Defense Mechanism Of

Gingiva
Presented to : Presented By
Dr Ashutosh Nirola Ishu Kansal
Dr Priyanka Batra
Dr Jyotsna Goyal
Dr Shivani Rathore
Dr Kanika Mohindra
Dr Bhavya Sharma
SULCULAR FLUID OR
GINGIVAL CREVICULAR
FLUID
 INTRODUCTION
• Gingival crevicular fluid is an exudate secreted by the gums
that can be found in the crevices located at the point where
the gum line meets the teeth.
• Concentrations of this fluid are usually low, but can spike
when an inflammatory process occurs in the oral cavity.
Patients with active gum disease tend to have more gingival
crevicular fluid.
• Gingival crevicular fluid (GCF) is treated as a window for
noninvasive analysis of periodontitis, taking into account
indicators and markers of connective tissue and bone
destruction so it could be a useful indicator in determining
the severity of gum disease.
Schematic figure indicating the flux
of gingival crevicular fluid through
epithelial cells in the presence of a
biofilm.
 Definition
• Gingival crevice fluid (GCF) is a complex mixture of substances derived
from serum, leukocytes, structural cells of the periodontium and oral
bacteria

Uitto, V.-J. (2003). Gingival crevice fluid - an introduction.

Periodontology 2000, 31(1), 9–11
 Gingival crevice fluid – a window to
periodontal disease. Gingival crevice fluid is
composed of substances derived from
serum, leukocytes, bacteria, activated
epithelial cells, connective tissue cells, and
bone cells.
 Tissue destruction during periodontal
inflammation results in production of tissue
fragments and growth factors released from
tissue stores. All these substances reflect
the periodontal disease process and can be
potentially used as indicators of periodontal
condition.
Studies on gingival crevice fluid (GCF) extend
over a period of about 50years.
• In 1952, Jens Waerhaug, while investigating the
physiological properties of the gingival pocket,
administered India ink into healthy gingival sulci of young
experimental dogs.
• After 1h, he observed increased fluid transudation and
emigration of leukocytes and within the next 48h this
‘transudate fluid’ had eliminated most of the ink particles
from the sulci .

• Delima, A. J., & Van Dyke, T. E. (2003). Origin and function of the
cellular components in gingival crevice fluid. Periodontology 2000,
31(1), 55–76.
• Brill & Krasse (1962) , also working with an
experimental canine model, injected fluorescein
dye intravenously into the animals’ hind legs and
discovered that it could penetrate into the
epithelial lining of the gingival sulcus.
• Fluorescein could also be collected on strips of
filter paper introduced into the sulcus in over 90%
of the animals, as quickly as one and a half
minutes after administration.
• However, no dye was detected on other intraoral
locations, such as the teeth, tongue, palate, and
oral mucosa, and even extra-oral mucous
membranes sites provided negative results.
• These findings suggested that the epithelium
lining of the gingival sulcus was permeable to small
molecular weight compounds and that the passage
of tissue fluid into the sulcus acts as a possible
defence mechanism that may play an important
role in the homeostasis of the crevicular
environment.
• The studies of Löe et al (1965) contributed to this
understanding and started to explore the use of GCF as an
indicator of periodontal diseases.
• Egelberg (1966) continued to analyse GCF and focused his
studies on the dentogingival blood vessels and their
permeability as they relate to GCF flow.
• The GCF studies boomed in the 1970s. The rationale for
understanding dentogingival structure and physiology was
created by the outstanding electron microscopic studies of
Schroeder(1969) and Listgarten(1966). Presence and functions
of proteins, especially enzymes in GCF were first explored by
Sueda, Bang and Cimasoni(1969).
• It was soon understood that enzymes released from damaged
periodontal tissue possessed an enormous potential for periodontal
diagnosis.
• Ohlsson, Golub and Uitto discovered that collagenase and elastase in
GCF are derived primarily from human cells, most notably
neutrophils, and that their activity is correlated with gingival
inflammation and gingival pocket depth.
• Migration of neutrophils and their function in gingival tissue and GCF
were clarified by the excellent investigations of Attström et al.
• In 1974 the first edition of the monograph The Crevicular Fluid by
Cimasoni was published.
 Methods of collection of GCF
Several techniques have been employed
1) Gingival washing methods
In this technique the gingival crevice is perfused with an
isotonic solution, such as Hanks’ balanced salt solution,
usually of fixed volume.
Two different techniques have been used :
a) The simplest involved the instillation and re-aspiration of
10ml of Hanks’ balanced salt solution at the interdental
papilla .This process was repeated 12 times to allow
thorough mixing of the transport solution and GCF.
 This technique could therefore be applied either to
individual interdental units or to multiple units which were
then pooled.
Advantages
The technique may be applied to individual sites or groups of sites
which may be categorized as healthy or inflamed.
Disadvantages
All fluid may not be recovered during the aspiration and reaspiration
procedure. Thus accurate quantification of GCF volume or composition
is not possible as the precise dilution factor cannot be determined.
Collection of crevicular fluid
by means of gingival
washings;
10 ml of fluid is ejected from
a microsyringe and re-
aspirated,
b. A more complicated method involved
 the construction of a customized acrylic stent which
isolated the gingival tissues from the rest of the mouth.
 The tissues were then irrigated for 15min, with a saline
solution, using a peristaltic pump, and the diluted GCF
was removed.
Advantages
It is valuable for harvesting cells from the gingival crevice region .
Disadvantages
GCF from individual sites cannot be analyzed
The production of customized acrylic stents is complicated and technically
demanding.
It has been restricted to the study of GCF obtained from only a few
individuals.
It has also usually only been applied to the maxillary arch, because of the
difficulties of producing a technically satisfactory appliance for the
mandibular arch.
 Capillary tubing or micropipettes
• Following the isolation and drying of a site, capillary tubes
of known internal diameter are inserted into the entrance
of the gingival crevice.
• GCF from the crevice migrates into the tube by capillary
action and because the internal diameter is known the
vol- ume of fluid collected can be accurately determined
by measuring the distance which the GCF has migrated.
This technique appears to be ideal as it provides an
undiluted sample of ‘native’ GCF whose volume can be
accurately assessed.
However, it is difficult to collect an adequate volume of
GCF in a short period, unless the sites are inflamed and
contain large volumes of GCF.
• To collect a reasonable volume of fluid may, mean that
collection times from an individual site may exceed 30min
and, even then, adequate samples from healthy crevices
may be impossible to obtain.
• A further complication of this technique is the difficulty
of removing the complete sample from the tubing. This
has either been forced out with a jet of air or, by passing
a larger fixed volume of a diluting solution through the
capillary or, more usually, by centrifugation of the tube.
Collection of crevicular fluid by
means of capillary tubing.
Capillary tubing of known
diameter is placed at the
entrance of the crevice and the
fluid migrates up the tube by
capillary action.
 Absorbent filter paper strips

Methods of collection
Intracrevicular and
The extracrevicular techniques
a. Intracrevicular Technique : The strip is inserted just at
the entrance of the crevice or periodontal pocket or
whether the strip is inserted to the base of the pocket or
‘until minimum resistance is felt’ .
b.Extracrevicular technique
The strips are overlaid on the gingival crevice region in an
attempt to minimize trauma.
The positioning of papers for
the
filter paper strip method of
collection: (a) extracrevicular
method;
(b) intracrevicular method
‘superficial’ [Löe & Holm-
Pederson],
(c) intracrevicular method
‘deep’ [Brill].
 Methods of estimating the volume
collected
The amount of GCF collected on a strip was assessed by
the distance the fluid had migrated up the strip.
A more accurate value was achieved by assessing the
area of filter paper wetted by the GCF sample.
• Further accuracy was achieved by staining the strips with
ninhydrin to produce a purple colour in the area where
GCF had accumulated.
• A similar result was shown with 2g fluorescein given
systemically to each patient 2hours prior to the collection
of GCF, following which the strips were examined under
ultraviolet light.
• Fuorescein labeling was 100 times more sensitive than
ninhydrin for staining protein.
• The staining techniques have a number of disadvantages;
Firstly, they are not easily applied at the chairside.
The inevitable delay in measuring the strip may result in
increased variation in the reported volume as a result of
evaporation.
Secondly, the staining of the strips for protein labeling
prevents further laboratory investigations of the components
of GCF, effectively limiting the technique to that of volume
determination.
• Measurements performed by Cimasoni showed that a strip of paper
1.5mm wide and inserted 1mm within the gingival sulcus of a slightly
inflamed gingiva absorbs about 0.1mg of GCF in 3 minutes.
• Challacombe used an isotope dilution method to measure the
amount of GCF present in a particular space at any given time.
• His calculations in human volunteers with a mean gingival index of
less than 1 showed that mean GCF volume in proximal spaces from
molar teeth ranged from 0.43 to 1.56µl.
• The introduction of an electronic measuring device, the
PeriotronA, has allowed accurate determination of the
GCF volume and subsequent laboratory investigation of
the sample composition.
• The instrument measures the affect on the electrical
current flow of the wetted paper strips. It has two metal
‘jaws’ which act as the plates of an electrical condenser.
• If a dry strip is placed between the ‘jaws’, the capacitance
is translated via the electrical circuitry and registers
‘zero’ on the digital readout.
• A wet strip will increase the capacitance in proportion to
the volume of fluid and this can be measured as an
increased value in the readout. The technique is rapid
and has no discernible affect upon the GCF sample.
• Three models of Periotron A have been produced (the
600, 6000 and now the 8000) and each one has been
shown to be an efficient means of measuring the volume
of fluid collected on filter paper strips.
Illustration of GCF sampling
sequence and use of
Periotron®. Once the sites are
isolated with cotton rolls and
gently air-dried, the Perio®
paper strips are inserted in the
gingival sulcus for 30 seconds.
The paper strips are then
inserted in the Periotron 8000®
device (Harco, Tustin, CA,
USA), previously calibrated to
each individual sample to
obtain the fluid volume. Paper
strips are then wrapped in
aluminium foil, transferred to
liquid nitrogen and stored at
−80°C until assayed.
• An alternative approach, involving the weighing of strips
before and after sample collection, has been adopted by
some workers. 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.
COMPOSITION OF GINGIVAL
CREVICULAR FLUID
Component Source Function
Bacteria Oral biofilm plaque Initiates the host immune response

Epithelial cells Oral sulcular and junctional Represents the high cell turnover
epithelium of the gingival sulcus

Leukocytes Gingival blood vessel plexus PMNs are involved with innate
immunity.
Monocytes/macrophages and
lymphocytes are involved with cell-
mediated immunity.

Erythrocytes Gingival blood vessels Results from small blood vessels

and capillaries damages.

Alkaline phosphatase Fibroblasts, osteoblasts, Play a role in superoxide

osteoclasts, and neutrophils generation and in the first line of
defense
Cathepsin B Macrophages Active enzyme in proteolysis

Collagenase-2 Neutrophils Active enzyme associated with collagenatic

(MMP-8) activity

Gelatinase Neutrophils Hydrolysis of intercellular matrix

(MMP-9)
Neutrophil Neutrophils Cleavage of elastin, collagen, and
elastase proteoglycans

Macrophage Macrophages Cleavage of elastin, collagen, and

elastase (MMP- proteoglycans
12)
ICTP (Collagen Fragment of bone type I Highly correlated with bone turnover
telopeptide collagen
pyridinoline
cross link)
Interleukin 1-beta Macrophages Regulates immune and
inflammatory reactions,
stimulates bone resorption

Interleukin 4 Basophils Anti-inflammatory,

macrophage inhibition, Th2
differentiation
Interleukin 6 T cells, macrophages, Regulator of T- and B-cell
osteoblasts growth, stimulate osteoclast
formation
Interleukin 8 Macrophages, epithelial cells Recruitment and activation of
neutrophils

Interferon gamma Leukocytes, lymphocytes Macrophage activation,

suppression of Th2

Immunoglobulin A (IgA) Plasma B cells Antigen neutralization

Immunoglobulin G (IgG) Plasma B cells Antigen neutralization

Immunoglobulin M (IgM) Plasma B cells Antigen neutralization

Lactoferrin PMNs, acinar cells Antibacterial, creates iron-

limiting environment

Lysozyme PMNs, macrophages Hydrolysis of peptidoglycans

of bacterial cell walls

Osteoprotegerin (OPG) Osteoblasts Decoy receptor for RANK-L,

inhibits osteoclast formation
Osteocalcin Osteoblasts Calcium binding
Prostaglandin E2 (PGE2) All cell types Proinflammatory and
immunomodulatory effects
Transforming growth factor- Macrophages, keratinocytes Regulation of tissue repair, cell
alpha proliferation, chemotaxis,
differentiation and matrix
synthesis

Transforming growth factor- Macrophages Modulates proinflammatory

beta cytokine production
TIMPs Neutrophils, macrophages, Inhibits MMPs
fibroblasts, keratinocytes

Tumor necrosis factor-alpha Neutrophils, macrophages, Delays neutrophil apoptosis

lymphocytes
 Significance of gingival crevicular fluid

• To assess the severity of gingival diseases, the

effectiveness of periodontal therapy and oral hygiene, the
healing following gingival surgery, and the effectiveness
of oral hygiene.
• To evaluate the rate of local destruction, to assess the
permeability of junctional and sulcular epithelium, and to
assess the relationship between periodontal and systemic
diseases.
 Factors stimulating gingival crevicular
fluid flow

• Gingival inflammation, mastication of coarse food, pocket

depth, intracrevicular scraping, scaling, and histamine topical
application.
• Enzymes and sex hormones: Female sex hormones increase the
gingival fluid flow because they enhance vascular permeability.
• Circadian periodicity: There is gradual increase in gingival
fluid amount from 6 am to 10 pm and a decrease afterward.
• Post-periodontal surgery, restorative procedure, strip
placement, mobility, increased body temperature, and salivary
contamination.
• Ovulation, hormonal contraceptives, and smoking.
Leukocytes in The Dentogingival
Area
• Leukocytes have been found in clinically healthy gingival
sulci in humans and experimental animals
• Leukocytes found are predominantly PMNs. They appear
in small numbers extravascularly in the connective tissue
adjacent to the bottom of the sulcus from where they
travel across the epithelium to the gingival sulcus, where
they are expelled.
• They are present even when histologic sections of
adjacent tissue are free of inflammatory infiltrate.
• Differential counts of leukocytes are :
PMNs 91.5%
Mononuclear cells 8.5 % to 8.8%
• B lymphocytes 58%
• T lymphocytes 24%
• Mononuclear Phagocytes 18%
• Ratio of T lymphocytes to B lymphocytes was found to be
reversed from 3:1 to about 1:3 in GCF.
• Leukocytes were reported in the gingival sulcus in
nonmechanically irritated (resting) healthy gingiva ,
indicating that their migration may be independent of an
increase in vascular permeability.
• Therefore leukocytes constitute a major protective
mechanism against the extension of plaque into the
gingival sulcus.
• Leukocytes are found in saliva. The main port of entry of
leukocytes into the oral cavity is the gingival sulcus.
 Gingival Crevicular Fluid and Implants

• The levels of neutral proteases were higher at moderately

to severely inflamed implant sites compared to mildly
inflamed sites. The levels of neutrophil elastase,
myeloperoxidase, and β-glucuronidase were significantly
higher around sailing implants compared to successful
implants.
• The levels of IL-1β were approximately three times higher
than those of the healthy sites in GCF around implants.
Significantly low activity of elastase and collagenase was
detected in osseointegrated implants than in adult
periodontitis.
 Dynamics of the Gingival Crevicular Fluid
composition and development of Periodontal
Disease
• The development of periodontitis is associated with a rise
in pH in the gingival sulcus to around 8.5, and this is
thought to occur by the bacterial degradation of proteins
supplied by GCF to produce NH4, assacharolytic
subgingival bacteria utilize proteins as primary nutrients
and produce NH4 as a by-product, which serves to
promote the precipitation of calcium salts from the GCF
or saliva resulting in the promotion of calculus formation
subgingivally or right at the free-gingival margin.
• In the transition from a ‘healthy’ to a ‘disease-associated’
subgingival microbiota a number of allogenic factors play
a role which includes pH, oxygen levels, temperature,
osmotic pressure and oxidation–reduction potential and
in addition, protein-based nutrients become available
within the GCF.
• Saccharolytic organisms, Fusobacterium
nucleatum and Prevotella intermedia, initially colonize the
shallow periodontal pocket. During these early stages, the
pH of the GCF in the pocket may be acidic but the
metabolism of nutrients, which corresponds mainly to
host proteins supplied by GCF, leads to the establishment
of a neutral environment.
• The shift towards lower pH likely is a consequence of the
downward extension of saccharolytic bacteria which are
adherent and utilize glucose to produce lactic acid.
• The pH range of the gingival sulcus has been reported to
vary between 6.5 and 8.5, and increased pocket depth and
inflammation have been associated with increased
alkalinization and the severity of the inflammatory host
response, likely associated with the shift to assacharolytic
subgingival organisms. The rise in localized pH facilitates
the emergence of acid-sensitive and more proteolytic
species, such as Porphyromonas gingivalis to colonize.
• The destruction of host tissues and increased efflux of
GCF caused by the proliferation of the so-called ‘red
complex’ group of bacteria leads to an increase in the
fermentation of amino acids into organic acids producing
the rise in pH above neutrality. In particular, ammonia
produced by the metabolism of amino acids found in GCF
and released by the breakdown of host tissues, leads to
an increase in pH above 8.0, thereby promoting the
proliferation of acid-sensitive pathogenic bacteria.
GCF as a Diagnostic Tool for Analysis of
Oral Diseases
• GCF, plays a constructive role in the diagnosis of oral
diseases, especially for periodontitis and gingivitis.
• The introduction of mass spectrometry, with highly
sensitive techniques, helps in the detection of protein
and their components.
• Various inflammatory factors have been isolated from
GCF, including cytokines, phosphatase, proteinase, local
tissue degradation products, and proteins. These factors
have been reported to be possible diagnostic markers in
periodontitis.
• The GCF proteome analyzed was found different in each
clinical condition suggests that this analysis can assist us
in knowing pathogenesis of the periodontal disease.
• Tsuchida et al. collected GCFs from a healthy patient
and compared them with patients having mild to
moderate periodontal disease and with a patient suffering
from the severe periodontal disease.
• The gel-free proteomic approach was used, and GCFs
were labelled by a reagent called as Tandem Mass Tags
(TMTs). In this method, 619 proteins were analyzed.
• Their reports showed, in severe periodontitis, higher
amounts of lipocalin2 (LCN2) and matrix
metalloproteinase-9 (MMP-9) were expressed.
• Furthermore, Pisano et al. worked on the peptides of
GCF by using HPLC-ESI-MS and detected a high
concentration of α-defensins, and small amounts of
cystatin-A, basic PB peptides, and statherin were also
detected .
• External root resorption is a universal phenomenon that is
associated with many factors such as trauma, orthodontic
tooth movement periodontal disease, and physiological
shedding of deciduous teeth.
• Rody Jr et al. worked to identify the biomarkers in GCF by
using LC-MS (liquid chromatography-mass spectrometry).
• He postulated 2789 proteins in a control group, and 2421
proteins in resorption samples were identified. Therefore,
these biomarkers can provide us with a new possible tool for
the early detection of external root resorption so that
orthodontic tooth movement should avoided, and other
conditions mentioned above will better be managed.
• Certain studies were conducted to assess changes within
GCF during orthodontic tooth movement.
• Initially, when orthodontic force is applied to allow tooth
movement to occur, certain metabolic changes occur
within periodontium along with the process of bone
remodeling that includes some osteoblastic and
osteoclastic activity.
• This biological and physiological process results in acute
inflammatory response that occurs within periodontal
space.
• Lactate dehydrogenase, an important enzyme that is
released out after cell death, was reported as a potential
marker, as its level was found to be elevated within GCF
during orthodontic treatment.
• Serra et al. demonstrated the LDH level in GCF during
the early stage of orthodontic treatment and formulated
that, when orthodontic force is applied, the
periodontium either goes under tension or compression,
resulting in cell death. As hypothesized, the LDH level in
GCF was elevated in those sites enduring orthodontic
force and was independent of age and sex.
• A longitudinal study was performed to assess the level of
alkaline phosphatase within GCF in a patient undergoing
orthodontic treatment. It was noted that the enzyme
level was elevated at the site of tension, as compared to
the site of compression. Hence, these enzymes represent
future novel markers and are associated with gingival
inflammation caused during forces applied by orthodontic
appliances.
 CONCLUSION
• Gingival crevicular fluid is a serum exudate that
originates from the periodontal sulcus or pocket and is
regarded as a promising biological fluid for the detection
of periodontal disease.
• Its composition resembles normal serum, but its volume
fluctuates in certain conditions such as those of
gingivitis, caries, external root resorption, and chronic
periodontitis, as well as during orthodontic tooth
movement.
• GCF is composed of variable substances that include
immunoglobulin, enzymes, local mediators, toxin cells,
protein peptides, tissue breakdown products, and
microorganisms.
• The level of this substance when fluctuating in the
above-mentioned conditions, as reported in many papers,
will mark as a future diagnostic tool in their non-invasive
analysis.
• Due to limitations in its collection, which includes
volume size and contamination, collecting methods need
further work, and a way to improve the ease for clinicians
must be found; such development would help us to better
demonstrate the pathogenesis of such diseases and to
determine better strategies for treatment and early
prevention.
 References
• Uitto, V.-J. (2003). Gingival crevice fluid - an introduction.
Periodontology 2000, 31(1), 9–11.
• Delima, A. J., & Van Dyke, T. E. (2003). Origin and
function of the cellular components in gingival crevice
fluid. Periodontology 2000, 31(1), 55–76.
• Griffiths, G. S. (2003). Formation, collection and
significance of gingival crevice fluid. Periodontology
2000, 31(1), 32–42.
• Gingival Crevicular as a Source of Biomarkers for Periodontitis Silvana
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• Gingival Crevicular Fluid: An Overview Krishnan
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• Gingival crevicular fluid – composition and clinical
importance in gingivitis and periodontitis Pol J Public
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• Human Gingival Crevicular Fluids (GCF) Proteomics: An Overview
Zohaib Khurshid, Maria Mali, Mustafa Naseem,Shariq Najeeb,
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• Carranza ‘ s Clinical Periodontology Tenth Edition.

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