Introduction
A skin wound is the result of compromised epidermal layer integrity.[1] A wound is defined as any tissue injury that disrupts anatomical integrity and leads to functional loss. The ability of an organism to repair or regenerate tissues is a definite advantage for survival.[2] In common parlance, the term wound healing typically refers to the process of healing skin wounds. This process begins immediately after an injury to the epidermal layer and may require years to complete fully. Wound healing is a dynamic process involving highly specialized cellular, humoral, and molecular mechanisms.[2] This process consists of 4 overlapping phases—hemostasis, inflammation, proliferation, and remodeling.[3] Any disruption of these processes can cause abnormal wound healing.[4]
Wound healing can occur through primary or secondary intention. Primary healing refers to the uncomplicated healing of a noninfected, non-draining wound with a close approximation of the edges of the skin. Surgical wounds are the best example of primary healing. If the healing course is disrupted by infection, dehiscence (separation of the edges of the wound), hypoxia, or immune dysfunction, healing by secondary intention begins. This process is characterized by granulation tissue formation and epithelialization over the new tissue. Wounds that heal by secondary intention healing are more prone to infections, yield poor cosmetic results, and exhibit reduced optimal tissue strength.[5][6]
Pathophysiology
The wound healing process consists of 4 overlapping phases—hemostasis, inflammation, proliferation, and remodeling.[3]
Hemostasis
Any significant injury to the skin immediately triggers the clotting cascade, leading to the formation of a temporary fibrin blood clot plug at the injury site.[1][2] In addition to clot formation, local vasoconstriction is immediately established around the wound area, lasting up to 10 minutes.[2] These temporary responses prevent further bleeding and protect the wound from pathogen invasion.[8][3] Following this brief vasoconstriction response, vasodilatation occurs, resulting in local hyperemia and edema.[2] Capillary dilation and leakage accelerate the migration of inflammatory cells into the wound bed.[11]
The endothelium normally releases a continuous stream of antiplatelet molecules, such as nitric oxide, prostaglandin I2, and prostacyclin.[12] A healthy endothelium is also covered by glycocalyx, a 0.5- to 5-µm-thick proteoglycan-rich structure that prevents endothelium-platelet interactions.[13] When a blood vessel is damaged, exposure of the subendothelium, collagen, and tissue factor stimulates platelet adherence to the vascular wall through different receptors. Initially, the platelet receptor glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the immobilized von Willebrand factor on subendothelial matrix and endothelial surfaces. This interaction leads to the binding of platelet collagen receptor GP-VI to its ligand in the exposed subendothelial matrix, triggering intracellular signals that activate integrins and allow platelet adhesion, activation, and aggregation.[14] Platelet-endothelium adhesion activates platelet degranulation, releasing chemical mediators that activate more platelets.[15][16] Within minutes, platelet aggregation forms a primary fibrin or hemostatic plug. The secreted microparticles and the trans-bilayer movement of negatively charged phospholipids offer binding sites for coagulation system components. The fibrin plug recruits more platelets, erythrocytes, and leukocytes, and the primary fibrin plug transforms into the secondary hemostatic plug. Synchronously, counter-regulatory pathways limit the extension of the plug.[17] The fibrin plug acts as a temporary matrix, serving as a scaffold for further healing processes by supporting the migration of leukocytes, keratinocytes, fibroblasts, and endothelial cells. Additionally, it functions as a reservoir for growth factors essential for wound repair.[2]
Inflammation
The inflammation phase begins shortly after hemostasis. After an injury, pattern-recognition receptors on tissue-resident macrophages in the dermis are activated by danger-associated molecular patterns released by damaged and necrotic cells and pathogen-associated molecular patterns on pathogens.[18] Danger-associated molecular patterns are believed to be the initial signals for neutrophil recruitment to the injury site.[19] These danger signal molecules can activate neutrophils.[18][19] Activated platelets and tissue-resident macrophages release pro-inflammatory chemokines, cytokines, and growth factors, thereby initiating the recruitment and activation of other immune cells, primarily neutrophils and bone marrow-derived monocytes.[3] Furthermore, reactive oxygen species (ROS), such as hydrogen peroxide, contribute to neutrophil recruitment and macrophage polarization.[20][21] Neutrophils and monocytes are recruited to the injury site through microhemorrhages or transendothelial migration.[22]
Neutrophils are recruited to the wound site within the first 24 hours and remain for 2 to 5 days.[2][6][15] Neutrophils are the most abundant cells in the wound site during the early stages of inflammation.[3] Neutrophils initiate phagocytosis, which is later continued by macrophages.[2][15] These phagocytic cells release ROS, nitric oxide (NO), and proteases to kill local pathogens and debride necrotic tissues.[2] Neutrophils can also eliminate pathogens in the extracellular environment by deploying neutrophil extracellular traps, defined as web-like structures comprising strands of decondensed chromatin bound to neutrophil-produced bactericidal proteins. These structures can either directly kill microorganisms or immobilize pathogens to facilitate phagocytosis.[23][24] In addition to clearing pathogens, neutrophils act as a chemoattractant for other cells, such as macrophages, T-cells, and additional neutrophils, and augment the inflammatory response by releasing many pro-inflammatory cytokines.[2][25] Neutrophils also increase the expression of cytokines, promoting angiogenesis, proliferation of fibroblasts and keratinocytes, adhesion of keratinocytes, and tissue remodeling.[26][27][28]
Macrophages are phagocytic monocyte-derived cells that scavenge and remove debris, necrotic tissue, and toxic metabolites from the injury site.[29] These cells typically arrive approximately 3 days after the injury.[2] Danger-associated molecular patterns and pathogen-associated molecular patterns—released from necrotic tissue and pathogens—and interferon γ (IFN-γ)—released from natural killer cells—polarize macrophages into a pro-inflammatory phenotype. These macrophages secrete pro-inflammatory cytokines and chemokines, stimulating natural killer cells, macrophages, and helper T-cell responses.[30][31][32] This inflammatory amplification is necessary because uninjured, healthy skin holds relatively few resident macrophages at baseline. However, after injury, these homeostatic functions are amplified by various stimuli to facilitate this highly complex wound-healing process. Different macrophage subsets are required to perform specific roles in each wound-healing phase.[3][29]
Macrophages are also responsible for neutrophil apoptosis. Neutrophil persistence is associated with delayed wound healing. After foreign debris clearance, neutrophils normally undergo apoptosis and are removed by pro-inflammatory macrophages. This process is called efferocytosis.[33][34] Macrophage-neutrophil physical interactions promote direct neutrophil reverse migration back into circulation.[34] Efferocytosis is a crucial signal for the local immune milieu, facilitating the transition from a pro-inflammatory macrophage-dominant wound microenvironment into an anti-inflammatory macrophage-dominant one. This shift marks the onset of a pro-repair state, initiating the proliferation phase of wound healing.[34][11][35]
Proliferation
As inflammation resolves, the proliferative phase begins, characterized by vascular network restoration, re-epithelization, and granulation tissue formation.[2][11] This phase starts approximately 3 to 10 days after injury and may require days or weeks to complete.[2][15] At this stage, the wound microenvironment is transformed into a pro-healing environment, accompanied by a phenotype shift of pro-inflammatory macrophages into anti-inflammatory macrophages.[36] These anti-inflammatory macrophages have highly immunosuppressive and regenerative properties, releasing various angiogenic and growth factors, anti-inflammatory cytokines, and chemokines, including metalloproteinases (MMPs), platelet-derived growth factor (PDGF), resistin-like molecule-alpha, vascular endothelial factor (VEGF), interleukin-8 (IL-8), transforming growth factor-beta (TGF-β), IL-10, and arginase. In addition to their role in extracellular matrix deposition, these factors induce the proliferation and differentiation of endothelial cells, keratinocytes, and fibroblasts.[33] Various cytokines and growth factors involved in this phase include the TGF-β family, such as TGF-β1, TGF-β2, and TGF-β3; IL family; and angiogenesis factors.[15] Fibroblasts and endothelial cells are the primary mediators of proliferation.[10] Fibroblasts proliferate and contribute to angiogenesis and the formation of granulation tissue.[37]
An adequate blood supply is essential for cell proliferation, facilitated by the angiogenic response.[8] This response is primarily stimulated by local hypoxia, VEGF, PDGF, fibroblast growth factor-basic, and serine protease thrombin.[2][8] New vessels are formed through two mechanisms—angiogenesis and vasculogenesis.[38] Angiogenesis is a sprouting process in which neo-vessels grow into the avascular site from resident endothelial cells of the adjacent mature vascular network.[2][38] In contrast, vasculogenesis is a de novo process during which progenitor stem cells differentiate and form new vessels without sprouting from any mature vascular network.[38] These endothelial progenitor cells are stem cells typically found in the bone marrow. Endothelial progenitor cell recruitment into the circulation begins immediately post-injury. NO, VEGF, and MMPs—primarily MMP-9—all stimulate endothelial progenitor cell mobilization.[8] Likewise, stromal-derived factor 1-α is the primary signal guiding endothelial progenitor cells to gather to areas of ischemia.[38] Finally, a new vascular network is formed, which provides nutrient delivery, gas, and metabolite exchange.[8] The administration of antiangiogenic medications such as bevacizumab may disturb this phase and lead to chronic wound formation.[6]
Keratinocyte reepithelialization is influenced by fibroblasts found in the granulation tissue and pro-repair macrophages. This process is initiated by epidermal growth factor, keratinocyte growth factor, and TGF-α, which are produced by platelets, keratinocytes, and activated pro-repair, anti-inflammatory macrophages.[39] Keratinocytes also activate fibroblasts through a feedback loop by producing fibronectin, tenascin C, and laminin 332.[40] During normal wound healing, keratinocytes begin migrating centrally from wound edges within hours of tissue injury, and epithelial stem cells begin proliferating from the basal layer of the epidermis and hair follicle root sheaths within 3 days.[41] The keratinocytes migrate over the wound edge until they make physical contact with each other. This contact inhibition from neighboring keratinocytes halts migration.[6]
The final step of the proliferation phase is granulation tissue formation.[2] Fibroblasts migrate to the wound site and proliferate in response to growth factors produced by macrophages and other immune cells.[2][8] These fibroblasts produce MMPs that degrade the present fibrin clot.[42] Fibroblasts then synthesize a provisional matrix containing collagen type III, glycosaminoglycans, and fibronectin.[10] This process replaces the fibrin clot with a new provisional matrix supporting keratinocyte migration for reepithelialization.[42] The newly-formed granulation tissue comprises fibroblasts, granulocytes, macrophages, capillaries, and loosely organized collagen bundles. This new tissue has a classically red appearance due to incomplete angiogenesis.[2]
Remodeling
Remodeling is the final phase of wound healing, which begins on day 21 and may continue for up to 1 year.[2][15] This phase requires a precise balance between the synthesis and degradation of new tissue. Any disruption causes the formation of a chronic wound.[15] During the remodeling phase, the granulation tissue formation ends, and the maturation of the wound begins. Extracellular matrix components are stimulated by various signals to form a stronger and more organized extracellular matrix.[2][10] An example is the replacement of collagen type III by stronger type I, which gradually increases the tensile strength of the wound.[2][6] Collagen synthesis continues for at least 4 weeks. However, the collagen in the wounded area is never as organized as in healthy skin.[10] During collagen synthesis, hydroxylases require oxygen and vitamin C. Thus, hypoxia and vitamin C deficiency can affect the strength of the repaired tissue.[6][43] Matrix remodeling enzymes, particularly MMPs, have significant roles during cellular migration, proliferation, and angiogenic processes to remodel the local matrix microenvironment.[8] The remaining cells of the previous phases undergo apoptosis.[2]
In addition to new tissue formation, wound contraction begins during remodeling. TGF-β1 stimulates the fibroblasts to differentiate into myofibroblasts.[10] Besides synthesizing major extracellular matrix proteins such as collagen types I to VI and XVIII, glycoproteins, and proteoglycans, myofibroblasts also participate in wound contraction.[10][44] Myofibroblasts resemble smooth muscle cells, express α-smooth muscle actin, and can generate traction and strong contractile forces throughout the wound site.[1] This contraction aids the approximation of wound edges, enabling wound closure. After the wound is fully epithelialized, myofibroblasts undergo apoptosis. Persistent or excessive myofibroblast activity may result in fibrosis and scar formation.[44] Apoptosis of the fibroblastic cells significantly contributes to the formation of a mature wound, which is relatively acellular.[2][8] However, the apoptotic mechanisms in wound healing are not well understood.[8] Recent investigations have revealed that these wound-bed myofibroblasts may further differentiate into fat cells to replenish subcutaneous adipose tissue.[45]
The angiogenic response ceases at the end of the entire process, and blood flow diminishes to pre-injury levels. These processes provide complete closure for injured tissue sites and restoration of the mechanical strength of the wound.[2] Wound healing ends with scar formation, a process that can be significantly influenced by inflammation. This scar tissue has some defects. For instance, the wound strength can never catch up with the normal skin strength. At 3 months and beyond, the wound strength reaches only about 80% of that of the original tissue.[10] Similarly, subepidermal appendages such as hair follicles or sweat glands do not heal after a severe injury. Scar tissue also lacks rete pegs, which allow for a tight connection between the epidermis and dermis.[2]
Clinical Significance
Disruption during any wound-healing phase can lead to excessive wound healing or chronic wound formation.[4][15]
Excessive Wound Healing
The pathogenesis of excessive wound healing is not fully understood. This condition is an abnormal form of wound healing characterized by continuous localized inflammation, excessive collagen synthesis, abnormal collagen turnover, and exaggerated extracellular matrix accumulation. Examples of excessive wound healing include keloid and hypertrophic scars.[15]
Chronic Wound Formation
A chronic wound is any wound that has failed to heal within 4 weeks. However, some professionals prefer to wait 3 months before diagnosing a chronic wound.[9]
The primary risk factors for chronic wound formation include age, immune status, malnutrition, infection, insufficient oxygenation or perfusion, smoking, diseases, medications, radiation, and chemotherapy.[6] The most common examples of chronic wounds include vascular ulcers (venous or arterial ulcers), diabetic ulcers, and pressure ulcers.[8]
Wound healing in fetal and older populations requires additional and different considerations.[6] The first difference is that inflammation is not apparent in fetal wounds, with fewer inflammatory cells. Similarly, IL-6, IL-8, IL-10, TGF-β1, TGF-β2, and TGF-β3 concentrations in a fetal wound differ from an adult wound. In addition, extracellular matrix content differs in fetal wounds with a higher extracellular matrix production rate, a higher ratio of type III to type I collagen, and a higher amount of hyaluronic acid. Furthermore, few, if any, myofibroblasts are present in fetal wounds. These conditions may explain the scarless healing typical of fetal wounds.[15] Human skin structure changes dynamically during pregnancy, and scarless wound healing is typically not observed after the 24th week of gestation.[2] In contrast, wound healing is significantly less effective in older adults. The total collagen amount in the dermis and the epidermal turnover time decreases during aging. These changes lead to slower wound healing as the patient ages, which various comorbidities may exacerbate.[6]
Patients with immunosuppression are more susceptible to infections and have a diminished ability to fight infections, leading to delayed wound healing due to an extended inflammatory phase. Anti-inflammatory drugs, particularly during the first 3 days of healing; corticosteroids; immunosuppressants; and chemotherapy agents can alter wound healing.[6] Similarly, some comorbidities negatively influence wound healing, with diabetes mellitus being one of the most significant diseases associated with chronic wound formation. Poor perfusion, neuropathy, immunosuppression, slower collagen synthesis and accumulation, and inadequate angiogenesis often cause an increased risk of infection and delayed wound healing in patients with diabetes mellitus.
Adequate perfusion and oxygenation of the wounded area are also critical for normal wound healing. Oxygen significantly affects successful wound healing due to its role in inflammation, bactericidal activity, angiogenesis, epithelization, and collagen deposition.[6] Smoking, peripheral arterial insufficiency, prolonged local pressure, and radiation exposure can cause chronic wounds by impeding adequate oxygenation.[6][8] Radiation causes microvascular obliteration, fibrosis, and alterations in cellular replication, leading to delayed wound healing.
Chronic venous insufficiency leads to chronic wound formation due to ischemia. Venous hypertension causes edema, which disrupts the metabolite diffusion of the tissues. Edema leads to an ischemic environment, which is relieved during walking or elevation of the extremities. Repeated ischemic cycles cause chronic reperfusion injury and inflammatory changes.[46] Inadequate protein and carbohydrate intake and vitamin deficiencies can result in delayed wound healing. Fibroblast proliferation, collagen synthesis and remodeling, and angiogenesis are affected by protein status. Poor carbohydrate reserves may cause protein catabolism. Vitamin C and thiamine (B1) participate in collagen formation and can significantly affect wound strength. Vitamin A is involved in inflammatory processes, and zinc is an important cofactor in wound healing.[6]
