Basic Skin Histology The skin is divided into two main regions, the epidermis, and the dermis. The dermis is attached to an underlying hypodermis, also called subcutaneous connective tissue. Introduction to Skin Histology The skin is considered the largest organ of the body and has many different functions. By: Mohamed Antar Aziz Mohamed

**Epidermis Skin layers The most superficial layer of the skin The first barrier of protection from the invasion of foreign substances Keratinocyte The epidermis is subdivided into four layers or strata, the stratum germinativum (SG), the stratum spinosum(SS), the stratum granulosum(SGR), and the stratum corneum(SC)

(1)Stratum germinativum Provides the germinal cells necessary for the regeneration of the layers of the epidermis. Separated from the dermis by a thin layer of basement membrane. After a mitotic division a newly formed cell will undergo a progressive maturation called keratinization as its migrates to the surface.

(2)Stratum spinosum The cells that divide in the statum germinativum soon begin to accumulate many desmosomes on their outer surface which provide the characteristic prickles of the stratum spinosum (SS), which is often called the prickle-cell layer.

(3)Stratum granulosum The progressive maturation of a keratinocyte is charcterized by the accumulation of keratin, called keratinization. The cells of the stratum granulosum (SGR) accumlate dense basophilic keratohyalin granules . These granules contain lipids, which along with the desmosomal connections, help to form a waterproof barrier that functions to prevent fluid loss from the body.

(4)Stratum Lucidum The stratum lucidum is normally only well seen in thick epidermis and represents a transition from the stratum granulosum to the stratum corneum.

(5)Stratum corneum The stratum corneum is the outermost layer of the epidermis, consisting of dead cells (corneocytes) that lack nuclei and organelles. Desquamation, the process of cell shedding from the surface of the stratum corneum, balances proliferating keratinocytes that form in the stratum basale.

Types of skin Thin skinThick skin *5 layers * Prominent stratum corneum * Well developed stratum granulosum * Palms of the hands and soles of the feet * Thinner dermis * No hair and sebaceous glands *4 layers *less Prominent stratum corneum * Less developed stratum granulosum * Dominant and lines most of the body surface * Thicker dermis * hair and sebaceous glands

TYPES OF EPIDERMAL CELLS

(1)-keratinocytes: Formed of many layers that continuously shed and regenerate every 2-4 weeks They are responsible for keratin formation They are arranged In many layers

They form melanin by tyrosinase from tyrosine amino acid by converting it to dioxyphenyl alanine DOPA. (2)-Melanocytes: Found inbetween cells of the basal layer Branched cells with centeral nuclei by EM contains organells for protein synthesizes (rER, Golgi, mitochondria &melanosomes).

(3)- Langerhans cells: Langerhans cells are dendritic cells (antigen-presenting immune cells) of the skin. Found in upper layers of st.spinosum Have branched shape &central nuclei Represent 3-8%of epid. Cells

(4)-Merkel cells Found in basal cell layer Function as touch receptors They are modified epidermal cells Sensory nerve fibers form terminal disk under Merkels cells

The dermis contains mostly fibroblasts which are responsible for secreting collagen, elastin and ground substance that give the support and elasticity of the skin. **Dermis Thermoregulation Supply the avascular epidermis with nutrients The dermis is typically subdivided into two zones, a papillary dermis and a reticular layer.

(1)-Papillary dermis The papillary dermis (PD) contains vascular networks that have two important functions. The first being to support the avascular epidermis with vital nutrients and secondly to provide a network for thermoregulation. The papillary dermis also contains the free sensory nerve endings and structures called Meissners corpuscles also called mechanreceptor which is responsible for light touch

(2)Reticular dermis The reticular layer of the dermis (RD) consists of mainly loose connective tissue The reticular layer of the dermis is important in giving the skin it overall strength and elasticity, as well as housing other important epithelial derived structures such as glands and hair follicles.

Components of the Dermis The dermis is composed of The dermis is composed of three major types of cell: Fibroblasts , Mast cells, and Adipocytes. Apart from these cells, the dermis is also composed of matrix components such as collagen (which provides strength), elastin (which provides elasticity), and extrafibrillar matrix, an extracellular gel-like substance primarily composed of glycosaminoglycans (most notably hyaluronan), proteoglycans, and glycoproteins) A Mast cell contains many granules rich in histamine and heparin. Although best known for their role in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing and defense against pathogens.

Fibroblast Fibroblast is a type of cell that synthesizes the extracellular matrix and collagen and plays a critical role in wound healing. Fibroblasts have a branched cytoplasm surrounding an elliptical, speckled nucleus having two or more nucleoli. Active fibroblasts can be recognized by their abundant rough endoplasmic reticulum. Inactive fibroblasts, which are also called fibrocytes , are smaller and spindle shaped. They have a reduced rough endoplasmic reticulum.

FUNCTIONS OF SKIN Protective function : It is the first line of defense. It protects our body from infection, pathogens, and harmful UV irradiation. Sensory function: Free nerve endings on the skin are sensitive to pain, touch, heat and cold, resulting in either voluntary or reflex activities. Secretory function: Sweat help in temperature regulation and sebum makes skin smooth. Heat regulatory function: Sweating and cutaneous blood flow help in temperature regulation Excretory function: Through the secretion of glands of the skin water, salt, fatty substances and urea are excreted. Synthetic function : Suns ultraviolet rays help in synthesis of natural vitamin D. skin can also manufacture melanin pigment. Water balance: Skin serve a useful means in regulating water balance of the body by perspiration.

Skin Appendages 1-Hair Follicles and hair 2-Sweat Glands Eccrine or merocrine sweat glands Apocrine sweat glands 3-Sebaceous glands 4-Nails

(1)-Hair and hair Follicles Hair -Produced by hair follicle which are made of hard keratinized epithelial cells Melanocytes provide pigment for hair color Structure of Hair Follicle

Hair Anatomy Hair anatomy Central medulla Cortex surrounds medulla Cuticle on outside of cortex Most heavily keratinized

Associated hair structures Hair follicle Dermal and epidermal sheath surround hair root Arrector pili muscle Smooth muscle Pulls hairs upright when cold or frightened Sebaceous gland

(2)-Sweat Glands Empty into hair follicle Location: armpits, groin, nipples Viscous, cloudy secretion good nutrient source for bacteria (odor !!) Secretion may contain Pheromones Secretion begins at puberty and is stimulated during emotional distress Apocrine sweat gland Merocrine secretion Empty directly onto skin surface Location: most all over body (esp. abundant on palms & soles: ~ 500/cm2) Clear, watery secretion (99% H2O; rest NaCl + some waste products Eccrine sweat gland

(3)-Sebaceous glands Sebum discharged mostly into hair follicles (lubrication & bactericidal)

4-Nail Nails Scale-like modifications of the epidermis Heavily keratinized Stratum basale extends beneath the nail bed Responsible for growth Lack of pigment makes them colorless Nail Anatomy Nail structures Free edge Body is the visible attached portion Root of nail embedded in skin Cuticle is the proximal nail fold that projects onto the nail body

Keratinocytes Culture in Vitro Dermal Fibroblast DMEM medium (high glucose) supplemented with 2 mM L- glutamine ,10% fetal bovine serum and and antibiotics (100 unit/ml penicillin, 100 g/ml streptomycin). Culture Medium: Subculturing: Remove medium, add 0.05% EDTA solution and incubate for 5 min at 37C. Add fresh medium, aspirate and dispense into new flasks. 3 times weeklyDoubling time FGM-2 medium (containing 2% fetal bovine serum , 0.1% recombinant human broblast growth factor (rhFGF), 0.1% insulin, 5 units/ml heparin, 100 units/ml of penicillin, and 100mg/ml of streptomycin. Remove medium, add 0.05% EDTA solution and incubate for 2 min at 37C. Add fresh medium, aspirate and dispense into new flasks. 2 times weekly Cell stock: 5 : 4 : 1 Free DMEM : FBS : DMSO

Field to be used (cell therapy) Keratinocytes and dermal fibroblasts cells have allowed the characterization of several processes, such as their utilization as a model system for Vitamin D metabolism in the skin, Skin ageing ,wound healing, skin cancer model, the study of growth factor behavior, toxicity/irritancy studies and other skin diseases like atopic dermatitis and psoriasis.

Isolation of human keratinocytes from skin (1)-Place skin tissues in cold HBSS (or EpiLife medium) containing antibiotics and antimycotics and keep at 4 C until use. (2)-Place the tissues in an uncoated 100-mm bacterial Petri dish and keep moist with some medium . (3)-Remove subcutaneous fat and loose connective tissues (hypodermis) using fine tweezers and a scalpel (4)Use the edge of the scalpel to scrape away tissues until only the thin epidermis and the dense dermis remain . With foreskin samples, cut the piece into strips about 34 mm width. (5)-Place the pieces with the dermal side down in a 60- mm dish containing 56 ml of HBSS (or EpiLife) with antibiotics and dispase . Cover and store the pieces in a sterile place at 4 C for 1216 h (overnight).

(6)-Take the overnight digested tissue pieces and grab the edge of the dermal part and the epidermal part with tweezers and slowly peel off the epidermis . Immediately transfer the epidermis (almost transparent) into another dish with either HBSS or EpiLife medium . (7)-Cut the epidermis using a scalpel into small pieces (8)-Place the minced tissue pieces in a Falcon tube containing TrypLE Select (9)-incubate at 37 C for 4045 min. Mix the sample gently every 5 min. The solution should become turbid. (10)-Add 2030 ml of the medium containing a minimum of 10% serum (vol/vol) (e.g., DMEM or RM + ) and pipette the solution vigorously up and down for 1015 times (11)-Pass the solution through a 70-m mesh filter into a new Falcon tube to remove undigested pieces of tissue. (12)-Centrifuge at 200g for 5 min and remove the supernatant and resuspend in 5 ml EpiLife medium. (13)-Count the number of cells and seed out about 2.5 106 cells

Isolation of dermal fibroblasts from skin (1)-Place skin tissues in cold HBSS (or EpiLife medium) containing antibiotics and antimycotics and keep at 4 C until use. (2)-Place the tissues in an uncoated 100-mm bacterial Petri dish and keep moist with some medium . (3)-Remove subcutaneous fat and loose connective tissues (hypodermis) using fine tweezers and a scalpel (4)Use the edge of the scalpel to scrape away tissues until only the thin epidermis and the dense dermis remain . With foreskin samples, cut the piece into strips about 34 mm width. (5)-Place the pieces with the dermal side down in a 60-mm dish containing 56 ml of HBSS (or EpiLife) with antibiotics and dispase . Cover and store the pieces in a sterile place at 4 C for 1216 h (overnight). (6)-After overnight digestion with dispase and removal of the epidermis, the dermis is placed in DMEM culture medium.

(7)-cut the dermis into 0.5- to 1.0-mm pieces (8)-Dip each piece in DMEM culture medium and place directly in tissue culture plates , Incubate in a 37 C, 5% CO2, 90% humidity incubator for a minimum of 4 h up to overnight (9)-Replace with fresh medium every 34 d and remove any tissue pieces that are floating (10)-Within 710 d, outgrowths of fibroblast should appear. (11)_After 1421 d, aspirate the medium and wash twice with PBS. Add 45 ml of a 0.05% Trypsin/EDTA solution and incubate at 37 C for 45 min. (12)-Once fibroblasts have rounded up and some have detached, tap the tissue culture dish on the side to detach the rest of the cells and then immediately add 10 ml of complete DMEM medium (13)-Centrifuge at 200g for 5 min, then cutlure fibroblast cells by using growth medium

Identification of keratinocytes after isolation

Dermal fibroblasts specific markers Specific Markers: P4HB and Collagen I/III Staining of frozen rat skinsections using mouse anti ratProlyl-4-Hydroxylase beta (fibroblastmarker) antibody 6-9H6 (AF5110-1)