**2. Reactive oxygen species production and physical response**

The production and scavenging of ROS may be initiated by adverse environmental factors. Research has shown that intra-cellular levels of ROS may rapidly rise and ROS may be generated by the activation of various oxidases and peroxidases in response to certain environmental changes [23]. ROS forms through energy transfer or through electron transfer reactions. ROS formation causes the formation of singlet oxygen, which results in sequential reduction to superoxide, H2O2 and hydroxyl radicals [24]. Mitochondria are a crucial source of ROS production in most cells. This ROS production contributes to mitochondrial stress and plays a critical role in redox signalling from the organelles [25]. Mitochondria have a 4-layer structure composed of the outer mitochondrial membrane, intermembrane space, inner mitochondrial membrane and matrix [26]. NADPH oxidase is an enzymatic source in the mitochondrial structure that generates ROS and plays a fundamental role in maintaining normal cell functions. Recent research has focussed on the influence of this enzyme to cellular oxidative stress that may contribute to various pathophysiological conditions and diseases [27, 28]. A crucial function of NADPH oxidase is modulating multiple redox-sensitive intra-cellular signalling pathways; NADPH modulates these pathways by generating ROS molecules, inhibiting protein tyrosine phosphatases and activating certain redox-sensitive transcription factors. Moreover, the ROS consist of numerous molecular species, including H2O2, oxide ions (O2) and hydroxide (OH− ) [29]. Molecular oxygen is a biradical, containing two unpaired electrons in the outer structure; because these two electrons have the same spin, oxygen can only react with one electron; therefore it is not very reactive when these two electrons have the same spin. Oxygen's unpaired electrons can become excited and can change the spin of one electron. This transforms oxygen into a powerful oxidant because the two electrons with opposing spins can rapidly react with other pairs of electrons [30]. Electrons can be contributed from NADH and FADH2 enzymes and can pass through the electron transport chain, generating superoxide (O2 − ) at complexes I and III. This generated superoxide can be reduced to H2O2 by superoxide dismutase and can be completely reduced into water by glutathione peroxidase, as presented in **Figure 1**.

species (ROS) and reactive nitrogen species (RNS) that can initiate changes in cell functions, including cell signalling pathways, transcription factor activation, mediator release and apoptosis. However, whether the ROS and RNS that are produced and released by neutrophils or macrophages are sufficient to diffuse through the extra-cellular matrix, enter epithelial cells and cross the cytoplasm is not clear [5–7]. Even the physiological roles of ROS and RNS in the cellular response are not clear [8–11]. The results obtained from experiments performed on the livers of tilapia showed that extra-cellular hydrogen peroxide (H2O2) attracted cell migration. These results suggested that ROS is a crucial factor in initiating the migration of

In the microenvironment of inflammation, the platelet-derived growth factor (PDGF), the tumour necrosis factors (TNF)-α and TNF-β, the hepatocyte growth factor, transforming growth factor (TGF)-β2, the epidermal growth factor (EGF) and the fibroblast growth factor all play an important role in physiological immune response. The interleukins (IL)-1, IL-6, IL-8, IL-10, and the interferon gamma (INF-γ) also detain key functions in the natural inflammatory response [12–16]. These factors hold a primordial function in fibroblast activation and regulation, also concerning reactive fibrosis that follows their continuing activation. Although these growth factors are also related to fibroblast migration and activation, particular research was recently focused on the PDGF family of growth factors and their relative receptors [17, 18]. Research has documented that PDGF exerts autocrine, mitogenic effects on keratinocytes to support epidermal proliferation and stabilisation of the epidermal junction during wound closure. In addition, it stimulates vessel maturation by recruiting and differentiating pericytes to the immature-endothelial channel [19–22]. According to these references, we investigate whether the produced ROS/RNS is related to the released factors and (if so) what type of

macrophages that trigger cascades of phagocytic activity.

168 Wound Healing - New insights into Ancient Challenges

relationship exists among ROS/RNS and these factors.

**2. Reactive oxygen species production and physical response**

The production and scavenging of ROS may be initiated by adverse environmental factors. Research has shown that intra-cellular levels of ROS may rapidly rise and ROS may be generated by the activation of various oxidases and peroxidases in response to certain environmental changes [23]. ROS forms through energy transfer or through electron transfer reactions. ROS formation causes the formation of singlet oxygen, which results in sequential reduction to superoxide, H2O2 and hydroxyl radicals [24]. Mitochondria are a crucial source of ROS production in most cells. This ROS production contributes to mitochondrial stress and plays a critical role in redox signalling from the organelles [25]. Mitochondria have a 4-layer structure composed of the outer mitochondrial membrane, intermembrane space, inner mitochondrial membrane and matrix [26]. NADPH oxidase is an enzymatic source in the mitochondrial structure that generates ROS and plays a fundamental role in maintaining normal cell functions. Recent research has focussed on the influence of this enzyme to cellular oxidative stress that may contribute to various pathophysiological conditions and diseases [27, 28]. A crucial function of NADPH oxidase is modulating multiple redox-sensitive intra-cellular signalling pathways; NADPH modulates these pathways by generating ROS molecules,

**Figure 1.** ROS are produced from the electron transport pathway to form superoxide (O2 − ) at complex I and complex III released into the matrix and reduced O2 to form H2O at complex IV. Following, the generated O2 − is transferred to the form of H2O2 by superoxide dismutase (SOD) and completely reduced to water (H2O) by glutathione peroxidase (GPX).

Research has shown that ROS consist of numerous molecular species, including H2O2, oxide ions (O2 − ) and OH−29. These molecular species act as signalling molecules in the migration of profibrogenic cells [31] and peripheral blood monocytes [23, 32]. One of the crucial physiological functions of ROS is the modulation of ion channels. Research has illustrated that ROS may act through Ca2+ as an intra-cellular second messenger involved in regulating diverse functions, such as fertilisation, electrical signalling, contraction, secretion, memory, gene transcription and cell death [33, 34]. Furthermore, studies have reported that H2O2 may affect cell energy stores [35], induce DNA strand breaks [36], enhance cell adhesion [37], increase endothelial tissue permeability [38] and stimulate the release of cytokines.

In the research presented in **Figure 2**, the concentration of ROS seems to be considered the concentration of a crucial signalling molecule. Low concentrations of generated ROS are believed to be critical for metabolic adaptation in the organelle. Moderate concentrations of ROS can be produced and released by stress; pathogen-infected and bacterial endotoxin lipopolysaccharide (LPS) are involved in the inflammatory response. The high concentration of ROS in the induced apoptosis/autophagy process can cause cell death [39] and initiate selfhealing [40].

**Figure 2.** Concentration of generated ROS may involve in the different physiological response. At the low concentration, the ROS regulate in the redox signalling, and at the moderate concentration of induced ROS which participated in the inflammation process. At the high level of ROS concentration increased and was to be involved in the cellular apoptosis.
