Histochemist

ry of
Gingiva
Dr. Sandip Ladani
 What is histo
chemistry?
logy?
• It is a histological technique used for
studying chemistry of tissues and
cells
– Histochemistry
– Enzyme histochemistry
– Immunocytochemistry
– In situ hybridization
 Need for Histochemistry
• The microscopical identification -
largely morphological.

• Histochemical techniques - chemical

components, enzyme systems,
physiologic processes, and the
changes that occur in disease.
 Gingiva
• Epithelium
• Basement
membrane
• Lamina Propria
 Gingival Epithelium
• Keratinocyte
• Enzymes
• Non-Keratinocytes
IMMUNOHISTOCHEMISTRY
 Keratins
• Different polypeptide subunits
• High sulfhydryl and disulfide
content: the performic acid-alcian
blue technique. The disulfide stains
Blue.
• Stains red with H&E
• Keratin retains the red phloxine
stain avidly, so Lendrum’s phloxine-
tartrazine technique is a suitable
method for its demonstration.
• Keratohyaline granules stains deep
blue with H&E stain.
CYTOKERATINS
 KERATOLININ & INVOLUCRIN
• Produced during maturation process.
• These are precursors of chemically resistant
structure, envelope located below cell membrane.

FILAGGRIN
• Precursors of filaggrin are packed into
keratohyaline granules.
• It forms the matrix of the corneocyte.
Cytoplasmic organelles & Enzymes
• Mitochondria are more numerous in deeper strata
and decrease toward the surface of the cell.
• Altmann’s acid fuchsin-picric acid technique for
mitochondria in which mitochondria stains red and
background tissue yellow.

• Basal & Parabasal cells show :

Succinic Dehydrogenase (SD)
Nicotinamide adenine dinucleotide
Cytochrome oxidase

• More active TCA cycle

Cytoplasmic organelles & Enzymes
SURFACE LAYER
• Enzymes of pentose shunt such as G6PD

• This pathway produces larger amount of

intermediate products which are used for the
production of RNA, which in turn can be used for the
synthesis of keratinization proteins.

• This histochemical pattern is in accordance with the

increased volume & amount of tonofilaments
observed in cells reaching the surface; the intensity
of the activity is proportional to the degree of
differentiation.
• SD content was greater in attached gingiva than in all
other zones. Its concentration decreases from basal
to superficial layers.

• This indicates a reduction in oxidative activity in

superficial layers. It may mean that these epithelial
areas require less energy release due to lowered
functional metabolic requirements.

• G-6-PD had its maximum content in epithelium of

marginal gingiva & lower content in oral mucosal
epithelium, crevicular epithelium & JE.

• This indicates need for higher energy release in areas

of heavier keratinization & greater mechanical
requirements.
- Iotiz & Carranza,1972
 Glycogen
• Glycogen can accumulate intracellularly when it is not
completely degraded by any of the glycolytic pathways.
• Thus, its concentration in normal gingiva is inversely related to
the degree of keratinization and inflammation.
• McManus’ PAS method of staining.
• Some researchers consider it to be a normal component of
epithelium; others find it only in acanthosis, usually associated
with inflammation.
• Phosphorylase activity generally occurs in the epithelium where
glycogen is located.
 Nucleic Acid
• RNA
– large quantities in the basal cells
– in lowest concentration in the crevicular epithelium.
• DNA
– normally present in the nucleus of all gingival cells
– is increased in gingival hyperplasia.

• The DNA and RNA activity of the epithelium at the gingival

margin and junctional epithelium is greater than in the
remaining oral mucosa.
• Feulgen’s reaction for DNA:
– hydrolysis of DNA by hydrochloric acid
– this process leads to the formation of aldehyde groups
– free aldehyde groups react with the Schiff reagent
– result: insoluble red substance
• RNA-rich organelles are stained with basic dyes
i.e.: toluidine blue, methylene blue
 Acid phosphatase
• The uppermost cells of the stratum spinosum contain
numerous dense granules, keratinosomes or Odland
bodies, which is modified lysosomes.

• They contain a large amount of acid phosphatase, an

enzyme involved in the destruction of organelle
membranes, which occurs suddenly between the
granulosum and corneum strata and during the
intercellular cementation of cornified cells.

• Acid phosphatase can be detected by

– The Gomori lead method – black color of acid
phosphatase activity.
– Azo dye method – red color
 Melanocytes
• They synthesize
melanin in organelles
called permelanosomes
or melanosomes.
• Melanin granules are
phagocytosed and
contained within other
cells of the epithelium
and connective tissue
called melanophages or
melanophores.
• Masson-Fontana
method – black color
• DOPA reaction
 Langerhans Cells
• Antigen presenting cells

• Birbeck’s granules

• Marked ATPase activity

• Therefore, are stained by

Wachstein lead method-
Brown or Black precipitate.
 Basement membrane
• The basal lamina consists of
– Lamina lucida
• Glycoprotein laminin
– Lamina densa
• Type IV collagen

• The anchoring fibrils

• The complex of basal lamina

and fibrils is the periodic acid-
Schiff (PAS) positive and
argyrophilic line observed at
the optical level.
• Periodic acid-methenamine
silver microwave method : BM-
black, Background-Green
 Lamina Propria
• Fibers
• Ground substance
 Staining reactions of
collagen
• Type I collagen stains strongly with acid
dyes, due to the affinity of the cationic
groups of the proteins for the anionic
reactive groups of the acid dyes.
• They may be demonstrated more
selectively by compound solutions of acid
dyes e.g. van Gieson or by sequential
combinations of acid dyes e.g. Masson’s
trichrome, Lendrum’s MSB, etc.
 Reticular Fibers
• Reticular fibers may be
demonstrated distinctly, in paraffin
sections, using one of the many
argyrophil-type silver impregn ation
techniques available or in frozen
section, by the periodic acid-Schiff
technique.

• Both methods of demonstration are

dependent upon the reactive groups
present in the carbohydrate matrix
and not upon the fibrillar elements of
the fiber.

• Reticulin fibers are present at the

epithelium-connective tissue and
the endothelium-connective tissue
interfaces.
 Elastic Fiber System
• Oxytalan
• Elaunin
• Elastin
• Stains:
– Verhoff’s method – black color
– Resorcin-fuchsin method – Brown to purple
– Methyl violet/ethyl violet-resorcin method
for elastic fibers – Blue-black
 Oxytalan Fibers
• Oxytalan fibers are scarce
in the gingiva.

• Long thin fibrils with a

diameter of approximately
150 A.

• These connective tissue

fibers can be demonstrated
light microscopically only
after previous oxidation
with peracetic acid,
potassium permanganate, &
performic acid.
 Elaunin
• Stain with orcein, aldehyde fuchsin
and resorcin-fuchsin without prior
oxidation.
 van Masson H & E PAS
Gieson Trichrome

Collagen Red Blue Deep +

green pink
Elastin Yellow Pink -

Reticulin Yellow Blue ++

green
Basement Yellow Blue Pink +++
membranes green
 Ground Substance
• It consists of:
– proteoglycans
• hyaluronic acid
– chondroitin sulfate
– Glycoproteins
• fibronectin

• Glycoproteins account for the faint PAS-

positive reaction of the ground substance.
 Saccharides
• PAS reaction (periodic acid-Schiff)
• It is based on oxidative action of
periodic acid (HIO4) → aldehyde groups
• These aldehyde groups react with
Schiff’s reagent (as in Feulgen’s
reaction)
• A new compound with a purple colour
(PAS-positive substances)
 Clinical application
• PAS-positive substances are:
– polysaccharides (glycogen)
– glycosaminoglycans /mucopolysaccharides/
(hyaluronic acid, chondroitin sulphate)
– proteoglycans
– glycoproteins (thyreoglobulin, collagen)
– glycolipids (lipofuscin)

• Biopsies of tissues from patients with diseases

that store glycogen (glycogenosis),
glycosaminoglycans (mucopolysaccharidosis), etc.
 Blood Vessels
• Blood vessels are easily evidenced in tissue sections by
means of immunohistochemical reactions against proteins
of endothelial cells (factor VIII and adhesion molecules).

• Before these techniques were developed, vascularization

patterns of periodontal tissues had been described using
histoenzymatic reactions for alkaline phosphatase and
adnosine triphosphatase because of the great activity of
these enzymes in endothelial cells.
 Blood Vessels
• In experimental animals the
perfusion with India ink also was
used to study vascular distribution.
The injection and subsequent
demonstration of peroxidase allow
blood vessel identification and
permeability studies.
• The PAS reaction also outlines
vascular walls by a positive line in
their basal membrane.
 References
• Carranza’s Clinical Periodontology, 7th
, 10th edition.
• Clinical Periodontology and Implant
Dentistry by Jan Lindhe, 4th edition.
• Theory and Practice of histological
techniques by John D Bancroft &
Marilyn Gamble, 6th edition.
• Various Internet sources.