Download Chapter 1 General introduction and outine of the thesis

Chapter 1
General introduction and outine of the thesis
Introduction
Skin anatomy
Skin covers the entire body and provides a barrier between the body and the outside world
through its mechanical as well as immunological properties. The skin protects the body
form dangers such as dehydration, UV radiation and pathogens. The skin consists of two
layers: the epidermis and the dermis. The epidermis consists of keratinocytes, but also
contains Langerhans cells and melanocytes. The dermis is composed of extracellular matrix (ECM), blood vessels, ECM producing fibroblasts and several types of immune cells.
The dermis also contains skin appendices and nerve endings (figure 1). ECM consists of
proteoglycans (glycosylated proteins) and several fibrous proteins including collagen and
elastin1. Collagen, elastin and most of the other proteins are produced by fibroblasts1,2. After assembly, collagen undergoes some changes in order to mature and stabilize. The triple
helix is first stabilized by means of hydrogen bonds3. Subsequently, several types of crosslinks are formed within the triple helices and between triple helices4,5. Cross-links are also
formed in elastin fibres4,5. Collagen and elastin fibre content and composition (including
cross-linking) and several other proteins including proteoglycans determine the mechanical
properties of skin6.
Figure 1: anatomy of the skin Adapted from Nestle OF, Di Meglio P, Qin JZ, Nickoloff BJ
(2009)17.
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Skin resident immune cells include dermal Tcells and macrophages (Mφ) and epidermal Langerhans cells7,8. These cells are important for immune surveillance, but also
prevent the immune system from reacting to harmless agents7.
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Cutaneous wound healing
When the skin is damaged, its barrier function is compromised. Therefore, swift sealing
and successive repair of the defect are mandatory. This is achieved through wound healing, where the defect is not resolved by regeneration, but rather by deposition of fibrotic
tissue, resulting in scar formation9. Fibrosis occurs in many other tissues of the human
body besides skin, such as lung and liver9. The complex process of wound healing can be
subdivided in roughly four successive, but partially overlapping phases: the hemostasis and
inflammation phase, the proliferation phase and the remodeling phase10. In order to minimize blood loss, hemostasis is achieved directly after wounding through the formation of a
fibrin clot by activated platelets and the coagulation cascade10. Both activated endothelium
and activated platelets kick start inflammation by secreting a variety of inflammatory and
chemotactic cytokines10. Neutrophils are the first inflammatory cells to arrive in the wound
in response to the chemotactic stimuli1. These cells scavenge the wound for pathogens and
produce cytokines that attract Mφ1. Then, the neutrophils undergo apoptosis as Mφ start to
enter the wound area after approximately two days1. Mφ dispose the wound of dead cells,
including neutrophils, resolve the inflammatory phase and direct wound healing towards
the granulation phase1. After three days, re-epithelialization and neovascularization begin
to occur and fibroblasts start to deposit immature (type III) collagen to fill the defect1. Subsequently, the newly formed ECM is remodeled to become stronger and more organized1.
The remodeling phase lasts until approximately one year after trauma (figure 2)1.
Excessive cutaneous wound healing
Derailment of the wound healing cascade results in deposition of excess scar tissue. Two
forms of excessive skin scarring exist: keloid- and hypertrophic scars (HTS). This thesis
focuses mainly on HTS formation. These types of scars frequently develop as a result of
burn wounds, but also after surgical trauma with incidences varying from 40 to 70% after
surgical trauma and 91% after burn trauma10. HTS is defined as a scar which is raised
above skin level, but remains within the boundaries of the original wound, while keloids
exceed the wound margins10. Other clinical characteristics of HTS include esthetically undesirable features such as erythema and rigidness and co-morbidity such as pain, itch and
diminished range of motion when positioned over a joint11. Histological properties of HTS
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Introduction
include increased amounts of collagen compared to normal scars and myofibroblasts which
persist in mature HTS as opposed to normal mature scars10.
Figure 2: normal wound healing First, hemostasis occurs and immediately starts inflammation,
followed by proliferation after approximately 3 days and remodeling which lasts for months.
Regarding possible mechanisms involved in excessive scarring, several risk factors for
HTS formation have been suggested, comprising both systemic and local factors. Systemic
factors include ethnic background and possibly genetic predisposition; an example of local
factors is mechanical force in the form of stretch12-15.
With respect to the wound healing cascade itself, the current opinion states that HTS
results from excessive and/or prolonged inflammation, overabundant proliferation and abnormal remodeling10. Alterations of the inflammatory response are thought to play a major
role in this derailment of wound healing11. Indeed, altered immune cell populations have
been observed in HTS before. For example, HTS contain more Langerhans cells, which is
thought to contribute to excessive inflammation in HTS formation11. Mahdavian Delavary
and colleagues proposed that a prolonged inflammatory phase in hypertrophic wound
healing causes Mφ to accumulate and induce overabundant ECM production8. Although
Niessen and colleagues did not observe differences in Mφ numbers between HTS and normal human scars of three and 12 months old, this observation does not exclude increased
Mφ numbers during earlier stages of hypertrophic wound healing11.
Despite extensive research on the subject, the exact cause of HTS formation remains
unknown and to date no optimal treatment is available. Even though literature suggests
that the immune system plays an important role, it is unknown whether systemic or local
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immunological predisposing factors (or both) induce or contribute to HTS formation. Next
to that, just a few researchers have studied human hypertrophic wound healing as early as
two weeks after injury, but most studies focus on later time points in the process of hypertrophic scar formation. Thus, the exact time of onset of the derailment of wound healing
leading to HTS formation has not been established yet.
Outline of the thesis
Several risk factors for HTS formation have been described of which ethnic background
is probably the best known risk factor. Chapter 2 discusses currently known risk factors
for HTS formation, their levels of evidence and their influence on the individual phases
of the wound healing process. In addition, risk factors for HTS are studied in chapter 3 in
patients who received presternal incisions for the purpose of open heart surgery. Most open
heart surgeries are performed using extra corporal circulation (ECC; heart lung machine). Surgery as well as ECC induces a systemic inflammatory response16. This systemic
inflammatory response may affect wound outcome, where a stronger response would result
in HTS more frequently. The effect of different types of ECC on scar outcome is examined
in chapter 4.
Besides in skin, fibrosis occurs in many other tissues, for example in coronary arteries9,17. HTS and coronary sclerosis are both forms of excessive fibrosis and have several
features in common17. The possible relation of coronary sclerosis with HTS formation is
examined in chapter 5.
Next to several risk factors, the immune response is thought to play an important role
in HTS formation, but the precise mechanisms and moment of derailment of the wound
healing process are unknown. In order to elucidate whether early inflammation could play
a role, the immune response of early hypertrophic wound healing versus normal wound
healing on genetic-, protein- and cellular level is examined in chapter 6. No research has
been performed previously on alterations of the very early processes of wound healing in
association with HTS formation.
With reference to local factors, skin of different anatomic sites is subjected to different influences, for example UV radiation, pathogens, but also mechanical forces such as
stretch. Since excessive scarring occurs more frequently in certain anatomic locations,
histological and molecular properties of skin of different anatomic sites and predilection
sites for excessive scarring are studied in chapter 715.
A general discussion of the results is provided in chapter 8.
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Introduction
References
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de Rijn M, Chang HY. A systems biology approach
to anatomic diversity of skin. J Invest Dermatol.
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3. van der Slot-Verhoeven AJ (2005). Telopeptide
lysyl hydroxylase: a novel player in the field of
fibrosis (Unpublished doctoral dissertation). University of Leiden, The Netherlands.
4. Eyre DR, Paz MA, Gallop PM. Cross-linking in collagen and elastin. Annu Rev Biochem.
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5. Muiznieks LD, Keeley FW. Molecular assembly
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11. Niessen FB, Schalkwijk J, Vos H, Timens W.
Hypertrophic scar formation is associated with an
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12. Bayat A, Bock O, Mrowietz U, Ollier WE, Ferguson MW. Genetic susceptibility to keloid disease
and hypertrophic scarring: transforming growth
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18. Nestle OF, Di Meglio P, Qin JZ, Nickoloff BJ.
Skin immune sentinels in health and disease. Nat
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Oude Rijn, Leiden; adventurous girl on pedestian bridge