There are multiple signalling pathways and cascades that traverse through the various stages of wound healing in a structured manner, which results in tissue replacement and wound healing. In chronic wounds, normal signalling pathways are disrupted which prevents the normal progression of healing. The normal wound-healing process may be divided into four continuous phases, namely vascular response (haemostasis), inflammatory phase (inflammation), proliferative phase (proliferation) and maturation phase (maturation or remodelling). A schematic of the different phases of cutaneous wound healing is given in Figure 4.1 [1].
4.2.1 Haemostasis
Haemostasis is the process that causes bleeding to stop within the damaged blood vessel, which begins within seconds after tissue damage. Haemostasis has three major steps as shown in Figure 4.2 [2]. Upon injury, the blood vessels are disrupted and the blood cells move from the capillaries to the nearby tissues. The first step is the constriction of blood vessels to limit the amount of blood loss, called vascular spasm, which is more effective in small blood vessels. It is triggered via the chemicals released by endothelial cells and platelets, and as a reflex initiated by the pain receptors. The platelets present in the exposed blood are activated by the exposed collagen fibres and form a gel via the action of a prostaglandin called thromboxane A2; this gel contains platelets and forms a temporary plug, constricting blood vessels to stop bleeding. The temporary plug that forms also acts as a provisional ECM for cell migration. The clotting cascade finally forms fibrin, a mesh-like structure which acts as a framework to trap cellular elements of the blood and reinforces the platelet plug. The third step in this process of haemostasis is called the blood coagulation phase. The platelet plug secretes wound-healing mediators such as PDGF which activates macrophages and fibroblasts. In some trauma cases natural haemostasis is difficult to achieve, particularly for large injuries. Applying topical haemostatic agents and/or direct pressure to a bleeding wound may stop bleeding or slow down the bleeding process; some wounds require suturing to control bleeding. In surgical wounds surgeons use topical agents such as microfibrillar collagen to stop bleeding, which attracts the patient’s platelets and helps to initiate the clotting process. Heavy blood flow wounds, inflicted during combat operations, require pressure bandages to slow down the process of blood loss until medical attention is available. It is reported that about 50% of combat fatalities, and a significant amount of civilian trauma fatalities, are attributed to uncontrolled haemorrhaging. These fatalities may be prevented by prompt application of a tourniquet or any product that can achieve haemostasis. Several haemostatic agents are currently used in combat operations,
such as TraumaDEXTM particles, QuickClot® powder, dry fibrin sealant dressings, rapid deployment haemostats and HemCon® chitosan bandages.
4.2.2 Inflammation
Inflammation is considered to be the first of several overlapping processes that constitute the actual process of wound healing. The inflammatory process begins immediately following initial vasoconstriction with the release of prostaglandin and activated complement proteins, which causes widespread vasodilation and inflammation. Once haemostasis is achieved, the blood vessels start to dilate allowing essential cells such as white blood cells, growth factors, enzymes, nutrients and antibodies to reach the wounded area, which leads to a rise in exudate levels. This stage normally lasts up to four days. The body’s early defence system against microbial invasion initiates at this stage of the healing process. The infiltrating leukocytes are the principal cellular component of the inflammatory process. The polymorphonuclear leukocytes (polymorphonuclear neutrophils) entrapped and aggregated in the blood clot release a wide variety of growth factors and cytokine signals, and act as chemoattractants for cells involved in the inflammatory phase. Neutrophils begin the process of debridement of the tissue and phagocytosis of infectious agents. Excessive leukocyte infiltration can prolong the release of proteolytic enzymes, oxygen-free radicals and proinflammatory cytokines, which can lead to chronic wounds. However, the impairment of leukocyte recruitment is associated with delayed wound healing;
therefore, a dynamic balance between changes in systemic neutrophil availability and their recruitment to the wound is important in normal wound healing [3]. Neutrophil infiltration over the time course of wound healing is demonstrated in an animal model shown in Figure 4.3. Neutrophil influx, after a wound occurrence, increased most rapidly over the initial 12 h and reached a maximum value between days 1 and 2, the level plateaued up to day 3, and decreased rapidly at day 5. Neutrophil accumulation in the wound was 6-fold greater than that contained in the entire blood circulation volume on day 2. It returned to normal, upon wound closure, by day 9. Macrophages, the critical cells in wound healing, secrete an angiogenesis factor which stimulates the formation of new blood vessels. Leukocytes and macrophages serve as phagocytes that act to clear debris by ingesting it, and subsequently destroy the ingested material, and are the most important cells in the early phase of wound healing. They release collagenases and elastases, that breakdown injured tissue, and also release cytokines.
Platelet-derived growth factor is released by the macrophages which stimulates the chemotaxis and proliferation of fibroblasts and smooth muscle cells. Macrophages also secrete substances that attract endothelial cells to the wound and stimulate their proliferation to promote angiogenesis. T-lymphocytes that migrate into the wound secrete a heparin-binding epidermal growth factor and basic fibroblast growth factor, which interact with the processes of and promote wound healing.
Platelet aggregation and activation Platelets adhere to site of vascular injury
Neutrophil Red blood cell
Platelet Endothelial
cell
Basement
membrane Collagen and
ECM proteins
ADP Thromboxane A2
Thrombin Fibrin
Activation of coagulation cascade
Smooth muscle cell
Haemostatic plug formation
Figure 4.2 Haemostasis schematic. The process of coagulation depends on a complex interplay of enzymatic and cellular activity, culminating in the formation
of a stable vascular ‘plug’. ADP: Adenosine diphosphate and ECM: extracellular matrix. Reproduced with permission from J.W. Semple, J.E. Italiano, Jr., and
J. Freedman, Nature Reviews Immunology, 2011, 11, 4, 264.
©2011, Nature Publishing Group [2]
4.2.3 Proliferation
The proliferation phase occurs 3−5 days following injury which is primarily the formation of granulation tissue (actual tissue repair starts here) and may last up to 3 weeks. Granulation tissue consists of a combination of cellular elements, fibroblasts and inflammatory cells, along with new capillaries embedded in a loose extra cellular matrix of collagen, fibronectin and hyaluronic acid. The three stages of the proliferative phase can be termed as granulation, contraction and epithelialization. Angiogenesis induces the formation of new vessels; oxygenated blood reaches the wound bed and the wound becomes less hypoxic and nutrient deficient. The macrophages recruit a new cell type, i.e., the fibroblast, which lays down a network of collagen fibres surrounding the neovasculature of the wound. Fibroblasts proliferate and produce the matrix proteins fibronectin and hyaluronic acid (HA), and later collagen and proteoglycans.
Fibronectin plays a major role in cell adhesion, growth, migration and differentiation, and is important for wound healing. Fibroblasts secrete proteases, including matrix metalloproteinases, which digest the plasma fibronectin, and then the fibroblasts secrete cellular fibronectin and assemble it into an insoluble matrix. Fibronectin links the components of the ECM to one another and to the cells. Collagens synthesised by fibroblasts are the most abundant family of proteins in the body and provide strength and integrity to the tissue. Collagen synthesis is induced by the PDGF, basic fibroblast growth factor, TGF-β, interleukin (IL)-1 and tumour necrosis factor. Proteoglycans, such as heparansulfate, keratansulfate, chondroitin sulfate, hyaluronic acid and so on, help to regulate the structure and permeability of the ECM, and can modulate the
growth and differentiation of cells. Re-epithelialization occurs upon the migration of cells from the periphery of the wound to the centre and is initiated within one day of injury; the division of peripheral cells occurs between 48−72 h, resulting in a thin epithelial cell layer which bridges the wound. A moist environment is very important during this stage as it accelerates this process; the epithelial cells finally differentiate until they form a continuous layer of the epidermis.
c) a)
EGFP neutrophils in wound 0.0
0 4 8 12 18 24 1.0
2.0 3.0 4.0 5.0 6.0 7.0
Time post-wounding (h)
0 h 12 h 24 h Day 3
Day 9
6 5
x109
(x106 cells)
4 3 2 1 Day 6
Day 5 Day 4
Photon per second per cm2 per steradian b)
EGFP neutrophils in wound 0.0
0 1 2 3 4 5 6 7 8 9 1.0
2.0 3.0 4.0 5.0 6.0 7.0
Time post-wounding (day) (x106 cells)
Figure 4.3 Dynamics of neutrophil infiltration over the time course of wound healing. a) Time course of wound EGFP fluorescence during initial 24 h after wounding (n = 4). b) Time course of wound EGFP fluorescence during initial 10 days after wounding (n = 5). c) Representative fluorescent images of EGFP
neutrophil infiltration during the entire wound-healing process. Data were expressed as means ± standard error of mean (SEM). EGFP: Enhanced green
fluorescent protein. Reproduced with permission from M.H. Kim, W. Liu, D.L. Borjesson, F.R.E. Curry, L.S. Miller, A.L. Cheung, F.T. Liu, R.R. Isseroff and
S.I. Simon, Journal of Investigative Dermatology, 2008, 128, 7, 1812.
©2008, Nature Publishing Group [3]
4.2.4 Maturation
This final phase of wound healing starts from the 3rd week and can last up to a year or more. It is the process of remodelling the collagen fibres laid down during the proliferation phase. Blood vessels formed in the granulation tissue are not required during this phase of healing and are removed by apoptosis. Type III collagen, a soft gelatinous collagen laid down in the proliferation phase, is replaced with the more highly structured type I collagen. The tensile strength continues to increase up to 80% of normal tissue during this process. The differentiation of collagen is a dynamic process and although it takes place predominantly during the maturation phase it may continue to be remodelled indefinitely. The tissue is realigned along the lines of the stress and this process is mostly regulated by PDGF and TGF-β, and fibroblast growth factors. Maintaining a delicate balance between degradation and synthesis is required for normal wound healing which is controlled by regulatory mechanisms.
Hyaluronic acid and fibronectin are degraded as collagen bundles become organised.
Metalloproteinase activity decreases via the action of tissue inhibitors upon the accumulation of new tissue. Finally, a matured scar with a decreased number of cells and blood vessels, which has a high tensile strength, is formed. Since remodelling is a long process, closed wounds can quickly breakdown and reopen if the initial causes of the wound were not properly addressed; this can happen more often in the case of chronic ulcer wounds.
Coagulation Inflammation
Polymorphonuclear neutrophils predominant
Vasodilation Vasoconstriction
Epithelialization
Angiogenesis Fibroplasia and granulation tissue formation
Maturation and remodelling 50% of normal tissue strength
- incomplete basement membrane - complete basement membrane
Contraction
minutes hours days weeks months 1 years2
1 12 1
1
1 1
7 14 7 102
2 2
2 10 2030 60 24
48 3 2 3
3
3 45 456 12 456 3 456
Macrophages predominant
Figure 4.4 Timeline of different phases of normal wound healing. Limits vary within faded intervals, mainly by wound size and healing conditions. Reproduced
with permission from M. Häggström, Wikiversity Journal of Medicine, DOI:10.15347/wjm/2014.008. ©2014, Wikiversity [4]
The timeline of the major phases of normal wound healing is provided in Figure 4.4.
The normal wound-healing process is initiated within seconds of injury via haemostasis and coagulation resulting in fibrin deposition; this may last from 10 min to 1 h. Fibrin deposition activates inflammation, with neutrophils, macrophages and lymphocytes
seen predominantly within the tissue, and may last 1-4 days; this stage is followed by migration and proliferation of fibroblasts resulting in collagen deposition. The proliferation phase can last from day 4 up to day 21. Finally, the remodelling phase starts via the crosslinking of collagen and scar maturation, which can continue for up to 2 years. These ordered sequences of events are responsible for normal wound healing; however, if any part of this healing sequence is altered the pathological response leads to chronic wounds.