Introduction/Definitions:
The term “skin” refers to the outer protective covering of the body, consisting of the corium and the epidermis, including sweat and sebaceous glands, as well as hair follicle structures. Skin acts as a natural barrier between internal and external environments and therefore plays an important role in vital biological functions such as protection against mechanical and chemical injury, microorganisms, and ultraviolet damage. Human skin is made up mainly of two main layers, the outer epidermis and the underlying dermis.
Skin and oral musosae as well are composed to two primary layers, the outer epidermis, which is stratified epithelial tissue mainly composed of multiple layers of keratinocytes and the dermis, which is the layer beneath the epidermis consisting of connective tissue, blood vessel, immune cells, collagen and other components.
The skin, together with the hair, nails and sweat and oil glands, forms the integument.
The skin is the largest organ in the human body and functions as the first line of defense by providing a protective barrier between the environment and inner body. The skin harbors several hundreds of resident microorganisms, which function in communities and protect the body from invasion of pathogens. Several studies have shown that shifts in the skin microbiota are associated with various skin diseases.
Human skin in particular is one of the most complex structures wehn compared to similar organisms, qualifying it as the largest and one of the most complex organs of the human body. Special derived structures including hair follicles, eccrine seat glands, sebaceious glands, and apocrine glands, along with heterogenous cells and extracellular ocmponents make the skin a physically diverse entity. Variations of skin type on different locations on the body, as well as between different individuals, include skin thickness, composition, density of structures and biochemical differentiation. (Suhail, “Engineered skin tissue equivalents for product evaluation and therapeutic applications” Biotechnol J. 2019, 14(7))
Desquamation is the normal shedding of corneocytes from the outer surface of the skin.
Epidermis:
Introduction:
Human skin is composed of several morphologically distinct layers. The outer-most layer of the skin, the epidermis, is composed fo 4 to 5 sub-layers depending on hwere on teh body the skin is lcoated. Tehse sub-layers form the outer-most layer to the inner-most layer, include the stratum corneum, the stratum lucidum (which is present only in thick skin, such as the soles of feet and palms of hands), the stratum ganulosum, the stratum spinosum and the stratum basale. (Eberting, US2016/0184245A1)
The epidermis of humans and animals is composed of three layers: the stratum basale, the stratum spinosum and the stratum corneum. The stratum basale, the innermost layer, is the only layer in which active cell division occurs. When division transpires, a daughter cell, called a keratinocyte, begins to undergo terminal differentiation. By this process, the keratinocyte is sequentially transformed into a cell of the spinosum and then into one of the corneum. Among the changes occurring during differentiation are increased synthesis of ceramide, loss of the nucleus, cell death and replacement of the cytoplasm with keratin. As keratinocytes reach the outermost stratum corneum layer, they are shed to the environment. (Kligerman, US 7402323 B2)
The epidermis is the outermost skin layer. As skin cells migrate to the surface, farther away from their source of nourishment, they flatten and shrink. They lose their nuclei, move out of the basal layer to the horny layer (stratum corneum), and die. This process, called keratinization, takes about 4 weeks. About 10 percent of epidermal cells are melanocytes that pigment the skin. The epidermis is differentiated into five layers: horny layer (stratum corneum); clear layer (stratum lucidum); granular layer (stratum granulosum); prickle-cell layer (stratum spinosum); and the basal layer (stratum basale).
Like the intestine, the epidermis of skin and oral musoca are self-renewing. The layers of the epidermis reflect the stages along the continuous process of keratinocyte maturation, from the basal layer adjacent the dermis which contains the proliferating stem and progenitor cells, to the outer protective layer of dead, cornified keratinocytes which are being sloughed form the surface. The cell division that occurs in the basal layer of the epidermis provides a continuous source of keratinocytes which fully replenish the epidermis at rates that are estimated to be up to several weeks.
Cells constituting the epidermis are delimited by a lipid domain. In the course of differentiation, phospholipids, the role of which consists in producing the fluid structure of the cell membranes of the living layers of the epidermis, are gradually replaced by a mixture composed predominantly of fatty acids, cholesterol and ceramides (sphingolipids). These lipids are responsible for the “barrier” properties of the epidermis, particularly of the outermost layer of the epidermis, the stratum corneum.
Stratum corneum is the outermost layer in the skin’s physiology. It is produced through the terminal differentiation of epidermal cells. It is a complex multi-layered structure that provides effective cellular barrier between external enviornment and internal miliu of living cells. It includes, what is traditionally known as a brick and mortar structure. Where bricks are formed of protein rich cells, called corneocytes, which are embedded into a mortar of multi-layered lipid structure. The wall of corneocytes is made of highly crosslinked proteins that is tightly bound to lipids forming cornified lipid envelope. (Suhail, “Engineered skin tissue equivalents for product evaluation and therapeutic applications” Biotechnol J. 2019, 14(7))
Lipid organization in the intercellular matrix of human SC plays a key part in the creation of the SC barrier. These lipids include ceramides (CER), free fatty acids (FFA), and cholesterol (CHOL), which are present in nearly equimolar ratios. In certain skin diseases in which the barrier function is affected, aberrations in lipid composition have been described. (Pilgram, “Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis” Society for Investigative Dermatology, 2001)
The normal pH of the stratum corneum is somewhat acidic, 5.80 in men and 5.54 in women (Ehlers, et al. Skin Research and Technology, 7(2):90-4, 2001). Maintenance of normal skin flora is pH dependent, and even modest increases in pH (0.1 to 0.2 pH units) are sufficient to foster growth of undesirable microorganisms such as Candida albicans (Runeman, et al. Acta Dermato-Venereologica 80(6): 421-4, 2000). Therefore, it is important that products designed for application to the skin have carefully controlled pH levels. (Kligerman, US 7402323 B2)
Epidermal lipids are mainly synthesized in living epidermis. They are made up mainly of phospholipids, sphingolipids, cholesterol, free fatty acids, and triglycerides. Ceramides are a class of sphingolipids that play a paramount role in cellular signaling and are linked to cell proliferation, differentiation and apoptosis in human epidermis. Epidermal lipids are necessary for maintaining the multilamellar structure of the intercorneocytic lipids. They also contribute to the “barrier” function of the epidermis and for overcoming water loss/moisturization problems.
There are several genetic skin diseases with known defect in lipid metabolism that have scaly or ichthyotic skin as part of their clinical picture.
—Ceramides are a class of lipids (fats) that occur naturally in the human skin, making up about 50% of the skin’s intercellular lipids. There are at least 12 different types of ceramides found in the skin (e.g., Ceramide NP, AP, EOP), each with a slightly different structure based on its specific sphingoid base and fatty acid chain length. They are essential for maintaining the skin’s structural integrity, acting as the “grout” between skin cells to form a protective, water-retaining barrier. They are “skin-identical” ingredients and highly effective in reinforcing the natural skin barrier.
Although ceramides, which are one class of sphingolipids, are lipids that are available only in an extremely small amount with respect to the whole body, in the stratum corneum which is the outermost layer of the skin, ceramides are contained more than half of the amount of lipids, and play the important roles in the water holding function and barrier function of the skin. These ceramides are produced in the epidermal cells, then secreted to form a lamellar structure in intercellular spaces of the stratum corneum, and they maintain the stratum corneum functions. It has been extensively reported that, in skin diseases such as dry skin, rough skin, atopic dermatitis, senile xerosis and psoriasis, normal metabolism of ceramides is interfered and the amount of ceramides in the stratum corneum decreases, followed by occurring deterioration of the water holding function, barrier function and the like of the skin. (US US 20120196841)
Ceramides are composed of a long-chain base (LCB) and a FA linked by an amide bond. Vie types of LCBs (sphingosine [S], dihydrosphingosine [DS], 6-hydroxysphingosine [H], phytosphingosine [P], and 4,14-sphingadiene [SD]) and five types of FAs (non-hydroxy FA [N], alpha-hydroxy FA [A], w-hydroxy FA [O], esteried w-hydroxy FA [EO] and protein-bound FA [PB]) exist in human ceramides. Accordingly, human ceramides are classified into 25 classes based on the different combinations of FAs and LCBs, and each class is represented by a combination of the abbreviations for the FA and LCB. Each ceramdie class contains various ceramide species composed of LCBs and FAs with different carbon chain lenghts and/or number of double bonds and there are over 1500 ceramide species in the human SC. Ceramides with EO-type FAs are called w-O-acylceramides. In acylceramides, linoleic acid is mianly esterifed at the w-position of the w-hydroxy FA moeity. Acylceramides are important for the formation and maintenance of the lipid lamellae. Mutations of thee genes invovled int he production of acyceramides or protein-bound ceramides cause congenital ichthyosis. (Akyama, “Correlations between skin condition parameters and ceramide profiles in the stratum corneum of healthy individuals” Int. J. Mol. Sci. 2024)
—-Ceramide PC-104 (N-(3-hexadecyloxy-2-hydroxypropyl)-N-2-hydroxyethyl hexadecanamide): is a synthetic pseudoceramide designed to mimic the function of natural ceramides in the skin. The primary difference is its origin and specific chemical structure, which offer better formulation stability and cost-effectiveness compared to some natural or animal-derived ceramides. It is synthetically manufactured, though its components may be derived from natural fatty acids.
Corium or Dermis:
Introduction: Dermis refers to the layer of the skin deep to the epidermis, consisting of a dense bed of fascular connective tissue, and containing the nerves and terminal organs of sensation. The hair roots (follicles), and sebaceous and sweat glands are structures which are also embedded in the dermis.
The dermis is the connective tissue layer beneath the epidermis and it is well connected with epidermis via dermal-epidermal junctioon that includes lamina densa, lamina lucida and several proteins such as collagen anoring fir. Dermis is the primary source of elasticity, flexibility, and tensile strenght of the skin. (Guiliana, US US 20110301091)
The dermis is the layer just below the outer keratinized epidermal layer. The dermis contains cells, water, collagen fibers, glycosaminoglycans and fibronectins that form a hydrated gel and are responsible for the high elasticity and tensile strength of the dermis. Embedded in this layer are lymph channels, blood vessels, nerve fibers, muscle cells, hair follicles, sebaceous glands, and sweat glands.
The dermis is a rich matrix of fibroblast cells and fibers such collagen, and it contains macrophages and mast cells. The dermis also harbors a dense network fo nevers, blood vessels, and lymphatic vessels. Damage to the epidermis generally does not result in bleeding, whereas damage deep enough to penetrate the dermis results in broken blood vessels.
The dermis also contains collagens. Type I collagen is the most abundant protein in skin connective tissue, which also contains other types of collagen (III, V, VII), elastin, proteoglycans, fibronectin, and other extracellular matrix proteins. Newly synthesized type I procollagen is secreted into the dermal extracellular space where it undergoes enzymatic-processing, arranging itself into a triple helix configuration. The triple helix complexes associate with other extracellular matrix proteins such as leucine-rich small proteoglycans, to form regularly arranged fibrillar structures. This process, called fibrillogenesis, results in formation of collagen bundles that are responsible for the strength and resiliency of the skin. (Guiliana, US US 20110301091)
Dermal-epidermal Junction (DEJ): DEJ is a critical component of the skin and is composed of a network of structural proteins that provide a firm connection between the basal keratiocytes of the epidermis and the dermis. This structural network is made up of (1) the hemidesmosome-anchoring filament complex; (2) the basement membrane comprising two layers, the lamina lucida and the lamin densa and (3) anchoring fibrils.
Hypodermis:
The hypodermis is the third skin layer and its primary funciton is that of protection and cushioning. Unlike the primarily fibrous composition of the dermis above it, the hypodermis is an adipose tissue and lipid rich layer and integrates with epidermis and dermis through a complex network of nerves and vessels. Sweat glands and the bulbs of hair follicles extend into teh hypodermis as well. The blood supply provided to the skin varies depending on region and functionality of that region.
Sebaceious (oil) glands and scent glands:
Sebaceious (oil) glands are associated with the hair follicle. All of these glands have opening on the surface of the skin, so they pass through the epidermis as well. The sebasceous glands secretion, celld sebum, has a low pH, hwicch makes the skin inhospitable to many microorganisms. Sebum is oily due to its high concentraiton of lipids. The lipids can serve as nutrients for normal microbiota, but breakdown of the fatty acids contained in lipids, leads to toxic by products that inhibit the growth of microorgansms not adapted to the skin environment. Lysozyme is an enzyme found in sweat (and tears and saliva) that specifically breaks down peptidoglycan, found in bacterial cell walls.
Blister formation, the result of friction trauma or burns, represents a separation between the dermis and epidermis.
Cell Types Present in Skin:
Various cell types are present in the skin. Keratinocytes is the most abundant cell type in the epidermis. These cells produce keratin proteins. Fibroblasts differentiate into cells that form the dermis and produce collagen and elastin. Melanocytes produce the pigment melanin that accumulates around the nuclei of the keratinocytes absorbing harmful ultraviolet (UV) light. Langerhans cells (macrophages) reside in the dermis mediating humoral and cellular immune functions. Merkel’s cells, which are present in small numbers but are more numerous in the skin of the palms and soles of the feet, are sensory mechanical receptors that respond to certain stimuli such as pressure or touch.
Cells of the Epidermis:
–Introduction:
Epidermis refers to the outermost and novascular layer of the skin, derived from the embryonic ectoderm, varying in thickness from 0.07-1.4 mm. The epidermis is a complex epithelial tissue containing keratinocytes that are proliferating, differentiating and desquamating, and is stratified such that morphological functional changes in the keratinocytes occur in an orderly progression. The normal epidermis is maintained in a dynamic steady state as proliferation of keratinocytes continually compensates for the loss of cells which are shed from the surface of the skin. Within the epidermis, proliferation takes place in the basal layer of keratinocytes that are attached to the underlying basement membrane, and cells undergo differentiation as they migrate through the suprabasal layers, finally being shed from the tissue surface as dead, cornified squames. Three subpopulations of basal keratinocytes have been defined by cell kinetic analysis: stem cells, transit-ampliyfing cells, and committed cells. (Guiliana, US US 20110301091)
Skin cells which make up the epidermis include Merkel cells, keratinocytes, melanocytes and Langerhans cells.
Langerhans cells are specialized antigen-presenting immune cells found primarily in the stratum spinosum of the epidermis. They extend dendritic processes between keratinocytes, where they can capture antigens that enter through the skin.
The human epidermis is a pluristratified tissue made up of basal, spinous, granular, upper-granular and cornified layers, in ascending order. The keratinocytes in the basal layer express keratins K5 and K14, as well as K15, which are then substituted by markers of epidermal differentiation such as K1, K10, IVL in the spinous layer. With progression of differentiation, other markers such as FLG and loricrin are expressed in the granular layer. The keratinocytes at the level of the cornified layer are called corneocytes as they are devoid of organelles and nuclei and are finally sloughed off.
Lamellar granules (also called lamellar bodies or Odland bodies) are small lipid-containing organelles found primarily in keratinocytes in the upper layers of the epidermis. They’re extremely important for forming the skin barrier.
As keratinocytes mature and move toward the skin surface, lamellar granules accumulate substances including ceramides and their precursors, cholesterol, free fatty acids, and enzymes involved in lipid processing.
When a keratinocyte reaches the boundary between the stratum granulosum and stratum corneum, the lamellar granules fuse with the cell membrane and release their contents into the extracellular space. Those lipids then organize into the lamellar lipid layers between corneocytes.
The process is as follows: Keratinocyte → produces lamellar granules → granules carry barrier lipids → contents secreted between cells → lipids organize into lamellae → functional stratum-corneum barrier.
Lamela granules are small organelles with a bounding membrane, most prominent in the granular cell layer of the epidermis and visible only by electron microscopy. They contain stacks of lipid lamellae composed of phospholipids, cholesterol and glucosylceramides. that are the precursors of the SC intercellular lipids. Late in epidermal differentiation, at the transition form ganular cell to corneocyte, LG are thought to fuse with the PM of the ganular cell and discharge theri lipid membranes in to the intercellular space. Madison, Barrier Funciton of the Skin: “La Raison d’Etre” of the Epidermis” The Society for Investigative Dermatology, Inc., (2003).
LG are particulalry enriched in a lipid unique to keratinizing epithelia, acyglucosylceramide (AcylGlcCer). This unusual lipid has a very long chainehydroxy fatty acid moeity (C28-36) with linoleic acid (an essential fatty acid) ester-linked to the hydroxyl group. The interior lipid lamellae of LG have been suggested to arise form the flattening and stocking of lipid vesicles and AcylGlcCer has been proposed to funciton as a molecualr revet to accomplish this process. Madison, Barrier Funciton of the Skin: “La Raison d’Etre” of the Epidermis” The Society for Investigative Dermatology, Inc., (2003).
–Keratinocytes: Keratinocytes, which are the main cell type in the epidermis, are subject to cycles of proliferation, differentiation and death that allow preservation of epidermal homeostasis as well as of epidermal barrier function. They maintain their proliferative capacity in the basal epidermal layer and when starting to differentiate they cease dividing and move to occupy the suprabasal spinous, granular, upper-granular and cornified layers. In the suprabasal layers, the keratinocytes express markers of terminal differentiation, until they ultimately loose nuclei and organelles and become corneocytes.
The keratinocytes, which are the main cell type in the epidermis, are subject to cycles of proliferation, differentiation and death that allow preservation of epidermal homeostasis as well as of epidermal barrier function. They maintain their proliferative capacity in the basal epidermal layer and when starting to differentiate they cease dividing and move to occupy the suprabasal spinous, granular, upper-granular and cornified layers. In the suprabasal layers, the keratinocytes express markers of terminal differentiation, until they ultimately loose nuclei and organelles and become corneocytes. Corneocytes are essentially bundles of filaments surrounded by cross-linked proteins and insoluble lipids and are ultimately shed out as dead cells.
Among the proteins that keratinocytes express during the distinct stages of differentiation, there are keratins, which are usually organized in heterodimers to constitute the keratin intermediate filaments (KIFs) that extend from the cell nucleus to the cell-cell desmosomes junctions. The basal keratinocytes typically express keratin 5 (K5) and 14 (K14). With differentiation, K5 and 14 are replaced by K1 and K10 and other “specialised” keratins such as K9 in the palms and soles’ skin and K2 in thickened skin areas. Keratin 15 (K15), which in the hair follicles (HFs) is associated with bulge stem cells, is also expressed by undifferentiated keratinocytes and lost when keratinocytes differentiate. Other proteins that mark epidermal terminal differentiation include, but are not limited to, involucrin (IVL), loricrin, filaggrin (FLG), trichohyalin (TCHH) and small proline-rich proteins (SPRRs).
—Corneocytes: are highly specialized terminally differentiated cells of the epidermis. Corneocytes form the stratum corneum, the outermost layer of the skin. During differentiation, they lose their nucleus and most organelles, become packed with keratin, and acquire a tough cornified envelope. So they are unusual cells: they’re essentially dead, flattened, anucleate cells, but they are critically important to the skin barrier. A useful analogy for the stratum corneum is the “bricks and mortar” model: Corneocytes = bricks and Intercellular lipids (ceramides, cholesterol, free fatty acids) = mortar.
Corneocytes are essentially bundles of filaments surrounded by cross-linked proteins and insoluble lipids and are ultimately shed out as dead cells. Among the proteins that keratinocytes express during the distinct stages of differentiation, there are keratins, which are usually organised in heterodimers to constitute the keratin intermediate filaments (KIFs) that extend from the cell nucleus to the cell-cell desmosomes junctions.
Anucleate coreocytes contain keratin filaments bound to a peripheral cornified envelope composed of cross-linked proteins. The many layers of these specialized cells in the stratum corneum provide a tough and resident framework for the intercellular lipid lamellae. The laemlaae are derived from disk-like lipid membranes extrded from lamellar ganules into the intercellular spaces of the upper granular layer. Lysosomal and other enzymes present in the extracellular compartment are responsible for the lipid remodeling required to generate the barrier lamellae as well as for the reactions that result in desquamation. (Madison, Barrier Funciton of the Skin: “La Raison d’Etre” of the Epidermis” The Society for Investigative Dermatology, Inc., (2003).
Each corneocyte has about 10 nm thick tough peripheral protein envelope, called the cornified envelope, this is composed of several structural proteins, notably involucrin and locricrin, cross-linked by sulfhydryl oxidases and transglutaminases. The interior surface of the cornified envelope is linke to the bunldes of keratin filaments that fill the intracellular compartment of corenocyte. Madison, Barrier Function of the Skin: “La Raison d’Etre” of the Epidermis” The Society for Investigative Dermatology, Inc., (2003).
Corneocytes are normally shed in small enough groups that they are not visible on the skin surface; when this process is distrubed corneocytes collect in visible clubs (scales) that produce a roughg texture and appearance. Flaky skin, often called “dry” skin is a cutaneous reaction pattern reflecting abnormal desquamation of diverse etiologies.
The lipid envleop acts as an interface between the stacked layers of corneocytes, forming a lipophilic and non-polar layer between the hydrophilic corneocytes. The intercellular lipid layers between layers of corneocytes are a complex matric sonsisting of a wide variety of ceramides, cholesterol, cholesterol esters, and free fatty acids. (Eberting, US2016/0184245A1)
Cells of the Dermis:
—Dermal fibroblasts are cells found primarily in the dermis, the connective-tissue layer of your skin underneath the epidermis. Their major job is to make and maintain the skin’s extracellular matrix—especially collagen and elastin. They’re also important in wound healing. When skin is injured, fibroblasts become activated, migrate toward the injury, proliferate, and produce collagen and other matrix material to help repair the wound.
Fibroblasts can be experimentally reprogrammed into pluripotent cells. This is the basis of induced pluripotent stem cells (iPSCs).
Skin Models:
Introduction: When designing a skin model there are various chemical and physical considerations that need to be considered to produce a biomimetic design. This includes designing a structure that mimics the structural characteristics and mechanical strengh needed for tribological property measurement and toxicological testing. (Suhail, “Engineered skin tissue equivalents for product evaluation and therapeutic applications” Biotechnol J. 2019, 14(7))
In Vitro Models: Current in vitro models are largely epidermal, and include EpiSkin, EpiDerm and SkinEthic. (Suhail, “Engineered skin tissue equivalents for product evaluation and therapeutic applications” Biotechnol J. 2019, 14(7)). Epidermal Skin Test 1000 (CellSystems Biotechnologic GmbH) is an epidermal reconstructed model composed of primary human keratiocytes, resulting in differentiated cornified skin layers and is primarily sued for skin corrosiveness testing.
Episkin is a model that was acquired by L’Oreal in 1997 and is marketed as a 12 well plate system. It is comprised of a type 1 bovine collagen matrix, type IV human collagen, and passaged human keratinocyte cells.
EpiDerm is an in vitro skin model marketed by MatTek Corporation from Ashland, MA. It is a normal human derived epidermal keratinocytes whcih have been culstured to form a multilayered, highly differentiated model of the human epidermis. EpidermFt featrues neonatal human derived dermal fibroblasts and neonatal human derived epidermal keratinocytes in co-culture that form a multi-layered differentail epidermal-dermal model.
Skin Ethic is an artificial human skin model originating from the Martin Rosdy Laboratories in Nice, France. The SkinEthic model consists of a stratum corneum, stratum granulosum, and stratum spinosum.