**3.1 Hemostasis and inflammation phase (begins soon after injury and continues for 3 days)**

Hemostasis and inflammation occur soon after tissue injury and constitute the first and the foundation phase of wound repair. It leads to the formation of a platelet plug to prevent blood loss, removal of dead tissues and to prevent infection, and employs the components of the coagulation, inflammatory, and immune cascade to accomplish these tasks (**Figure 1**) [31, 32]. Further, the fibrin matrix acts as a scaffold for the infiltrating cells that are essential for later phases of wound healing. The clot consists of collagen, fibrin molecules, fibronectin, platelets, and thrombin. The fibrin clot operates as a scaffold hence aiding the migration of neutrophils, monocytes, leukocytes, keratinocytes, fibroblasts, and endothelial cells, and simultaneously forms a matrix for concentrating cytokines and growth factors [31, 33, 34]. The soluble factors released by these initiate the inflammatory phase aimed at establishing an immune barrier against infections [10].

In response to complement system activation, the next step is the recruitment of neutrophils to the wound site [35]. Following the formation of the clot, a stress signal is transmitted with neutrophils being the first cells to respond. This causes nearby vessels to vasodilate and accumulation of inflammatory mediators to facilitate the sudden rise in cellular traffic. Neutrophils release proteolytic enzymes such as proteases, which clean up the wounded area by removing cellular debris and invading bacteria [18, 31]. Neutrophils lead to the creation of reactive oxygen species that in combination with chlorine lead to wound sterilization [31]. *In vitro* studies have indicated the possibility that neutrophils could change the cytokine expression and phenotype of macrophages, leading to an innate immune response during wound healing [36]. This phase is sometimes called as the "lag phase," where in the absence of tensile strength in the wound, the recruitment of the migrating cells and various factors must be managed for the healing process [33].

Approximately 2–3 days post-injury, the monocytes from the neighboring tissue migrate to the wound site and finally differentiate into macrophages, which are seemingly crucial for the healing cascade. They cause phagocytosis of cell debris, apoptotic cells, and pathogens and lead to the secretion of various soluble factors [37, 38]. Despite their importance, the role of neutrophils and macrophages has not been well elucidated in wound repair. Reports have suggested that deficiency of one of these cells can be compensated *via* redundancy in the inflammation phase [39], whereas when cells of both types are absent, wound repair still takes place, with a lesser scarring response [40]. Macrophages synthesize various enzymes such as collagenases, cytokines, and growth factors like epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), interleukins (ILs), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF), leading to the promotion of cell proliferation and extracellular matrix (ECM) synthesis (**Figure 1**) [31, 41]. The inflammatory phase essentially supplies growth factors and cytokines that keep the wound-healing process intact and are quite crucial in the later stages of wound repair [25, 42]. Past evidence suggests that the extent of scarring depends on the amount of inflammation. This is validated by reports that show that the hypothesis behind scarless healing of fetus wounds is the lack of intrauterine inflammation [43].

#### **3.2 Proliferative phase (Day 3–10)**

Proliferation is the second phase of wound healing, which takes place from 3 to 10 days post-injury. This stage is characterized by new tissue formation, cellular

migration, and proliferation (**Figure 1**). In this phase, the focus is on covering the injured surface, the synthesis of granulation tissue, and the reformation of the vascular network [33, 44, 45]. It indicates the start of angiogenesis and the synthesis of the ECM components. In the first step, keratinocytes migrate to the injured dermis and angiogenesis occurs. New capillaries sprout replacing the fibrin clot with granulation tissue, resulting in a new substrate for migration of keratinocytes during later stages of the healing (**Figure 1**). Capillaries help in nutrient supply during the granulation and tissue deposition phase, in absence of which a chronic wound will develop [31]. The keratinocytes proliferate and mature to restore the epithelial barrier. There is also secretion of cytokines such as ILs, TNF-α, enzymes like matrix metalloproteinase (MMPs), and growth factors like EGF, PDGF, TGF-β, and VEGF by the activated macrophages and de-granulated platelets (**Figure 1**) [46]. The concentrations of these factors vary and are dependent on the immunological state of the individual [42]. Excessive synthesis of granulation tissue and collagen causes scar formation [47]. Several pathways play an important role in restoring the normal anatomy, physiology, and functional status of the injured tissue with mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, TGF-β, and Wnt/β-catenin being the major ones [48].

Vascular system restoration is a complex cascade in the injured tissue involving various cellular and molecular events. The most important angiogenesis upregulators are VEGF and FGF [49]. In a study, VEGF application to diabetic animal wounds normalized the healing process [50]. Later in this phase of repair, fibroblasts differentiate into myofibroblasts upon stimulation by macrophages [51]. Myofibroblasts being contractile in nature bring the wound edges together over time. Further, in combination with myofibroblasts, they produce extracellular matrix and lead to collagen and scar formation [52]. Fibroblasts secrete IL-6 and keratinocyte growth factors (KGFs) that act as stimuli causing migration of neighboring keratinocytes to the injury site, leading to their proliferation and differentiation into the epidermis [31]. Keratinocytes present at the wound edges cause re-epithelialization of the wound [53, 54].

Proliferative phase ends with the formation of acute granulation tissue [31]. During this phase, the proliferating endothelial cells get activated by VEGF, leading to the formation of new capillaries. By this time, the remodeling phase has already been initiated. The fibrin-based wound matrix is replaced with scar tissue characterized by high-cellular density due to fibroblasts, granulocytes, and macrophages, as well as capillaries and collagen bundles, hence termed granulation tissue [25, 33, 44, 53, 55]. This tissue is highly vascular because angiogenesis is still incomplete. By the end of this phase, myofibroblasts differentiation reduces the population of maturing fibroblasts, which are further terminated *via* apoptosis [56].
