What to Know About Epithelialization in Wound Healing

The process of wound healing restores the skin’s protective barrier after injury. A key component of this repair is epithelialization, where new epithelial cells migrate and proliferate to cover the wound. Efficient epithelialization prevents infection and minimizes scarring.

Biological mechanisms regulate this process, involving immune responses, signaling molecules, and extracellular matrix changes. Understanding these factors can improve wound care strategies and therapeutic approaches.

Phases Of Re-Epithelialization

Restoring the epidermal layer after injury is a coordinated process occurring in overlapping phases. Re-epithelialization begins immediately as keratinocytes at the wound margins reorganize their cytoskeleton and loosen attachments to neighboring cells and the basement membrane. This transition to a migratory phenotype is driven by intracellular changes, including downregulation of E-cadherin and upregulation of integrins that aid adhesion to the wound matrix. Leading-edge keratinocytes extend lamellipodia and filopodia, enabling directed movement across the wound bed.

As migration continues, keratinocyte proliferation increases, particularly in the basal epidermal layer. Stimulated by local biochemical cues, these cells re-enter the cell cycle, ensuring enough keratinocytes cover the wound. Proliferation and migration are tightly regulated to prevent excessive cell accumulation, which could disrupt tissue architecture. Studies show keratinocyte proliferation peaks within the first few days post-injury before subsiding as the wound closes.

Once migrating keratinocytes meet at the wound center, contact inhibition halts movement. The new epithelial sheet then undergoes stratification and differentiation to restore epidermal function. Basal keratinocytes deposit basement membrane components like laminin-332 and type IV collagen, providing structural support and biochemical signals for epidermal stability. Tight junctions and desmosomes reinforce tissue integrity, enabling the epidermis to function as a protective barrier.

Dynamic Role Of Immune Cells

Immune cells actively regulate keratinocyte behavior and wound closure beyond their role in pathogen defense. Neutrophils, the first responders, release proteolytic enzymes and reactive oxygen species to clear debris. They also secrete cytokines like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), which influence keratinocyte migration by modulating integrins and matrix metalloproteinases, facilitating movement.

As inflammation resolves, monocytes enter the wound and differentiate into macrophages, which shift from a pro-inflammatory (M1) to an anti-inflammatory (M2) state. M2 macrophages secrete transforming growth factor-beta (TGF-β) and vascular endothelial growth factor (VEGF), which suppress inflammation, promote keratinocyte proliferation, and enhance angiogenesis. Studies show macrophage depletion delays re-epithelialization, highlighting their essential role in wound healing.

Dendritic cells and innate lymphoid cells also modulate immune-epithelial interactions. Langerhans cells, a specialized dendritic cell subset, produce IL-10 to control inflammation while preserving keratinocyte viability. Group 2 innate lymphoid cells (ILC2s) secrete amphiregulin, a growth factor that enhances epithelial survival and differentiation. Research indicates ILC2-deficient mice exhibit impaired epithelialization, emphasizing their role in optimizing keratinocyte function.

Growth Factors And Signaling Pathways

Epithelialization relies on growth factors and signaling pathways that regulate keratinocyte migration, proliferation, and differentiation. Epidermal growth factor (EGF) plays a central role by binding to its receptor (EGFR) and triggering intracellular cascades that enhance cytoskeletal reorganization and cell motility. EGF stimulation increases phosphorylation of focal adhesion kinase (FAK), essential for keratinocyte adhesion turnover during migration. The mitogen-activated protein kinase (MAPK) pathway further supports cell cycle progression, ensuring sufficient proliferating keratinocytes for wound coverage.

Transforming growth factor-beta (TGF-β) has a dual role depending on its concentration and timing. Early in epithelialization, TGF-β1 enhances keratinocyte migration by inducing transient epithelial-mesenchymal transition-like (EMT-like) features, reducing cell-cell adhesion and increasing mesenchymal marker expression. However, prolonged TGF-β signaling inhibits proliferation by promoting terminal differentiation. This balance is mediated through the Smad-dependent pathway, where phosphorylated Smad2/3 complexes regulate gene transcription. Disruptions in this pathway contribute to chronic wounds, such as diabetic ulcers, where TGF-β signaling is often dysregulated.

Fibroblast growth factors (FGFs), particularly FGF7 and FGF10, are secreted by dermal fibroblasts to stimulate keratinocyte proliferation and migration. These ligands activate fibroblast growth factor receptors (FGFRs) on keratinocytes, triggering downstream signaling through the PI3K/Akt and Ras/MAPK pathways. Research shows FGF10-deficient mice experience delayed wound closure, underscoring its role in epithelial repair. Cross-talk between FGFR and EGFR pathways enhances keratinocyte responses, ensuring efficient wound coverage.

Extracellular Matrix Remodeling

The extracellular matrix (ECM) serves as a structural scaffold and signaling hub during epithelialization, undergoing remodeling to support keratinocyte migration and tissue regeneration. After injury, a provisional matrix of fibrin and fibronectin forms, providing an initial substrate for migrating cells. This temporary network is later replaced by a more structured ECM as keratinocytes deposit laminins, collagen IV, and nidogens to re-establish the basement membrane. Matrix metalloproteinases (MMPs) regulate this transition by breaking down ECM components to clear pathways for advancing epithelial cells while activating bioactive fragments that influence cell behavior.

Balancing ECM degradation and synthesis is crucial for proper wound closure. MMP-1 and MMP-9 break down fibrillar collagen and denatured matrix proteins, ensuring migrating keratinocytes are not obstructed. Tissue inhibitors of metalloproteinases (TIMPs) prevent excessive ECM breakdown, maintaining structural integrity and preventing abnormal remodeling. Imbalances in MMP/TIMP activity are linked to chronic wounds, where excessive degradation hinders epithelialization, or fibrosis, where unchecked ECM accumulation leads to rigid scarring.

Novel Insights From Advanced Methods

Advances in imaging, molecular profiling, and bioengineered models have provided deeper insights into epithelialization. High-resolution live-cell imaging allows researchers to visualize keratinocyte dynamics in real time, revealing that keratinocytes migrate collectively rather than as isolated units. Leader cells extend lamellipodia while follower cells maintain intercellular adhesions. Traction force microscopy shows keratinocytes adjust adhesion strength based on ECM stiffness, indicating that mechanical properties influence wound closure efficiency.

Single-cell RNA sequencing has further clarified keratinocyte heterogeneity during epithelialization, identifying distinct transcriptional states linked to migration and proliferation. This specialization ensures migration and proliferation occur in a coordinated manner rather than in competition. Organotypic skin models derived from patient cells provide a physiologically relevant platform for studying epithelialization in chronic wounds and diabetic ulcers. These models help test novel therapeutics, including bioengineered growth factors and ECM-modulating compounds, aimed at enhancing re-epithelialization in patients with impaired wound healing.