Physiology, Epithelialization

Issues of Concern

As previously mentioned, epithelization is the process of repairing epithelial wounds, with impairment of this process leading to improper wound healing, wound reoccurrence, and chronic wounds.[3] Impaired epithelialization can be caused by diabetes, trauma, and burns, as well as by bacterial infections, tissue hypoxia, local ischemia, and excessive inflammation.[4] In some instances, impaired epithelialization can also lead to excessive wound healing, resulting in hypertrophic scars with decreased tensile strength.[5]

Cellular Level

The process of epithelialization is vital for skin renewal and wound healing. The epithelial layer is made primarily of keratinocytes, which proliferate in the basal layer, differentiate as they rise through the spinous and granular layer, and then lose their nucleus and flatten to become the outer layer of skin known as the stratum corneum.[3]

Multiple anatomical connections between epithelial cells exist, including tight junctions, anchoring junctions, and gap junctions, which are important in epithelialization. Tight junctions split epithelial cells into apical and basal sections and are impermeable, preventing molecules from passing through the intercellular space. Anchoring junctions include desmosomes, hemidesmosomes, and adherens junctions. They are found on the basal and lateral surfaces of the epithelial cells and function to provide strength and flexibility. Desmosomes hold adjacent cells together using adhesion molecules such as cadherin. Hemidesmosomes link cells to the extracellular matrix using adhesion molecules such as integrins.[6] Adherens junctions use actin, along with either cadherin or integrins, to influence the shape of the epithelium. Gap junctions are unique in that they allow for intercellular communication and the transfer of ions and small molecules between adjacent cells.[7]

Development

Epithelial cells are derived from each of the three embryonic layers: ectoderm, mesoderm, and endoderm. Ectoderm develops into the epithelial lining of the skin, nose, mouth, and anus. Mesoderm creates endothelium, composed of epithelial cells that line vessels and lymphatics. Endoderm forms the epithelium lining the airways and most of the digestive system.

Organ Systems Involved

Classification of epithelial tissue is based on the shape of the cells and the number of cell layers. Cell shapes can be squamous (flat and thin), cuboidal (box-shaped), or columnar (rectangular). Additionally, there may be a single layer of cells, termed simple epithelium, or multiple layers of cells, termed stratified epithelium. There also exists pseudostratified epithelium, which describes a single layer of irregularly shaped cells that appear to be more than one layer. Transitional epithelium refers to a specialized form of stratified epithelium in which cell shape varies.[1] 

Simple Epithelium (Single Layer of Epithelial Cells)

• Simple Squamous
  • Thin, flat, horizontal, and elliptical-shaped
  • It makes up the endothelium lining the lymphatic and cardiovascular system, lung alveoli, kidney tubules, lining of capillaries, and mesothelium lining the serous surface of body cavities and internal organs.
• Simple Cuboidal
  • A box-shaped cell with a round nucleus; actively absorbs and secretes molecules.
  • Found in kidney tubules and gland ducts
• Simple Columnar
  • Rectangular shaped with elongated nuclei at the basal end of the cells; absorbs and secretes molecules.
  • Found in the digestive tract and female reproductive tract
  • Ciliated simple columnar epithelial cells are found in the respiratory tract and fallopian tubes, aiding in the movement of mucous and other substances via the beating of their cilia.
• Pseudostratified Columnar
  • Irregularly shaped with nuclei at different locations in the cell
  • Pseudo = "false" - appears stratified but is a single layer of epithelial cells attached to the basal lamina

Stratified Epithelium (Multiple Layers of Epithelial Cells) 

• Stratified Squamous Epithelium
  • The most common type of stratified epithelium
  • The basal layer is cuboidal or columnar, and apical layers are squamous
  • The top layer may be dead cells filled with keratin
  • Found in the skin (keratinized) and mouth (non-keratinized)[1]
• Stratified Cuboidal and Columnar Epithelium
  • Uncommon - found in ducts and glands
• Transitional Epithelium (Urothelium)
  • Changes shape in response to stretch
  • Found only in the bladder, ureters, and proximal urethra
  • It appears cuboidal when relaxed and squamous when stretched

Function

Epithelium serves as an essential barrier, protecting internal organs from external pathogens, preserving adequate fluid levels in the body, and controlling the permeability of substances across the epithelial barrier.[3] The mechanical barrier properties of epithelium aid in protecting our bodies from microbes. Injured or infected epithelial cells lead our bodies to be exposed to invading agents or pathogens. The function of epithelialization is to aid in epithelial wound repair, specifically through the proliferation and differentiation of keratinocytes.[8]

Mechanism

Epithelialization

The process of epithelialization has been studied primarily in the context of skin reepithelialization during wound healing. Skin is composed of three main layers, from superficial to deep: epidermis, dermis, and subcutaneous tissue. The epidermis is created by stratified squamous epithelium and can further be broken down into multiple layers, again from superficial to deep: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale.[9]

Keratinocytes are the primary component of the epidermis and play a key role in wound healing and maintaining the outer skin barrier. Keratinocytes proliferate in the basal layer, differentiate as they rise through the granular layer, lose their nucleus and flatten to become the superficial layer of skin, the stratum corneum. Deficits in this process can result in delayed wound healing.[3]

Keratinocytes proliferate in the basal layer and are composed of keratin intermediate filaments K5 and K14. As the keratinocytes rise into the suprabasal layers, they characteristically differentiate into K1 and K10. The lamellar granules produce lipids and proteins in the granular layer and fill in the crevices between the keratinocytes in the stratum corneum. High molecular weight polymers form via the crossing of cornified envelope proteins (loricrin, involucrin, filaggrin). The cornified envelope is formed through the process of terminal differentiation, where the keratinocyte becomes dehydrated and flattens into a polyhedron referred to as terminal corneocyte. The lipid layer fills in between the corneocytes, functioning as the "mortar" and corneocytes as "bricks." This lipid layer is essential for keeping water in the body.[3]

The cells change from dividing to non-dividing cells during keratinocyte differentiation as they migrate to the surface through the granular layer. Three major MAP kinase pathways regulate this process. These pathways are activated by calcium influx, epidermal growth factor, and tumor necrosis factor. The process also uses protein kinase C isoforms. Once keratinocytes turn profilaggrin into filaggrin, they undergo changes into late terminal differentiation and are irreversibly committed to the process of differentiation. Differentiation ends when proteolytic and nucleolytic activity destroys the cellular organelles and DNA. Increased intracellular calcium forms the cornified envelope by activating transglutaminase, which covalently crosslinks structural proteins such as loricrin, involucrin, and filaggrin. Finally, insoluble lipids attach, and the outermost layer of the epidermis is complete.[3]

The epidermal barrier is maintained by skin calmodulin-related factor (Scarf), which senses calcium and regulates the protein function in the skin barrier. Keratinocytes constantly renew to maintain this barrier. The renewing capabilities of the skin are driven by the population of epidermal stem cells (ESC). ESCs are found in three distinct niches: the bulge of hair follicles, the base of sebaceous glands, and the basal layer of the interfollicular epidermis. Each ESC niche replenishes its compartment; therefore, the depth of a wound and damage to these structures affects the skin’s ability to re-epithelialize.[3]

Reepithelialization in Wound Healing

There are four distinct but overlapping phases of wound healing: hemostasis, inflammation, proliferation, and remodeling (maturation).[2] Tissue injury triggers hemostasis and the inflammatory phase, dominated by the release of inflammatory mediators, neutrophils, and macrophages traveling to the wound site to phagocytize bacteria and initiate the proliferative phase. The proliferative phase refers to the formation of new granulation tissue filling the wound, followed by the remodeling phase, where collagen fibers become more organized and strengthen the wound.[3]

The hemostasis and inflammatory phase of wound healing are closely linked and begin with the release of cytokines and vasoactive substances, such as histamine and serotonin, causing increased vessel permeability. This increased permeability allows for the recruitment of lymphocytes, neutrophils, and macrophages to the wound site. Blood constituents also leak into the area and activate the clotting cascade. These blood clots serve as a matrix for cells to adhere and migrate into the wound and become a source of growth factors for fibroblasts and inflammatory cells. The first 48 hours following a tissue injury are predominated by the inflammatory phase, with neutrophils cleaning the wound and phagocytosing bacteria.[3] Important cytokines for wound repair are also released, which initiate the proliferative phase, such as IL-1, IL-6, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), and TGF-beta.[10]

As the inflammatory phase of wound healing ends, the proliferative phase begins, and granulation tissue forms. The onset of the proliferative phase is marked by the proliferation of fibroblasts and endothelial cells. Fibroblasts form structural proteins that make collagen and increase the strength of the wound. Angiogenesis also occurs to provide oxygen and nutrients to the new granulated tissue. Once the bed of granulation tissue is laid, reepithelialization can take place.[3]

Within hours of skin trauma, keratinocytes along the edges of the wound, around hair follicles, and surrounding sebaceous glands are activated by monocytes and neutrophils to migrate and proliferate to re-epithelialize the area with skin cells.[10] Basal cell keratinocytes release their attachment to the underlying dermis and migrate across the wound to cover its surface, going through rapid mitotic division as the cells move across each other in a "leapfrog" fashion, advancing in a sheet across the wound.[3]

Keratinocytes must undergo a structural transformation at the cellular layer to migrate across the wound. The cells lengthen, flatten, and develop actin filaments and pseudopodia, which serve as temporary protrusions for movement.[11] The keratinocytes then lose their hemidesmosome attachment to the surrounding cells. Integrins (proteins for attachment) are released and relocated to actin filaments that pull the keratinocytes into the wound. The keratinocytes migrate into and adhere to the newly formed wound matrix. These changes in the keratinocyte cytoskeleton allow for increased cell flexibility and migration of keratinocytes and are classically marked by changes in the expression of keratin-6 and keratin-16.[1] 

Activated keratinocytes and fibroblasts in the dermis release growth factors such as keratinocyte growth factor and hepatocyte growth factor that help to regulate this process. Keratinocytes continue proliferating at the wound edges and migrating to cover the wound until they meet in the middle. Some sources report that well-approximated wounds can re-epithelialize within 48 hours; others report the process of epithelialization generally takes 2 to 3 weeks.[10] The faster this process occurs, the less scarring there is. Thick scabs over a wound can inhibit the ability of the keratinocytes to migrate across the wound and, in turn, increase healing time.

At two weeks, the wound is only at 10% total wound strength. At one month, the wound is at 50% of total wound strength, and, by one year, the maximum scar strength of 80% is achieved.[12]

Clinical Significance

In understanding the epithelialization process, one can begin to understand the clinical importance of using moisture-retaining occlusive dressings to enhance wound healing. A moist environment enhances epidermal cell migration, especially in reepithelialization. In a dry environment, cells dehydrate and die, leading to scab or crust formation over the wound and impairing reepithelialization.[13] Epidermal cells undergo an easier migration on a moist surface, faster epithelialization, and a prolonged presence of proteinases and growth factors. Fluid presence in wound healing promotes keratinocyte proliferation, fibroblast growth, and preservation of growth factors. Studies have shown an acceleration of the inflammatory phase of repair in moist wounds, allowing the late phase of inflammation to begin more rapidly.[14] Dry wounds lead to delayed wound healing and poor epidermal migration. There is also more wound debris than moist wounds, which impedes epidermal cell migration.

In summary, wounds exposed to air and allowed to dry tend to heal slowly and result in poor cosmesis. On the other hand, moist wound environments improve wound healing, enhance angiogenesis, and reduce necrosis.[14] This knowledge has led to the production of various moist wound dressings, such as foams, hydrocolloids, alginates, and hydrogels.[15]