Journal of Clinical Periodontology 19S3: 10: 364-369

Key words: Humans - iniiiof gin^ivilis.

Accepted for publicaiion August 31, I9S2

Histopathology of initial gingivitis

in humans
A pilot study

BJORN KLINGE', LARS MATSSON" AND ROLF ATTSTROM'

Departments of 'Periodontology and ^Pedodontics, School of Dentistry, Malmo, Sweden, and ^Department
of Periodontology, School of Dentistry, Aarhus, Denmark

Abstract. The present study concerns the inflammatory alterations in the gingival margin during initial
gingivitis in l ] - i 3 year old human subjects.
At day 0 of the experiment, all participants had clean teelh and healthy gingiva. Ali active oral hygiene
measures were excluded for 4 days. From upper and lower premolars, which were extracted for
orthodontic reasons, contralateral gingival biopsies, including the tooth and the adjacent gingiva, were
obtained on days 0 and 4. The presence of inflammatory cells in the junctional epithelium and the
adjacent connective tissue was determined quantitatively in semi-thin sections. The collagen content of
the gingival margin was also determined.
From day 0 to day 4 there was only a slight increase in the number of neutrophilic granulocytes in the
junctional epithelium and adjacent connective tissue, while a more pronounced increase was found in
the number of mononuclear leukocytes. A loss of collagen was noticed in 4 of the subjects, while 2 did
not show any changes in collagen content.
The inflammatory reaction seen in the present study differs somewhat from that observed in adult
humans and adult dogs. The results correspond more to the reaction seen in juventJe dogs.

Research in recent decades has shown that
gingivitis and periodontitis are caused primarily
by bacterial substances released from bacteria
colonizing the tooth at or below the gingival
margin (Loe et al. 1965, Lindhe et al. 1973,
Lindhe & Rylander 1975, Payne et al. 1975; for
review see Page & Schroeder 1976).
In dogs, strictly normal gingiva can be
achieved by careful, prolonged mechanical
cleaning of the teeth. The gingival tissues of the
adult dog are characterized histologically by a
smooth junctional epithelium without rete pegs,
and by a gingival connective tissue with dense
collagen fiber bundles. Single inflammatory
cells can be observed in the junctional epithe-
lium and the connective tissue, but these cells do
not form an infiltrate (Attstrom et al. 1975,
Lindhe & Rylander 1975, Matsson & Attstrom
1979), When such gingiva is re-subjected to
accumulation of dental plaque, an acute exuda-
tive inflammatory process will be initiated.
Clinically an increased flow of gingival fluid
and an enhanced migration of dento-gingival
leukocytes are noted, Histopathologically,
dense accumulations of neutrophilic granulo-
cytes and mononuclear leukocytes are observed
in the coronal part of the junctional epithelium
and adjacent connective tissue. Exudation and
deposition of fibrin can also be seen in the
affected connective tissue area. The reaction
involved a 5-10% portion of the gingival con-
nective tissue and there is in this portion a loss
of up to 70% of the collagen fibers (Lindhe &
Rylander 1975, Matsson & Attstrom 1979). The
 HISTOPATHOLOGY OF INITIAL GINGIVITIS IN HUMANS
mechanisms behind the coHagen loss are not
known, but they probably include enzymatic
breakdown (Schroeder et al. 1975). In the
animal studies referred to, methods were used
which allowed preservation of the entire dento-
gingival region and the changes in the gingival
tissues were determined on the light micro-
scopic level, utilizing morphometric methods.
Payne et al. (1975) studied the initial gingiva!
alterations during developing gingivitis in adult
humans. The inflammatory changes during the
first 4 days were similar to the initial changes
seen in adult dogs, i.e. edema, collagen break-
down and a large increase in the number of
neutrophils in the junctional epithelium and
adjacent connective tissue. However, the biopsy
technique used in that study limited the pos-
sibilities of a thorough analysis of the gingival
changes, due to disruption of the gingivai
tissues.
The aim of the present study was lo determine
the presence of inflammatory cells in the junc-
tional epithelium and adjacent connective tissue
during initial gingivitis in 11-13 year old chil-
dren, starting from strictly normal gingiva and
employing a biopsy technique which preserves
the dento-gingival junction intact. Aiso, the
collagen content in the infiltrated portion was
determined.
Material and Methods
6 humans, aged 11-13, who, for orthodontic
reasons, needed extraction of 2 fully erupted
contralateral upper or lower premolars were
selected. Prior to the experiment the subjects
were instructed in meticulous oral hygiene
procedures, using toothbrush and floss. The
individual tooth cleaning was supplemented by
professional cleaning every second to third day
for a period of 2 months.
The condition of the buccal gingiva of the
experimental teeth was assessed once weekly
during the second pre-experimental month by
determining the gingival index {GI, Loe &
Silness 1963) and the gingival fluid flow (Loe &
365
Holm-Pedersen 1965). Before the start of the
experiment the gingival index score was 0 and
on two consecutive occasions only minor
amounts of gingival fluid were present.
On day 0 of the experiment 1 tooth in each
contralateral pair was randomly selected for
extraction. Following extraction of this tooth
the experimental subjects were told to abstain
from all oral hygiene procedures and after 4
days the contralateral tooth was extracted. At
extraction, the buccal gingiva was removed
along with the tooth as described by Schroeder
(1970). After fixation (Karnowsky 1965), the
tooth was demineralized according to War-
shawsky & Moore (1967). Following demin-
eralization the blocks were divided bucco-
linguaily into slices 1-2 mm thick, postfixed in
OsO4 and embedded in Epon. Sections 1 //m
thick were cut and double stained with me-
thylene blue and basic fuchsin (Sato & Shamoto
1973).
From each block, I section was randomly
selected for light microscopic morphologic
analysis of the gingival tissues.
All analyses were performed at a magnifica-
tion of XIOOO (objective XIOO, ocular XIO)
using a grid in the ocular of the microscope
(Matsson & Attstrom 1979). The grid covered
an area of 0.1X0.1 mm and comprised 100
squares and 121 cross-points, the latter formed
by the intersections between the lines and the
grid.
Junctional epithelium. Based on the mean length
of the junctionai epithelium of all sections, the
junctional epithelium was divided into 3 por-
tions of equal length (Fig. 1). Within each
portion, the numbers of neutrophilic granulo-
cytes and mononuclear cells were determined.
As the width of the junctional epithelium varies
from portion to portion and from specimen to
specimen, the number of inflammatory cells
was expressed as the number per unit area
(0.1X0.1 mm).
Connective tissue. By means of the grid, 3 areas
366 KLINGE, MATSSON AND ATTSTROM

Counts were madeof ail cells residing within the

Fig. 1. Schematic ilJustration of the various tissue
compartments subjected to quantitative analysis of
the presence of inflammatory cells and collagen
contetit.
Schematische Wiedergabe der verschiedenen Gewebs-
abschnitte, in denen Entziindungszellen sowie der Kol-
lagengehah quantitativ analysieri worden ist (JE: Epi-
ihelansaiz: ICT infUtriertes und NCTnichi infiltriertes
Bindegewebe).
Schema des divers comparttmenis tissulaires soumis a
I'analyse quantitative de la presence de cellules inflam-
matoires et de la teneur en collagene (JE: epithelium
de jonction: tissu conjonciif infihre ICT el non infillre
NCT).
3 grid areas, and the data were expressed as the
number of cells per unit area (0.1X0.1 mm).
Intravascular neutrophilic granulocyles and
endothelial cells were excluded. The percentage
number of intersections between collagen
bundles and grid cross-points was also calcu-
lated. The relative positions between the grid
areas are shown in Fig. 1. The distance between
the areas was 0.1 mm.
In addition, a connective tissue area opposite
the cemento-enamel junction, and adjacent to
the oral epithelium, was analysed (Fig. 1). This
area was defined as non-infiltrated connective
tissue (NCT).
Counts of cells were performed according to
the light microscopic cell morphology described
by Leeson & Leeson (1970). The following
classification of the ceils was used: N = neutro-
philic granutocytes, M / M — monocytes/macro-
phages, L —lymphocytes, P = plasma cells,
M—mast cells, F — fibroblasts,U~unidentified
cells. Cells which did not show any defined
nucleus were classified as unidentified cells.
Statistical comparisons were performed using
the t-test for paired observations. Differences at
the 5% level of probability were considered
statistically significant.

(each area comprising 0.1X0.1 mm) in the

connective tissue adjacent to the junctional Results
epithehum (ICT 1-3) were analysed (Fig. 1). The average numbers of neutrophilic granulo-

Table L Average number of neutrophils (N) and mononuelear leukocytes (M) in different portions of the
junctionat epithelium. Expressed as the number/unit area (O.IXO.I mm)
Mitilere Anzahl Neutrophiler (N) und mononuklearer Leukozyten (M) in den verschiedenen Abschnitlen des
Epithelansaizes. Die Angaben werden in Anzahl pro Niveau-Abschnitt (OJXO.I mm) ausgedriicki
Nombre moyen de neulrophiles (N) et de leucocytes mononucleaires (M) dans differentes portions de repithelium
de jonciion. Exprime en nombre par unite de surface (0.1X0,1 mm)
Level 1 Leve! 2 Level 3
Day N M N M N M

X±SD X±SD X±SD X±SD X±SD X±SD

0 1.1 1 .2 2.2 2.3 2.4 4.5 3.5 1 .5 1.8 2.0 2.4 2.4
4 1A 2 .1 5.7 2.0 2.4 2.5 5.7 3 .8 0 0 5.2 3.3
t -0.24 -2.70 -0.01 -1.26 1.80 -0.35
P N.S. P<Q.O5 N.S. N.S. N.S. N.S.
 HISTOPATHOLOGY OF INITIAL GINGIVITIS IN HUMANS 367

cytes and motionuciear leukocytes in different Table 3. Density of collagen (expressed as the per-
portions of the junctional epithelium are given centage part of the surface superimposed by lattice
points) in iCT and NCT at days 0 and 4 of the
in Table 1. At day 0 low numbers of these cells
experiment
were observed in all regions of the junctional
Kollagendichle (amgedriickt in prozentualem Anteil
epithelium. At day4 during initial gingivitis, the der Oberflache zwischen kollagenen Bundeln und den
number of neutrophilic granulocytes was still Kretizungspunktei! des MessgiUers) in den Ahschnitien
low, whereas mononuclear leukocytes had in- JCT=infiltrierter Bindegewebsabschniii und NCT—
nichl-infiUrienes Bindegewebe (siehe Fig. I), an den
creased around twofold in the junctional epithe- Versuchsiagen 0 und 4
lium. At level 1 the increase in the number of
Denxiie du collagene (exprimee en pourcenlage de
mononuclear leukocytes was statistically sig- surface recouverie par des points de croisemenij dans
nificant. le lissu conjonctif infiltre (ICT) el non infilire (NCT)
aux jours 0 et 4 de /'experience
The average numbers of inflammatory cells
in the connective tissue adjacent to the junc- NCT ICT
tional epithelium are presented in Table 2. Subject Day 0 Day 4 Day 0 Day 4
These figures represent the average number of
i 46.7 48.3 33.0 17.8
cells found per unil area in ICT 1-3. There was a 2 56.7 63.3 33.4 14.6
slight increase in netitrophilic granulocytes from 3 65.0 62.5 36.8 24. S
day 0 to day 4. The increase in the number of 4 58.3 62.5 33.2 36.5
monocytes/macrophages was more pronoun- 5 60.0 55.8 28.5 30.7
6 56.7 61.2 32.7 21.2
ced than that of the neutrophils. Lymphocytes
and plastna ceUs and the number of unidentified X 57.2 58.9 32.9 24.3
SD 6.0 5,9 2.6 8.2
cells also increased. However, for all cell types t -0.9! 2.30
only numerical differences between days 0 and 4 P N.S. N.S.
were seen, and the increments were not statisti-
Subject (Proband. sujel).
cally significant. The collagen content in ICT
1-3 and in the non-infiltrated connective tissue
(NCT) adjacent to the oral epithelium and
opposite to the cemento-enamel junction is NCT was about the same on day 4 as on day 0
shown in Table 3. The density of collagen in the and did not vary significantly from subject to

Table 2. Average number of neutrophils (N), monocytes/macrophages ( M / M ) , lymphocytes (L), plasma cells
(P), mast cells (M), fibroblasts (F), and unidentified cells (U), in ICT (ICT I, 2 and 3) at days 0 and 4
of the experiment
Mittlere Anzahi Neutrophiler (N). der Monozylen/Makrophagen (M/M), Lymphozyten (L). PlasmazeUen (P).
Mastzellen (M), Fibroblasten (Fj und nicht-idenlifizierter Zellen (U). Die Angaben werden in ICT= mfiltrierier
Bindegewebsabschnill (ICT 1, 2 und 3 - siehe Fig. }). am Versuchstage 0 und 4 angegeben
Nombre moyen de neutrophiles (N), monocytes/macrophages (M/M). lymphocytes (L). plasmocytcs (P),
mastocytes (M). fihroblastes (F) el cellules non idenlifiees (I!) dans le tissu conjonctif infiUre (ICT I. 2 et 3)
aux jours 0 el 4 de /'experience
Day N M/M M
X + SD X + SD X + SD [±SD 1
X + SD X±SD X±SD
0 1.2 2.2 9.5 1.7 2.1 1. 5 0 .9 1.4 0 .5 0.6 29.4 5. 9 2.4 0.6
4 2.2 1.5 17.7 10 4.7 4. 6 2 .3 1.9 0 .6 0.5 32.2 8. 0 4.9 3.4
t -0.76 -2.16 -I.I4 -1.29 -0.09 -0.82 -1.86
P N.S. N.S. • N.S. N.S. N.S. N.S. N.S.
Day (Tag. jour).
368 KLINGE, MATSSON AND A T T S T R O M
subject. In ICT 1-3 the density of collagen
dropped from day 0 to day 4 in 4 subjects. In
these subjects the collagen loss varied between
35-55%. In 2 subjects no decrease was found in
the coitagen content from day 0 to day 4.
Discussion
In the present experiment the infiatnmatory
reaction in the gingival margin during an initial
period of developing gingivitis was studied in
young humans. At the start of the experiment
the gingiva was in excellent health and a biopsy
technique was employed which preserves the
dento-gingival region intact. The gingival in-
flammatory process was characterized by an
increase of mononuclear leukocytes in the junc-
tional epithelium. In the adjacent connective
tissue there was only a slight increase in neu-
trophils while a more pronounced increase in
monocytes/macrophages, lymphocytes and
plasma cells was seen. The intensity of the
reaction and the type of cellular response
observed in the present study differed in part
from that known from studies on experimental
gingivitis in adult dogs (Lindhe & Rylander
1975, Schroeder et al. 1975, Matsson & Att-
strom 1979) and adult humans (Payne et al.
1975). In those studies the initial stage of
developing gingivitis was characterized by an
extensive connective tissue reaction and a heavy
neutrophilic response. The limited increase in
the number of neutrophilic granuiocytes ob-
served in the present study agrees more with
findings in studies of the initial stage of devel-
oping gingivitis in juvenile dogs (Matsson &
Attstrom 1979).
The collagen content in ICT 1-3 dropped
from day 0 to day 4 in 4 of the subjects. The
remaining 2 subjects showed no loss of collagen.
In the former 4 subjects, the decrease was lower
than that observed in adult dogs (Lindhe &
Rylander 1975, Schroeder et al. 1975, Matsson
& Attstrom 1979). In the latter studies a col-
lagen loss of 60-70% was found in the corona!
region of the gingiva! connective tissue during
the initial 4 days of developing gingivitis. The
lower degree of co!!agen loss found in the
present study corresponds more to that in
juvenile dogs, where only a slight decrease was
seen from day 0 to day 4 of developing gingivitis
(Matsson & Attstrom 1979).
In general the gingival reaction to the devel-
opment of dental plaque in young humans
seems to correspond more to the type of
reaction reported for juvenile dogs than to that
occurring in adult dogs. Clinical studies in
humans have shovi'n that gingivitis develops
more slowly in pre-school children than in
adults (Matsson 1978). A similar difference
exists between juvenile and adult dogs (Matsson
& Attstrom 1979). Therefore, it may be that
11-13 year old children have a gingivitis devel-
opment pattern which corresponds more to that
of pre-school children than to that of adults.
The reason for this might be differences in the
bacterial flora between young and mature indi-
viduals, or that young individuals have a dif-
ferent inflammatory and immune reaction.
Studies using bacteriological analysis of the
dento-gingival plaque, combined with simul-
taneous determinations of the inflammatory
reaction in the adjacent gingiva, could give
further insight into the mechanisms involved in
the tissue destruction occurring during initial
gingivitis.
Zusammenfassung
Die Hisiopathologie beginnender Gingivitis beim Men-
schen
Eine orientierende Siudie
Diese Studie befasst sich mit entziindlichen Veran-
deruiigen des Zahnfleischsaumes wiihrend beginnen-
der Gingivitis, bei 11-13 jahrigen menschiichen Pro-
band en.
Am Versuchstage 0 hatten alle Probanden saiibere
Zahne und eine gesunde Gingiva. Dann wsirde jeg-
liche aktive Mundhygiene 4 Tage tang abgesetzl. Am
Versuchstage 0 und 4 wurden an oberen und unteren
Pramolaren, die aus orthodontischen Grtinden ex-
trahiert wurden, kontraiaterale Biopsien entnommen,
die den Zahn und die ihn umgebende Gingiva um-
fassten. Das Vorhandensein von Entziindungszellen
 HISTOPATHOLOGY OF INITIAL GINGIVITIS IN HUMANS
im Epiihelansatz und in den umgebenden Binde-
geweben wurde mit Hilfe halbdiinner Schniite quan-
titativ erfassl. Ausserdem wurde dcr Kollagengehall
des Zahnfleischsaumes ermitteit.
Vom Tage 0 bis zuni Tage 4 wurde nur eine ieichte
Zunahme neutrophiler Granulozyten im Epithelan-
salz und in dem umgebenden Bindegewebe gesehen,
wahrenddessen eine deutliche Zunahme der Anzahl
mononukJearer Leukozyten gefunden wurde. Bei 4
Probanden wurde Kollagenverlust beobachtet, wiih-
rend bei 2 Probanden der Kollagengehalt unverandert
verblieb.
Die in dieser Studie gefundene entziindliclie Reak-
tion unterschcidet sich etwas von denen, die bei
erwachsenen Menschen und bei erwachsenen Hunden
bcobaehtet wurden. Die Resuitate entsprechen denen,
die bei juvenilen Hunden beschrieben woiden sind.
Resume
Hiatopaihologie de la gingiviie inhialc chez I'homme
Etude piloie
La presente etude porte sur Ics modifications inter-
venant an niveau de la gencive marginale durant la
gingivite initialc chez les enfants de II a !3 ans.
Au jour 0 de I'experience, les 6 sujets avaient les
dents propres et la gencive saine. Toute mesure
d'hygiene buccale a ete suspendue pendant 4 jours.
Aux jours 0 et 4, des biopsies contralaierales com-
prenant !a dent et la gencive adjacente ont ete
preleveessurdespremolairessuperieuresctinferieures
extraites pour des raisons orthodontiques. La pre-
sence de cellules inflammatoires dans repithelium de
jonction et dans le lissu conjonctif adjacent a ete
detcrminec quantitativement dans des coupes semi-
fines. La tcneur en collagene dc la gencive marginale a
egalement ete determinee.
Du jour 0 au jour 4 on n'observait qu'un ieger
accroissement du nombre de granuiocytes neutro-
phiies dans I'epithelium dc jonction et le tissu con-
jonctif adjacent, mais une plus forte augmentation du
nombre de leucocytes mononucleaires. Une perte de
coilagene etait enregistree chez 4 sujets, tandis qu'au-
cune modification de la leneiir en collagene n'ap-
paraissait chez les 2 autres sujets.
La reaction infiammatoire decrite dans la presente
etude diflere quelque peu de ceiie observee che?,
I'homme adulte et le chien aduhc. Les resultats cor-
respondent plus a la reaction observee chez le jeune
chien.
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Address:
Bjorn Klinge
School of Dentistry
University of Lund
Car! Gustafs vag 34
S-214 21 Malmo
Sweden •-.