, JiaoYun Dong1 and Ming Tian1
(1)
Medical School, Rui Jin Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of China
Abstract
In the following paragraph we will discuss the differences of the cell biology in the repair process of wound and refractory wound surface . In the repair process of wound surface the cell biology in hemostasis phase, in inflammation phase, in proliferation, angiogenesis, fibroplasia and epithelialization phase and in contraction, maturation and remodeling phase in the normal organ or tissue such as skin after injury will be shown. The cell biology in the repair process of refractory wound surface, we mainly discuss the cell biology in refractory wound surface of the diabetes such as the effect of diabetes on the biological function of fibroblasts, M1/M2 macrophage imbalance in the repair process of refractory wound surface of diabetic, the effect of glycosylated extracellular matrix on fibroblasts and so on.
Keywords
The cell biologyRepair processWound surfaceRefractory wound surfaceDiabetesSkin repair after injury such as scald includes a complex programmed sequence of cellular and molecular progresses that involves hemostasis, inflammation, proliferation, and maturation, which include multiple cell populations, the extracellular matrix (ECM) and the action of soluble mediators such as cytokines (including growth factors). In this paragraph we mainly talk about the differences of cell biology in the repair process of wound and refractory wound surface .
1 The Cell Biology in Hemostasis Phase
The platelets that can release cytokines (including growth factors), chemokines, and hormones play a crucial role in clot formation during hemostasis after aggregation and attachment to exposed collagen surfaces and activated in the initial stage of injury. Cytokines (including growth factors) have emerged as important mediators in repair process. As we know, cytokine is released from various cells, which can bind to target cell surface receptors to stimulate a cell response by endocrine, paracrine, autocrine, or intracrine routes. Platelets elaborate a number of proinflammatory substances and growth factors such as platelet-derived growth factors (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and so on. PDGF is released from the alpha granules of platelets and is responsible for the stimulation of neutrophils and macrophages, and also a mitogen and chemotactic agent for fibroblasts and smooth muscle cells, which can stimulate angiogenesis, collagen synthesis, and collagenase. VEGF contributes to angiogenesis by stimulating the mitosis of endothelial cells. TGF-β promotes proliferation of fibroblasts, regulates its own production in an autocrine manner and produces proteoglycans, collagen, and fibrin. It also promotes accumulation of ECM and fibrosis. All these cytokines (including growth factors) act on surrounding cells and stimulate chemotaxis of neutrophils, monocytes, and fibroblasts to the area of injury. So chemokines released by platelet activation attract inflammatory cells to the area, leading to the next phase i.e. inflammatory phase in the repairing process in the adult body.
1.1 The Cell Biology in Inflammation Phase
Neutrophils, monocytes/macrophages and lymphocytes are the main cells in the inflammatory phase in the wound surfaces area.
Neutrophils cleanse the wound site of bacteria and necrotic matter and release the chemotaxis such as interleukin 8 (IL-8) that chemotactic the macrophages and other cells involved into the wound site. Chemokines (or chemotactic cytokines) are small heparin-binding proteins that direct the movement of circulating leukocytes to sites of inflammation or injury via interaction with specific membrane-bound receptors and, as such, contribute to the pathogenesis of a variety of diseases [1]. Depending on the spacing or presence of four conserved cysteine residues, chemokines are classified into CC, CXC, CX3C, and XC families. CXC chemokines primarily attract neutrophils and lymphocytes and are believed to orchestrate the early phases of wound healing [2].
On the other hand, neutrophils produce reactive oxygen species (ROS) and proteases and also function to debride devitalized tissue. These functions are required in a timely manner. Neutrophils are produced in the bone marrow from stem cells that proliferate and differentiate to mature neutrophils fully equipped with an armory of granules. Neutrophils are dormant in the blood circulation. Once trauma and infection occurred the neutrophils are activated and the first to arrive the wound site. Neutrophils can release the particles of granules not only to fight microorganisms but also to cause great tissue damage. In the burn wounds, neutrophil infiltrates in the skin tissue in 4 h after the injury and reached the peak level 24 h later. The number of neutrophils decreased after 48 h of injury. At sites of infection and trauma, endothelial cells capture bypassing neutrophils and guide them through the endothelial cell lining whereby the neutrophils are activated and tuned for the subsequent interaction with microbes.
Neutrophils are the predominant cell type in the first inflammation phase (48 h after injury) and begin to wane after 24–36 h by apoptosis in the time of circulating monocytes enter the wound and mature into tissue macrophages that play the very important role in the wound site. In the adult body, no macrophages, no wound repair.
Following the neutrophils the monocytes involve in the wound site and become the macrophages. Macrophages play a central role not only in the inflammatory phase but also in all stages of repairing. Their functional phenotype is dependent on the wound microenvironment. During the early and short inflammatory phase macrophages phagocytose debris and bacteria and produce and orchestrate inflammatory cytokines (including growth factors) such as Tumor Necrosis Factor (TNF), Interleukin-6 (IL-6), Interleukin -1 (IL-1) and basic fibroblast growth factor (bFGF) and so on. IL-1 stimulates inflammatory cell proliferation and promotes angiogenesis. TNF-α is secreted from macrophages and as a mitogen for fibroblasts. bFGF is a chemotactic and mitogenic factor for fibroblasts and endothelial cells and other mesenchymal cells and also is an important stimulus for angiogenesis, that facilitate the repairing process.
Then let’s talk about macrophages. Macrophages are known to produce collagenases and elastases, which remove the damage tissue by phagocytosis and make the wound clean.
Depending on the stimulus in vitro, activation of macrophages has been classified into two populations. The classical (M1) activation results in a highly pro-inflammatory macrophage phenotype, with microbicidal activity and pro-inflammatory cytokine production, and is mediated by like Toll-like receptor (TLR)-4 ligands and interferon-γ (IFN-γ). The alternative (M2) activation can reduce inflammatory reaction, promote tissue repair and humoral immunity, and is mediated by IL-4 and/or IL-13.
The phenotype of wound macrophages in this phase is probably the classically activated or the so-called M1 phenotype. During the proliferative phase, macrophages stimulate proliferation of connective, endothelial and epithelial tissue directly and indirectly. M2-type macrophages release some growth factors such as PDGF, acid fibroblast growth factor (αFGF) and bFGF, transforming growth factor α (TGFα), macrophage-derived growth factors. Especially fibroblasts, keratinocytes and endothelial cells are stimulated by macrophages during this phase to induce and complete ECM formation, reepithelialization and neovascularization. Subsequently, macrophages can change the composition of the ECM both during angiogenesis and in the remodeling phase by release of degrading enzymes and by synthesizing ECM molecules [3, 4].
Besides, M1 and M2 promote T help 1 cells (Th1) that play the main role in cellular immunity and T help 2 cells (Th2) that play the main role in humoral immunity responses, respectively. Products of Th1 such as interleukin-2 (lL-2), interferon (IFN) and Th2 such as interleukin-10 (IL-10), interleukin-4 (IL-4) responses also down regulate M1 and M2 activity, respectively. The balance of the products of Th1 and Th2, is the balance of cellular immunity and humoral immunity. Thus, M1/M2 also demonstrated the importance of Innate Immunity [5, 6]. This suggests an important role for alternatively (M2) activated macrophages in this phase of wound healing.
Recent studies have been showed another factor, autophage, may play role of the cell biology in the repair process , because autophage has a lot of functions that influence infection, inflammation and immunity. Autophage is induced by pattern recognition receptors and, through autophage adaptors, provides a mechanism for the elimination of intracellular microorganisms. Autophage regulates inflammation through controlling interactions with innate immune signaling pathways, by removing endogenous inflammasome agonists and through effects on the secretion of immune mediators. At the same time, autophage participate in antigen presentation and to T cell homeostasis, and it can affect T cell polarization and repertoires [7].
During the inflammatory phase, lymphocytes migrate into the wound area approximately 72 h post injury. Lymphocytes produce lymphokines such as bFGF, heparin-binding epidermal growth factor (EGF) and so on. T lymphocytes arrive to wound through IL-1 induced, which also contributes to the regulation of collagenase. Therefore, lymphocytes also play an important role in antibody production and cellular immunity. As mononuclear cells, T lymphocytes continue to replace macrophages and other inflammatory cells, their proliferation phase begins. They take wound repair into the end of inflammatory phase, the evolving milieu of eicosanoids in the wound interact with the cell types present, resulting in fibroblast synthesis of collagen and other substance. Additionally, the macrophage-derived growth factors are now at optimal levels, strongly influencing the influx of fibroblasts and then endothelial cells and keratinocytes into the wound.
2 The Cell Biology in Proliferation, Angiogenesis, Fibroplasia and Epithelialization Phase
Angiogenesis, fibroplasia and epithelialization occur during the proliferation phase. Formation of granulation tissue is a central event during the proliferation phase. Its formation occurs 3–5 days following injury and overlaps with the preceding inflammatory phase. A rich blood supply is vital to sustain newly formed granulation tissue. The macrophage is essential to the stimulation of angiogenesis and produces macrophage-derived angiogenic factor in response to low tissue oxygenation. This factor functions as a chemoattractant for endothelial cells. Besides, the macrophages secrete bFGF and vascular endothelial growth factor (VEGF), which are also important to angiogenesis. Endothelial expansion contributes to angiogenesis, as intact vessels generate buds in granulation tissue. Neovascularization facilitates growth of the advancing line of fibroblasts into the wound, providing them with necessary nutrients and cytokines. The fibroblasts is a critical component of granulation tissue, which grow in the wound as the number of inflammation cells decrease. Two to three days after injury, the fibroblasts migrate inward from wound margins over the fibrinous matrix, which has been established during the inflammatory phase. During the first week, fibroblasts begin to migrate, proliferate and produce glycosaminoglycans and proteoglycans, the ground substance for granulation tissue, as well as collagen, in response to macrophage-synthesized growth factors such as PDGF, FGF, VEGF, TGF-α, TGF-β and etc. Type III collagen is the primary component of early granulation tissue. Fibroblasts soon become the dominant cell type, peaking at 1–2 weeks. The synthesis and deposition of collagen is a critical event in the proliferation phase and to wound healing in general. They generate not only collagen molecules but also growth factors such as PDGF, TGF-ß, bFGF, insulinlike growth factor-1(IGF-1), keratinocyte growth factor (KGF) and so on. Angiogenesis results in greater blood flow to the wound and, consequently, increased perfusion of repairing factors. Degradation of the fibrin clot and provisional matrix is accompanied by the deposition of granulation tissue (ground substance, collagen, capillaries), which continues until the wound is covered. Angiogenesis ceases as the demand for new blood vessels ceases. New blood vessels that become unnecessary disappear by apoptosis.
Fibroplasia starts on 3–5 days following injury and may sustain as long as 14 days. Fibroblasts produce the collagen, fibronectin, glycosaminoglycans, and other components of ECM. Fibroblasts are able to assemble cross-linked and fascicular fibers using collagen molecules. This synthesis work would last about 2–4 weeks. In normal skin there is approximately 80 % of the collagen identified type I collagen; the remaining is mostly type III. Collagen is the major component of acute wound connective tissue, it will continue to produce in the next 6 weeks. The accumulation of wound collagen is related to the increase of tensile strength. Collagen is rich in hydroxyproline and hydroxylysine moieties, which promote to form a strong cross-link structure. The hydroxylation of lysine and proline residues depends on the presence of oxygen, vitamin C, ferrous iron, and α-ketoglutarate. Particularly, deficiencies of vitamin C and oxygen result in under-hydroxylated collagen that is less capable of forming strong cross-links and is easier to breakdown. The formation of collagen is carried out on extracellular. First cells secret procollagen. Then procollagen is cleaved of its terminal segments and called tropocollagen. Collagen filaments can be formed through aggregate of tropocollagen molecules. Moreover, the cross-linked structure of intermolecular makes collagen fiber stabilize and resistant to destruction. Collagen fibers are deposited in a framework of fibronectin, which is closely connect with fibronectin In addition, fibronectin can paly a role of an anchor to make the myofibroblast migrate into the wound. At the moment, granulation tissue is gradually formed, and the wound begins to contract.
Epithelialization is the formation of epithelium that involves cell migration and covering the wound area. Firstly, epidermal cells at the wound edges, under their structural changes, detach from their basement membrane. Cellular movement relies on the establishment of physical forces by means of protrusive forces that lead to membrane extensions and traction forces allowing the cell to contract and slide forward [8]. The polar change of actin cytoskeleton intracellular causes the cell generate these deformations. Protrusions rely on polymerization and depolymerization of actin filaments while the traction is generated by myosin-based motors which pull actin filaments past one another. In a word, cell movement is based on the direction of polarity of the cells.
The initial step of cell polarization is that intracellular actin polymerizes to form ruffles or leading pseudopodia. The Rho family small guanosine triphosphate (GTP)-binding proteins (GTPases) are pivotal regulators of actin organization and control the formation of lamellipodia and filopodia. At the sites where contact with the extra cellular matrix (ECM) occurs, big protein complexes are assembled through the recruitment and the clustering of receptors of the integrin families. These large protein molecule structures are known as focal adhesions or focal contacts. There are known two types of migration mode: “Integrin/MMP dependent mode” and “Integrin/MMP-independent mode”.
“The dependent mode of cell migration” is called “mesenchymal”. Surface proteases, such as MT1-MMP, break down pericellular matrix molecules locally to provide sufficient space of cell expanding. Shortly after integrin binding with ECM, contractile proteins connect with cytoplasmic actin filaments, such as myosin II, which can stabilize and shorten the membrane-tethered actin filaments. This results in local cell contraction, generally at the opposite pole respect to the leading edge. Another mode is called “ameboid”. Cells also can migrate across connective tissue within pre-existing ECM pores by simply squeezing.
The formation of Intracellular actin microfilaments makes the epidermal cells crawl across the wound surface. Epidermal cells can secrete collagenases and plasminogen activator, collagenases break down collagen, plasminogen activator stimulates the production of plasmin, which promotes clot dissolution along the pathway of epithelial cell migration. Migrating epithelial cells interact with a provisional matrix of fibrin cross-linked to fibronectin and collagen. In particular, fibronectin seems to promote keratinocyte adhesion to guide these cells across the wound base. This epithelial layer provides a seal between the underlying wound and the environment. Besides, as the cells migrate, they dissect the wound and separate the overlying eschar from the underlying viable tissue. The stem cells are found in the deep rete ridges, leading them to propose that this site may provide protection for the long-lived stem cell population from harmful environmental mutagens. The sebaceous glands and hair follicles contribute to reepithelialization.
When epithelialization is complete, the epidermal cell restores its original morphology, and forms new desmosomal linkaging to other epidermal cells, and hemidesmosomal linkages to the basement membrane are restored. At the same time, epithelial cells continue to migrate inward from the wound edge until the defect is covered. The transformation of fibroblasts into myofibroblasts which contain contractile actin fibers, is contact inhibition induced. Then the new tissue replaces injured tissue volume.
2.1 The Cell Biology in Contraction, Maturation and Remodeling Phase
Contraction, defined as the centripetal movement of wound edges that facilitates closure of a wound defect, is maximal 5–15 days after injury. The result of contraction is decreased wound size which depends on the degree of tissue laxity and shape of the wound. The process of wound contraction is usually accompanied by collagen synthesis. During this phase, collagen remodeling depends on continued collagen synthesis in the presence of collagen destruction. For the first 6 weeks, new collagen production dominates the wound healing process, deposited randomly in acute wound granulation tissue. As the wound matures, collagen is remodeled into a more organized structure with increased tensile strength. With collagen synthesis, matrix metalloproteinase collagenolysis achieves a steady state.
Collagen forms tight cross-links to other collagen and with protein molecules, increasing the tensile strength of the healing wound. Stress, age, pressure and tension affect the rate of collagen synthesis. Loose tissues contract more than tissues with poor laxity, and square wounds tend to contract more than circular wounds. Wound contraction does not seem to depend on collagen synthesis but depends on the myofibroblast located at the edge of the wound, its connection to myofibroblast proliferation and components of the ECM.
In remodeling phase, collagen becomes organized increasingly. During this phase, a balance exists between formation of new collagen and removal of old collagen depending on collagenases and matrix metalloproteinases in the wound to assist removal of excess collagen while synthesis of new collagen persists. Fibronectin gradually disappears, and proteoglycans instead of hyaluronic acid and glycosaminoglycans. Gradually, type I collagen replaces type III until the normal skin ratio of 4:1 is achieved. The cross-links of Intramolecular and intermolecular collagen result in increased wound bursting strength. Remodeling begins approximately 21 days after injury, when the net collagen content of the wound is stable. Remodeling may continue indefinitely. Bursting strength varies with skin thickness. The tensile strength of the wound reached its peak at about 60 days after injury.
2.2 The Biology of Stem Cell in the Repair Process of Wound
Stem cell research has become one of the hot points in the repair process of wound because the stem cells have the characteristic of self-renewing, differentiated into multiple types of total specialized cells of the body [9]. But it still exist many problems because we still don’t know how many stem cells which include many types still existing in our organism when we leave the uterus. Such as what kind of damage and microenvironment can “home” the stem cells and induce them to differentiate into the appropriate cells to remodel damaged tissue. Nevertheless, the stem cell research has helped our mankind to understand how single cell can grow and develop into tissue and organ, and how the damaged cells can be replaced by healthy cells in adult body, which guides us to explore the cytological pathway to treat disease.
Kucia et al. have found and identified a population of stem cells in the BM, they are small (about 2–4 µm), but have large nuclei surrounded by a narrow rim of cytoplasm, and contain open-type chromatin (euchromatin), express several markers such as SSEA-1, Oct-4, Nanog and Rex-1, they are typical embryonic stem cells (ES). These cells can differentiate into all three germ-layer lineages in vitro. So they are also called very small embryonic-like (VSEL) stem cells. These cells have the characteristics of age-dependence. With the increase of age, the number of them is gradually reduced. They are barely detectable in 1 year old mice which correspond to a 50 year old human. This feature may be one of the reasons why the regeneration of young individuals is more effective than aged. It has been provided that as the organ is damaged, non-hematopoietic stem cells (including VSELs) are enter the peripheral blood circulation from the BM to “home” to the damaged tissues and participate in tissue repair. These cells may efficiently differentiate and regenerate into special tissue cells to replace the damaged cells in injuries. During this time damaged tissues up-regulate the expression of several chemotactic factors, which may participate in the homing and inducing differentiation of VSELs. But, if these cells migrate to the wrong place or/and migrate at the wrong time, they may lead to the formation of pathological diseases, such as tumor formation.
Adult stem cells are present in tissues and organs of the body, which have the potential to self-renew and differentiate into various types of cells. The process of differentiation is regulated by multiple genes. So that they can develop into specific structures and perform special biological functions. Some local adult stem cells are differentiated to supply new cells that effectively replace senescent ones or those undergoing apoptosis such as epidermal stem cells differentiated and developed into several layers of epidermis in maintaining normal metabolism condition of our skin [10]. In some injuries such as second-degree burns, some adult stem cells that are located in the wound or wound edge can be rapidly differentiated, proliferated and migrated with still healthy terminated cells to replace the damaged tissue cells, even in the microenvironment of chemoattractants that may express and secrete by damaged tissue cells or/and healthy cells, and finally restore the damaged tissue to the normal condition. Because there are no healthy cells, structures and even adult stem cells in site used to wound repair, such as the local area of severe burns or three-degree burns, bone marrow stem cells as precursor cells of other stem cells, play an important role in the reconstruction of various kinds of trauma. For example: many fibroblasts are derived from the blood delivered cells harvested from BM in the early stage of granulation formation of the repairing process in the local severe wound surfaces area.
Now the hypothesis of “Stem cell Niche” has become the hot point to stem cell research. Some scholars believe that “Niche” may be a habitat, such as the limbal SC niche, in which SCs could remain stable in this environment or microenvironment that not to differentiation. Adult SCs are regulated by microenvironment of their “niche”, i.e., the adult-specific SCs in “niche” are maintained in undifferentiated state, and their biological functions, consisting of other cellular and extracellular components have been adjusted accordingly in the vicinity of the area. So, the differentiation of stem cells may be synergistically regulated by various factors of micro-environmental, such as gene expression, cell-cell contacts, cell-matrix interactions and etc. The existence of niche and surrounding cells may guard stem cells to have a stable reserve force, by avoiding stimulation of differentiation and apoptosis. Stem cells, in the niche, not only have the ability of self-renewal, but also when the niche or the surrounding environment is stimulated from outside, they are able to differentiate. At that time the niche would modify to ensure that SC activity parallels the organism’s needs for particular differentiated cell types. However, if the adult stem cells are differentiated or/and differentiated cells migrate and proliferate into the inappropriate region, such as defection or without of “dermal template” or niche, it may result in “abnormal repair”, e.g. scar formation. But there are still a lot of issues that need to be explored, such as the multiple signal-pathways relative to stem cell differentiation, and the corresponding microenvironment of niche and surrounding that is suit for stem cell biological changes.
In view of the characteristics of bone marrow stromal cells (BMSCs), which are easily harvested from bone marrow, easy to culture in vitro and could be re-introduced into patients as autografts without serious ethical and technical problems, many researchers have used them as the ideal seed cells to transplant onto various medias such as denuded human amniotic membrane (AM). It has been used in many surgical procedures, such as skin equivalent and vaginal reconstructive surgery, and also for human embryonic SC differentiation into neural cells as well as for supporting chondrocyte proliferation and phenotype maintenance in vitro and the regeneration of osteochondral defect in rabbits [9]. Some researchers have confirmed that the amnion-derived cellular cytokine solution can promote the migration of macrophages during wound repair [11]. So the bio-scaffold with appropriate three-dimensional structure e.g. niche or “dermal template” and their components such as collagen of ECM may have a special influence on SC differentiation or “homing” SC and then assisting them to differentiate to form a functional tissue or organ. And the problem we are facing now is that due to the structure and content of bio-scaffold is always suffered a certain degree of damage in the process of production, we can’t create an ideal bio-artificial scaffold that is completely consisted with natural structure and environment.
Above all shows the cell biology in normal organ or tissue such as skin after injury. But many common chronic wounds such as diabetic foot ulcer, pressure ulcer, venous stasis ulcer and etc. Considers specific type of nonhealing wounds such as pressure ulcer, leg ulcers, diabetic foot wounds, surgical and malignant wounds as well as lymphoedema and dermatological conditions associated with skin breakdown [12]. Here we mainly discuses the diabetes and cell biology in refractory wound surface .
3 Diabetes Has Multiple Effects on Cell Biology in Refractory Wound Surface
Chronic wounds include vasculitis, non healing ulcer, pyoderma gangrenosum, and disease that cause ischemia. There are so many physiologic factors which contribute to wound healing deficiencies in individuals, such as decreased, hyperglycemia or impaired growth factor production, cytokine receptor, angiogenic response, macrophage function, collagen accumulation, epidermal barrier function, quantity of granulation tissue, keratinocyte and fibroblast migration and proliferation, number of epidermal nerves and balance between the accumulation of ECM components and their remodeling by MMPs [13–18].
Diabetic patients with spontaneous rupture of skin, such as diabetic lower extremity ulcers, wound or trauma is difficult to heal, all is a hotspot and difficulty in clinical research.
In recent years, the research on the mechanism of diabetic wound healing focus on harmful substances deposited, such as advanced glycation end products (AGE), high glucose deposition, cell signal transduction, cell apoptosis and cell function, blood vessels and extracellular matrix etc. Histological observation also showed that the thickness of the epidermis and the dermis of skin tissue of diabetic rats was significantly thinner, the epidermal cell layer is not clear, partial epidermis lack of multiple layer arrangement, significant reduction in the number of heckle cells, dermal collagen arrangement disordered, partial collagen degeneration, fracture, focal infiltration of chronic inflammatory cells was seen in the area of collagen degeneration. This shows that the skin tissue of diabetic patients in the absence of injury has been the existence of the changes in histology and cell biology behavior; this is a kind of “Underling Disorder”, that does not result in the integrity and continuity damage of the skin. This damage is endogenous, although it does not cause skin defects or damage to the visibility of the damage, but because of the change of histology and cell function, can make the skin tissue to increase the vulnerability of exogenous damage [19].
3.1 Neutrophils
The normal healing process can be defined by a number of overlapping events: clot formation, inflammation, reepithelialization, angiogenesis, granulation tissue formation, wound contracture, scar formation, and tissue remodeling. Diabetic wounds are characterized by functional defects in the majority of these events, leading to impaired wound healing, in addition to local ischemia caused by well-recognized macro- and microvascular occlusive disease. Usually, impaired wound healing in diabetic patients is accompanied by decreased early inflammatory cell infiltration but persistence of neutrophils and macrophages in the chronic, impaired wounds [20].
3.1.1 The Biological Characteristics of Neutrophils in the Diabetic Impaired Wound Healing
A series of function changes of neutrophil will occur in diabetic state. Insulin levels in patients with diabetes have a certain effect on the function of neutrophils. In the research [21] of 8 healthy volunteers showed that after treated with insulin, neutrophil chemotaxis, phagocytic ability and sterilizing ability were improved, and the control group showed significant differences. At the same time, Okonchi’s [22] research found, high concentrations of insulin can promote neutrophil transmembrane swimming and the expression of the platelet endothelial cell adhesion molecule-1 increased. When used with Gliclazide drug, can inhibit the abnormal function of neutrophils. From the above research, it can be speculated that the level of insulin in the body can directly affect the function of neutrophils. Then, the secretion of insulin in patients with type 1 diabetes is insufficient. This is one of the reasons that diabetes patients are susceptible to infection and impaired wound healing. Although there is no reduction in insulin secretion in patients with type 2 diabetes, neutrophil receptor glycosylation also affects the binding of neutrophils to insulin, which affect neutrophil function.
Tennenberg et al. [23] found that neutrophils from patients with diabetes are prone to apoptosis, the authors believe that this may be related with hyperglycemia. This would cause decreased functional longevity of neutrophils and increased neutrophil clearance from infectious sites, possibly contributing to the increased susceptibility and severity of infections in diabetic patients.
Long term hyperglycemia may lead to the production of a large number of advanced glycation end products (AGEs) in the body. Study of Collison et al. [24] found that AGEs could be high affinity with the human neutrophil AGER (AGE Receptor) and lead to increase in intracellular calcium and actin polymerization, which will depress the transendothelial cell migration and sterilization ability of neutrophil. Tian et al.’s research found [25, 26] that neutrophils couldn’t reach the basal part of the wound in time and form a dense inflammatory zone. A large number of neutrophils were scattered around the wound. Immunohistochemistry showed that AGE was distributed in the skin tissue of diabetic rats. Neutrophil migration test is shown in vitro that AGE can inhibit the migration of the neutrophil by binding its receptor on the surface of the neutrophil. At the same time, neutrophil and AGE combined outside the vascular tissue leads to a large number of inflammatory cytokines are released and oxidative stress burst by neutrophils. This release and burst are delayed and lasts longer than the normal wound.
Some studies have also similar views, Osar et al. [27] confirmed that the neutrophil oxidative burst index decreased significantly compared with the control group (p < 0.05), coenzyme I (NADPH) activity decreased by studying 30 type 2 diabetes patients. Gustke et al. [28] found that the average neutrophil chemotaxis index was significantly lower than that of the control group (p < 0.02) in type 1 diabetic patients. This changed cell function of neutrophil were dependent on HLA-DR3, DR4, and DR5 genes.
Wound healing involves many complex, interrelated processes that involve multiple cell types. Neutrophil plays an important role in the normal healing process, but abnormal neutrophil function may contribute to the pathogenesis of nonhealing wounds present in diabetic patients. A better understanding of the molecular mechanisms and cellular interactions of neutrophil in diabetic patients, is critical for the development of novel therapeutic strategies to promote diabetic wound healing.
3.2 Macrophage
Abnormal macrophage function in process of wound healing may not be conducive to the normal development of wound and lead to adverse results, such as the formation of ulcers, chronic wounds, hypertrophic scars and keloids. During the process of impaired wound healing, macrophage activation phase and degree were abnormal, wound repair process cannot be in accordance with the conversion from severe inflammation to mild inflammation state. Compared with the normal repair of acute wound, impaired wound is usually stuck in inflammatory phase and it was found that there was an in situ retention of macrophages.
3.2.1 M1/M2 Macrophage Imbalance in the Repair Process of Refractory Wound Surface of Diabetic
Impaired wounds such as diabetic wounds and chronic venous ulcer were found abnormal inflammatory retention and reduced granulation tissue state [29]. The first evidence is the number of aaM in wound area more than caM in diabetic wound healing model using db/db mice. Miao et al. [30] found a decrease in iNOS level, which is marker of caM(M1), on days 1 and 3 after wounding in STZ-induced diabetic rat lesion, especially on day 3, compared with the normal rats. The expression of Arg-1, which is marker of aaM(M2), in the diabetic group was lower than in normal group on day 7, but increased sharply and significantly higher on day 13. The study showed that the M1 in the non diabetic SD rats mainly appeared in the inflammatory phase and gradually replaced by the M2 in the repair of the proliferative phase. The infiltration of macrophages (CD68+) in the scald wound of STZ-induced diabetic rats was “slow in and slow out” which insufficient at early stage, and detained at late stage. Compared with normal rats, the expression of iNOS in the early stage of diabetic rats was decreased, Arg-1 was increased in expression, IL-4, IL-10 and other anti-inflammatory factors were relatively higher, indicating that Th1/Th2-M1/M2-iNOS/Arg-1 adjustment mechanism of normal healing was inclined to the side of Th2-M2-Arg-1 in diabetic wound, that is, changes in performance for insufficiently pro-inflammatory at early stage, at late stage the pro-inflammatory and anti-inflammation disordered, and with anti-inflammatory as the main.
So the balance of caM/aaM is very important in the process of wound healing. It is found Th2-aaM-Arg-1 increased in streptozotocin-induced diabetic wounds. But, abnormal caM may also lead to bad results. Unrestrained proinflammatory caM induced by iron and too many TNF-a positive macrophages, which are considered as caM cells, impairs wound healing in humans and mice [31]. An imbalance of caM/aaM in wound healing may delay and even hinder skin defect restoration. It appears that successful healing requires the activation of macrophages at an appropriate phase and a suitable extent.
There is large number of accumulated AGEs founded in diabetes mellitus, and AGEs might induce macrophages to product TNF-α to influence wound healing. Goren et al. [32] found that in the dorsum of ob/ob mice full thickness concise was observed in the number of abnormal TNF-a positive caM, and at the late stages of inflammation (post injury 7, 9, 11 days) in removal wound caM secreting TNF-a, thus launched a fast impaired wound epithelialization process. Dong et al. [33] also found that activating α7 nicotinic acetylcholine receptor (α7nAChR) can promote diabetic wound healing by suppressing AGE-induced TNF-α production, which may be closely associated with the blockage of NF-κB activation in macrophages. Suggesting that, there are inflammatory disorders during the process of some impaired wound healing such as diabetes, in this environment due to anomaly time phase of activated macrophages, macrophages cannot successfully complete from the early inflammation M1 based active state to late inflammatory M2 based activation state transition. Trem2 is a cell surface receptor that is specifically induced in macrophages by IL-4/IL-13 and is important in injury responses. Wound healing in Trem2−/− mice showed an increased expression of caM markers, decline aaM markers. This wound also demonstrated diminished burst of epithelial proliferation and wound closure rate [34].
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