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Cutaneous and oral wound closure in vitro; Role of salivary peptides and cytokines
Boink, M.A.
2017
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Boink, M. A. (2017). Cutaneous and oral wound closure in vitro; Role of salivary peptides and cytokines.
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CHAPTER 1 GENERAL INTRODUCTION 9 8 Chapt er 1 General introduction
Difficult-to-heal wounds are a major issue in our society and significantly impair the quality of life of millions of people. Difficult-to-heal wounds are therapy-resistant wounds such as leg ulcers, pressure ulcers and diabetic foot ulcers. They have a prevalence of approximately 1% of the population, exist for prolonged periods of time and as a result have a large socio-economical impact. It is expected that the incidence will increase dramatically due to an ageing population and increasing incidence of health-related disorders (e.g. vascular diseases and diabetes), which are associated with ulcer development (1). Trauma-induced wounds (e.g. those caused by automobile accidents, deep burns, or animal bites) and some wounds resulting from surgical procedures are also difficult to heal. To achieve optimal healing of all these types of wounds, the primary aim is to close the wound as quickly as possible in order to prevent infection and dehydration. A secondary aim for optimal wound healing is to create a cosmetically acceptable and functional scar that does not limit joint mobility. Unfortunately, current treatments of difficult-to-heal wounds are suboptimal. Even though 50% of treated venous leg ulcers will heal, recurrence rates are as high as 26%-56% (2). Experimental studies have indicated that physiological growth factors can positively influence wound healing, and might have therapeutic value (3). However, large-scale production of these compounds via recombinant DNA technology is complex and expensive. Therefore, alternatives to these growth factors are required, particularly those that function via new and/or additional mechanisms, to provide better treatment options at acceptable costs.
The skin
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Before describing the process of cutaneous wound healing, first some background of the skin (Figure 1) is given. The skin is the largest human organ, forming a first line of defense. Acting as a barrier, it protects the body from water loss and prevents hazardous substances from getting in. The skin also has an important role in thermo- regulation, by vasodilation or vasoconstriction and excretion via sweat. It is colonized by many commensal bacteria that together form a resident skin flora, which hampers colonization with other, potentially more pathogenic, bacteria. Furthermore, the skin produces host defense peptides, which can kill many micro-organisms (bacteria, yeast, and some viruses). It protects against UV radiation, by melanin producing melanocytes and it contains many nerve ends, which make it possible to perceive heat, cold, pain, vibrations, pressure and itch.
These are more differentiated and contain keratohyalin granulae filled with proteins involved in the organization of keratin filaments. In the skin of the palms of hands and feet there is also a stratum lucidum present. The outermost layer of the epidermis is the stratum corneum, which consists of dead, flattened keratinocytes that have lost their cellular structures and are filled with keratin. The cells within the stratum corneum contain proteins lining the cell membrane to form an impermeable cornified envelope. These dead cornified cells together with extracellular lipids form the stratum corneum, which has an extremely important barrier function. Keratinocytes from the stratum corneum eventually shed from the skin, which is called desquamation.
The epidermis consists of mainly keratinocytes (95%), the other 5% consists of melanocytes, Langerhans cells and Merkel cells. Melanocytes produce melanin, a pigment that effectively absorbs UV radiation, thereby preventing the skin from radiation damage. Langerhans cells are immunocompetent cells that can present antigens from the skin to the immune system. Merkel cells are described as touch cells.
Underneath the epidermis lays the basement membrane, which connects the epidermis to the dermis. Important cells in the dermis are fibroblasts, which produce extracellular matrix, such as collagen and elastin, giving the dermis its strength and elasticity. Furthermore, the dermis contains infiltrating immune cells, such as dendritic cells, mast cells, macrophages and lymphocytes. Blood vessels supply the skin with nutrients. The dermis consists of two distinct layers, the superficial stratum papillare and the deeper stratum reticulare, the latter contains larger fibers and less cells. Underneath the dermis is the subcutaneous adipose tissue, consisting of connective tissue and adipocytes.
Cutaneous wound healing (4)
When the skin is injured the body starts the repair process immediately. Normal wound healing can be divided in four partly overlapping stages: hemostasis (minutes), inflammation (days), proliferation (weeks) and remodeling (months) (Figure 3).
Figure 2: the different layers (strata) of the epidermis. Adapted from: http://spaces.imperial.edu/thomas.morrell
CHAPTER 1 GENERAL INTRODUCTION
13 12
Chapt
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During hemostasis the bleeding of a wound is stopped by vasoconstriction, the formation of a thrombus of aggregated platelets and blood coagulation. Activated platelets chemotactically recruit inflammatory cells such as neutrophils, monocytes and macrophages. In the inflammatory phase local vasodilatation, increased permeabilization and blocked lymph drainage cause rubor (redness), tumor (swelling), calor (heat) and dolor (pain). In the early inflammatory phase many neutrophils enter the wounded area to phagocytose debris and bacteria. They also produce pro-inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α), which maintains the inflammatory process and activates fibroblasts and keratinocytes. In the late inflammatory phase monocytes arrive in the wound area, which then differentiate towards macrophages. Macrophages phagocytose micro-organisms and tissue debris, and produce cytokines, chemokines and growth factors (such as IL-1α, TNF-α, TGF-β and FGF), that attract and further stimulate fibroblasts, endothelial cells and keratinocytes.
Once the neutrophils and macrophages have decontaminated the wound the inflammation is downregulated. Then the proliferation phase starts, in which fibroblasts synthesize a provisional matrix, mainly consisting of collagen type III and elastin. At the same time some fibroblasts differentiate into myofibroblasts, causing contraction of the wound. Re-epithelialization occurs by keratinocytes, while endothelial cells form new blood vessels (angiogenesis). In the remodeling phase new matrix is still being produced. Simultaneously collagen III is replaced by collagen I, which is cross-linked and re-aligned to improve tensile strength. Furthermore, the amount of cells (fibroblasts, immune cells and endothelial cells) and vascularization is decreased. This remodeling phase can take up to a year after
injury. Table 1 gives an overview of the different factors that are important in wound healing (cytokines, chemokines and growth factors).
Table 1: Overview of important cytokines, chemokines and growth factors in wound healing
Name Function in wound healing Ref.
Interleukin 6 (IL-6) Immune response during infection and after trauma
Neutrophil chemoattractant
Fibroblast proliferation, keratinocyte migration and proliferation
(3;5)
CXCL8 Mediator in innate immune response
Neutrophil and macrophage activation and chemotaxis Keratinocyte proliferation
(5;6)
CCL2 / MCP-1 Macrophages, T-cell and mast cell chemoattractant (3)
CCL5 / RANTES Fibroblast migration (7)
CCL27 / CTACK Effector cell recruitment to sites of epithelial injury
Keratinocyte chemoattractant (8) (7)
CCL28 / MEC Effector cell recruitment to sites of epithelial injury (8)
Hepatocyte growth factor
(HGF) Keratinocyte migration and proliferation Angiogenesis
Anti-fibrosis
(3;9)
Vascular endothelial growth
factor (VEGF) Stimulation of vasculogenesis and angiogenesis (5)
Basic fibroblast growth factor
(bFGF / FGF-2 / FGFβ) Fibroblast proliferation, keratinocyte migration and proliferation Endothelial growth and migration
Collagen remodeling
(5)
Oral wound healing
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The oral mucosa consists of a differentiated epithelium on a lamina propria (equivalent to epidermis and dermis in skin). The epithelium can be keratinized, e.g. gingiva and upper palate, but also non keratinized, e.g. buccal (cheek) and soft palate (10). Gingiva epithelium (parakeratinized) consists of a stratum basale, stratum spinosum and stratum granulosum. Parakeratinized epithelium is characterized by incomplete disintegration of the nucleus and some cytoplasmic organelles in the terminally differentiated keratinocytes, and a less complete and uniform cornification than orthokeratinized epithelium (such as skin epidermis). In general, oral mucosa epithelium has faster cell renewal and contains a higher number of cell layers compared to the epidermis. Wound healing is more rapid in oral mucosa than it is in the skin. In an experimental setup, wounds created in the skin took 4-5 weeks to heal (11), while wounds of the same size in oral mucosa healed in just one week (12). In pigs a direct comparison of wound closure was made between skin and oral mucosa, in which oral mucosa closed faster (13). Another difference was the quality of the scar, which was better in oral mucosa than in skin. There are at least two differences between the oral cavity and the skin that could account for superior oral wound healing. The first is a better microcirculation in oral mucosa, that enhances nutrient and inflammatory cell access at the wound site, as wells as removal of waste and debris. The second is that the mouth is bathed with saliva, containing many compounds that are beneficial for wound healing.
Saliva
The ancient Greeks already used saliva from snakes to heal skin wounds (14). The function of saliva in wound healing has been scientifically proven by experiments in rats, in which oral wound closure was delayed when salivary glands were removed (15). In human saliva a number of factors that promote wound healing have been identified, although in a much lower concentration than found in rodent saliva (Table 2).
Table 2: Possible wound healing factors present in human saliva and their physiological concentrations.
Growth factors Human saliva ng/ml Murine saliva ng/ml
EGF ~ 0.9 ~ 20,000 NGF ~ 0.9 ~ 40,000 VEGF ~ 1.4 FGF < 0.001 IGF ~ 0.4 ~ 75 TGF-α ~ 5.6 ~ 560 TGF-β ~ 0.024 TNF-α ~ 0.003
Other factors Human saliva μg/ml
LL-37 ~ 0.14
Defensins ~ 0.3
Histatins ~ 100
Modified from M.J. Oudhoff (16)
Human saliva is a rich source of tissue factor, which activates the coagulation cascade. Although the warm, moist and nutrient-rich environment is beneficial for wound healing, these conditions are also beneficial for bacterial growth. The total number of bacteria in saliva is estimated to be between 108 to 109 and therefore it can be expected that saliva plays a role in regulating the balance between commensal and pathogenic bacteria Saliva contains a variety of proteins that protect against microbial infections and damage. Secretory leukocyte protease inhibitor (SLPI) is found in human saliva; it inhibits serine proteinases, but it has antimicrobial properties as well (17). Furthermore human saliva contains β-defensins, which are antimicrobial peptides active against many bacteria, fungi, and enveloped viruses. They are widely distributed throughout the body; besides being secreted by salivary glands, they are also secreted by a wide variety of leucocytes and epithelial cells, and their expression can be upregulated upon injury, infection or inflammation. β-defensins stimulate migration and proliferation of epidermal keratinocytes and thus might promote cutaneous wound healing (18).
CHAPTER 1 GENERAL INTRODUCTION
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It was shown that histatins are major wound-closure stimulating factors in human saliva (20). Histatins are a class of histidine-rich peptides that are present in saliva only of humans and primates that have antifungal activities (21;22). There are many different variants of histatin, but Hst1, Hst3 and Hst5 make up 85% of total histatin concentration in human saliva (22). All histatins are derived from the genes HTN1 and HTN3, which give rise to Hst1 and Hst3 respectively. Hst2 is a proteolytic cleavage product of Hst1, while cleavage of Hst3 gives rise to the other 23 variants. In 2008 Oudhoff discovered another function of histatins, which is wound closure. Hst3 and 5 have the most potent antibacterial and antifungal activity, while Hst1, 2 and 3 enhance wound closure the most (20). Histatins beneficially affected migration of buccal epithelial cell-lines as wells as primary dermal and gingival fibroblasts in a wound scratch assay (20;23). Furthermore re-epithelialization was enhanced in an epidermal skin equivalent with freeze burn wound (24). Recently, it was described that histatins promote cell-substrate and cell-cell adhesion (25;26).
Histatins are small peptides which do not adopt a well-defined secondary or tertiary structure in solution, in contrast to EGF and TGF-β for example, which do have a highly structured three-dimensional conformation. As a consequence, histatins can withstand heat-sterilization without loss of activity. Furthermore, the simple structural features of histatins make large-scale production by standard organic-chemistry synthesis commercially feasible. This offers interesting opportunities from the perspectives of clinical application. In table 3 the sequences of the different peptides used in this thesis are shown. The question arising from Oudhoff’s work was whether histatins would also have a beneficial effect on cutaneous wound healing, which was the basis for this thesis.
Table 3: Peptide sequences
Peptide Amino acid sequence MW (Da)
Hst1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN 4848
c-Hst1 GGDSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDNLPET 5383
Hst2 RKFHEKHHSHREFPFYGDYGSNYLYDN 3445
Mad-Hst1 SHREFPFYGDYGS 1560
LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 4493
c-Hst1: cyclic form of Hst1 peptide, mad-Hst1: minimal active domain of Hst1
Thesis outline
The aim of this thesis was to investigate differences in cutaneous and or mucosa wound healing and to determine the role of saliva histatin peptides and cytokines in the wound healing process. This being in mind for developing better therapeutic strategies for cutaneous wounds. Chapter 2 describes in vitro methods to study differences in cell mobility
of keratinocytes, melanocytes, fibroblasts and endothelial cells during skin wound healing. These methods included cultured cell monolayers in the scratch assay and chemotactic assay, as well as in organotypic tissue cultures. A fibrin gel was used to study endothelial sprouting and tissue-engineered skin models were used to investigate expansion of the differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis. Chapter 3 shows the chemokine receptor expression profile of skin keratinocytes
and the effect of the chemokine ligands on migration and proliferation of keratinocytes. A reconstructed epidermal wound model was used to investigate chemokine secretion after wounding. In chapter 4 the differences between mesenchymal stromal cells that were
isolated from adipose tissue, dermis and gingival was studied, as well as the effect of histatin, on different wound healing processes. Proliferation and migration was studied in cell monolayers, while matrix contraction was studied in organotypic tissue cultures. In chapter 5 intrinsic differences between human skin and gingiva tissue cultures that are fully compliant
with the European regulations for advanced therapy medicinal products were investigated. Information on characterization and mode of action is described. Migration and proliferation of keratinocytes was studied as well as their cytokine and growth factor secretion. Chapter 6 describes the stability of different variants of histatins and the cytokines IL-6 and CXCL8
in chronic wound extracts in order to gain insight on their potential therapeutic properties. To investigate whether peptide degradation occurred and was caused by proteases, a protease inhibitor cocktail was added to the chronic wound extracts. Furthermore, it was studied whether a short peptide exposure of fibroblasts to these peptides was sufficient to activate prolonged cell migration. Chapter 7 describes the secretion of inflammatory and
antimicrobial mediators after histatin and LL-37 exposure. The response of fibroblasts and keratinocytes was investigated separately, but also in co-culture to investigate the cross-talk between the two cells types of both skin and gingiva. In the concluding chapter 8 the
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