Jan Lindhe. Clinical Periodontology

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HOST-PARASITE INTERACTIONS IN PERIODONTAL DISEASE •

Fig. 5-3. Leukocytes in the junctional epithelium. Ob-

serve that the volume of leukocytes decreases in apical

direction and approaches 0 in the most apical portion.

Within the junctional epithelium, the mononuclear leu

kocytes are located in more basal layers, while the

neutrophilic granulocytes are present primarily in the

superficial portions of the junctional epithelium.

Fig. 5-4. Immunostained section showing neutrophils

in the junctional epithelium of healthy gingiva.

transudative fluid and plasma proteins arrive in the

gingival crevice region having left the vessels and

travelled through the tissues to create the gingival

crevicular fluid (GCF) (Egelberg 1967, Cimasoni 1983)

(Figs. 5-1 and 5-2). The infiltrate at this stage may

occupy as much as 5% of the connective tissue volume

and is composed of monocytes, macrophages, lym-

phocytes and neutrophils. These cells are found in the

junctional epithelium as well as in the connective

tissue of clinically healthy gingivae (Fig. 5-3). Neutro-

phils predominate in the crevice region and appear to

migrate continuously through the junctional epithe-

lium into the sulcus (Fig. 5-4). The recruitment of

leukocytes (predominantly PMNs) from the tissues to

the crevice is due both to the chemoattractant actions

of the host systems (interleukin-8, complement com-

ponent C5a, leukotriene B4 etc., see below) and prod-

ucts derived from the biofim (formyl methionyl leucyl

phenylalanine, lipopolysaccharide etc). With further

deposition of plaque and the development of overt

gingivitis, there is a marked increase in leukocytes

recruited to the area (Attstrom, 1971, Moughal et al.

1992). One additional effect of the inflammation

which encourages rapid accumulation of leukocytes

is the pro-inflammatory cytokine-mediated upregula-

tion of adhesion molecules on the endothelial cells.

This encourages leukocytes, particularly PMNs, in the

early stages to adhere to postcapillary venules and

begin migrating through the vessel and chemotacting

to the gingival crevice. This upregulation of adhesion

molecules (ICAM-1 and ELAM-1) occurs during "ex-

perimental gingivitis" (Loe et al. 1965) with a con-

comitant increase in leukocyte infiltration which cor-

responds with days of plaque accumulation (Kinane

et al. 1991, Moughal et al. 1992) (Fig. 5-5).

Clinically healthy gingiva appears to deal with

microbial challenges without progressing to a dis-

eased state, probably because of several defensive

factors which include:

1. Regular shedding of epithelial cells into the oral

cavity

2. Intact epithelial barrier

3. Positive fluid flow of the gingival crevice which

may remove non-attached microorganisms and

noxious products

154 • CHAPTER 5

Fig. 5-5. (a) ICAM-1 immunohistochemical staining of a gingival biopsy sample during an experimental gingivitis

study in humans after day 7. ICAM-1 positive blood vessels and junctional epithelium can be clearly seen.

(b) Higher magnification of Fig 5-5a showing the extensive junctional epithelium staining. (c) Higher magnification of

Fig 5-5a showing the ICAM-1 positive vessels within the connective tissue.

4. Antimicrobial effect of antibodies

5. Phagocytic function of neutrophils and macro-

phages

6. Detrimental effect of complement on the micro-

biota.

All of these factors may operate at the same time to

reduce the bacterial load and thus prevent an over-re-

sponse of the tissue defense systems which could

result in the formation of a lesion. The host-microbial

interplay which constitutes the clinically healthy situ-

ation must clearly change if gingivitis and periodon-

titis is to follow. Gingivitis will follow if there is suffi

cient plaque accumulation such that microbial prod-

ucts will initiate a substantive inflammatory response.

This response can be modified by hormones as in the

case of puberty or pregnancy (resulting in edematous

gingivitis) or by drugs such as phenytoin, cyclosporin

or nidefipine (which induce gingival overgrowth).

Gingivitis lesions are accompanied by more pro-

nounced loss of collagen albeit in discrete areas. The

gingivitis response will also initiate and perpetuate

immune responses to the oral microorganisms but the

level of this early response, particularly at the local

site, will be considerably less than the gross tissue

destruction and bone loss seen in more advanced

periodontitis lesions. Gingivitis may persist at sites for

many years without appreciable loss of periodontal

attachment, destruction of periodontal ligament or

evidence of bone loss. Clearly certain individuals (and

sites) go on to develop periodontitis from gingivitis

lesions whilst others remain resistant and merely ex-

hibit gingivitis responses to the accumulating plaque

microorganisms. Investigations of periodontal mani-

festations of systemic disease suggest that individuals

with obvious defects of the inflammatory system, e.g.

neutrophil depletion or dysfunction, may rapidly de-

velop severe periodontitis. In addition, there appears

to be a genetic predisposition to both aggressive and

chronic forms of periodontitis (Michalowicz et al.

2000, Hodge & Michalowicz 2001). There is an accu-

mulating body of evidence which suggests that the

host's immune response to periodontopathogens may

be quite different in those affected by chronic perio-

HOST-PARASITE INTERACTIONS IN PERIODONTAL DISEASE •

Fig. 5-6. Gingival alterations which occurred during a 28-day period of plaque accumulation and gingivitis devel-

opment in beagles. (a) Normal gingival. (b) Day 4. (c) Day 7. (d) Day 14. (e) Day 21. (f) Day 28 of undisturbed

plaque accumulation. Note the gradually developing plaque on the tooth surfaces and the inflammatory changes

in the gingiva. The vascular reaction is illustrated by a gradually increasing number of vessels in the gingival mar

-gin. (g) Gingival index (GI), plaque index (PLI) and gingival exudate alterations (exudate) that occurred during

the experimental gingivitis period. (h) In gingival biopsies obtained at various time intervals it can be seen that

the inflammatory cell infiltrate (ICT) in the gingiva gradually increased in size.

dontitis and those resistant to this disease, who would

not progress beyond gingivitis. There is also evidence

that "protective" antibodies increase following a

course of initial periodontal therapy, whereas those

individuals with a poor outcome following periodon-

tal therapy have antibodies which are less functional (

Mooney et al. 1995).

Initial, early, established and advanced

lesions

Introduction

Within 10-20 days of plaque accumulation, clinical

signs of gingivitis are established in most individuals,

although this varies greatly with some individuals

being intrinsically more resistant and others more

prone to overt gingivitis (Van der Weijden et al. 1993).

This gingivitis appears as gingival redness, swelling

and an increased tendency of the soft tissue to bleed on

gentle probing (Fig. 5-6). Even at this stage clinical

signs are reversible following removal of microbial

plaque by effective plaque control measures (Loe et al.

1965, Lindhe & Rylander 1975).

Histopathological features of gingival inflammation

The clinical changes may appear subtle in the early

stages of gingivitis but the underlying histopathologi-

cal changes are quite marked. Alterations in the vas-

cular network occur with many capillary beds being

opened up. Exudative fluid and proteins swell the

tissues and an influx of inflammatory cells in the

connective tissue occurs subjacent to the junctional

epithelium. The inflammatory cell infiltrate mainly

156 • CHAPTER 5

Table 5-1. Size (% volume) of various components of the normal connective tissue (NCT) and the infiltrated

connective tissue (ICT) at various days (4, 14 and 28) after onset of plaque formation (from Lindhe & Rylan-

der 1975)

comprises lymphocytes, macrophages and neutro-

phils. As the cellular infiltrate develops, the structural

and cellular composition of the tissues changes. An

experimental gingivitis study in dogs has compared

the cellular and structural composition of the affected

area before and during the development of gingivitis

over a period of 28 days (Lindhe & Rylander 1975).

Plaque was allowed to accumulate on the teeth of dogs

with initially normal gingiva, and biopsy samples

were taken at various times. The normal tissue (Day

0) is referred to below as non-infiltrated connective

tissue (NCT) and the altered area as the infiltrated

connective tissue (ICT) (Table 5-1).

At Day 0 of this dog experiment the normal gingival

unit has virtually no inflammatory cells (Fig. 5-7) and

is comprised of approximately 40-45% epithelium and

55-60% connective tissue. The NCT zone consists of

collagen (60%), fibroblasts (13%), vessels (7%) and

other tissue constituents, such as intercellular matrix

and nerves (20%). Following plaque accumulation,

neutrophils and mononuclear leukocytes readily mi-

Fig. 5-7. Composition of the gingiva (GT), non-infil-

trated (NCT) and infiltrated (ICT) gingival connective

tissue on day 0 (normal gingiva) and on day 28 of gin-

givitis in beagles. Note that the infiltrated connective

tissue portion amounts to 17% of the free gingival mar

-gin on day 28 and that the collagen content is reduced

from approximately 60% on day 0 to 30% on day 28 in

the area where the inflammatory infiltrate has become

established. Note also that in this area (on day 28) a re-

duction in fibroblast proportion has occurred, as well

as an increase of vessels and residual tissue. OE: oral

epithelium, JE: junctional epithelium, NCT: non-infil-

trated connective tissue, ICT: infiltrated connective tis-

sue, CO: collagen fibers, FI: fibroblasts, V: vascular

structures, L: leukocytes, R: residual tissue. (From

Lindhe & Rylander 1975)

grate to this area and the ICT begins to form and

increase in volume over the 28-day period. At this 28-

day interval the ICT is comprised of lymphocytes,

plasma cells and macrophages (Fig. 5-7) which adhere

to the collagen matrix and remain in the tissue,

whereas neutrophils continue to migrate into the gin-

gival sulcus. With the extensive influx of leukocytes, a

marked reduction in the amount of collagen and

fibroblasts occurs and the volume of residual tissue (

intercellular matrix, degraded collagen, exudate ma-

terial, degenerated or dead cells) and small blood

vessels increases.

In 1976, Page and Schroeder classified the progres-

sion of gingival and periodontal inflammation on the

basis of the then available clinical and histopathologi-

cal evidence. They divided the progressing lesion into

four phases: initial, early, established and advanced stages

or lesions. The initial and early lesion descriptions

were thought to reflect the histopathology of clinically

early stages of gingivitis, while the established lesion

reflected the histopathology of more

HOST-PARASITE INTERACTIONS IN PERIODONTAL DISEASE • 157

"chronic" gingivitis. The description of the his-

topathology of the advanced lesion was considered to

reflect the progression of gingivitis to periodontitis.

The evidence on which these descriptions were based

was the prevailing information gleaned predomi-

nantly from animal biopsy material and some human

adolescent samples. Therefore the following account

by Page and Schroeder (1976) of lesion progression is

based very much on data from non-human experi-

ments.

The classical phases of "acute" and "chronic" in-

flammation are not easily applied in periodontal dis-

ease, probably because in most clinically healthy situ-

ations a lesion similar to an acute lesion occurs. Sub-

sequently, chronic inflammatory changes become su-

perimposed so that both acute and chronic elements

co-exist in early, established and advanced lesions. It

is important to repeat that in most clinically "normal"

human gingival biopsies a similar infiltrate can be

seen to that noted in the initial and early gingival

lesions of dogs. The Page and Schroeder system will

be utilized as a framework to outline the histopatho-

genesis of periodontal disease and modern conflicting

views will be outlined. A rational synthesis of opin-

ions will be attempted and a new classification based

on our current understanding of lesion progression

will be presented (Table 5-2).

Fig. 5-8. Schematic illustrations depicting the terminal

vascular bed and the mechanisms of increased vascu-

lar permeability. Under normal conditions the terminal

vessels are freely permeable to small molecules, salt

and water. The intercellular junctions between the en-

dothelial cells are closed. Via the influence of vasoac-

tive mediators the endothelial cells become separated

and increased vascular permeability occurs. Large

molecules and plasma proteins leak into surrounding

tissue. A number of inflammatory mediators present in

plasma are activated when they enter the perivascular

tissue.

Clinical condition Histopathologic condition

Histologic perfection

Initial lesion of Page & Schroeder

Early lesion of Page & Schroeder (few

plasma cells)

Established lesion with no bone loss nor

apical epithelial migration (plasma cell

density between 10% and 30% of

leukocyte infiltrate)

Established lesion with bone loss and

apical epithelial migration from the

amelocemental junction (plasma cell

density > 50%)

The initial lesion

Inflammation quickly develops as plaque is deposited

on the tooth. Within 24 hours marked changes are

evident in the microvascular plexus beneath the junc-

tional epithelium as more blood is brought to the area.

Dilation of the arterioles, capillaries and venules of the

dentogingival plexus is evident histopathologically.

Hydrostatic pressure within the microcirculation in-

creases and intercellular gaps form between adjacent

Pristine gingiva

Normal health

gingiva

Early gingivitis

Established

gingivitis

Periodontitis

Table 5-2. A new classification is outlinec

158 • CHAPTER 5

Fig. 5-9. Schematic illustration of the process whereby neutrophils are attracted into the junctional epithelium and

crevice region.

Fig. 5-10. Alterations in number of crevicular leuko-

cytes and in gingival fluid during a period of develop

ing gingivitis in beagles. Note the gradual increase

of leukocytes and fluid flow during the experimental

period. (From Attstrom & Egelberg 1970.)

capillary endothelial cells. An increase in the perme-

ability of the microvascular bed results, so that fluids

and proteins exude into the tissues (Fig. 5-8).

As the lesion enlarges, and gingival crevicular fluid

flow increases, noxious substances from microbes will

be diluted both in the tissue and the crevice. Bacteria

and their products may thus be flushed from the

sulcus. Plasma proteins escaping from the microcircu-

lation include defensive proteins such as antibodies,

complement and protease inhibitors and other macro-

molecules with numerous functions, which will be

discussed below. This gingival crevicular fluid (GCF)

can be readily sampled by placing filter strips at the

gingival margin to absorb the exudate. The volume of

the exudate is proportional to the severity of the gin-

gival inflammation present (Fig. 5-6). The absolute

amounts and the concentration of various plasma

proteins, tissue proteases, inhibitors and breakdown

products and leukocyte enzymes in the gingival sul-

cus have been studied extensively. GCF components

are considered as very useful markers of the inflam-

matory process and are currently being developed as

diagnostic markers of periodontal disease.

Simultaneously with these vascular alterations,

PMN cell migration from the dentogingival vascular

system is enhanced by the adhesion molecules, inter-

cellular adhesion molecule-1 (ICAM-1) and endothe-

lial leukocyte adhesion molecule-1 (ELAM-1) and

other adhesins. These molecules assist PMNs binding

to the post capillary venules and help the cells to leave

the blood vessel (Fig. 5-9). The leukocytes migrate up

a chemoattractant gradient to the crevice and are prob-

ably further assisted in their movement by the adhe-

sion molecules uniquely present on the junctional

epithelial cells (Fig. 5-5) (Moughal et al. 1992) and by

the presence of host and microbial chemotactic factors.

Lymphocytes may be retained in tissues on contact

with antigens, cytokines or adhesion molecules and

thus are not so readily lost through the junctional

epithelium and into oral cavity, as are PMNs. Most

lymphocytes have the ability to produce CD44 (CD =

cluster determinant) receptors on their surfaces,

which permit binding of the cell to the connective

tissue framework. This is consistent with the T and B

cell requirement to remain within the tissues and to

perform cell mediated and humoral immune func-

tions locally.

Probably within 2-4 days of plaque build-up the

HOST-PARASITE INTERACTIONS IN PERIODONTAL DISEASE •

Fig. 5-11. Buccolingual section of the gingiva on day 4 of developing gingivitis in a beagle. Note plaque (P) in the

sulcus region and increased cellularity in the coronal part of the connective tissue below the junctional

epithelium. Note the presence of leukocytes in the coronal part of the junctional epithelium and at the surfaces of

the subgingival plaque.

cellular response is well established and is helped by

chemotactic substances originating from the plaque

microbiota as well as from host cells and secretions.

PMNs move through the connective tissue and the

majority seem to accumulate in the junctional epithe-

lium and gingival sulcus region (Figs 5-9 and 5-10).

The early lesion

The early gingival lesion occurs after approximately

one week of plaque accumulation (Fig. 5-11). Only an

approximation of the time required can be given as

marked subject variation occurs in humans although

this may well be less variable in animal models. The

variation seen amongst humans could be due to dif-

ferences in plaque accumulation, both at the site and

subject level, or to differences between individuals in

features such as hormonal levels. Histologically the

vessels below the junctional epithelium remain di-

lated, but their numbers increase due to the opening

up of previously inactive capillary beds (compare Fig.

5-1 and 5-12). The course, size and quantity of mi-

crovasculature units are reflected in the clinical ap-

pearance of the gingival margin during this phase (

Egelberg 1967, Lindhe & Rylander 1975).

Lymphocytes and PMNs are the predominant infil-

trating leukocytes at this stage and very few plasma

cells are noted within the lesion (Listgarten & Elle-

gaard 1973, Payne et al. 1975, Seymour et al. 1983,

Brecx et al. 1987). The inflammatory cell infiltrate may

at this stage comprise as much as 15% of the connec-

tive tissue volume. Within the lesion fibroblasts de-

generate. This probably occurs by apoptosis and

serves to remove fibroblasts from the area, thus per-

mitting more leukocyte infiltration (Page & Schroeder

1976, Takahashi et al. 1995). Similarly collagen de-

struction occurs in the infiltrated area and is necessary

in order that the tissues can be pushed apart to accom-

modate the infiltrating cells and thus could be consid-

ered as a space creating process. Inflammatory

changes are detectable clinically at this stage and near-

ing the end of the second week of plaque accumula-

tion, a subgingivally located biofilm can be found.

The basal cells of the junctional and sulcular epithe-

lium have now proliferated. This represents an at-

tempt by the body to enhance the innate barrier to

plaque (Fig. 5-13). Epithelial rete pegs can be seen

invading the coronal portion of the lesion (Schroeder

1970, Schroeder et al. 1973).

The duration of the early lesion in humans has not

been determined. The early lesion may persist for

much longer than previously thought and the vari-

ability in time required to produce an established

160 • CHAPTER 5

Fig. 5-12. (a) Buccolingual section of chronically in-

flamed gingiva in a dog. The vessels have been filled

with a contrasting carbon suspension. Note the wide

and tortuous vessels below the junctional epithelium

to the left in the illustration. Compare with the vascula-

ture in healthy gingiva in Fig. 5-10. The pronounced

vascular reaction in this area is a result of plaque accu-

mulation. (b) Mesiodistal section of a gingiva illustrat-

ing the vessels below the junctional epithelium of

chronically inflamed gingiva. Note vascular dilation

and proliferation at the level of the gingival margin (

GM). Note also that vascular proliferation has oc-

curred in the apical part of the vascular plexus (from

Egelberg 1967).

Fig. 5-13. Buccolingual section of the gingiva on day 7

of developing gingivitis in a beagle. Note the subgingi-

val extension of plaque (P), as well as the increased

number of inflammatory cells in the connective tissue

below the dentogingival epithelium.

HOST-PARASITE INTERACTIONS IN PERIODONTAL DISEASE • 161

Fig. 5-14. Buccolingual section of the gingiva on day 21

of developing gingivitis in a beagle. Compare with Fig.

5-15. Note the extension of the subgingival plaque (P)

as well as the apical extension of the infiltrate (the le-

sion) in the connective tissue.

Fig. 5-15. Buccolingual section of the gingiva on day 28

of gingivitis development in a beagle. Note the exten-

sion of the subgingival plaque (P) and the increased

number of leukocytes in the connective tissue below

the junctional epithelium. Note rete pegs in the den-

togingival epithelium.

lesion may reflect susceptibility variance within and

between subjects.

The established lesion

Generally there is a further enhancement of the in-

flammatory state as exposure to plaque continues.

There is increased fluid exudation and leukocyte mi-

gration into the tissues and the gingival crevice. Clini-

cally this lesion will exhibit more edematous swelling

than the "early gingivitis" lesion and could be consid-

ered as "established gingivitis".

The established lesion as defined by Page and

Schroeder is one dominated by plasma cells. This

conclusion was based mainly on data from animal

experiments. However, Brecx et al. (1988) demon-

strated that even following 6 months of oral hygiene

neglect, the plasma cell fraction in human biopsies

comprised only 10% of the cellular infiltrate and was

clearly not the dominant cell type. Thus, the human

established lesion apparently requires much more time

to "mature" than its animal counterparts.

In the established lesion described by Page and

Schroeder (1976), plasma cells are seen situated pri-

marily in the coronal connective tissues as well as

Fig. 5-16. Diagram showing the increase in size of the

infiltrated connective tissue during the development of

gingivitis in beagles (from Mattsson & Attstrom 1979)

around vessels. Collagen loss continues in both lateral

and apical directions as the inflammatory cell infil-

trate expands, resulting in collagen depleted spaces

extending deeper into the tissues which are then avail-

able for leukocytic infiltration (Figs. 5-14, 5-15, 5-16).

During this time the dentogingival epithelium contin-

162 • CHAPTER 5

Fig. 5-17. Schematic illustration of the changes in the gingival tissues during the development of gingivitis and

periodontitis. The most significant differences are in the extent and composition of the inflammatory infiltrate and

the epithelial proliferation in gingivitis, and the apical migration of epithelium and bone loss seen in periodontitis

lesions.

ues to proliferate and the rete pegs extend deeper into

the connective tissue in an attempt to maintain epi-

thelial integrity and a barrier to microbial entry. The

junctional epithelium is changed and is no longer

closely attached to the tooth surface. The pocket epi-

thelium that now has formed has a heavy leukocyte

infiltrate, predominantly of PMNs which eventually

migrate across the epithelium into the gingival pocket.

In comparison to the original junctional epithelium,

the pocket epithelium is more permeable to the pas-

sage of substances into and out of the underlying

connective tissues and may in places be temporarily

ulcerated. Fig. 5-17 schematically illustrates the altera

tions which occur in the epithelium and the connec-