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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.
3
ORAL MUCOSA
-Epithelial barrier
-epithelial renewal
-keratinization
-mucosal permeability
-non-keratinocytes
GCF
SALIVA
MOVEMENTS OF LIPS AND CHEEKS
4
Oral mucosa
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
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.
10
A complex mixture of substances derived from serum, leukocytes,
structural cells of periodontium and oral bacteria.
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.
Exudate Transudate
14
Is GCF an inflammatory exudate?
Early studies
15
Vital evans blue dye Bound to albumin will not pass until permeability
No explanation !!
16
Studies of Egelberg et al (1966)
17
2) increased permeability of blood vessels:
18
Is GCF a transudate of interstitial fluid?
On stimulation an inflammatory
exudate
19
Very early or pre-inflammatory fluid may be
osmotically mediated.
2⁰ inflammatory exudate.
21
Based on Starling’s factors governing fluid distribution across capillaries.
23
Factors modulating filtration & uptake:
24
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.
25
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
26
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
27
Collection of GCF
28
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
29
A. Intra crevicular method:
B. Extracrevicular Method:
30
B. Extracrevicular
31
Advantages Disadvantage
Quick and easy to use
Introduces degree of irritation
that can trigger the fluid flow
Can be applied to individual
sites
32
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.
33
Ths method was first utilized by
KRASSE and EGELBERG.(1962)
• Mann 1963: micropipettes
35
• 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.
36
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)
37
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
38
• Used by Weinstein in 1967
Plastic strips
Transparent strips
Platinum loops or microspatulas
39
1. Appreciation by direct
viewing or staining
40
Paper strip after collection
41
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
43
Periotron is an instrument designed to quantify submicrolitre
volumes of fluid sampled on a filter paper strip.
44
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.
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 –
46
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.
48
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
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.
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
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
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)
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:
62
Significance
Healthy
gingiva
gingivitis
63
B. ELECTROLYTES
(Matsue 1967 – 1st quantitative study)
64
65
Presence of fluoride in dental plaque is well established, but
its origin remains uncertain.
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
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).
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
71
D.Metabolic and bacterial products:
LIPOPOLYSACCHARIDES
• Lactic Acid: + correlation (LPS) (ENDOTOXINS)
degree of inflammation
intensity of flow found in the outer membrane of the
cell wall of Gram-negative bacteria.
• Hydroxyproline:
Breakdown product of Collagen. The presence of endotoxin has been
Rate of progress of periodontal disease. positively correlated with gingival
After periodontal therapy. inflammation (Simon et al, 1971)
Source is doubtful.
The level of endotoxin is related to
• Prostaglandin: the number of Gram-negative
Component of inf. Reaction. bacteria.
PGE2 : Vasodilatation
bone resoption. LPS is highly toxic to gingival
collagen synthesis. tissues, a potent stimulator of bone
Rate of progression of PDL disease. resorption in vitro (Hopps, 1992)
Significantly higher in GCF collected
from site with periodontitis compared to
that of gingivitis (Offenbacher et al 1981)
• Urea :
Inversely related to inflammation. 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)
77
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.
78
Capable of destroying structural periodontal tissues.
Includes :
79
Periodontal bacteria also produce enzymes capable of
degrading non-proteinacious elements of periodontal CT
80
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.
82
1. Presence of disease severity and test should be able to
distinguish between periodontally healthy,gingivitis and
periodontitis sites.
83
Clinical judgement based on …..
84
The amount of GCF produced is directly
related to the increased vascular permeability
and ulceration of the pocket epithelium.
86
• Mainly IL-1α, IL-1β,IL-6, IL-8 and TNF-α
SOURCE • IL-1 released by activated macrophages,
PMNs, lymphocytes and fibroblasts.
87
• Derived from arachidonic metabolism
SOURCE and found abundance at sites of
inflammation.
88
• Found in all sites where there is active
acute inflammation.
SOURCE
• Includes α2macroglobulin, α1antitrypsin
and C- reactive protein.
89
• Is a neuropeptide released from
SOURCE primary sensory afferent nerves.
90
•Nina-Li-Avellan(2008), conducted one study to observe
whether tooth stimulation would release neuropeptide
substance P in GCF.
91
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.
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)
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)
95
BONE RESORPTION MARKERS
97
All the enzymes evaluated so far have yielded high false
positive results.
98
• 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.
99
They reflect process occurring in gingival connective tissue
and tell little about attachment loss and bone loss .
100
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.
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.
102
Thankyou !
103