## 2 Hyaluronan Role in Wound Healing
_Phase II: inflammation:_ At the molecular level, HMW-HA is metabolized from platelets, producing LMW-HA fragments (-2.0 \(\times\) 10\({}^{4}\)) that initiate inflammation. LMW-HA binds to TLR-2 and TLR-4 and promotes activation, infiltration, and maturation of immune cells, including neutrophils and monocytes, which further trigger pro-inflammatory cascades [33]. HA stimulated the function of neutrophils both in vitro and in vivo. After subcutaneous administration of HA to healthy volunteers, an enhanced rate of phagocytosis was observed in the neutrophils. HA injections to patients with increased susceptibility to bacterial infections and impaired neutrophil function demonstrated an improved function [34]. VEGF is one of the most potent proangiogenic growth factors in the skin and promotes repair in cutaneous wounds [35]. HMW-HA (1.5 \(\times\) 10\({}^{6}\) g/mol) enhances intracellular ROS production and apoptosis in neutrophils (the most abundant immune cells) in the presence of inflammatory stimuli such as TNF-\(\alpha\). Thus, HA favors a fast neutrophil response and finishes inflammation [36]. Macrophages are classified into (M1) or classically activated, which is pro-inflammatory, and M2 (\"alternatively activated\"), which is anti-inflammatory and pro-healing. Macrophages participate in host defense by the production of cytokines, nitric oxide (NO), and toxic oxygen metabolites, contributing to collagen degradation. HMW-HA and LMW-HA influence macrophage activation differently. HMW-HA plays a role in the phenotype transition from pro-inflammatory M1 to reparative M2 or the switching of the inflammation to the proliferation phase [37; 38]. M1 macrophages secrete pro-inflammatory cytokines, such as IL-6, tumor necrosis factor-alpha (TNF-\(\alpha\)), and reactive oxygen species (ROS). Particularly, HMW-HA up-regulates the production of pro-inflammatory cytokines such as TNF-\(\alpha\), interleukin-1\(\beta\) (IL-1b), and IL-6 [37]. LMW-HA modulates the toll-like receptor-4 (TLR-4) and activates the nuclear factor kappa B (NF-kB), promoting an anti-inflammatory effect via CD44 [39]. HMW-HA inhibits angiogenesis, whereas LMW-HA stimulates it [40]. LMW-HA promotes angiogenesis [41]. Anti-inflammatory cytokines, such as IL-4 and IL-13, lead to M2 macrophage subset formation, which regulates inflammation by expressing mediators such as decoy IL-1 receptor type II and IL-10, as well as several growth factors that promote tissue growth and regeneration, fibroblast proliferation, extracellular matrix synthesis, deposition of collagens, and angiogenesis [42]. In chronic wounds such as diabetic foot ulcers, the wound is under hypoxia with elevated levels of pro-inflammatory cytokines (IL-6, IL-8, TNF-\(\alpha\), and MMP-2/9) [43], damaging cells and growth factor-binding receptors, causing prolonged inflammation and impeded angiogenesis. Hypoxia affects wound healing, including granulation, re-epithelialization, infection clearance, angiogenesis, and tissue regeneration. In this part, ROS scavengers or O\({}_{2}\) generators that oxygenate wound tissue are required. Figure 3: Hylauronan role observed during wound healing. Phase III: proliferation: In this phase, oligomeric HA reduces inflammation. Fibroblasts migrate to facilitate re-epithelialization. Fibroblasts in the wound are stimulated by TGF-\(\beta\) and CXCL8 to differentiate into myofibroblasts [44]. During the first phase, granulation tissue fills the wound bed with type III collagen with neo-vascularization. As new tissue is formed, the wound begins to contract. The proliferative phase lasts up to 2 or 3 weeks, starting about 4 days after injury. Oligomeric-HA binds to CD44 and RHAMM receptors to regulate keratinocyte proliferation, further facilitating epithelial formation and wound closure [45].