Basic Skin Histology

Introduction to Skin Histology

The skin is considered the largest organ of the body and
has many different functions.
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.

By:
Mohamed Antar Aziz
Mohamed

**Epidermis

Skin
layers

he 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),
thestratum granulosum(SGR),andthe stratum corneum(SC)

1)Stratum germinativum
Provides the germinal cells necessary for the regeneration of the
layers of the epidermis.
Separated from thedermis 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
Thestratum corneumis the outermost layer of theepidermis,
consisting of deadcells(corneocytes) that lacknuclei and
organelles.
Desquamation, the process of cell shedding from the surface of
thestratum corneum, balances proliferatingkeratinocytesthat
form in thestratum basale.

Types of
skin
Thick
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

Thin skin
*4 layers
*less
Prominent
stratum
corneum
* Less
developed
stratum
granulosum
* Dominant
and lines
most of the
body surface
* Thicker
dermis
* hair and
sebaceous

TYPES OF EPIDERMAL CELLS

(1)-keratinocytes:

They are responsible for keratin

formation
Formed of many layers that
continuously shed and regenerate
2-4 weeks
eyevery
are arranged
In many layers

2)-Melanocytes:

nd inbetween cells of the basal layer

Branched cells with centeral nuclei by EM
contains organells for protein
synthesizes (rER, Golgi, mitochondria
&melanosomes).
They form melanin by tyrosinase from
tyrosine amino acid by converting it
to dioxyphenyl alanine DOPA.

3)- Langerhans cells:

Langerhans cells aredendritic cells (antigen-presenting

immune
cells)
of theskin.
ound
in upper
layers
of st.spinosum

ave branched shape &central nuclei

Represent 3-8%of epid. Cells

(4)-Merkel cells

Found in basal cell layer

They are modified epidermal cells Sensory nerve fibers
form terminal disk under
Merkels cells
Function as touch receptors

**Dermis

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

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, andAdipocytes.

A Mast cell contains manygranules rich inhistamine andheparin.

Although best known for their role inallergyandanaphylaxis,
mast cells play an important protective role as well, being
intimately
involved
in
wound
healing
and
defense
againstpathogens.
Apart from thesecells, the dermis is also composed ofmatrix
components
such
ascollagen(which
providesstrength),elastin(which
provides
elasticity),
andextrafibrillar matrix, an extracellular gel-like substance
primarily
composed
ofglycosaminoglycans(most
notablyhyaluronan),proteoglycans, andglycoproteins)

Fibroblast
Fibroblastis a type ofcellthat synthesizes theextracellular
matrixandcollagen and plays a critical role inwound healing.
Fibroblasts have a branchedcytoplasmsurrounding an elliptical,
specklednucleushaving two or morenucleoli. Active fibroblasts
can be recognized by their abundant roughendoplasmic
reticulum. Inactive fibroblasts, which are also calledfibrocytes ,
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

Eccrine sweat gland

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

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

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

Culture in Vitro
Dermal Fibroblast

Keratinocytes

FGM-2
medium
(containing 2%
fetal
Culture
bovine serum , 0.1%
Medium:
recombinant
human
fibroblast growth factor
(rhFGF), 0.1% insulin, 5
units/ml
heparin,
100
units/ml of penicillin, and
Remove
medium,
add
Subculturing:
100mg/ml
of
0.05% EDTA solution and
streptomycin.
incubate for 2 min at 37C.
Add fresh medium, aspirate
and dispense into new
times weekly
Doubling time 2 flasks.

Cell stock:
:
1

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).
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 weekly

Free DMEM :
:

DMSO

4
FBS

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 forVitamin 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
(7)-Cut the
medium
. 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.

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.

-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

trifuge at 200g for 5 min, then cutlure fibroblast cells by using growth m

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)