Histochemistry of

GingivaDr. Sandip Ladani

What is histo logy?chemistry?

• 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

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.

Cytoplasmic organelles & Enzymes

• 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 Gieson

Masson Trichrome

H & E PAS

Collagen Red Blue green Deep pink

+

Elastin Yellow Pink -

Reticulin Yellow Blue green ++

Basement membranes

Yellow Blue green Pink +++

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.