defensive mechanisms of the gingiva (saliva, sulcular fluid, epithelial surface, immune response)

1. Defensive mechanisms of the gingiva
Presented by: Toleen Mazloum
Periodontology

2.  Outline
Defensive mechanisms of the gingiva:
 Gingival crevicular fluid
 Saliva
 Immune response (leukocytes)
 Epithelial barrier
 Sulcular fluid
 Methods of collection
 Permeability of junctional & sulcular epithelia
 Amount
 Composition
 Clinical significance
 Cellular and humoral activity in GCF
 Drugs in GCF
 Immune response
 Leukocytes in dentogingival area
 Saliva
 Role of saliva
 Antibacterial factors
 Salivary antibodies
 Salivary buffers & coagulation factors
 Leukocytes
 Enzymes
 Role in periodontal pathology
 Epithelial surface

3.  Introduction:
– The gingival tissue is constantly subjected to mechanical &
bacterial aggressions
– Thus the gingiva defend itself through 4 vital components:
 Epithelial barrier
 Sulcular fluid
 Saliva
 Immune response (leukocytes)
 Sulcular fluid (1,2)
– Also known as gingival crevicular fluid (GCF)
– It is a complex mixture of substances derived from serum,
leukocytes, structural cells of the periodontium & oral bacteria
– It was first discovered by Waerhaug, Brill & Krasse during the
1950s
– Brill considered that the GCF is a continuous transudate
– However, others like Waerhaug considered that GCF is an
inflammatory exudate
֍ Methods of collection
1- The use of absorbing of paper strips
 The absorbent paper strips are placed:
 Placement of the filter paper strip in relation to the sulcus or
pocket is important
within the sulcus (intra-sulcular method)
at its entrance (extra-sulcular method)

4. A) Intra-sulcular method (Brill's technique)
B) Extra-sulcular method (Löe and Holm-Pedersen)
2- Twisted threads (Weinstein)
3- Micropipettes
insert the end of the strip
into the sulcus until
minimum resistance is felt
thus producing some
degree of irritation of the
sulcular epithelium
triggering the flow of fluid
insert the filter paper
strip just at or over the
pocket entrance
thus minimizing the
irritation caused by the
previous technique
the strip picks the fluid
that seeps out while the
sulcular epithelium isn't in
contact with the paper
preweighted twisted threads
are placed in the gingival
crevice around the tooth
the amount of fluid collected
is estimated by weighing the
sample thread
- capillary tubes of
standardized length &
diameter are placed in the
pocket
- later their contents are
centrifuged & analyzed

5. 4- Intracrevicular washing
 Crevicular washings can be used to study GCF from clinically
normal gingiva
 1st
method:
 2nd
method:
֍ Permeability of junctional & sulcular epithelia
– Substances shown to penetrate the sulcular epithelium are:
– thus substances of molecular weight up to 1000 kD can
penetrate the sulcular epithelium
use a hard acrylic plate that covers the maxilla with soft borders &
a groove that follows the gingival margins
these are connected to 4 collection tubes
washing is obtained by rinsing the crevicular areas from
one side to the other with the use of a peristaltic pump
use 2 injection needles that are fitted one within the other
during sampling the inside (ejection) needles is at the bottom of
the pocket & the outside (collecting) needle is at the gingival
margin
the collecting needle is drained into a sample tube via
continuous suction
albumin endotoxin thymidine histamine phenytoin
horseradish
peroxidase

6. ֍ Amount
– The amount of GCF collected on a strip paper can be evaluated
by:
1- Staining
 The wetted paper strip is stained by Ninhydrin
 Then it is measured planimetrically on an enlarged
photograph or with a magnifying glass or a microscope
2- Electronic method
 Through the use of an electronic transducer that measures
the fluid collected on a blotter (Periopaper)
 The wetness of the paper strip affects the flow of an electric
current & provides a digital readout
֍ Composition
1- Cellular element s:
2- Electrolytes:
Electronic device (Periotron 8000) for measuring the
amount of fluid collected on filter paper
leukocytes
•neutrophils,
lymphocytes &
monocytes
•numerous at the
base & decrease
coronally
Epithelial cells
•Shed from
junctional or
sulcular epithelium
Bacteria
•Either free
(floating in the
GCF) or attached
to the host cells or
engulfed by
neutrophils
sodium
potassium
(exceeds that
of the serum)
fluoride calcium magnesium phosphate

7. 3- Organic compounds:
4- Metabolic & bacterial products:
5- Enzymes & enzyme inhibitors:
֍ Cellular and humoral activity in GCF
– The analysis of GCF has identified cellular & humoral responses
in both healthy individuals & those having periodontal disease
lactic acid
hydroxyproline
(breakdown of
collagen)
Prostaglandins
(PgE2)
urea
endotoxins
cytotoxic substances
(hydrogen sulphide)
antibacterial
factors
Carbohydrates
•Glucose (3-6 times
that of the serum)
•hexosamine
•hexuronic acid
Proteins
•much less than
that of the serum
•Alpha, beta, alpha
1, alpha 2 globulin
& albumin
Lipids
•GCF contains many
classes of
phospholipids as
well as neutral
lipids
acid
phosphatase
alkaline
phosphatase
pyrophosphatase
beta
glucouronidase
lyzosymes hyaluronidase
proteolytic
enzymes
collagenases
lactic
dehydrogenase

8.  cellular immune response:
- includes cytokines (no clear evidence of a relationship
between them & the disease)
- IL-1α & IL-1β
- Also there is interferon-α in GCF >>> inhibit the bone
resorption activity of IL-1β >>> thus having a protective
role in periodontal disease
 Humoral immune response:
- Includes antibodies secreted against bacteria
֍ Clinical significance
– GCF is an inflammatory exudate which positively correlates
the amount & severity of inflammation
– Its presence in normal sulcus can be explained by the fact that
gingiva appears normal exhibits inflammation when examined
microscopically
– GCF is not increased by trauma from occlusion
IL-1α &
IL-1β
increase the binding of PMNs
& monocytes/macrophages
to endothelial cells
stimulate
the
release of lysosomal
enzymes
stimulate bone
resorption
stimulate the
production of
prostaglandin E2

9. – Factors that affect the amount of GCF:
1- Circadian periodicity
 There is a gradual increase in the amount of GCF from
& then it decreases
2- Sex hormones
 Female sex hormones >>> enhance vascularity >>>
increases GCF flow
 Pregnancy, ovulation & hormonal contraceptive >>>
increases GCP production
3- Mechanical stimulation
 Chewing & vigorous gingival brushing >>> stimulates the
GCF flow
 intrasulcular placement of paper strips >>> minor stimuli
>>> increases the production of GCF.
4- Smoking
 Smoking >>> immediate transient increase in GCF flow
 On the long term >>> smoking decreases the salivary &
GCF flow
5- Periodontal therapy
 After periodontal surgery (during the healing period) >>> there
is an increase in the GCF production
֍ Drugs in GCF
– Some drugs can be excreted through the GCF >>> thus they can
be used in periodontal therapy
– Examples:
metronidazole
tetracycline

10.  Immune response(1)
֍ Leukocytes in dentogingival area
– PMNs are the most common leukocytes present in the gingival
sulcus
– Neutrophils are the first line of defense in the dentogingival
area
– Gingival sulcus is the port of entry of leukocytes into the oral
cavity
– Leukocytes are present in the gingival sulcus even when
histologic sections of adjacent tissue are free of inflammatory
infiltrates
– Mononuclear cells were identified as:
90%
10%
PMNs Mononuclear cells
58%24%
18%
B Lymphocytes
T Lymphocytes
Mononuclear phagocytes

11. – The ratio of T lymphocytes to B lymphocytes is:
– Leukocytes constitute a major protective mechanism against
the extension of plaque into the gingival sulcus
 Saliva(1)
– Saliva is an important physiological fluid that contains a highly
complex mixture of substances
– It is secreted by:
֍ Role of saliva
Role of saliva Salivary components Probable mechanism
Lubrication Glycoproteins; mucoids Coating similar to gastric
mucin
Physical protection Glycoproteins; mucoids Coating similar to gastric
mucin
Cleansing Physical flow Clearance of debris &
bacteria
Buffering Bicarbonate &
phosphate
Antacids
3:1 • in the peripherical blood
1:3 • in the GCF
major salivary glands
parotid gland
submandibular salivary
gland
submental salivary gland
minor salivary glands
located below the
mucosal surfaces of the
mouth

12. Maintaining tooth
integrity
Minerals; glycoproteins;
mucoids
Maturation,
remineralization,
mechanical protection
Antibacterial
action
Immunoglobulin A Control of bacterial
colonization
Lysozyme Breaks bacterial
colonization
Lactoperoxidase Oxidation of susceptible
bacteria
֍ Antibacterial factors
– Saliva carries organic & inorganic factors that affects bacteria &
their products in the oral cavity
 Inorganic factors:
 Organic factors:
 Lysozyme:
- It is a hydrolytic enzyme that breaks the linkage of cell wall of
both Gram positive & Gram negative bacteria
- its targets Veillonella species and Actinobacillus
actinomycetemcomitans
 lactoperoxidase- thiocyanate system:
- it is bactericidal to Lactobacillus & Streptococcus strains
- it prevents the accumulation of lysine & glutamic acid that
are essential for bacterial growth
 lactoferrin:
- effective against Actinobacillus species
ions & gases bicarbonate sodium potassium phosphates
calcium fluorides ammonium
carbon
dioxide

13.  myeloperoxidase: (similar to salivary peroxidase)
- released by leukocytes
- it is bactericidal to Actinobacillus
- it inhibits the attachment of Actinomyces to hydroxyapatite
 defensins
 peptides
 agglutinins (antibodies, fibronectin, β2-macroglobulins, mucins,
glycoproteins…)
֍ Salivary antibodies
– Major &minor salivary glands >>> contributes to all the
secretory IgA (sIgA)
– GCF >>> contributes most of the IgG, complement & PMNs
– salivary antibodies are synthesized locally as they react with
strains of bacteria indigenous to mouth but not that of intestinal
tract
– antibodies in saliva impairs the ability of bacteria to attach to
mucosal or tooth surface
֍ Salivary buffers & coagulation factors
– Salivary buffers maintain the pH at the mucosal surface as well
as the tooth surface
– The most important buffer in saliva is bicarbonate- carbonic
acid system
IgM

14. – Saliva also contains coagulation factors (factors VIII, IX, X,
plasma thromboplastin antecedent & Hageman factor) >>>
accelerate blood coagulation & protect wounds from bacterial
invasion
֍ Leukocytes
– Saliva contains all type of leukocytes, but mainly PMNs
– The number of PMN s increases in cases of gingivitis
– PMNs reach the oral cavity through gingival sulcus
– Living PMNs in saliva = orogranulocytes & their rate of
migration is referred to as orogranulocytic migratory rate
֍ Enzymes
– Enzymes found in saliva are derived from
– The major enzyme present is parotid amylase
– Certain enzymes increase in periodontal disease such as
(hyaluronidase, chondroitin sulfate, aspartate aminotransferase,
alkaline phosphatase ….)
– Saliva also contains tissue inhibitor metalloproteinases (TIMP)
that inhibit collagenases
֍ Role in periodontal pathology
– Saliva affects plaque initiation, maturation & metabolism
– Salivary flow & composition also affects calculus formation,
periodontal disease & dental caries
How?
The removal of salivary glands >>> increased the incidence of
dental caries & periodontal disease. It also delayed wound
healing(3)
salivary
glands
bacteria leukocytes oral tissues
ingested
substances

15. – Xerostomia (reduced salivary flow) increases the prevalence &
severity of periodontal disease
Sjogren's
syndrome
sialothiasis
sarcoidosis
post-
radiotherapy
Mikulicz's
disease
Xerostomia

16.  Epithelial surface (2)
– The epithelium is the first line of defense
– The junctional epithelium is firmly attached to the tooth >>>
forming epithelial barrier against plaque bacteria
– It allows the access of GCF, inflammatory cells as well as
components of the immunoglobulin host defense to the gingival
margin
– Epithelial cells exhibit rapid turnover >>> contributes to host-
parasite equilibrium & rapid repair of damaged tissue
Upon prolonged exposure to bacterial plaque
amplification of the inflammatory response
protective function of the junctional epithelium
starts to erode
intercellular spaces allow the passage of bacteria
& their by-products into the host tissues

17.  References:
1- Newman M, Takei H, Klokkevold P, Carranza F. Newman and
Carranza's clinical periodontology. 13th ed.
2- Subbarao K, Nattuthurai G, Sundararajan S, Sujith I, Joseph J,
Syedshah Y. Gingival crevicular fluid: An overview. Journal of
Pharmacy And Bioallied Sciences. 2019;11(6):135.
3- Rodrigues Neves C, Buskermolen J, Roffel S, Waaijman T, Thon M,
Veerman E et al. Human saliva stimulates skin and oral wound
healing in vitro. Journal of Tissue Engineering and Regenerative
Medicine. 2019