1

CONTENTS
1. Introduction
2. Oral defense mechanisms
3. Anatomy of gingival crevice
4. Mechanism of GCF production
5. Functions of GCF
6. Methods of GCF collection
7. GCF volume
8. GCF flow
9. Composition of GCF
10. GCF as a diagnostic marker
11. Limitatons of GCF as a marker
12. Peri-implant sulcular fluid
13. conclusion

2
 Oral cavity and periodontal tissues are exposed to many
factors that may cause disease and tissue destruction such
as bacteria, viruses, masticatory forces, pH differences,
temperature differences, physical irritation, trauma due to
food particles.

 These challenges to homeostasis are met with a variety of

host immune responses.

 Often distinct immune mechanisms are encountered in

different oral environments or different stages of disease.

3
ORAL MUCOSA
-Epithelial barrier
-epithelial renewal
-keratinization
-mucosal permeability
-non-keratinocytes
GCF
SALIVA
MOVEMENTS OF LIPS AND CHEEKS

4
 Oral mucosa

Epithelial cells play an active role in innate host defense by

responding to bacteria in an interactive manner.
EPITHELIAL BARRIER
Mechanical barrier to bacterial penetration
Protects against mechanical damages like mastication and
brushing.
Basement membrane barrier to bacterial penetration

5
 KERATINIZATION
 The protection afforded by the epithelium depends to a great
extent on keratinization.
 Keratins are a group of fibrous protein resistant to hydrolysis
and enzymatic action.

 EPITHELIAL RENEWAL
 High rate of tissue turnover .
 Constant renewal contribute to self cleansing tendency of the
sulcular area.
 The rate of epithelial replacement varies b/w various regions
of oral mucosa. Turnover rate of JE is approximately 4-6 days.
 Superficial injuries are repaired by the rapidity of cell turn-over
also removes the colonized bacteria

6
MUCOSAL PERMEABILITY
The epithelial components of the skin & oral mucosa form the
primary barrier by being selectively permeable to necessary
molecules e.g. leukocytes

NON-KERATINOCYTES

A.Melanocytes
serve to protect the tissue from effect of actinic radiation.

B.Langerhans cell:
dendritic, intra-epithelial cells which are the peripheral arm of
the immune system.
Have functional & cell surface features common with
macrophages, serving as antigen presenting cells.

7
C. Odland Body / Keratinosome

The cells in the stratum spinosum contain numerous dense

granules, keratinosomes.

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.

8
 The gingival sulcus
is the shallow crevice or
space around the tooth ,
bounded by the surface
of the tooth on one side and
the epithelial lining the
free margin of the
gingiva on the other.

9
• Presence of some fluid in this sulcus was known since the 19th
century.

Gingival crevicular fluid /Sulcular Fluid / Gingival Fluid

10
A complex mixture of substances derived from serum, leukocytes,
structural cells of periodontium and oral bacteria.

Fluid occurring in minute amounts in

the gingival crevice believed by some
authorities to be an inflammatory
exudate and by others as transudate to
cleanse material from the crevice,
containing sticky plasma proteins which
improve adhesion of the epithelial
attachment, have antimicrobial
properties and exert antibody activity.
(Jablonski, Illustrated Dictionary of Dentistry, 1982)

Gingival crevice fluid – a unique window to analysis of periodontal condition.

Diagnostic marker of periodontal disease
11
12
 Gingival vasculature :
Arterioles, A repetitive pattern of
Pre capillary arterioles, “capillary units “
Arterial & Venular capillaries
Postcapillary venules
Small venules.
Width and length increases.
INFLAMMATION Twisting and looping of the
vessels.

Network underlying sulcular & junctional epithelium .

(Imp. In production of GCF)
Egelberg (1966): Vessels are arranged in flat layer.
Epithelium does not posses ridges in the connective tissue.
Vasculature is located in a very superficial position irt surface of epithelium

13
Original investigation of BRILL and EGELBERG have shown that
production of fluid is essentially related to an inflammatory
increase in permeability of vessels underlying sulcular & junctional
epithelium.

The fluid produced can simply represent interstitial fluid which

appears in the crevice as a result of osmotic gradient.(Alfanos
hypothesis and Pashleys mathematical model

Exudate Transudate

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Is GCF an inflammatory exudate?

Early studies

Monitored the Monitored the

appearance in GCF of
substances introduced effect on the tissues by
into the parenteral studying the histology
circulation. in gingival biopsies

BRILL & KRASSE: (1958)

Conclusion : Differences in permeability must exist between these

oral epithelia and the epithelium lining the gingival pocket.

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 Vital evans blue dye Bound to albumin will not pass until permeability

Strip was colorless until histamine was injected / mechanical stimulation by

brushing

Brill (1959):recovered a minute but definite amount of blue coloured

substance on the strips even in the absence of inflammation or stimulation

No explanation !!

Egelberg (1966): Introduction of a filter paper in healthy crevice : mechanical stimuli.

Drying of gingiva

Conclusion : Some irritation, whether chemical or mechanical, necessary

to induce the production of GCF (a pathological phenomenon.)

16
Studies of Egelberg et al (1966)
(compared the Measurement of gingival fluid flow with altered permeability of
1) blood vessels)
Suspension of carbon particles
(known particle size)
Injected intravenously into dogs
Sacrificed
Gingival tissue examined
histologically
Mucous membranes show gray color.
Dissappears after 1hour.
As particulate matter is removed
from circulating blood by the R.E.S
“Vascular labeling”
Histological examination:
- healthy tissue: carbon particles
adhered to the walls of blood
vessels.
- acute inflammed tissue: particles
seen in intercellular spaces.
17
 2) increased permeability of blood vessels:

Greater flow of GCF & an increase in vascular permeability. (penetration of

endothelium by carbon particles )

3)Introduction of paper strip

Labeling was observed corresponding to the area of paper strip

Different susceptibilities of chronically inflamed & healthy gingiva

air drying & histamine
Healthy gingiva
Occ. Responded to these stim.
In resting : chronically inflamed gingiva no fluid seen in paper strips.

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Is GCF a transudate of interstitial fluid?

• 2 theories proposed: ALFANO (1974)

PASHLEY (1976)

Initial fluid produced

Interstitial fluid which appears as a result of an osmotic gradient.

Pre-inflammatory fluid a transudate

On stimulation an inflammatory
exudate

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Very early or pre-inflammatory fluid may be
osmotically mediated.

Subgingival Limited qty of Absorbed into

desqamating
plaque in strictly macromolecular epithelial
healthy gingiva by products cells/phagocytosis

Osmotic Reach basement

gradient attracts membrane Permeate
interstitial fluid (Limiting intercellularly
towards sulcus barrier)
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This osmotically modulated fluid is not an inflammatory exudate.

It may progress from an initial osmotically modulated transudate

2⁰ inflammatory exudate.

 When more macromolecules

reach the basal membrane an
inflammatory reaction will start

 Osmotic gradient will be greater

but the fluid now penetrating
the weakened basement
membrane and junctional
epithelium is typically
inflammatory exudate.

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Based on Starling’s factors governing fluid distribution across capillaries.

The passage of fluid from

capillaries into the tissues
(capillary filtration)
&
Removal of the interstitial
fluid by the lymphatics of the
gingiva (lymphatic uptake)

When the production of fluid

from capillaries is greater than
the lymphatic uptake

Fluid will accumulate as

oedema or leave the area as
gingival fluid
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 In health : In inflammation
If the oncotic pressure of the
Oncotic pressure of
sulcular compartment > tissue fluid
Sulcular compartment = Tissue fluid
Because of accumulation of
Oncotic pressure effect cancelled
bacterial by products
Exudation of fluid depend upon
net increase in the flow of GCF.
capillary pressure =(Alfano’s hypothesis)

23
 Factors modulating filtration & uptake:

-Filtration co-efficients of lymphatics & capillary endothelium.

-Hydrostatic & osmotic pressure within the different compartments
-Filtration co-efficients of junctional epithelium & sulcular epithelium
-Difference is oncotic pressure between oncotic pressure of
interstitial fluid and sulcular fluid

• Tissue compliance is “low” Gingival fluid

(capacity of gingival tissue to adapt to increased pressure)

Pashley’s mathematical model support Alfano’s hypothesis

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1) cleanse material from the sulcus
2) contain plasma proteins that may
improve adhesion of the epithelium to the
tooth.
3) Possess antimicrobial properties.
4) Exert antibody activity in defense of the
gingiva.

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I.Effects of Histamine
-Increases after administration
-fluid is derived from plasma
-rate of production depends on gingival
microcirculation.
II.Inflammatory changes of the basement
membrane
-basement membrane may become
thinner or partially disappear
-decrease in coefficient of filtration
,more fluid flow towards sulcus

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III.Morphology of junctional epithelium
-loose organisation of junctional epithelium
allowing large molecules or even cells to penetrate.
IV. Mechanical stimuli and GCF
-Pressure sources such as mastication, tooth
brushing massage may cause increase in GCF
production.
V. Factors such as ovulation ,OCPs ,smoking increases
GCF
VI. Periodontal therapy
-increases during healing period after periodontal
surgery

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Collection of GCF

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 Absorbent Capillary
paper
Several techniques employed fororthe
tubings
collectionstrips micropipettes
Depend upon the objectives of the study.
Upper anteriors : contamination least
possible

Gingival Other
washings methods

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A. Intra crevicular method:

B. Extracrevicular Method:

BRILL TECHNIQUE; 1958 LOE HOLM-PEDERSEN TECHNIQUE; 1965

Until the Entrance

“maximum of
resistance” the
is felt. crevice

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 B. Extracrevicular

Absorbing paper strips are placed

at the entrance of the sulcus.

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Advantages Disadvantage
Quick and easy to use
Introduces degree of irritation
that can trigger the fluid flow
Can be applied to individual
sites

Least traumatic when used

correctly

- - Whatman no 1 or Munktell no.3

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MODIFICATIONS OF INTRA-CREVICULAR TECHNIQUES
1 Rudin et al (1970) utilized paper strips with a standardized
notch at their tips.

The tip of the paper was applied at the sulcus entrance and the
notch could be used as a safeguard against any deeper penetration
and for checking dislocations.

2 Mann (1963) :
Proposed modification which permits the collection of
fluid from a limited area of the crevice , but assuring that
the sample is uncontaminated by saliva.

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 Ths method was first utilized by
KRASSE and EGELBERG.(1962)
• Mann 1963: micropipettes

Isolation and drying of sites

Capillary tubes of known diameters

inserted into the entrance of gingival
crevice

 GCF migrates by capillary action

 Because the internal diameter is
known, the volume of fluid can
be accurately determined by
measuring the distance which
GCF has migrated
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Advantages Limitations
 Ideal technique  Difficult to collect an
adequate volume of GCF
in a short period.
 Provides an undiluted
sample of “native”  Viscosity makes aspiration
difficult
GCF
 Holding the capillary tube
 Volume can be at the entrance of the
gingival crevice for a long
accurately assessed time periods may lead to
traumatic collection.

 Difficult to remove the

complete sampling from
the tubing.

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• Gingival crevice is perfused with an isotonic solution, such as Hanks’
balanced salt solution, usually of fixed volume.
• The fluid collected represents a dilution of GCF and contains both cells
and soluble constituents such as plasma proteins.

Two different techniques have been used:

1. Method of Oppenheim (1970) : modification of Takamori(1963)

2. Method of Skapski and Lehner(1976)

Advantage:Useful in clinically normal gingiva

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 Method of Oppenheim (1970)
• Consists of hard acrylic plate covering the
maxilla with soft borders and a groove along
the gingival margin which is connected to 4
plastic tubes.
• (Isolates the gingival margins from the rest
of the mouth)

Widely used, useful for longitudinal studies.

Valuable in harvesting cells from G.C.F.

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Method of Skapski & Lehner (1976)
This method uses two injection needles fitted one within the other
such that during sampling the inside, or ejection, needle is at the
bottom of the pocket and the outside, or collecting, one is at the
gingival margin interdentally on the buccal surfaces of teeth just
above the interdental papilla.
Involves the instillation and re-aspiration of 10 µl of Hanks’
balanced salt solution at the interdental papilla.( 12times to allow
thorough mixing of the transport solution and GCF.)
Useful for studying number and functional state of cells & bacteria
from the crevicular area
Does not permit absolute quantitative assessments, as dilution
factor can not be determined

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• Used by Weinstein in 1967

• Threads are placed in gingival crevice around the tooth.

• Amount of fluid collected is estimated by weighing the sample

thread.

Plastic strips
Transparent strips
Platinum loops or microspatulas

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1. Appreciation by direct
viewing or staining

2. Weighing the paper

strip

3. The use of Periotron

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 Paper strip after collection

Viewed under a microscope fitted with a graticule for the

determination of the wetted area.

The area of the wetted surface is made more visible by

staining the strip with an alcoholic solution of ninhydrin
at concentration varying between 0.2 to 2 %

Stained area can then be measured with an ordinary

transparent ruler, sliding caliper, calibrated magnifying
glass, microscope with an eye piece graticule, photometric
planimetric techniques or a specially designed inexpensive
paper strip viewer

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Staining techniques has following limitations:
•Not easily applied at chair side
•Inevitable delay may result in increase variation due to
evaporation
•Staining limits the technique for volume determination only,
prevents further laboratory investigation

42
Paper strips are weighed before the collection and repeated
after the collection.
1st by Weinstein 1967

This has been successful but requires a very sensitive balance

to estimate the very small amounts of fluid which may be
collected from a healthy crevice.

Limitations- weighing errors

-evaporation

43
 Periotron is an instrument designed to quantify submicrolitre
volumes of fluid sampled on a filter paper strip.

To date, 3 models have been manufactured

1. Periotron 600 (1976)
2. Periotron 6000 (1983)
3. Periotron 8000 (1995)

Harco electronics (Dental product division

Winnipeg, Manitoba, Canada).

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 Functioning units are a pair of upper and lower
counterparts which can be opened and closed in order to
insert or remove the wetted paper strips.

o Measures the affect on the electrical current flow of the

wetted paper strips

When the wet strip is placed If a dry strip is placed between

between the jaws the ‘jaws’

Increase in capacitance in
proportion to volume of fluid The capacitance is translated via
the electrical circuitry and
and this can be measured as
registers ‘zero’ on the digital
an increased value in the
readout.
readout
45
• Periotron, allowed accurate determinations of the GCF volume
and subsequent laboratory investigation of the sample
composition.
Advantage – Disadvantage –

• Compatible with • inability to measure

subsequent chemical greater than 1.0 µl
analysis
• Keeps evaporation to
minimum

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1. Contamination
 Major source of contamination would be blood, saliva or plaque
 Presence of plaque can have marked effect on volume recorded
 Alpha-amylase was used in an assay to confirm, or refute, the presence of
the contamination of GCF samples with saliva

2. Sampling time
 May take a longer time to collect the GCF from the required area.
 This may irritate the gingival sulcus
 Nature of the GCF sample collected is likely to change
3. Volume determination
• Evaporation is considered to be a significant problem in
accurate volume determination
• Total volumes collected are usually < 1ul and > o.5ul.
• Accuracy of the Periotron®, particularly with respect to small volumes
47
 4. Recovery from Strips
• For various investigation (composition of GCF)
• Initial work : protein recovery was close to 100% using a
centrifugal elution technique.
• A variety of other methods of elution employed (to determine
the % recovery from the original samples.)
• Recent studies sig. diff. in the % recovery of proteins from
filter papers dependent on: 1) the type of paper
2) the concn. of the original protein sample
• Possibly because of entrapment within, or binding of GCF proteins
to the filter papers.
5. Data Reporting
• Constituents found within GCF samples have either been
reported as absolute amount (mg), concentrations (mg/ml)
or either of these two measurements with reference to
pocket depth or duration of sample collection.
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Challacombe(1980) –first to estimate.
In human volunteers with GI<1
Mean GCF volume –anteriors: 0.24 to 0.43µl/tooth
Mean GCF volume –molars : 0.43 to 1.56µl
Also calculated total amount of fluid collected/day 0.5 to
2.4ml

also suggested : it is possible to make an estimation of the total

vol. of gingival fluid secreted into the mouth /day by determining the
conc. in saliva of proteins known to be derived from gingival crevice
. ( Ig G & C3).

49
 Definition: It is the volume that crosses the defined
boundary over a given time
 process of fluid moving into and out of gingival
crevice or pocket.

 Important determinant of the ecology of

periodontal pocket or sulcus.
 Creates flushing action and isolation effect.
 Probably determines the growth level of
subgingival microorganism and is a potential
marker of periodontal disease activity

50
 GCF flow and experimental gingivitis:
 The GCF sample volume collected increased
linearly with development of experimental
gingivitis
 The gingival flow, would be expected to increase
dramatically as inflammation becomes more
severe and vascular permeability increases
 GCF flow and therapeutic response:
 Deep pockets with periodontal disease can exhibit
high GCF flow rates.Therefore it is reasonable to
expect that effective therapy should substantially
reduce the flow rate bringing the pocket closer to
the flow rates measured at healthy sites

51
GCF flow and clinical status
GCF flow can be used as a chair side measure to differentiate healthy sites from
sites with mild disease within the same month.

52
Composition

53
 GCF composition

Metabolic Enzymes and

Cellular Organic
electrolytes and bacterial enzyme
elements compounds
products inhibitors

54
A. Cellular Component of GCF

55
 Smears of GCF with Gram staining technique consistently
showed the presence of variety of microrganisms (Cimasoni
et al 1968, Ishikawa et al 1972)
 Bacteria cultured from GCF were found to be similar to
those grown from adjacent dental plaque (Kaess et al
1972)
 Count of microorganisms did not increase when more
supra-gingival plaque was present.
 Frick (1977) established a poor association between
bacterial counts within the GCF and both the degree of
gingival inflammation and corresponding pocket depth.

Bacteriological quantification studies of gingival fluid are probably inadequate for the
study of the complex bacterial flora in the sulcus.

56
 plaque

Plasma escapes from circulation

Exudate serves as an excellent source of nutrients for subgingival microbes and may
actually contain factors that are necessary for the proliferation of some pathological
bacterial species.
Release large quantities of metabolites that diffuse throughout the dentogingival
space, penetrate JE and contribute to further bact. Colonization & tissue destruction.
57
 Oral Sulcular epithelium and JE are constantly renewing
and the shed cells will be found in the gingival crevice.

Morphological diff. between the two types of cell present. (Lang &
Shroeder 1971)

 JE has higher desquamation rate than that of oral

epithelium (Listgarten 1972, Attstrom 1975)
 Rate of renewal is higher when inflammation is present
(Muhlemann & Hart 1955).
 In contrast , a small percentage of epithelial cells were found
in the GCF collected from inflamed sulci as compared to
healthy (Egelberg 1963)

58
ORAL SULCULAR EPITHELIUM
• An intact epithelial barrier prevents frank bacterial invasion of periodontal
tissues by bacterial components and metabolites
• Oral epithelium has a high turnover rate:
 rapid replacement of damaged tissue components
 Removal of bacteria that have colonized the cellular surface
 Regeneration of intact epithelial barrier

JUNCTIONAL EPITHELIUM
• In inflammation intercellular spaces increase significantly in size
• Contain:
 Abundant migrating leukocytes
 GCF reservoir
• Serves as a diffusion pathway for GCF and its components

59
KERATINOCYTES
• Play a critical role in the recruitment of leukocytes into and through the JE

60
EPITHELIAL ANTIMICROBIAL PEPTIDES
• Β defensins
• Protect the host against bacterial infiltration
• 2 isoforms: Hbd-1, hBD-2
• Localized in the upper supra basal layers of stratified epithelium

61
 Major site of entrance of Leukocytes into the oral
cavity is gingival sulcus (Loe et al 1961)

DLC in sulcus:

o Neutrophil- 95-97% oT Lymphocytes- 24%

o Lymphocytes- 1-2% oB lymphocytes- 58%
o Monocytes- 2-3% oMononuclear phagocyte-18%
T:B :: 1 : 2.7
In blood T:B::2.7:1
Attstrom(1970)
Wislon et al(1976)

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Significance
Healthy
gingiva

gingivitis

• Migration of leukocytes from the gingival connective tissue through the

junctional epithelium and into the sulcus is an active and continuous process
directed along gradients of chemo attractant.
• In the sulcus the conc of leukocytes far exceed the conc in peripheral blood.
• They create a “leukocytic wall” forming aggregates of inflammatory cells along
the margins of advancing plaque front

63
B. ELECTROLYTES
(Matsue 1967 – 1st quantitative study)

64
65
Presence of fluoride in dental plaque is well established, but
its origin remains uncertain.

It is now established that a possible source of fluoride to the

cervical enamel region is represented by gingival fluid( MAC
Fadyen et al 1979)

Concentration of fluoride in GCF is similar to that of plasma

(Whitford et al 1981)

Other ions:
Calcium , Magnesium , Phosphate , Iodine.

66
C.Organic compounds :
1. Carbohydrate:
Sueda et al 1966 : Presence of glycoproteins
Hara & Loe 1969 : glucose , hexosamine , hexuronic acid
( no relation with inflammation)

Exudate glucose is 3-6 times higher than that of serum and decrease in
case of non inflamed gingiva( Hara & Loe 1969)
Increased amount of carbohydrate in inflamed gingiva (Biswas et al 1977)

67
• Histologic specimens from diabetic pts show no difference in severity of
inflammatory infiltrate.
• Vascular biomodifications are observed.
• In both healthy & diabetic pts glucose values in GCF much lower than in serum(
kjellman 1970)
• Similar conc. Of glucose in GCF and serum in both groups. ( Ficara 1975)

Strong positive correlation between blood glucose & GCF glucose has been found in
diabetic patients but not in a comparable groups of healthy individuals (Ficara
et al 1975)
The glucose content of both the GCF and blood of the diabetics was significantly
elevated above those seen in control groups

Dual origin : Reflect altered metabolic activity of adjacent tissues

Influenced by the immediate local flora.

68
 The total protein content of is much less than that of serum. No
significant correlations have been found between the concentration
of proteins in the gingival fluid and the severity of gingivitis,
pocket depth and extent of bone loss. (Bang & Cimasoni 1971).

 Average protein concentration of 8 g/100ml in GCF collected from

inflamed gingiva (Biswas et al 1979).

 Lack of correlation between concentration of protein and

importance of gingival inflammation confirm that GCF from
inflamed gingiva represents a classical inflammatory exudate.

69
 Immunoglobulins
IgG, IgA and IgM
Concentration comparable to those with serum
The IgG content plays a major role in the host defense in the oral
cavity and may provide a means of identifying different forms of
periodontal diseases.
Complement components.
• Local synthesis of C3 & C4 has been detected in gingiva of
individuals with periodontal disease showing that serum is not
the only source of complement in GCF.
Proteins namely , , 2 and 1 globulins, transferrin, albumin,
lactoferrin.
Acute phase proteins: α2macroglobulin, α1antitrypsin, C- reactive
protein

70
 Sudan black material – Sueda et al 1966

 Gingival fluid contains many classes of phospholipids as well as

neutral lipids(Pellat and Kohaut, 1981)

 Serum seems to be the most probable source of lipids in GCF,

although salivary, bacterial & tissue sources can not be excluded

71
D.Metabolic and bacterial products:
• Lactic Acid: + correlation
degree of inflammation
intensity of flow
• Hydroxyproline:
Breakdown product of Collagen.
Rate of progress of periodontal disease.
After periodontal therapy.
Source is doubtful.
• Prostaglandin:
Component of inf. Reaction.
PGE2 : Vasodilatation
bone resoption.
collagen synthesis.
Rate of progression of PDL disease.
Significantly higher in GCF collected
from site with periodontitis compared to
that of gingivitis (Offenbacher et al 1981)
• Urea :
Inversely related to inflammation.
LIPOPOLYSACCHARIDES
(LPS) (ENDOTOXINS)
found in the outer membrane of the
cell wall of Gram-negative bacteria.
The presence of endotoxin has been
positively correlated with gingival
inflammation (Simon et al, 1971)
The level of endotoxin is related to
the number of Gram-negative
bacteria.
LPS is highly toxic to gingival
tissues, a potent stimulator of bone
resorption in vitro (Hopps, 1992)
72
E.Enzyme and Enzyme inhibitors:
1. Acid Phosphatase
2. Alkaline Phosphatase
3. Pyrophosphatase
4. Β- Glucoronidase Central part in the control of
5. Lysozyme periodontal tissue turnover in health
and in the tissue destruction that
6. Hyaluronidase chatrecterizes diseases of the
7. Proteolytic enzymes periodontium

a. Mammalian Proteinases
 Cathepsin D
 Elastase
 Cathepsin G
 Plasminogen activator
 Collagenase
b. Bacterial Proteinases
c. Serum proteinase inhibitors 73
ACID PHOSPHATASE:
• Lysosomal marker
• Confined within the azurophilic granules of PMNs
• There high conc in gingival sulcus in cases of gingivitis and periodontitis is
contributed by desquamating epithelial cells.

ALKALINE PHOSPHATASE:
• Found in the specific or secondary granules of PMNs
• +ve correlation b/w pocket presence and A.P
• -ve correlation b/w supra-gingival calculus and A.P

PYROPHOSPHATASE:
• Plays a role in calculus formation by controlling the conc of pyrophosphate
• Conc of acid & alakaline pyrophosphatase in supra-gingival plaque was found to
be +vely correlated with the amount of calculus.

74
β GLUCORONIDASE:
• Found in the azurophilic or primary granules of PMNs.
• Used as a lysosomal marker to show the release of lysosomal hyrdolases from
phagocytosing cells in vitro.
• Release occurs from neutrophils in the presence of phagocytosable or non-
phagocytosable immune complexes and anti-neutrophil antibodies.

HYALURONIDASE
• Splits the bacterial cell wall linkages

75
CATHEPSIN D:
• A mammalian carboxyendopeptidase.
• Chief acid enzyme in lysosomes.
• Present in high conc in inflamed tissues.
• Abundant in mononuclear leukocytes while relatively less in PMNs.
• GCF conc is 10x more than serum conc; +ve correlation with periodontal
destruction.
• ph of 3.5
• Enzyme is capable of attacking various components of CT

ELASTASE:
• Serine endopeptidase proteinase
• Confined in the azurophilic granules of PMNs.
• ph 7.5-8.5
• Substrates: elastin, proteoglycans, hemoglobin, fibrinogen, collagen,
immunoglobulins and components of complement system.
76
 elastase enzyme
activity level and
Recent studies have indicated that elastase enzyme
defects in the PMN function
causes diabetics to be more prone concentration
to bacterial infections. were found to be
significantly
hyperglycemia and weak higher in
metabolic control increase the metabolic
risks of the periodontal disease, uncontrolled
both of which are attributed to the diabetic groups
defects of PMN function and
collagen synthesis in diabetes.
than controlled
groups. (A.
Dogru et al, 2006)

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CATHEPSIN G
• Second serine endopeptidase.
• Contained azurophilic granules of human PMNs
• 7.5 ph
• Hydrolyses hemoglobin, fibrinogen, casein, collagen and proteoglycans.

COLLAGENASES:
• Maybe derived from host cells or bacteria
• Increased in GCF in both gingivitis and periodontitis
• Levels of collagenase in crevicular fluid have been noted to correlate specific forms
of periodontitis including adult periodontitis and localized juvenile periodontitis
• Usefulness of this enzyme as a diagnostic marker is questionable because diff. b/w
gingivitis and periodontitis has been difficult.
• levels of this enzyme in GCF show marked fluctuations with regard to site, disease
status and treatment.

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Capable of destroying structural periodontal tissues.
Includes :

Collagenase degrading enzymes -P. ging., A.a & Spirochetes

Elastase like enzymes - Spirochetes & Capnocytophaga
Trypsin like protease - P. gingivalis, B. forthythus
Chymotrypsin like enzyme - T. denticola & Capn.
Aminopeptidase – Capnocytophaga & T. dentocola
Dipeptidylpeptidase – P. gingivalis, P. intermedia &
Capnocytophaga

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Periodontal bacteria also produce enzymes capable of
degrading non-proteinacious elements of periodontal CT

Hyaluronidase & chondroitinase activities are produced by F.

nucleatum, P. gingivalis & T. denticola

Neuraminidase produced by B. forsythus, P gingivalis, attacks

sialoproteins in the epithelium, thus increasing permeability
to bacterial products

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As a Diagnostic
Marker

81
GCF appears to be an ideal medium for monitoring
the changes occurring during development of
periodontal disease
- Can be collected non-invasively.

-Dynamics and composition is closely related to the periodontal

environment.- analysis of its constituent may provide an early
indicator of changes in the tissue.

-Saliva has a number of drawbacks.- as made up of components

derived from multiple sources ( Lamster & Grbic 1995) whereas
GCF contains products of host , plaque & their interactions
( Curtis et al 1989)

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1. Presence of disease severity and test should be able to
distinguish between periodontally healthy,gingivitis and
periodontitis sites.

2 To predict subsequent clinical course and prognosis.

3 To assess response to treatment after therapy.
4 To distinguish between active and inactive disease.

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Clinical judgement based on …..

1. Previous history of progression at a site.

2. Persistence of clinical signs of inflammation.
3. Persistence of probing depth ≥5mm
4. Teeth of high strategic importance in overall treatment
plan.

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 The amount of GCF produced is directly
related to the increased vascular permeability
and ulceration of the pocket epithelium.

 Over 65 GCF components have been

preliminarily examined as possible markers
for the progression of periodontitis.

1. Host derived enzymes and inhibitors

2. Inflammatory mediators & host response

modifiers

3. Tissue breakdown products

85
INFLAMMATORY
MEDIATORS AND
HOST RESPONSE
MODIFIERS

86
 • Mainly IL-1α, IL-1β,IL-6, IL-8 and TNF-α
SOURCE • IL-1 released by activated macrophages,
PMNs, lymphocytes and fibroblasts.

• Potential marker for active bone destruction.

SIGNIFICANCE • Cross sectional studies have indicated that

the levels of IL-1β are increased at periodontitis
sites compared to gingivitis and healthy sites.

87
 • Derived from arachidonic metabolism
SOURCE and found abundance at sites of
inflammation.

• PG-E2 concentration was significantly

higher in GCF collected from site with
SIGNIFICANCE periodontitis compared to that of
gingivitis. (Offenbacher et al 1981)

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 • Found in all sites where there is active
acute inflammation.
SOURCE
• Includes α2macroglobulin, α1antitrypsin
and C- reactive protein.

• These proteins are found to be increased

at the site of inflammation.
SIGNIFICANCE
• Does not show significant differences in
concentration in GCF between sites of
gingivitis or periodontitis.

89
 • Is a neuropeptide released from
SOURCE primary sensory afferent nerves.

• has significant pro-inflammatory

actions (Lembech & Holzer 1979) and
SIGNIFICANCE has been proposed to play a role in
neurogenic inflammation of the
periodontal tissues (Bartold et al. 1994).

90
•Nina-Li-Avellan(2008), conducted one study to observe
whether tooth stimulation would release neuropeptide
substance P in GCF.

•Result showed that the pulpal pain can contribute to local

elevation of substance P and MMP-8 levels in GCF.

•There is direct communication between pulp cavity and

PDL by the way of apical foramen, accessory lateral canals
and dentinal tubules (Seltzer, 1984, Towbridge et al 1998)

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TISSUE BREAKDOWN
PRODUCTS

92
 • The connective tissue breakdown which
occurs during periodontitis includes
SOURCE proteoglycan degradation and this process
involves proteolytic enzymes which release
GAGs from the protein core.

• The non-sulphated GAG, hyaluronic acid,

was found to be present in GCF and was
SIGNIFICANCE the only major GAG found in chronic
gingivitis patients.

93
 • An additional sulphated GAG, chondroitin-4-
sulphate, was detected in GCF from sites with
untreated advanced periodontitis. Embery G,
Oliver et al (1982) and Last K Set al (1985)

• This GAG was, however, not detected after

periodontal treatment of these sites with
SIGNIFICANCE subgingival scaling, pocket reduction surgery or
daily irrigation of the pockets with a
chlorhexidine solution.

• The presence of chondroitin-4-sulphate in GCF

may be a sensitive method of indicating active
phases of destructive periodontal disease.

94
 • Is major cytosolic protein of leukocytes
which exists in plasma and other body
fluids of healthy human subjects and
SOURCE recently identified in human dental
calculus and GCF. (Teruo Nakamura et al,
2000)

• Thought to be marker of inflammatory

disease activity.
• Concentration was found to be
SIGNIFICANCE significantly higher in patients with
periodontitis

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BONE RESORPTION MARKERS

1. Pyridinium cross links

2. Pyridinium crosslink collagen peptide
fragments
-serum C-terminal telopeptide (ICTP)
3. Tartrate-resistant acid phosphatase
(TRAP)
4. Galactosyl hydroxylysine
5. Hydroxyproline
6. N-terminal osteocalcin fragment
7. Glycosaminoglycans (GAGs)
96
 • Is one of the fragments of bone
typeI collagen released by
SOURCE digestion with trypsin or bacterial
collagenase.

• They are bone specific

• Potential markers of bone turnover

SIGNIFICANCE
• Significantly higher GCF ICTP levels were found
in periodontitis affected group and their levels
were strongly correlated with clinical parameters
of periodontal tissue destruction. (Talonpoika JT
et al,1994)

97
All the enzymes evaluated so far have yielded high false
positive results.

Potential markers-Chondroitin 4 sulphate.

Alpha 2 macroglobulin & alpha 1 antitrypsin –to distinguish

between gingivitis and periodontitis.

PG-E2-as a marker for active disease.

MMP1,Lysozyme-elevated in LJP.
MPO,Elastase –to assess treatment outcome

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• 1.Fluorometry…………….metalloproteinases
• 2.ELISA…………………….enzymes & IL1ß
• 3.Radioimmunoassay……cyclo-oxygenase derivatives
• 4.HPLC……………………...tinidazole
• 5.Direct and indirect immunodot tests for detection of
acute phase proteins.

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They reflect process occurring in gingival connective tissue
and tell little about attachment loss and bone loss .

Lack of gold standard with which it can be evaluated .

Complex gingival environment.

Presence of large number of molecules with interactions that

are not fully understood.

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The relevance of components of crevicular fluid around implants is
now recognized as diagnostically significant since the flow and volume
appears to be similar to GCF.

In particular levels of chondriotin sulphate were found to be high

immediately following exposure and also following occlusal loading.

Other connective tissue elements studied in peri-implant sulcular

fluid include proteolytic and lysosomal enzymes such as
cathepsins,elastase,,myeloperoxidase,ß-glucoronidase and trypsin….all
correlate positively with peri-implant inflammation and bone
resorption(Boutros et al 1996)

101
Although, a vast amount of work has been done to provide a
satisfactory method for diagnosing the progression of
periodontitis, none of the developed and suggested solutions
seem to be conclusive enough.

To the present day, therefore, the clinician is still forced to

rely heavily on the old conventional methods of assessing
periodontitis and gingivitis.

Moreover, none of the tests developed so far are capable of

indicating a risk of periodontal disease at any degree.
Nevertheless the interest in components of GCF as
potential markers continues to progress.

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Thankyou !

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