptosis. Increased dermal
proliferation and increased numbers of apoptotic keratinocytes
(Appleton et al., 1996) were only observed in the surrounding
normal skin, and absent from healthy skin. Lastly, Dohi et al.
(2019) proposed an even more prominent role for surrounding
normal skin as the driving force for keloid progression into
the normal skin, via local preferential increased mechanical
strain. Conversely, the surrounding normal skin has also been
reported to differ from keloids. Compared with keloid scars, the
surrounding normal skin has more blood vessels (Beer et al.,
1998) and showed increased expression of the proliferative PCNA
marker in the dermis, which was normally absent in both normal
skin and keloids (Appleton et al., 1996). The surrounding skin
also showed strong, increased levels of CD34 staining in contrast
with the CD34 absence in abnormal scars (Erdag et al., 2008).
It also lacks the abnormal thermosensory thresholds to warmth,
cold, heat and pain sensations reported for keloids (Lee S. S.
et al., 2004). All things considered, current literature suggests
that the surrounding normal skin shares several features with
the adjacent keloid and is therefore a relevant area to include for
further investigation.
In our histopathological analysis of keloids, we found that the
normal skin directly adjacent to the keloids mostly resembled
normal skin or mature normotrophic scar (Limandjaja et al.,
2019). An epidermis of normal thickness and rete ridge formation
with normal differentiation and proliferation could be seen,
together with a CD34+/α-SMA−/p16−phenotype instead of the
CD34−/α-SMA+/p16+phenotype associated with keloid scars.
However, the most interesting findings emerged from our in vitro
work (Limandjaja et al., 2018b), where changes were observed
which in and of itself were not statistically significant but overall
formed a clear pattern of intermediate abnormal expression.
Across all the abnormal scar parameters present in the in vitro
keloid scar model, the in vitro reconstructed surrounding normal
skin showed a phenotype more extreme than true normal skin but
less aggressive than the peripheral keloid models. With respect
to contraction, α-SMA immunoreactivity, HGF secretion and
collagen type IV α2 gene expression, the surrounding normal
skin showed intermediate between truly unaffected normal skin
and keloid scar.
Taken together, we and others have shown that heterogeneity
exists within keloid scars. For future studies it would therefore
be imperative to mention the shape and growth pattern of the
keloid (superficial spreading or bulging) and additionally, it
should always be mentioned where in the keloid any tissue for
experimentation was obtained from, preferably with a schematic
overview for unambiguous clarification. Additionally, we would
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argue that it is worth including the surrounding normal skin in
any keloid research study when possible.
KELOID SCAR PATHOGENESIS: WHERE
DO WE STAND?
The development of a human in vitro keloid scar model
resembling in vivo keloid tissue would not only benefit drug
development and testing, it would also greatly aid research into
the underlying mechanisms leading to keloid scarring. The fact
that keloid pathogenesis remains so poorly understood (Slemp
and Kirschner, 2006;Wolfram et al., 2009), has been the bane of
affected patients and clinicians alike. Despite the many theories
proposed by experts, no single unifying hypothesis has been
put forward (Seifert and Mrowietz, 2009). In Figure 2, adapted
from Wolfram et al. (2009), the various abnormalities reported
in keloid tissue and keloid-derived cells are organized in several
tiers: patient, topography or special skin sites, and environmental
factors all contribute to the expression of abnormal cellular
responses, which eventually lead to keloid scar formation. In the
following sections, recent and relevant findings for each of these
factors will be discussed.
Patient-Related Factors Influencing
Keloid Scarring
Ethnicity, genetic predisposition, gender and age are patient
characteristics which may influence keloid predilection (Wolfram
et al., 2009). There is no conclusive evidence in favor of
differences in occurrence based on gender. Some have reported
that keloids are more likely to occur in women than men (Bayat
et al., 2005;Burd and Huang, 2005;Seifert and Mrowietz, 2009;
Middelkoop et al., 2011;Young et al., 2014), but this may also
reflect at least in part, the overall greater awareness of unaesthetic
scarring in women and a consequent higher tendency to seek
medical assistance (Marneros et al., 2001;Burd, 2006). Young
age is also associated with increased risk of abnormal scarring
(Middelkoop et al., 2011). Keloids can develop at any age, but
incidence is highest between the ages of 10–30 years (Bayat
et al., 2005;Seifert and Mrowietz, 2009;Young et al., 2014;Lu
et al., 2015). Because of the peak in incidence immediately post-
puberty, exacerbations during pregnancy and resolution after
menopause, a potential role for endocrinological hyperactivity
in keloid pathogenesis has also been proposed (Rockwell et al.,
1998;Seifert and Mrowietz, 2009;Huang and Ogawa, 2013a;
Glass, 2017).
Perhaps the most relevant patient-related factor is the genetic
predisposition for keloid formation in certain individuals.
Ethnic differences in prevalence showed that darker pigmented
individuals are at higher risk of developing keloid scars
(Marneros et al., 2001;Al-Attar et al., 2006). Additionally, having
a family member with keloids is associated with increased keloid
prevalence (Bayat et al., 2005;Kiprono et al., 2015;Lu et al.,
2015). This is predominantly the case for first degree relatives,
as demonstrated by a heritability of 72, 41, and 17% for first,
second, and third degree relatives in the Chinese population
(Lu et al., 2015). Individuals with a family history of keloids
are also at higher risk of developing multiple keloids and
developing keloids of greater severity (Bayat et al., 2005;Lu
et al., 2015). The familial heritability, the increased prevalence
in certain ethnicities and common occurrence in twins, all
strongly support the concept of genetic susceptibility in patients
with keloid scars (Marneros et al., 2001;Shih and Bayat, 2010;
Glass, 2017). Other lines of evidence pointing to a genetic
influence on keloid predisposition include familial inheritance
patterns, linkage studies, case-control association studies, and
gene expression studies (Shih and Bayat, 2010;Glass, 2017).
Different modes of inheritance have been reported, varying
from autosomal recessive and X-linked to autosomal dominant
(Shih and Bayat, 2010;Glass, 2017). Shih and Bayat (2010)
have previously reviewed the available evidence and suggest that
most evidence points to an autosomal dominant inheritance
pattern with incomplete penetration and variable expression,
this then also explains why carriers do not always express
the keloid phenotype and why keloid patients do not always
respond to trauma with keloid scarring. Despite their valuable
contribution to our understanding of genetic predilection for
keloids, familial inheritance studies have not led to the discovery
of any particular predisposing genes (Glass, 2017). Multiple gene
mapping methods as well as targeted gene pathway investigations
have identified several gene polymorphisms (NEDD4, FOXL2,
MYO1E, and MYO7A also HLA) associated with keloids
(Nakashima et al., 2010;Zhu et al., 2013;Velez Edwards
et al., 2014;Glass, 2017), but the underlying mechanism is
still unclear. Similarly, various abnormalities in gene expression
have shown highly variable results between studies (Shih and
Bayat, 2010), but affected genes are known to be involved in
the ECM, inflammation and apoptosis (Shih and Bayat, 2010).
Aside from these inherited gene mutations, acquired altered
gene expression in the form of epigenetic modification may
also play a role in keloid pathogenesis and further complicates
matters (Glass, 2017;He et al., 2017). Ultimately, however, the
specific genetic variation responsible for keloid scarring has yet
to be identified, but likely involves more than a single gene.
Additionally, different keloid patients probably carry different
gene polymorphisms which can all lead to keloid scar formation,
this would explain the variations in keloid phenotype observed in
different people (Shih and Bayat, 2010;Velez Edwards et al., 2014;
Glass, 2017).
Topography-Related Factors Influencing
Keloid Scarring
It is important to note that patients with a history of keloid
scarring do not necessarily form keloids after every injury (Slemp
and Kirschner, 2006), two identical incisions can generate one
normal scar and one keloid in the same individual (Fong et al.,
1999;Fong and Bay, 2002;Al-Attar et al., 2006). Certain body
sites are more prone to keloid scarring, thus the location of
the wound influences risk of keloid scar formation (Wolfram
et al., 2009;Middelkoop et al., 2011). The earlobe, neck, sternum,
upper back, shoulders and upper limbs all constitute keloid-
prone anatomical sites (Murray et al., 1981;Bayat et al., 2005;
Burd and Huang, 2005;Bella et al., 2011;Middelkoop et al., 2011).
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FIGURE 2 | Pathogenesis of keloid scarring. Overview of the various factors involved in keloid pathogenesis, adapted from Wolfram et al. (2009). This figure is meant
to provide a provisionary framework to help organize the multitude of pathogenetic findings in a logical and systematic way. Abbreviations; ECM, extracellular matrix;
EMT, epithelial-mesenchymal transition; EndoMT, endothelial-mesenchymal transition; mϕ, macrophage; M2, alternatively activated, pro-fibrotic macrophage
subtype; PBMCs, peripheral blood mononuclear cells. Figure first published in Limandjaja (2019), used with permission.
Although the keloid-prone earlobe and anterior chest have
been described as tension-free areas (Brody, 1990;Ogawa et al.,
2003;Al-Attar et al., 2006), the most popular explanation for why
keloids occur more frequently at certain body sites remains the
theory that these are regions of increased skin tension that are
subject to constant stretching during normal movement (Peacock
et al., 1970;Rockwell et al., 1998;Butler et al., 2008a;Bux and
Madaree, 2012;Ogawa et al., 2012). There is no consensus on
whether elasticity may explain the differences between keloid-
prone and keloid-protected sites. Bux and Madaree (2012)
reported that keloid-prone sites are characterized by high tension
with low stretch and low elastic modulus. In contrast, Sano
et al. (2018) observed that with exception of the earlobes, sites
less prone to pathologic scarring (e.g., palmoplantar regions)
were “comparatively hard,” characterized by low distensibility
and reduced elasticity. In contrast, keloid susceptible sites showed
high distensibility and increased elasticity. Additionally, high
sebaceous gland density (Yagi et al., 1979;Fong et al., 1999;Fong
and Bay, 2002;Al-Attar et al., 2006), increased collagen and
decreased M1 macrophage numbers (Butzelaar et al., 2017), are
all characteristics of keloid-prone skin which may promote keloid
scar formation in genetically predisposed individuals.
Environmental Factors Influencing
Keloid Scarring
Although spontaneous keloid scar formation has been reported
(Jfri et al., 2015), it is a rare occurrence that has been reported
in association with certain syndromes such as Rubenstein-Taybi
and Goeminne syndrome (Jfri et al., 2015), or may simply
reflect a laps in memory (Robles and Berg, 2007;Jfri et al.,
2015). Environmental factors are therefore generally an essential
prerequisite to keloid scar formation as some form of assault to
the skin has to take place to incite keloidogenesis (Bran et al.,
2009;Wolfram et al., 2009;Shih and Bayat, 2010). These keloid-
inducing events vary from minor to major antecedent trauma,
as well as any process resulting in skin inflammation. Insect
bites or vaccinations are examples of minor insults to the skin
which may be so minor as to not be remembered by the patient
at all, while major trauma is usually observed in the setting
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of surgical and non-surgical wound healing. Examples of the
latter group include lacerations, abrasions, piercings, tattooing
or blunt trauma. Additionally, inflammatory skin conditions
such as acne, (peri)folliculitis, chicken pox, herpes zoster and
hidradenitis suppurativa, may also lead to the development of
keloids (Nemeth, 1993;Murray, 1994;English and Shenefelt,
1999;Bran et al., 2009). Isotretinoin is often used to treat acne
and has been suggested to act as an additional predisposing factor,
though this has not yet been proven conclusively (Guadanhim
et al., 2016). While burns have often been mentioned as one
of the many potential keloid-inducing events (Trusler and
Bauer, 1948;Nemeth, 1993;Murray, 1994;English and Shenefelt,
1999;Bran et al., 2009), they are usually associated with
the formation of widespread hypertrophic scars (Middelkoop
et al., 2011;Gold et al., 2014) rather than keloids. Fortunately,
venipuncture has not been reported to induce keloid scarring
(Yadav et al., 1995;Robles and Berg, 2007). Regardless of the
type of injury however, the resultant keloid scar response is
characteristically disproportionate to the original inciting injury
(Tuan and Nichter, 1998).
To summarize, keloid formation is most likely to occur after
an inciting stimulus such as dermal injury or inflammatory
process (environmental factor) at a keloid-prone anatomical
site (topological factor) in a genetically predisposed individual
(patient-related factor). The specific cellular abnormalities this
generates to ultimately result in keloid scar formation, are
discussed next (Figure 2).
Keloid Cellular Abnormalities
Although the keloid fibroblast is still considered the main culprit
responsible for keloid scar formation, recent studies have shifted
the focus to recognize the potential role of the epidermal
compartment and the immune system in keloidogenesis. This
section will address the trends in reported cellular abnormalities
across the entire spectrum of cells present in skin and/or involved
in wound healing (see Supplementary Tables S3A–D).
Abnormalities in Keloid Epidermal Cell Population
Often overlooked or even designated as ‘normal appearing’
(Butler et al., 2008b;Hollywood et al., 2010), the keloid
epidermis has only recently started garnering attention in
keloid research. A summary of the reported keloid epidermal
abnormalities has been listed in Supplementary Table S3A and
a summary of the histopathological epidermal abnormalities
can be found in the paragraph ‘Keloid Histopathology’ and
in Supplementary Table S1. The abnormalities of the keloid
epidermis are not limited to those visible by histopathology
alone, there is growing evidence that its barrier function is
also affected as measurements of transepidermal water loss
(TEWL) and high-frequency conductance suggest keloid scars
may show altered stratum corneum function compared with
healthy skin (Suetake et al., 1996;Sogabe et al., 2002). In
line with these findings, we found that the specific abnormal
overexpression of terminal differentiation marker involucrin was
not only associated with increased epidermal thickness, but
also with disorganization of the stratum corneum as visualized
by transmission electron microscopy (Limandjaja et al., 2017).
The following paragraphs will focus on abnormalities reported
for keloid keratinocytes and melanocytes. Langerhans cells also
reside in the epidermal compartment, but will be discussed in the
paragraph on keloid immune cells.
Keloid keratinocytes
Keloid keratinocytes may have a more direct role in keloid
scarring than previously assumed. Increased expression of
growth factors and cytokines such as CTGF (Khoo et al., 2006),
HGF and its receptor c-Met (Mukhopadhyay et al., 2010),
VEGF and PLGF (Ong et al., 2007b) have been demonstrated
in keloid-derived keratinocytes. Furthermore, cultured keloid
keratinocytes were found to differentially express 538 genes in
a study by Li and Wu (2016) and of these, further functional
analysis identified homeobox A7 (HOXA7), minichromosome
maintenance 8 (MCM8), proteasome subunit αtype 4 (PSMA4)
and proteasome subunit βtype 2 (PSMB2) as key differentially
expressed genes. In another gene expression study, Hahn
et al. (2013) found abnormal expression of genes involved in
differentiation, cell–cell adhesion and increased motility. Keloid
keratinocytes also contribute to keloid scarring by paracrine
regulation of ECM synthesis in fibroblasts, as evidenced by their
ability to induce a more profibrotic phenotype in vitro even in
fibroblasts of normal skin origin (Lim et al., 2002).
Lastly, several studies support a role for epithelial-
mesenchymal transition (EMT) in keloid scarring, a
phenomenon by which epithelial cells undergo phenotypic
changes and acquire more mesenchymal characteristics (Stone
et al., 2017). EMT has been found to occur in wound healing,
and plays a role in fibrosis by serving as a source of myofibroblast
generation (Stone et al., 2017). The changes associated with
EMT have been reported in keloid scars and involve the loss
of epithelial cell markers such as E-cadherin (Ma et al., 2015;
Yan et al., 2015;Hahn et al., 2016) and gain of mesenchymal
characteristics such as vimentin and FSP-1 (fibroblast specific
protein 1) expression (Yan et al., 2010, 2015;Ma et al., 2015;
Hahn et al., 2016;Kuwahara et al., 2016), combined with
changes in cell shape toward a more motile and migratory
phenotype (Hahn et al., 2013, 2016;Supp et al., 2014;Stone
et al., 2017). In short, the fundamental abnormalities found
in the keloid keratinocytes with respect to wound healing
mediator secretion, differentially expressed genes, paracrine
effects on co-cultured cells and epithelial-mesenchymal
transition, all support a more active role for keratinocytes
in keloidogenesis.
Keloid melanocytes
Little has been published on the role of melanocytes in
keloid pathogenesis (see Supplementary Tables S1, S3A),
despite the long observed increased keloid incidence in
individuals with darker pigmentation (Burd and Huang,
2005;Wolfram et al., 2009). To our knowledge, only Gao
et al. (2013) addressed the potential role of melanocytes
specifically in both hypertrophic and keloid scar formation
and proposed that during wound healing, a damaged
basement membrane allows the melanocytes to interact
with the dermal fibroblasts. The ensuing increase in
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fibroblast proliferation and collagen production together
with activation of the TGF-βpathway, promote abnormal scar
formation. They performed indirect co-culture experiments
in which melanocytes were able to induce increased levels
of proliferation, collagen I, TGF-β1 and its downstream
p-SMAD 2/3 expression in normal fibroblasts compared with
monocultured fibroblasts.
The increased melanin in keloid-prone patients may also
contribute to keloid scar formation by inhibiting the senescence-
inducing and anti-inflammatory effects of UVB radiation
(Wirohadidjojo et al., 2011) and vitamin D (Cooke et al.,
2005), respectively. However, variations in melanin levels
alone cannot fully explain the association of keloids with
darker pigmented individuals, as it has been reported that
the African albino population shows similar keloid prevalence
rates to the normally pigmented Africans (Kiprono et al.,
2015). Aberrations have also been reported in the steps
involved in melanogenesis, the process by which melanin is
generated (Koonin, 1964;Slominski et al., 1993;Song, 2014;
Wadhwa et al., 2016). For example, we now know that
polymorphisms in the MC1R gene are in fact responsible for
ethnic variations in pigmentation (Videira et al., 2013), and it
has been demonstrated that this receptor is not only expressed
on melanocytes but can also be found on dermal fibroblasts
(Stanisz et al., 2011). In fact, Luo et al. (2013) reported that
keloid scars and particularly keloid fibroblasts, showed reduced
expression of the melanocortin-1 receptor. They proposed that
this may negate the α-MSH-mediated suppression of collagen
synthesis and myofibroblast formation, thereby stimulating
keloid development. Additionally keloid fibroblasts have also
been found to be resistant to inhibitory effects of TGF-β1
on POMC expression (Teofoli et al., 1997;Lotti et al., 1999).
Thus, keloids association with increased pigmentation may very
well not reflect a primary abnormality in the melanocytes,
but a concomitant altered function of a shared receptor in
the fibroblasts.
Abnormalities in Keloid Dermal Cell Population
By their very nature, hypertrophic and keloid scars are defined
by the presence of raised, protruding scar tissue. The focus
of most studies has therefore understandably been on the
dermal component and more specifically on the extracellular
matrix (ECM) and the ECM-producing fibroblasts. Both keloid
and hypertrophic scars showed increased cellularity and were
in excess of all three primary ECM components of water,
collagen and proteoglycans. Notably, in keloids these processes
were significantly upregulated compared with normal skin and
hypertrophic scars (Ueda et al., 1999;Miller et al., 2003;
Meenakshi et al., 2005).
A summary of the reported keloid dermal abnormalities
has been listed in Supplementary Tables S3B–D, and a
summary of the dermal histopathological abnormalities
can be found in the paragraph ‘Keloid Histopathology’
and in Supplementary Table S1. The following paragraphs
will detail abnormalities reported for keloid fibroblasts and
keratinocyte/fibroblast interactions, myofibroblasts, fibrocytes,
endothelial cells, and nerve cells.
Keloid fibroblasts
An overwhelming number of studies have been devoted to
the keloid-derived fibroblast. However, the sheer multitude of
papers published on keloid fibroblasts have made it impossible
to discuss them all in this review and is outside our scope.
For this reason, the focus of this review was limited to
fibroblast abnormalities as they pertain to the main themes listed
by Marneros and Krieg (2004) and Robles and Berg (2007):
proliferation, ECM synthesis and degradation, expression of
wound healing mediators and apoptosis. A summary of these
publications is listed in Supplementary Table S3B, overall trends
in these in vitro monoculture findings will be summarized in the
following paragraphs.
There is an overall increase in the number of fibroblasts in
keloids, most likely mediated by increased proliferation rates.
Although some have also reported normal or even decreased
proliferation rates in keloid fibroblasts, the overwhelming
majority of in vitro monolayer studies support keloid fibroblast
hyperproliferation (Russell et al., 1988;Concannon et al., 1993;
Blume-Peytavi et al., 1997;Carroll et al., 2002;Carroll and
Koch, 2003;Giugliano et al., 2003;Hanasono et al., 2003,
2004;Meenakshi et al., 2005;Lim et al., 2006, 2009;Yeh
et al., 2006;Ghazizadeh et al., 2007;Ong et al., 2007a;Akino
et al., 2008;Witt et al., 2008;Zhang G. et al., 2009;Jing
et al., 2010;Romero-Valdovinos et al., 2011;He et al., 2012;
Syed and Bayat, 2012;Jurzak and Adamczyk, 2013;Wang
et al., 2013;Xin et al., 2017). In conjunction with increased
proliferation, reduced apoptosis by any means would also lead
to a cumulative net increase in the keloid fibroblast population.
Despite some papers reporting increased apoptosis (Akasaka
et al., 2000, 2005), overall, the majority of studies report
reduced apoptosis (Ladin et al., 1998;Messadi et al., 1999;
Chipev et al., 2000;Luo et al., 2001;Funayama et al., 2003;
Tucci-Viegas et al., 2010;Wang et al., 2013). Apoptosis is
also reduced in keloid fibroblasts by upregulation of apoptosis-
resistance (Ohtsuru et al., 2000;Messadi et al., 2004;Lu et al.,
2007b;Seifert et al., 2008), as well as telomere dysfunction
and defective senescence. Findings of telomerase upregulation
(Yu et al., 2016) and consequent telomere lengthening (Granick
et al., 2011;Yu et al., 2016) in keloid fibroblasts support
lifespan-prolonging effects of telomere dysfunction, although
telomere shortening as a result of oxidative stress has also
been reported in 30% of the keloids studied by De Felice
et al. (2009). In normal wound healing, fibroblasts eventually
become senescent and can then act as inhibitors in the regulation
of fibroblast proliferation and ECM synthesis. In this way,
defective senescence may also result in a net increase in fibroblast
density (Blaži´
c and Brajac, 2006), but literature on senescence
in keloid fibroblasts has been sparse and even counterintuitive
(Varmeh et al., 2011).
In line with their invasive nature, keloid fibroblasts also
show increased migration (Fujiwara et al., 2005a;Lim et al.,
2006;Witt et al., 2008;Wen et al., 2011;Syed and Bayat, 2012;
Wang et al., 2013;Fang et al., 2016;Jumper et al., 2017;Hsu
et al., 2018) and capacity for invasion in 3D invasion assays
(Dienus et al., 2010;He et al., 2012;Syed and Bayat, 2012;
Wang et al., 2018). Furthermore, increased metabolic activity
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(Meenakshi et al., 2005;Vincent et al., 2008), increased ECM
synthesis (McCoy et al., 1982;Abergel et al., 1987;Ala-Kokko
et al., 1987;Babu et al., 1989;Berman and Duncan, 1989;Suzawa
et al., 1992;Fujiwara et al., 2005a;Ong et al., 2007a;He et al.,
2012) and deposition (Abergel et al., 1985;Uzawa et al., 2003;
Fang et al., 2016) combined with reduced ECM degradation
(Abergel et al., 1985;Berman and Duncan, 1989;Uchida et al.,
2003;Yeh et al., 2006, 2009;Seifert et al., 2008;Russell et al., 2010;
McFarland et al., 2011;Suarez et al., 2013), all contribute to the
ECM overexpression and the resulting dermal protuberance that
defines these scars (see Supplementary Table S3B). Increased
levels of collagen I (Uitto et al., 1985;Ala-Kokko et al., 1987;
Lee et al., 1991;Friedman et al., 1993;Sato et al., 1998;Chipev
et al., 2000;Daian et al., 2003;Hasegawa et al., 2003;Lim et al.,
2003;Hsu et al., 2006, 2018;Xia et al., 2007;Zhang G. et al., 2009;
Dienus et al., 2010;McFarland et al., 2011;Lin et al., 2015;Suarez
et al., 2015;Fang et al., 2016;Luo et al., 2017), a major constituent
of the dermis, is likely responsible for the bulk of this increased
tissue mass. Additionally, there is an increased collagen I:III ratio
(Uitto et al., 1985;Abergel et al., 1987;Lee et al., 1991;Friedman
et al., 1993;Zhang G. et al., 2009), despite variable reports on
the levels of collagen III (Clore et al., 1979;Uitto et al., 1985;
Ala-Kokko et al., 1987;Lee et al., 1991;Sato et al., 1998;Lim
et al., 2002, 2003, 2013;Zhang G. et al., 2009;Lin et al., 2015;
Hsu et al., 2018). The most reported trend is that of increased
collagen III levels (Ala-Kokko et al., 1987;Lee et al., 1991;Sato
et al., 1998;Lim et al., 2003, 2013;Zhang G. et al., 2009;Hsu et al.,
2018). Other ECM constituents also expressed at higher levels in
keloid fibroblasts include fibronectin (Kischer and Hendrix, 1983;
Babu et al., 1989;Kischer and Pindur, 1990;Sible et al., 1994;
Blume-Peytavi et al., 1997;Chipev and Simon, 2002;Liang et al.,
2013;Suarez et al., 2015;Fang et al., 2016;Luo et al., 2017;Hsu
et al., 2018), elastin (Russell et al., 1989, 1995;Lee et al., 1991),
glycosaminoglycans (Berman and Duncan, 1989;Suarez et al.,
2013), and both small and large proteoglycans (Yagi et al., 2013).
The profibrotic characteristics of keloid fibroblasts are at
least in part, mediated by increased levels of several key
wound healing mediators and their associated receptors (see
Supplementary Table S3B). Major pathways upregulated in
keloid fibroblasts include TGF-β1 (Mccormack et al., 2001;
Mikulec et al., 2001;Carroll et al., 2002;Carroll and Koch, 2003;
Funayama et al., 2003;Hanasono et al., 2003;Xia et al., 2004;
Bock et al., 2005;Fujiwara et al., 2005b;Bran et al., 2010;Lim
et al., 2013;Wang et al., 2013;Jurzak et al., 2014;Yang et al.,
2014;Lin et al., 2015;Suarez et al., 2015;Fang et al., 2016), TGF-
β2 (Xia et al., 2005;Bran et al., 2010;Suarez et al., 2015;Fang
et al., 2016), and their receptors (Chin et al., 2001;Xia et al., 2004;
Tsujita-Kyutoku et al., 2005); CTGF (Xia et al., 2004, 2007;Khoo
et al., 2006;Russell et al., 2010;Jurzak et al., 2014;Fang et al.,
2016); VEGF (Wu et al., 2004;Fujiwara et al., 2005b;Ong et al.,
2007b;Dienus et al., 2010); interleukins IL-6 (Xue et al., 2000;
Ghazizadeh et al., 2007;Do et al., 2012) and IL-8 (Do et al., 2012);
as well as IGF-1 receptor (Yoshimoto et al., 1999;Ohtsuru et al.,
2000;Phan et al., 2003;Hu et al., 2014) and its binding-related
proteins (Phan et al., 2003;Seifert et al., 2008;Smith et al., 2008;
Russell et al., 2010). Moreover, keloid fibroblasts not only produce
higher levels of wound healing factors, they are also inherently
more sensitive to the effects of many of these factors (see
Supplementary Table S3B). Keloid fibroblasts showed increased
collagen secretion, PAI-1 and PDGFαreceptor expression, as well
as increased proliferation and migration in response to IL-18 (Do
et al., 2012), VEGF (Wu et al., 2004), TGF-β1 (Messadi et al.,
1998), HDGF (Ooi et al., 2010), and CTGF (Luo et al., 2017),
respectively, which was absent in normal fibroblasts. Similarly,
keloid fibroblasts exhibited a greater response in ECM synthesis,
proliferation, migration, invasion and inflammatory mediator
secretion to TGF-β(Bettinger et al., 1996;Daian et al., 2003), HGF
(Jin, 2014), PDGF (Haisa et al., 1994), and IL-18 (Do et al., 2012)
stimulation, respectively, compared with normal skin fibroblasts.
In a similar fashion to normal fibroblasts (Kroeze et al.,
2009), keloid-derived fibroblasts display mesenchymal stem cell
(MCS) markers and possess the multipotency to differentiate
into adipocytes, osteocytes, chondrocytes, smooth muscle cells,
endothelial cells, and neural lineage cells (Moon et al., 2008;
Iqbal et al., 2012;Plikus et al., 2017); thereby earning
the descriptor of multipotent precursor cells. Interestingly,
multipotency capabilities may differ between different scar
types as demonstrated by the ability of keloid fibroblasts,
but not their hypertrophic counterparts, to differentiate into
adipocytes either by stimulation with BMP4 or when co-
cultured with human scalp hair follicle cells (Plikus et al.,
2017). Iqbal et al. (2010) further differentiated between MSCs
of hematopoietic and non-hematopoietic origin, with the
majority comprising the non-hematopoietic subtype located
in the top and middle areas of the keloids. Regardless
of the MSC subtype, however, all MSC markers showed
progressive downregulation in culture with increasing cell
passage. Based on the similarly progressive loss of the keloid
phenotype with in vitro serial culturing and the abnormally
proliferative nature of keloid fibroblasts, Moon et al. (2008)
hypothesized that keloid fibroblasts may be stimulated by the
aberrant keloid cytokine milieu to remain in an undifferentiated
multipotent and proliferative stem cell state. By extension,
Qu et al. (2013) proposed that these keloid stem cells are
able to sustain themselves by asymmetric cell division due
to their drug resistant and high self-renewing abilities. The
continued generation of new aberrant keloid cells then sets
the typical tumor-like keloid growth in motion, and also
helps explain the high post-therapy recurrence rates. In
fact, the pathological keloid microenvironment may also be
responsible for generating the keloid stem cells in the first
place. Qu et al. (2013) also hypothesized that a pathological
niche exists in keloids that is the result of the pre-existing
abnormalities in keloid-prone patients, namely the enhanced
and persistent inflammatory response and the overexpression
of growth factors and their receptors. The multipotent keloid
fibroblasts, or rather keloid stem cells, are then transformed
from normal dermal stem cells after exposure to this pathological
keloid niche. Akino et al. (2008) co-culture experiments of
mesenchymal stem cells with keloid fibroblasts may support this
niche hypothesis, as mesenchymal stem cells showed similar
fibrotic and myofibroblast-like changes after exposure to keloid
fibroblasts in co-culture. Regardless of their cell of origin
however, the multipotent stem cell nature of the keloid fibroblast
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appears to play an important role in the genesis and maintenance
of keloid scars.
Although highly informative, findings from fibroblast
monolayer cultures are not without their limitations. Serial
culturing, methods of fibroblast cell isolation (enzymatic vs.
explant∗), presence or absence of serum in culture medium,
and 3D vs. monolayer culturing, are all potential culturing
artifacts which have differed across studies and may influence
outcome parameters significantly. It is important to consider
the potential confounding effects of these culturing differences
while interpreting different results. As a final consideration, it
should be noted that keloid fibroblasts are usually compared with
fibroblasts derived from healthy non-lesional skin, while in fact
normotrophic scars represent the true standard against which
keloids should be compared. This holds true for all the tissue and
cellular components studied in keloid research, and is not limited
to the comparison of keloid fibroblasts to normotrophic scar-
derived fibroblasts. In conclusion, keloid fibroblast monocultures
have generated a multitude of interesting findings, but it is
important to remember the inherent limitations associated
with monoculture model systems and consider the influence of
differences in cell isolation and culture methods.
Abnormal keloid keratinocyte-fibroblast interactions
We know that the interactions between keratinocyte and
fibroblasts are an integral component of the normal wound
healing process (Lorenz and Longaker, 2003;Broughton et al.,
2006) and findings from in vitro double chamber co-culture
experiments have been particularly informative in this regard, as
they allow us to study indirect paracrine interactions between
the two cell populations (see Supplementary Table S4D).
Co-cultures of keratinocytes with fibroblasts show increased
proliferation, levels of ECM and growth factor expression
compared with monocultures (Phan et al., 2002, 2003;Funayama
et al., 2003;Lim et al., 2003;Xia et al., 2004;Khoo et al., 2006;
Ong et al., 2007b;Ooi et al., 2010;Do et al., 2012), but this effect is
generally greatest with keloid-derived cells. Keloid keratinocytes
are able to induce the profibrotic keloid phenotype in normal skin
fibroblasts (Lim et al., 2001, 2002, 2003;Funayama et al., 2003;
Xia et al., 2004;Khoo et al., 2006;Ong et al., 2007b;Chua et al.,
2011), while keloid fibroblasts are able to the propagate fibrosis
markers even when co-cultured with normal keratinocytes (Lim
et al., 2001;Phan et al., 2003;Xia et al., 2004;Supp et al., 2012b;
Lee Y.-S. et al., 2015).
These findings all strongly support a role for abnormal keloid
keratinocyte/fibroblast interactions in the pathogenesis of keloids
and thereby provide a new point of interception for therapeutic
strategies. In this light, Burd and Chan (2002) described an
interesting case of a pediatric patient with a giant keloid covering
most of the upper right leg and buttocks. In a multistep
procedure, the keloid tissue was removed and an artificial dermal
matrix was placed on the wound bed. This was followed by the
addition of an autologous keratinocyte cell suspension fixated by
a fibrin glue spray. During the 18-month follow-up there was
no recurrence of keloid formation. This case report serves as
an excellent example of a bench to bedside approach to negate
the adverse keloid epidermis-dermis interaction, by removing the
diseased dermal matrix and introducing normal keratinocytes.
Keloid myofibroblasts
Although Ehrlich et al. (1994) put forward the absence of
myofibroblasts as a feature that differentiates keloids from
hypertrophic scars, the opposite has also been observed (Santucci
et al., 2001). In fact, the overwhelming majority of studies report
the presence of α-SMA+myofibroblasts in 33–81% of the keloids
analyzed (Santucci et al., 2001;Kamath et al., 2002;Amadeu
et al., 2003;Lee J. Y. Y.et al., 2004;Moshref and Mufti, 2009).
Particularly when cultured in vitro, keloid fibroblasts can be
shown to contain a significant portion of myofibroblasts (Chipev
et al., 2000;Chipev and Simon, 2002;van der Slot et al., 2004;
Ong et al., 2007a;Lee et al., 2012;Jin, 2014;Suarez et al., 2015;
Luo et al., 2017;Shang et al., 2018). In our histopathological
study on abnormal scar types and immature scars (3–5 weeks
old), we identified a CD34−/α-SMA+specific dermal cell
population, which were largely senescent in the abnormal scars
(p16+) but actively proliferating in the young scars (Ki67+)
(Limandjaja et al., 2019). See Supplementary Tables S2, S3B
for a summary of the histopathological results and cellular
abnormalities, respectively.
In wound healing by secondary intention, macrophages
stimulate wound bed-derived fibroblasts with TGF-β1 and PDGF
to transform them into myofibroblasts (Broughton et al., 2006).
In a recently published review, Lim et al. (2019) suggested
that the aberrant fibroblasts and myofibroblasts in keloids
may originate from an altogether different cell type, namely
the embryonal stem cell-like cell population located in the
endothelium of microvessels and on the perivascular cells
within keloid-associated lymphoid tissue. After injury, these
cells are thought to differentiate into abnormal fibroblasts and
myofibroblasts through the process of endothelial-mesenchymal
transition. Additionally, circulating fibrocytes or mesenchymal
stem cells from the bone marrow may also migrate to the
target site to generate the abnormal (myo-)fibroblast population.
In other words, myofibroblasts in the keloid environment
may have several sources of origin beyond the wound bed
fibroblasts. Alternatively, mesenchymal stem cells may also serve
as a myofibroblast source. Monolayer co-culture experiments
with mesenchymal stem cells and keloid fibroblasts have
shown that the latter are able to induce a myofibroblast-
like phenotypic switch of the mesenchymal stem cells (Akino
et al., 2008). In short, myofibroblasts may very well originate
from several different sources in addition to the wound-
bed fibroblasts.
In normal wound healing processes, we know that
myofibroblasts can produce significant wound surface reduction
through wound contraction (Lorenz and Longaker, 2003), but
Plikus et al. (2017) published an interesting new theory on how
myofibroblasts may contribute to the development of keloid
scars. They found that hair follicles are essential for inducing
myofibroblast-to-adipocyte reprogramming that allows for
regeneration rather than scar formation. As hair follicles are
absent from the keloid microenvironment, the myofibroblasts are
left unable to convert to adipocytes, thereby triggering the scar
response leading to keloid formation. In this way, hair follicles
and adipocytes may be involved in keloid scarring by effecting
myofibroblast dissipation from the wound bed, and serve as
interesting new potential therapeutic targets for further research.
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Keloid fibrocytes
Bucala et al. (1994) were the first to suggest that the surrounding
connective tissue may not be the sole source of new fibroblasts
in wound repair and described a blood-borne cell with
fibroblast-like properties that enter sites of tissue repair (see
Supplementary Tables S1, S3B). These so-called fibrocytes were
characterized by a collagen+/vimentin+/CD34+phenotype and
not only produced ECM proteins and wound healing mediators,
but were also capable of acting as antigen-presenting cells and
differentiating into myofibroblasts (Bucala et al., 1994;Quan
et al., 2004). Based on the limited available literature, there are
increased numbers of CD45RO+/35F9+/MRP8/9+fibrocytes
in keloids compared with normotrophic scars (Iqbal et al.,
2012), and moreover, PBMCs derived from keloid patients
produced more LSP-1+/collagen1+fibrocytes than PBMCs
from healthy controls (Naylor et al., 2012). In further support
of this, Mathangi Ramakrishnan et al. (2012) showed that
keloid fibroblasts expressed increased levels of fibrocyte markers
(CD34+/CD86+), which were absent in normal fibroblasts.
This suggests an at least partial fibrocyte origin of the keloid-
derived fibroblasts. Reduced fibrocyte numbers have also been
reported (Ueda et al., 1999), but this was based on the
presence of histologically identified slender nuclei rather than
immunohistochemical phenotyping, and may therefore not be as
reliable. Given the aforementioned findings of increased fibrocyte
presence in keloids (Iqbal et al., 2012;Mathangi Ramakrishnan
et al., 2012) and their potential differentiation into abnormal
keloid myofibroblasts (Lim et al., 2019), fibrocytes may be
significantly involved in keloidogenesis and therefore deserve
further investigation.
Keloid endothelial cells
Both increased (Ehrlich et al., 1994;Amadeu et al., 2003;Tanaka
et al., 2004;Materazzi et al., 2007;Ammendola et al., 2013;
Bakry et al., 2014) and decreased (Beer et al., 1998;Ueda et al.,
2004;Bux and Madaree, 2010;Kurokawa et al., 2010) vasculature
have been reported in keloid scars in approximately equal
measure (see Supplementary Tables S2, S3C). However, based
on reports of microvessel occlusion and increased expression
of hypoxia-induced factor 1α(HIF-1α) in abnormal scars, it
has been proposed that keloids are relatively hypoxic tissues
(Zhao et al., 2017). The ischemia hypothesis builds on this to
explain how hypoxia can contribute to keloid scar development.
Kischer et al. (1982) demonstrated that unlike normal skin, the
overwhelming majority of hypertrophic and keloid scars have
microvessels with occluded lumens and that this was likely due
to endothelial cell proliferation. The authors considered this
reaffirmation of their hypothesis that hypoxia “is an integral
factor in the generation of hypertrophic scar and keloid” (Kischer
et al., 1982), but whether or not this relative hypoxia promotes
fibroblast and endothelial cell proliferation has still not been
determined (Song, 2014).
Kischer (1984) also suggested another mechanism by which
the microvessel abnormalities could generate both hypertrophic
scars and keloids. They proposed that injury leads to regeneration
of the microvessels, and suggest that the pericytes of the
newly regenerating microvessels form the source of the
fibroblasts generating the excessive collagen which characterizes
these abnormal scars.
It has also been suggested that endothelial cell dysfunction
plays a role in keloidogenesis. Ogawa and Akaishi (2016)
proposed that local factors such as stretching tension together
with genetic factors both act to induce endothelial cell
dysfunction in the form of vascular hyperpermeability during
the inflammatory phase of wound healing. This prolongs
the influx of inflammatory cells and factors, thereby also
prolonging the inflammatory phase. Consequently, dysfunction
of the fibroblast cell population leads to the development of
either hypertrophic or keloid scars. Lastly, endothelial cells
may also contribute to keloid scar development by undergoing
endothelial-mesenchymal transition (EndoMT) to acquire a
mesenchymal phenotype (Lee Y.-S. et al., 2015). In this way,
endothelial cells may directly serve as a source of the abnormal
keloid fibroblasts.
Keloid nerve cells
Based on the symptoms of itching and pain, both sensations
carried by small nerve fibers, there does appear to be a role
for nerve cells in the development of keloid scars. Yet, thus
far little has been published on the presence of nerve cells
in keloid tissue (see Supplementary Tables S2, S3C). Sensory
nerve fibers have also been mentioned in the context of Ogawa’s
mechanobiology theory on keloid pathogenesis (Ogawa, 2011).
As part of the group of skin receptors perceiving mechanical
forces, information from the sensory fibers is then relayed to the
central nervous system leading to the release of neuropeptides,
which can then modulate scarring by altering skin and immune
cell functions. However, studies staining for nerve fibers in
keloid tissue have reported both increased (Hochman et al., 2008;
Drummond et al., 2017) and decreased (Saffari et al., 2018) nerve
fiber densities. As different markers (PGP9.5 and S100 protein,
respectively) were used to identify the nerve fibers, this could in
part explain the different outcomes.
Keloid Immune Cells
Although both increased and reduced levels of
certain immune cell types have been reported (see
Supplementary Tables S2, S3D), overall there appears to
be an increase in macrophages (Boyce et al., 2001;Shaker et al.,
2011;Bagabir et al., 2012a;Jiao et al., 2015), T-lymphocytes
(Boyce et al., 2001;Shaker et al., 2011;Bagabir et al., 2012a;Jiao
et al., 2015) and mast cells (Kamath et al., 2002;Moshref and
Mufti, 2009;Shaker et al., 2011;Bagabir et al., 2012a;Ammendola
et al., 2013) in keloids that have been found to interact with
each other, other immune cells and dermal fibroblasts on a
cellular level (Boyce et al., 2001;Santucci et al., 2001;Shaker
et al., 2011). Moreover, macrophages and T-lymphocytes from
keloids also showed intrinsic abnormalities compared with their
normal counterparts. Keloid-derived macrophages showed a
high activation status, increased M2 polarization and overall
increased expression of both M1 and M2 activation factors
compared with normal skin macrophages (Jin et al., 2018).
They were also more potent at inducing the regulatory T-cell
phenotype when co-cultured with CD4+T-lymphocytes from
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keloid patients’ blood. Thus, there was not just a general increase
in T-lymphocytes, but specifically the regulatory (Jin et al.,
2018) and memory (Chen et al., 2018) T-cells, as well as an
increased CD4+:CD8+ratio (Boyce et al., 2001;Bagabir et al.,
2012a) in keloids. Furthermore, the altered cytokine production
in keloid-derived memory T-cells (Chen et al., 2018) and the
reduced mitogenic response of circulating T-cells to known
mitogenic stimuli (Bloch et al., 1984), suggests that an abnormal
T-cell response may contribute to keloid scarring. In this way,
the previously discussed sebum reaction hypothesis is also
an extension of this concept as the intrinsically abnormal,
sebum-sensitive T-lymphocytes take center stage in this theory
(Song, 2014).
Mast cells have also been found in abundance in keloid scars.
Arbi et al. (2015) found that mast cells were closely associated
with fibroblasts in keloid scars and that the phagocytosis of
collagen fibrils by mast cells was a common ultrastructural
feature. They hypothesized that the abnormal collagen synthesis
observed in keloids and the consequent accumulation of collagen
fibers, are able to induce increased mast cell recruitment
and subsequent collagen phagocytosis. The resulting release
of mast cell-derived mediators (interleukins, mediators, and
growth factors) is then able to stimulate further collagen
production and thereby aids further keloid scar development.
Lastly, there have been variable reports on the presence of
Langerhans cells in the epidermal compartment of keloid
scars, both normal (Bagabir et al., 2012a) and increased (Jiao
et al., 2015) numbers have been observed in both hypertrophic
and keloid scars.
Several hypotheses centering on inflammatory processes
have also been put forward. In the chronic inflammation
hypothesis, Dong et al. (2013) posed that the presence of
chronic inflammation in keloids indicates that local inflammation
promotes keloid formation. The traumatic and inflammatory
stimuli that trigger keloid scar formation result in the
continuous upregulation of already highly sensitive pro-
inflammatory genes in the keloid microenvironment. This keloid
microenvironment fosters the development of abnormalities
in the resident keloid fibroblast, which in turn is considered
to be the driving force behind keloid scar formation. Ogawa
(2017) has further expanded on this notion and postulated
that chronic inflammation is responsible for the invasive
growth of keloids and even suggests that both hypertrophic
scars and keloids are principally inflammatory disorders of
the reticular dermis rather than being skin tumors. In
the neurogenic inflammation hypothesis, the inflammation
is thought to arise from mechanical stress, such as skin
stretching, which stimulates mechanosensitive receptors on
sensory fibers to release neuropeptides. These then bind to
receptors of various skin cell types including keratinocytes
and fibroblasts, mast cells and endothelial cells. Vasodilation
and vessel permeabilization, increased mast cell release of
histamines and cytokine production (including TGF-β) take
place as a result of this. Fibroblasts then become activated as
a result of the neurogenic inflammation and the upregulation
TGF-β, leading to keloid and hypertrophic scar formation
(Akaishi et al., 2008).
Other Proposed Hypotheses on Keloid
Scar Formation
The myriad of available treatment modalities is only matched
by the multitude of proposed hypotheses to explain keloid
scar formation. These are not mutually exclusive, and further
support the notion that keloid scarring has a multifactorial
genesis (Slemp and Kirschner, 2006). When appropriate, these
have already been discussed in the appropriate paragraphs of
the section on keloid cellular abnormalities. A brief discussion
of additional hypotheses that could not be categorized in the
previous paragraph, will follow next.
Keloid Triad Hypothesis
Perhaps one of the only proposed hypotheses encompassing
multiple risk factors in one, the keloid triad hypothesis
(Agbenorku et al., 1995) is defined as a group of three etiologic
factors: genetic links, infective agent (bacterial, viral, or other)
and surgery (e.g., sutures, tension of suture lines, location of
sutures in relation to the relaxed skin tension lines); which must
be simultaneously present and interact to develop keloid scarring.
These three factors are further subdivided into major factors
and minor etiological factors. Major factors include: African
ethnicity, age 10–30 years, familial susceptibility or keloid-prone
upper part of body site. Minor factors include: orientation
of incisions/sutures with respect to RTSLs, wound or sutures
under tension, healing by secondary intention, type of infection;
determines whether or not a keloid scar is likely to develop. At
least one major and two minor factors must be present for keloid
scars to develop. A keloid is unlikely to develop if all three factors
are minor or if only two factors are present, but a hypertrophic
scar may form instead.
Incomplete Malignancy Hypothesis
When Ladin et al. (1998) studied apoptosis in keloid scars, they
found that the level of apoptotic cells was significantly reduced
in keloid tissue and fibroblasts compared with normal foreskin
tissue and fibroblasts. However, keloid fibroblasts did show
increased apoptosis upregulation in response to treatment with
hypoxia, hydrocortisone or γinterferon, while normal fibroblasts
were only responsive to high doses of hydrocortisone. Because of
this, Ladin et al. (1998) suggested that keloids may represent a
type of incomplete malignancy that has undergone some, but not
all tumourigenic changes.
Viral Hypothesis
In this infection-based hypothesis (Alonso et al., 2008), the
authors proposed a role for a normally quiescent, unknown
virus in the bone marrow or lymphatic system which is
activated in a genetically susceptible person with a wound.
This virus can then reach the wound via fibrocytes that are
chemoattracted to the wound site or via infecting virions
in the saliva arriving at the wound bed. There the many
chemical stimuli from the wound healing processes allow the
virus to become activated, resulting in transcription of viral
proteins which derail wound healing and eventually lead to
keloid scarring.
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Stiffness Gap Hypothesis
Born out of recent findings on the role of mechanotransduction
in keloid scarring, this theory (Huang et al., 2017) proposes that
the enlarged gap between ECM stiffness and cellular stiffness
enables the constant and continued keloid progression. The
ECM is not only a cellular scaffold storing important wound
healing mediators, but its rigidity also influences fibroblast
function and can induce processes such as fibroblast migration,
proliferation and differentiation. When dermal fibroblasts sense
the stiffness gaps between the ECM and themselves via
mechanotransduction, alterations in fibroblast phenotype ensue
which promote proliferation, migration and ECM synthesis and
therefore contribute to keloid progression. However, systematic
mechanobiological experiments to verify this hypothesis have yet
to be performed.
Immunonutritional Hypothesis
It has been proposed by several authors that nutritional
imbalances can promote prolonged inflammation and cytokine-
mediated reactions which contribute to keloid scarring (Huang
and Ogawa, 2013a). Nutritional imbalances have been reported
for essential fatty acids and micronutrients in keloid-prone
patients. For example, the keloid-prone black South African
population were deficient in the essential fatty acids a-linolenic
acid, eicosapentaenoic acid and docosahexaenoic acid, but
showed an increased intake of linoleic acid and arachidonic acid.
The levels of micronutrients (calcium, copper, iron, vitamin B2,
vitamin C, vitamin A, and zinc) was also insufficient in their diet
(Louw and Dannhauser, 2000), and this was also reflected in the
serum as well as keloid scar tissue (Bang et al., 2002). Compared
with non-keloid forming patients, keloid-prone patients showed
higher levels of zinc in their serum, higher copper levels in non-
lesional normal skin and in the keloid scar tissue. The essential
fatty acid imbalance has also been observed in the membrane
of keloid fibroblasts with lower linoleic acid and higher neutral
lipid levels in the central keloid area compared with its periphery
(Louw et al., 1997). Lipids are not only essential cell membrane
constituents, but also have important roles in local inflammation
and intracellular signal transduction. As such they are also active
participants in the chronic inflammatory processes that result
in the development and continued growth of keloids (Huang
and Ogawa, 2013b). And finally, dietary compounds have been
shown to affect keloid-derived cells at the in vitro level as well.
Green tea and its extracts have been shown to reduce keloid
fibroblast proliferation, migration and collagen synthesis (Zhang
et al., 2006;Park et al., 2008).
Metabolic Hypothesis
It has also been theorized that the net overexpression of
ECM components in the keloid dermis is also the result of
both the increased number of fibroblasts and their increased
intrinsic metabolic activity, aside from the usual suspects
in abnormal signaling pathways (Butler et al., 2008a). Both
keloids and hypertrophic scars showed increased numbers of
fibroblasts compared with normal scars or normal skin (Ueda
et al., 1999), but only keloid fibroblasts showed increased
metabolic activity. Metabolic activity has also been measured
as levels of ATP (Ueda et al., 1999;Vincent et al., 2008) and
as total protein content synthesis together with endoplasmic
reticulum staining (Meenakshi et al., 2005), all of which have
proven to be upregulated in keloids compared with normal
skin, normotrophic and hypertrophic scars. Modulating keloid
metabolism may therefore be a way to reduce keloid scar growth
(Butler et al., 2008a;Huang and Ogawa, 2013a).
Nitric Oxide Activity
Using mathematical modeling, Cobbold (2001) proposed that
nitric oxide could be involved in both hypertrophic and keloid
scar formation. Nitric oxide is a known source of free radical
molecules which stimulates collagen synthesis in normal wound
healing, it therefore follows that excessive amounts of nitric
oxide can result in the elevated scar tissue characteristic of these
abnormal scar types. Cobbold further speculated that the source
of this excessive nitric oxide may be the basal epidermis, in
part because the generated free radicals also stimulate melanin
synthesis and melanocytes are located within this epidermal layer.
Psychoneuroimmune-Endocrine Hypothesis
Hochman et al. (2015) posed that the “brain-skin connection”
may play a role in keloid scarring. The psychogenic component is
a part of the pathogenesis in most skin diseases such as psoriasis
and atopic dermatitis, where it can trigger integrated responses
from the nervous, immune and the endocrine systems. Normal
wound healing also depends on neurogenic factors, which in turn
are influenced by psychological, immune and endocrine factors.
Any change in these factors may interfere with the scar formation
process. Hochman speculated that stressed patients themselves
exacerbate the neuro-endocrine system, leading to the release of
hormones, neural transmitters, and immune cells which creates
an inflammatory microenvironment that stimulates fibrotic
processes (Hochman et al., 2015). This has not been extensively
studied for abnormal scars but, stress as measured by the sweating
response during a stress-inducing task was found to be associated
with an increase in keloid recurrence rates after surgery with
postoperative radiotherapy (Furtado et al., 2012).
Hypertension Hypothesis
Dustan (1995) first hypothesized that the differences in growth
factor abnormalities that are conducive to keloid scarring in the
black population, could also be responsible for the development
of hypertension and therefore explain the differences in
hypertension severity between the black and white population.
A significant increase in hypertension rates has in fact been
reported in keloid-prone patients compared with non-keloid
formers in both the African–American (Snyder et al., 1996;
Adotama et al., 2016;Rutherford and Glass, 2017) and Caucasian
population (Snyder et al., 1996). Ogawa et al. (2013) also reported
that severe keloid cases (multiple or large >10 cm2) in Japan were
significantly more likely to have hypertension than patients with
mild keloids (<2 or <10 cm2). Furthermore, in patients with
keloids, those with more severe hypertension were significantly
more likely to have multiple and/or larger keloids than patients
with normotension or less severe hypertension (Arima et al.,
2015). The mechanisms by which hypertension could lead to
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FIGURE 3 | Keloid scar models. Overview of available keloid scar models. Keloid models can be further subdivided into in vivo and in vitro models, which can be of
human or animal origin. Human in vivo models include non-invasive testing methods (e.g., imaging and microscopy) and invasive methods, such as serial biopsy or
inducing keloid formation by wounding. Animal in vivo models include implantation of keloid tissue fragments, fibroblasts or full thickness skin equivalents, as well as
inducing keloid scar development by irritation or wounding. In vitro keloid models range from simple monolayers to 3D structures and co-culture systems. Indirect
co-culture systems include monolayer keloid fibroblasts combined with either monolayer keratinocytes or a fully differentiated epidermis. Lastly, explant cultures are a
combination of in vivo and in vitro as the method involves the maintaining of keloid tissue fragments in in vitro culture. Figure first published in Limandjaja (2019),
used with permission.
keloid development remain largely speculative for the time being,
but the changing blood flow likely affects the skin cellular
constituents in ways that promote fibrotic processes (Huang
and Ogawa, 2014). Angiotensin-converting enzyme (ACE) was
proposed as a potential common mechanism for the pathogenesis
of both keloids and hypertension, but Stewart and Glass (2018)
found no correlation between plasma ACE levels and keloids
or hypertension. Nevertheless, antihypertensives such as ACE-
inhibitors (e.g., captopril), calcium-channel blockers (e.g.,
verapamil) have been able to reduce keloid symptoms and are
part of the current therapeutic arsenal for keloid treatment.
Huang and Ogawa (2014) reviewed the evidence for the
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participation of systemic hypertension in hypertrophic and
keloid scar pathogenesis and although they could not establish
a strong causal relationship, hypertension was considered a likely
risk factor for abnormal scarring.
MODELS OF KELOID SCARRING
Keloid scar models can largely be divided into in vivo and in vitro
models, Supplementary Tables S4A–E and Figure 3 give an
overview of the currently available keloid scar models.
In vivo Keloid Models
In vivo keloid models are of the human or animal
variety. Currently available in vivo human keloid models
(Supplementary Table S4A) can be further subdivided into
non-invasive, invasive, and computational models. Non-invasive
in vivo modeling generally comprises live imaging whereby
certain tissue characteristics are visualized. Invasive keloid
modeling varies from the relatively minor FDG injections to
measure glucose metabolism (Ozawa et al., 2006), to serial
biopsies to evaluate keloid development over time (Lin et al.,
2015) and even includes attempts to induce keloid formation in
known keloid formers by wounding (Mancini and Quaife, 1962).
Despite their value especially in the clinical setting for follow-up
evaluation, human in vivo models post-1962 are inherently
limited in the ability to manipulate experimental variables and
yield primarily observational data. While Lebeko et al. (2019)
saw particular merit in the temporal data acquired from serial
keloid biopsy analysis as an in vivo ‘4D model’ in their review on
keloid models, it is important to recognize the potential risk of
exacerbating the existing keloid scars by continual provocation
with biopsy-associated injury.
In animal models, keloid formation is either induced
or involves the implantation of human keloid tissue (see
Supplementary Table S4C). Unfortunately, inducing keloid scar
formation has proven virtually impossible (Seo et al., 2013)
and more often than not, hypertrophic scars developed instead
of keloids (Morris et al., 1997;Khorshid, 2005). Implanting
human keloid cells or tissue fragments into animal models
has been more successful (Hillmer and Macleod, 2002) and
resulted in the development of a palpable nodule-like mass.
While implanted keloid tissue was generally able to retain the
keloid-specific keloidal collagen even within the animal model,
the use of already established keloid tissue does not allow us
to study de novo keloid development. However, when tissue
culture techniques were combined with IL-6 exposure to implant
a keloid fibroblasts-hydrogel suspension into nude mice, the
resultant mass not only showed the greatest increase in growth,
but also gave rise to de novo keloidal collagen formation (Zhang
Q. et al., 2009). The appeal of an in vivo animal model is
obvious, but there is an important limitation to the use of any
animal for the study of keloid scarring, in that keloids occur
exclusively in humans (Tuan and Nichter, 1998;Yang et al.,
2003). Though exuberant granulation tissue on horse limbs has
often been posed as the equine version of keloid tissue, a recent
study (Theoret et al., 2013) comparing keloids and exuberant
granulation tissue has shown they are not in fact one and the
same. Fundamental differences in essential skin physiology (hair
follicle density, epidermal, and dermal thickness) and wound
healing mechanisms between rodents and humans (Perez and
Davis, 2008;Ramos et al., 2008) pose additional important
limitations. The in vivo microenvironment in animal models may
therefore provide a less than human-like exposure to the keloid
cell population.
Keloid Explant Models
Keloid tissue explants do not necessarily require implantation
into an animal model in order to survive and be used as a keloid
model in and of itself, they can be maintained in culture after
removal from the human body for up to 6 weeks (Duong et al.,
2006;Bagabir et al., 2012b) (see Supplementary Table S4B).
Various culture methods have been tried, but keloid morphology
appears best conserved in explants embedded in collagen gel
that are cultured air-exposed (Duong et al., 2006;Bagabir et al.,
2012b;Mendoza-Garcia et al., 2015). Unfortunately, relying on
fresh keloid tissue for this explant model system has its obvious
logistical issues with limited availability and potential inter-
and intralesional variability between samples. Another important
limitation of this model type is the absence of circulatory system,
as is the case with all in vitro models to date. Ultimately, this
model system appears best suited for the testing of therapeutics
(Syed et al., 2012, 2013a,b) rather than studying the pathogenetic
mechanisms underlying keloid formation, as there is very limited
ability to manipulate any experimental variables.
In vitro Co-culture Keloid Models
As skin physiology, immunology and wound healing is markedly
different in animals (Hillmer and Macleod, 2002;Ramos et al.,
2008;Seo et al., 2013;Seok et al., 2013) and since keloids
are exclusive to humans (McCauley et al., 1992;Yang et al.,
2003), there is a need for relevant human in vitro models (see
Supplementary Tables S4D, E). What is more, in vitro co-culture
systems allow for extensive experimental manipulation, such
as the development of heterotypic models combining normal
keratinocytes with keloid fibroblasts to study the individual
contribution of keloid fibroblasts to keloid formation.
Indirect co-cultures consist of double chamber systems
with keratinocytes seeded (as a monolayer or differentiated
epidermis) onto an upper porous transwell insert and fibroblasts
cultured in a monolayer on the underlying bottom well
(see Supplementary Table S4D). These indirect co-cultures
are simple-to-execute experiments that are particularly suited
to study paracrine interactions, as previously discussed in
‘Abnormal Keratinocyte-Fibroblast Interactions’. However, the
monolayer fibroblast cultures and the artificial separation
between keratinocytes and fibroblasts, bear little resemblance to
the true in vivo situation.
Direct co-cultures represent the most in vivo-like in vitro
culture model and include either mixed monolayer cultures
or full thickness skin equivalents comprised of keloid-derived
cells (see Supplementary Table S4E). The full thickness skin
equivalents comprised entirely of keloid-derived keratinocytes
and fibroblasts most closely resemble the native keloid, but thus
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Limandjaja et al. The Keloid Disorder
far, these have only been developed in the context of implantation
into an animal model (Supp et al., 2012b;Lee Y.-S. et al.,
2015) and/or subjected only to limited experimental analysis
(Supp et al., 2012a). Overexpression of ECM components,
particularly collagen I, was the common denominator in these
keloid models. Recently, we were also able to reconstruct keloids
in vitro with keloid-derived keratinocytes and fibroblasts, using
a collagen-elastin scaffold (Limandjaja et al., 2018a). Compared
with in vitro reconstructed normal skin, the keloid model showed
a trend of increased dermal thickness, increased α-SMA and p16
expression, reduced HGF secretion and reduced collagen type IV
α2 chain, hyaluronan synthase 1 and matrix metalloproteinase
3 gene expression. More importantly, the keloid model behaved
differently from similarly reconstructed hypertrophic scars, and
was able to demonstrate intralesional heterogeneity within
keloids (Limandjaja et al., 2018b). In other words, a relatively
simple in vitro keloid model was already able to demonstrate
certain intrinsic abnormalities in keloid-derived keratinocytes
and fibroblasts and in doing so, identified certain aspects of keloid
behavior which could not have been deduced from ex vivo biopsy
analysis alone. In our opinion, this both illustrates and validates
the use of skin tissue engineering as an important adjunct to
furthering keloid scar research.
Ultimately, there is no single universal keloid model
that is able to satisfactorily answer every experimental
objective (Hillmer and Macleod, 2002;Marttala et al., 2016).
Overall, animal models are better suited for therapeutic
testing and particularly for safety testing prior to human
administration (Hillmer and Macleod, 2002;Marttala et al.,
2016), while tissue culture systems are best suited to study
keloid pathogenesis (Hillmer and Macleod, 2002;Marttala
et al., 2016). Given the exclusive occurrence of keloids in
humans (Tuan and Nichter, 1998;Yang et al., 2003) and
other serious limitations of using animal models for wound
healing studies, as well as the fact that research on its
pathogenesis cannot rely solely on (immunohistochemical)
analysis of an intact native specimen; it seems pertinent
to focus our efforts on further advancing tissue culture
models - in particular the in vitro, organotypic full thickness
keloid skin equivalents. Once proven faithful to actual
in vivo keloid biology, using this model to study keloid
formation will eventually aid in the development of superior
treatment methods by uncovering new mechanisms of
pathogenesis. In addition, such a model would have the
added benefit of serving as a test object for future therapeutic
modalities without the complication of ethical objections
(Yang et al., 2003).
THE KELOID DISORDER
The International Classification of Disease (ICD) represents
the international standard for reporting diseases and health
conditions and in the most recently released ICD-11 (2018),
keloids have been grouped under ‘fibromatoses and keloids,’
“a heterogenous group of disorders characterized by increased
deposition of fibrous tissue in the skin and subcutaneous tissues.”
This is departure from the previous ICD-10 (2016) in which
keloids were still categorized under ‘hypertrophic disorders of
skin,’ and is the result of recent international agreement to refer
to keloids as part of a ‘keloid disorder’ rather than a ‘keloid
scar.’ This change in ICD classification and nomenclature reflects
growing consensus among keloid researchers and clinicians alike
that keloids more closely resemble a benign, fibrous tumor
than scar tissue and can best be described in terms of a
fibrotic disorder.
By their very definition of continued, invasive horizontal
growth (Burd and Huang, 2005), keloids clearly share certain
characteristics of cancerous growths. Lu et al. (2007b) have
previously described keloids as behaving “clinically as
non-metastatic malignancies, although histologically they
are benign.” Similarly, Ladin et al. (1998) coined keloids
‘incomplete malignancies’ as keloid fibroblasts are still apoptosis-
responsive to in vitro treatment despite overall reduced
levels of apoptosis. The term refers to the manner in which
keloids show some, but not all the changes associated with
tumourigenesis. The absence of metastasis despite their invasive
growth and the aforementioned cancer-like characteristics,
separates keloids from true malignant tumors and lends
further support to the ‘incomplete malignancy’ term coined
by Ladin et al. (1998).
The change in nomenclature reflected in the newest edition
of the ICD may seem like an exercise in semantics, but it
should be noted that the cosmetic association with the word
‘keloid scar’ significantly complicates both insurance coverage
for treatment as well as funding for research. This is not to say
this nomenclature change must result in an altogether ban on
the association of the word ‘scar’ with keloids, merely that we
shift the focus away from a purely cosmetic association, toward
broadening our perspective of keloids for the fibroproliferative
disorders that they are and in this way recognize the severity of
this fibrotic disorder.
Keloid research remains plagued by significant controversy
and contradictory findings, which has been highlighted
throughout this manuscript where possible. Much of this could
be resolved by the standardization of study designs and general
research approach. Important considerations for future research
include (i) inclusion of a description of the keloid phenotype
(based on growth pattern), (ii) clarification of the location within
the keloid from which samples were taken (e.g., growing/bulging
area versus more quiescent region; peripheral versus central
areas), (iii) inclusion of scars of similar maturation stage (at
least 1 year old). Lastly, the inclusion of both hypertrophic
and normotrophic scars would be a valuable addition to keloid
research, as this provides a more comprehensive view of the
entire scarring spectrum.
AUTHOR CONTRIBUTIONS
GL, SG, and FN conceived the manuscript outline. GL performed
the literature study and wrote the manuscript with input from
all the authors. SG, FN, and RS supervised, helped shape the
manuscript, and offered final critical revision.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 17 May 2020 | Volume 8 | Article 360
fcell-08-00360 May 22, 2020 Time: 19:44 # 18
Limandjaja et al. The Keloid Disorder
FUNDING
This study was financed by a grant from the Dutch
government: Rijksdienst voor Ondernemend Nederland, project
number INT102010.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fcell.2020.00360/
full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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