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On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.84 no.3 Rio de Janeiro July 2009
Ricardo José de MendonçaI; Joaquim Coutinho-NettoII
IOngoing Ph.D. graduate course, Department
of Biochemistry and Immunology, Faculdade de Medicina de Ribeirão Preto
Universidade de São Paulo (USP) Ribeirão Preto (SP),
IIAssociate Professor, Department of Biochemistry and Immunology, Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo (USP) Ribeirão Preto (SP), Brazil
Wound healing is a dynamic interactive process that involves a sequence of molecular and cellular events. Recent advances in cellular and molecular biology have greatly expanded our understanding of the biological process involved in wound repair and tissue regeneration. From plasma extravasation, with coagulation and platelet aggregation, to reepithelialization and remodeling of injured tissue, the organism acts by trying to restore functionality tissue. Thus, the present study encompasses several cellular aspects involved in the wound healing process, as well as the main drugs used in treating the pathology related to wound healing complications. Economic aspects are also addressed, mainly related to chronic wounds of diabetic feet.
Keywords: Angiogenesis inducing agents; Wound healing; Capillary permeability
The costs of treating pathologies related with deficit in the healing process increase the importance of studies that search for drugs and dressings capable of interacting with the damaged tissue to speed up the healing process. For example, the delay in healing process, such as in the case of diabetic foot ulcers, is a world problem, both financial and social. Data from the United States stated that 15.5% of the world population aged over 30 years are diabetic and of this total, about 15% develop ulcers that are difficult to heal throughout life, especially in the lower limbs. Thus, 6% of the hospital admissions related with diabetic patients is due to these ulcers and in case of amputation the mean length of stay is about three weeks. These cases generate extra cost of about US$8,000 to US$12,000/ patient to the healthcare system. About 39 to 68% of amputated patients die within 5 years 1. In Brazil, it is estimated that there are two million cases, among retirees and those supported by diabetic-healthcare insurance. 2
Wound healing is a complex process that involves the organization of cells, chemical signs and extracellular matrix to repair the tissue. In turn, the treatment of wounds tries to quickly close the damage to obtain a functionally and esthetically satisfactory scar. To that end, it is indispensable to have greater understanding of the biological process involved in the healing of wounds and tissue regeneration 3.
With tissue destruction in vertebrate animals, the process of repair soon starts, which comprises a sequence of molecular events directed to restoring the damaged tissue. Only during the fetal phase, the repair of lesions happens without formation of scars, which occurs through real restoration of tissue by the process of tissue neoformation. After birth, the organism fails to repair by tissue neoformation, leading to the formation of scars after the repair 4-5.
CLASSIFICATION OF HEALING BIOLOGICAL PROCESSES
The healing process has been conveniently divided into three phases that overlap in a continuous and temporal way: inflammatory phase, proliferative phase and remodeling phase 6.
1- Inflammatory phase
After the occurrence of a wound, a process of blood extravasation starts to fill in the damaged area with plasma and cell elements, especially platelets. Platelet aggregation and blood coagulation generate a buffer, rich in fibrin, which restores hemostasis and forms a barrier against the invasion of microorganisms, organizes a temporary matrix required for cell migration. This matrix will also serve as a cytokine and growth factors reservoir to be released during the next phases of the healing process 7-8.
Platelets, essential to the formation of a hemostatic buffer, also secrete multiple mediators, including growth factors, released in the damaged area. Platelets, induced by thrombin, suffer platelet degranulation and release many growth factors, such as platelet derived growth-factor (PDGF), transforming growth factor beta (TGF-β), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), and vascular endothelial growth factor (VEGF), in addition to adhesive glucoproteins such as fibronectin and thrombospondin, which are important ingredients of the transient extracellular matrix 9-10. In addition, the activation of a cascade of coagulation and the complement, together with the release of growth factors and activation of parenchymatous cells by the lesion, produce innumerous vasoactive factors and chemotactic factors that support the recruitment of inflammatory cells at the wound site 6.
After the emergence of platelets from the vascular bed, neutrophils and monocytes, as a response to chemotactic agents, migrate towards the wound bed 6. However, the absence of neutrophils in the blood does not seem to affect the repair process in the absence of infection 8,11-12. In graph 1 we show cell specificity during healing 13.
In addition to the phagocytosis function of bacteria, cell fragments and foreign bodies, these inflammatory cells produce growth factors that prepare the wound to the proliferative phase, when fibroblasts and endothelial cells are also recruited. 3 (Graph 1)
Monocytes of peripheral blood, both at first and after the healing process starts, continue to infiltrate at the wound site in response to chemotactic agents to monocytes, such as PDGF, for example. The release of factors coming from platelets, as well as phagocytosis of cell components, such as fibronectin or collagen, contribute for the activation of monocytes, transforming them into macrophages that are the main cell involved in the control of the repair process 3,6.
The activated macrophage is the main effector cell in the process of tissue repair, degrading and removing components from the damaged connective tissue, such as collagen, elastin and proteoglycans. In addition to having a role in phagocytosis of cell fragments, macrophages also secrete chemotactic factors that attract other inflammatory cells to the wound site and produce prostaglandins that work as powerful vasodilators affecting the permeability of microvessels 3,10-11. Macrophages produce many growth factors, such as PDGF, TGF-α, fibroblast growth factor (FGF) and VEGF, which are highlighted as the main cytokines necessary to stimulate the formation of granulation tissue 3.
2- Proliferative Phase
The proliferative phase is responsible for actually closing the lesion. The phase comprises: reepithelialization, which starts few hours after the lesion and includes the movement of epithelial cells coming from the margins and the epidermal appendices located in the lesion core; fibroplasia and angiogenesis, that comprise the so-called granulation tissue responsible for taking over the area of the damaged tissue about 4 days after the lesion. Fibroblasts produce a new extracellular matrix required for cell growth, whereas new blood vessels carry oxygen and nutrients required for local cellular metabolism 3.
Epithelial proliferation phase, in case of the skin, starts by mitogenic and chemotactic stimulation of keratinocytes by TGF-β and EGF. Epithelialization, which starts at the repair process phase, is as important as the formation of granulation tissue, named after the granular characteristic given by the presence of new neoformed capillaries essential for the repair process 14.
However, before describing angiogenesis, it is necessary to emphasize that the increased microvascular permeability is the first phase of this process, representing an important phase, which allows formation of temporary extracellular matrix, by leak of proteins, cytokines and cell elements, necessary to migration and proliferation of endothelial cells 15-16.
2.1- Vascular Permeability
The production of new blood vessels from preexisting vessels is most of the time followed by increase in vascular permeability 15-17. In pathological angiogenesis, the increase in vascular permeability to water and to macromolecules present important function in the process, which is directly responsible for the formation of edema. This increase in capillary permeability seems to have less effect during physiological angiogenesis, but it causes considerable damage in specific pathologies, such as diabetic retinopathy, for example 18.
Many cytokines produced during the healing process directly interfere in vascular permeability. VEGF-A, for example, discovered in tumor ascitic fluid, was originally described by its capacity to increase the permeability of microvessels and leak of macromolecules, including fibrinogen and other coagulation proteins. The extravasation of these proteins result in extravascular deposits of fibrin, favoring the healing process in tumoral development 15.
The basic mechanisms of regulation of vascular permeability, caused mainly by growth factors, have not been completely understood yet 15,19. However, some researchers proposed that endothelial cells would have contraction induced by permeability agents, forming intercellular gaps of sufficient size that would enable extravasation of plasma proteins 20-21. More recently the discovery of a venous endothelial structure, vesiculo-vacuolar organelle, provides an alternative trans-endothelial pathway for plasma protein extravasation in response to permeability factors 21-23.
Angiogenesis is an essential phase of the healing process, in which new blood vessels are formed from preexisting vessels 24. New vessels participate in the formation of temporary granulation tissue and supply nutrients and oxygen to the growing tissue 25. Vasculogenesis refers to the first stages of vascular development, through which precursor cells of vascular endothelium suffer differentiation, expansion and coalescence to form a network of primitive tubules in the organism 26.
In an adult organism, under normal conditions, angiogenesis occurs only in the reproductive cycle of females (in the uterus, with the formation of the endometrium, and in the ovaries, in the formation of corpora lutea). Therefore, the vascular network remains quiescent, but it has the capacity to start angiogenesis, especially during healing 25.
Angiogenesis, in response to tissue damage, is a dynamic process, fine regulated by signs present both in serum and in the local extracellular matrix 26. During the healing process, the formation of new blood vessels becomes necessary to form new granulation tissues, in which the blood vessel cells correspond to about 60% of the repair tissue 10. Angiogenesis occurs in the extracellular matrix of the wound bed with the migration and the mitogenic stimulation of endothelial cells.
Induction of angiogenesis was initially attributed to acid or base FGF. Subsequently, many other molecules were identified as angiogenic, including VEGF, TGF-ΰ, angiogenin, angiotropine and angiopoetin-1.27 Low tension of oxygen 28 and high levels of lactic acid and bioactive amines 29 can also stimulate angiogenesis. Many molecules mentioned above are proteins and seem to indirectly induce angiogenesis, stimulating the production of acid or basic FGF and VEGF by macrophages and endothelial cells, direct inductors of angiogenesis.
2.3. Extracellular Matrix
The migration of endothelial cells and development of new capillaries of tubular structure depend on the cells and cytokines present, in addition to the production and organization of extracellular matrix components, including fibronectin, collagen, vibronectin, tenascin and laminin, both in the granulation tissue and in the basal endothelial membrane. The extracellular matrix is important for the growth and normal maintenance of vessels, because in addition to acting as the support platform for cell migration, they also act as a reservoir and modulator of the release of growth factors, such as FGF2 and TGF-β.30
The proliferation of endothelial cells inside and round the wound leads to transient build-up of large amounts of fibronectin on the vessel walls 6. Thus, angiogenesis requires the expression of fibronectin receptors by the endothelial cells 31, organizing fibronectin as a channel that enables the movement of endothelial cells. Protease expression and activity are also necessary to angiogenesis, especially in the remodeling phase 6.
3- Remodeling Phase
In this phase of the healing process there is an attempt to recover the normal tissue structure. It is a phase marked by maturation of elements and affections to the extracellular matrix, leading to proteoglycan and collagen deposits. In a later phase, fibroblasts of granulation tissue are transformed into myofibroblasts and behave as contractile tissue that responds to agonists that stimulate the smooth muscles. At the same time, there is extracellular matrix reorganization, which transforms the transient matrix into a definitive one, whose phenotypic intensity, observed in scars, reflects the intensity of the phenomena that occurred and the level of balance between them. 32
As a result of maturation and remodeling processes, most vessels, fibroblasts and inflammatory cells disappear from the wound site through a process of migration, apoptosis or other unknown death cell mechanisms, which means a scar with fewer cells. Conversely, if there is persistent cellularity at the site, there will be formation of hypertrophic scars or keloids 10.
The main cytokines involved in this phase are tumor necrosis factor (TNF-α), interleukin (IL-1), PDGF and TGF-β produced by fibroblasts, in addition to those produced by epithelial cells such as EGF and TGF-b.33
Reepithelialization, which is wound recovery with new epithelium and consists in both migration and proliferation of keratinocytes from the lesion periphery, also occurs during the proliferative phase. These events are regulated by three main agents: growth factors, integrins and metalloproteases 34.
During the inflammatory phase, the release of growth factors, fibroblasts and macrophages/ neutrophils by the plasma activates keratinocytes located on the margins and inside the wound bed. The main growth factors are PDGF, which induces proliferation of fibroblasts leading to production of extracellular matrix during wound contraction and matrix reorganization; KGF7, which is considered the main regulating factor of keratinocyte proliferation, and TGF-β, the agent responsible for initial stimulus of the migration of epithelial cells. The activation of integrin receptors by keratinocytes enables the interaction of a variety of proteins of extracellular matrix on the margin and the wound bed. On the other hand, the expression and activation of metalloprotease promote the degradation and modification of proteins of extracellular matrix at the wound site, facilitating cell migration. The proteolytic activity of these enzymes may release growth factors linked to the extracellular matrix, so as to have a constant stimulus to the proliferation and migration of keratinocytes, speeding up the process of reepithelialization 34.
There are many diseases that negatively interfere in the process of tissue repair, such as diabetes, systemic sclerosis, anemia, malnourishment, among others. There are many conditions that make this process difficult to solve, preventing or delaying complete restoration of tissues. Among those conditions we may emphasize extensive resections of abdominal wall, such as those required in cases of peritoniostomy. Given that they somewhat hinder tissue repair, these diseases potentially contribute to the increase in morbidity and mortality 35-36.
In the past decades, many studies have been carried out to identify substances capable of favoring the repair process. The search for substances with angiogenic activity has been significant owing to their great potential for clinical application.
Among the substances that have direct action in the process of repair there are some growth factors that when applied topically to a wound demonstrate good capability to speed up tissue repair in animal models 37-39. In this group, we should highlight the action of REGRANEX¿, a product based on human recombinant PDGF that directly interferes and favors the repair process, presenting good results in the healing of diabetic patient ulcers 39-40. However, they are high cost dressings that are not affordable to most patients who have chronic ulcers. Another substance that contains enzymatic agents such as DNAse and collagenase ointments act by promoting wound debridement 41 and support the process of tissue repair in a smooth and indirect way. Those drugs are widely used in clinical practice but they have low efficacy in healing of chronic wounds.
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Ricardo José de Mendonça
Universidade de São Paulo - Faculdade de
Medicina de Ribeirão Preto - Departamento de
Bioquímica e Imunologia
Av. Bandeirantes, 3900 -
14040 900 Ribeirão Preto SP
Tel./fax: 16 3602-3321 16 3633-6840
Conflict of interest: None
Financial funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Capes e Fundação de Apoio ao Ensino, Pesquisa e Assistência Faepa.
How to cite this article: Mendonça RJ, Coutinho-Netto J. Aspectos celulares da cicatrização. An Bras Dermatol. 2009;84(3):257-62.