Epidermal Growth Factor:
With the rapid development of the economy, the great changes in people’s lifestyles and the accelerated aging of the population, problems caused by various kinds of skin wounds have multiplied during the past decades. The treatments for these wounds include surgical and non-surgical ones. As a non-surgical treatment, the application of exogenous growth factors (eGFs) is one of the indispensable methods to promote wound healing or to provide healthy wound beds for surgical treatments. eGFs have been used in many countries all over the world and the first report on the successful treatment of wounds with commercial eGFs was published as early as 30 years ago. To date, no obvious toxicity or severe adverse reactions have been reported in the treatment of wounds with eGFs [1]. Recombinant human epidermal growth factor (rhEGF) stimulates the proliferation and migration of epithelial cells in human cell culture systems and animal models of partial-thickness skin wounds. Investigation on the effect of a topical rhEGF ointment on the rate of wound healing and skin re-epithelialization in a rat full thickness wound model, and verified whether or not the rhEGF treatment affected both myofibroblast proliferation and collagen synthesis in the dermis. When rhEGF (10 microgram/g ointment) was applied topically twice a day for 14 days, there was significantly enhanced wound closure from the 5th to the 12th day compared with the control (ointment base treatment) group [2].
Fig.1. The degree of wound healing in the rhEGF (10 µg/g) treatment group and control (ointment base) group. Residual Wound Area (%) = [R(2~12)/R(1)]×100, where R(1) and R(2~12) represent the area remaining at postoperative days 1 and day 2~12, respectively. The wound-healing curve was fitted using the Boltzman equation and the half heal time (HT50). Each bar represents the mean ± SE. *p < 0.01 compared with the control (ointment base) group
A visible cutaneous scar develops from the excess formation of immature collagen in response to an inflammatory reaction. This study examined the role of epidermal growth factor (EGF) in the formation of cutaneous scars. Twenty Crl:CD-1 (ICR) mice were used and 2 full-thickness skin wounds were made on the dorsum of each mouse. One of the wounds was treated with recombinant human EGF by local application and the other was treated with saline for control until complete healing was achieved. The EGF-treated group’s wounds healed faster than the control group’s. The width of the scar was smaller by 30% and the area was smaller by 26% in the EGF-treated group. Inflammatory cell numbers were significantly lower in the EGF-treated group. The expression of transforming growth factor (TGF)-beta(1) in the EGF-treated group was increased. It was observed that the amount of collagen in the EGF-treated group was larger than the control group. In the EGF-treated group, the visible external scars were less noticeable than that in the control group. These results suggest that EGF can reduce cutaneous scars by suppressing inflammatory reactions, decreasing expression of TGF-beta(1), and mediating the formation of collagen [3].
Insulin Growth Factor (IGF):
Insulin-like growth factor-I (IGF-I) plays a crucial role in wound healing and tissue repair. IGF-I significantly improved maximum force and ultimate stress in tissues with significant increases in matrix organization and type-I collagen expression [4]. IGF-1 has also been described as an agent that acts anabolically and anti-catabolically in a wide variety of cell types. In the context of wound healing, the evidence suggests that IGF-1 is mitogenic for keratinocytes and that fibroblasts inhibit the process of apoptosis, thereby reducing the production of inflammatory cytokines and stimulating extracellular matrix production. IGF-1 expression is modulated during wound healing. Expression is increased in wound fluid caused by burns and is also significantly increased during the healing process of skin cells, which ordinarily express only small amounts of this protein. This supports the idea that successful healing is associated with IGF-1 levels. During granulation, tissue fibroblasts develop various structural and biochemical characteristics similar to ultra-smooth muscle cells, including the presence of microfilaments and the expression of α-smooth muscle actin. When the granulation tissue develops into a scar, the myofibroblasts containing α-smooth muscle actin disappear, probably as a result of apoptosis. The mechanisms leading to myofibroblast development have been investigated elsewhere. Recent studies have reported the mitogenic action of myofibroblast differentiation and proliferation by IGF-1. During the repair of normal tissues, the controlled activation of myofibroblasts contributes to the restoration of tissue integrity10. The most widely used marker for myofibroblast differentiation is α-smooth muscle actin. Under normal conditions, fibroblast cells exhibit little or no short actin and, in the extracellular matrix produced by an injury, fibroblasts are activated by cytokines released locally from resident and inflammatory cells migrating into the damaged tissue to synthesize extracellular matrix components [5].
Fig3) Effects of IGF1 on glucose disposal and cutaneous wound healing in control (Cont), diabetic (Diab) and hypercortisolemic (Dexa) animals
Vascular endothelial growth factor (VEGF):
Angiogenesis, the growth of new blood vessels from existing vessels, is an important aspect of the repair process. Restoration of blood flow to damaged tissues provides oxygen and nutrients required to support the growth and function of reparative cells. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenic growth factors in the skin, and the amount of VEGF present in a wound can significantly impact healing [6, 7].
Fig.4)Topical VEGF accelerates wound healing in db/db mice. Depicted are the gross appearance of wounds treated with either VEGF ( left wounds) or PBS vehicle ( right wounds). A: Wounds at day 5. B: Wounds at day 12 after wounding. The VEGF-treated wound ( left ) has completely resurfaced, while the wound treated with PBS ( right ) is only beginning to heal. C: This graph demonstrates the kinetics of closure in VEGF-treated wounds ( D ) shows early histological evidence of granulation tissue deposition; epithelialization of the wound bed is apparent. The VEGF-treated wound ( E ) has abundant granulation tissue completely covering the wound; the epithelial layer is multilayered and the wound is completely epithelialized[8].
Fibroblast growth factor (FGF) 2:
Fibroblast growth factor (FGF) 2, also called basic FGF, is a member of a large FGF family of structurally related proteins that bind heparin sulfate and modulate the growth, differentiation, migration and survival of a wide variety of cell types1. FGF2 strongly activates not only fibroblasts but also other mesoderm-derived cells, including vascular endothelial and smooth muscle cells, osteoblasts and chondrocytes. Administration of recombinant FGF2 to skin wounds accelerates acute and chronic wound healing. Therefore, topical recombinant FGF2 has been approved in Japan for the treatment of skin ulcer since 2001. Local FGF2 administration also has an anti-fibrotic effect for the wound to antagonize myofibroblast differentiation and dampen fibrosis which has been evidenced by decreased SMA2 and fibronection in the wound tissue. In addition, FGF2 is considered to accelerate reepithelization, which is especially prevalent in an epidermis-defect wound model. However, most in vitro studies have reported that FGF2 is less active in keratinocytes that are derived from primitive ectoderm, relative to mesoderm-derived cells [9].
Fig.5) FGF2 accelerated wound healing, epidermal hypertrophy and keratinocytes migration at wound edge. (a) Murine wounds were created on the dorsal skin using 6 mm punch, and FGF2-treated wounds were significantly smaller on day 4. (b) Days required for wound closure were significantly decreased by FGF2 treatment. (c,d) FGF2-treated wounds demonstrated a more thickened epidermis (double arrow) than vehicle-treated wounds. Scale bar: 40 μm. (e) At the wound edge, spindle-shaped keratinocytes (arrow heads) were observed in FGF2-treated wounds (higher magnification images at the corner). Scale bar: 20 μm. (f) Cells migrating in fibrin clots (arrow heads) were confirmed to be keratinocytes by immunofluorescent staining with anti-pan keratin antibody. Scale bar: 40 μm. (g) The number of migrating keratinocytes was significantly higher in FGF2-treated wounds than in control wounds. Bar graphs are presented with the mean values ± standard error. FGF2: fibroblast growth factor 2, HE: hematoxylin and eosin staining. **p < 0.01, Mann–Whitney U test.
Extracellular matrix proteins: Collagen, Fibronectin and Elastin
The extracellular matrix is a component of all mammalian tissues and provides a bioactive environment that controls the behavior of cells using chemical and mechanical signals. However, with the expansion of data from various basic and clinical studies, it became clear that the components of the extracellular matrix are the main components of the cellular microenvironment and are actively involved in wound healing, due to their ability to influence cell behavior (proliferation, adhesion, and migration). In addition, the extracellular matrix is involved in the regulation of cell differentiation and death through integrins, cytokines, and growth factors. Remodeling of the extracellular matrix occurs under the action of MMPs and growth factors. Remodeling, in turn, is involved in the regulation of cell differentiation processes (maintenance of stem cell niches, angiogenesis, bone remodeling, and wound healing)[10]. The most abundant protein in the skin is collagen. Collagen accounts for up to 30% of the total protein in the skin. It provides tensile strength to the skin and binds to elastic fibers, which provide tissues with the ability to recover from stretching. The natural form of collagen fiber provides the necessary mobility of the skin during stretching. However, in scar tissue, collagen fibers are straighter and thinner. Other extracellular matrix proteins (fibronectin, laminins, and matrix cell proteins) are involved as connectors or binding proteins. Fibrin, fibronectin, and fibronectin are key mediators of hemostasis and cell migration in wound healing. The most common proteoglycans are hyaluronan, decorin, versican, and dermatopontin. Proteoglycans are composed of large carbohydrates (usually glycosaminoglycans (GAGs)) attached to a protein. Anionic GAGs allow to bind water and other cations (for example, calcium ions)[11]. Different types of GAGs can bind to each other. Another function of GAGs is to fill extracellular space and lubricate. Fibronectin is the second most abundant extracellular matrix protein. This protein is a dimeric molecule containing binding domains for many other molecules of the extracellular matrix (for example: collagen, heparin sulfates, integrins). Laminin is a trimeric cross-linked glycoprotein commonly found in the basement membrane. Laminin facilitates interactions between skin cells and other components of the extracellular matrix (for example, between heparin sulfate and collagen). Laminin can be a natural inhibitor of TGF-β1, and it can reduce the fibrosis of scars. In addition, laminin binds collagen at the β1-integrin binding sites, which is necessary for normal fibrillogenes [12].
Multi peptides Miracle:
In the field of wound healing, using stem cells conditioned medium contains multiple growth factors, for different types of wounds has been reported. Applying stem cells in wounds promoted more effective re-epithelialization and angiogenesis by their proliferative effect on keratinocytes and blood vessels. Moreover, this effect of stem cells conditioned medium was found to be mediated by keratinocyte growth factor-1 (KGF-1) and platelet derived growth factor-BB (PDGF-BB). A study showed that bone marrow mesenchymal stem cell in artificial dermal substitutes promote wound healing. Using secretome-containing conditioned medium (CM) has several benefits compared to the use of stem cells, as CM can be manufactured, freeze-dried, packaged, and transported more easily and it does not need to match the donor. Thus, there is no possibility for rejection. Therefore, stem cell-derived CM has great potential as a pharmaceutical for regenerative medicine[13].
