Histological observations on gingival mucosa level

determined by fixed partial dentures

Authors: Lucia-Elena Moldoveanu, Ph.D., D.M.D, Assistant Professor, Faculty of Dental

Medicine, Ovidius University, Constanta, Romania

Mehedinti Teofil, Ph.D., Professor, Faculty of Medicine, Ovidius University, Constanta,

Romania

Gheju Anca, Ph.D., MD, Anatomical Pathology Laboratory, No.2 Clinic, Municipal

Hospital Constanta, Romania

Dima Loredana, Ph.D., Lecturer, non-affiliated

Corresponding author: Lucia-Elena Moldoveanu, 92 Crinului Street, Constanta, Romania.

Phone: +40. 724.871.338; +40.241.630.868

Email address: lucia_moldo@yahoo.com

Key Words: Fixed Partial Dentures, Oral Mucosa, Fibrous chorion, Gingival Epithelium,

Papillomatous Lesions.

ABSTRACT

Background

The study aims to present the results of the microscopic tests obtained by prelevating

gingival mucosa from patients with fixed dentures. It used conventional histological techniques

for overall examination of tissues.

We should mention from the very beginning that this is the first part of a more detailed

study which covers a larger number of preparations and more articulated observations.

Methods

The cytohistopatological study was performed based on the known fact that the

interaction between biomaterial and tissue occurs both directly in a very narrow area in the

proximity of the prosthetic biomaterial, and indirectly in secondary reactions and interactions

situated at a distance and deep in these areas. Cells can interact through molecular groups located

on their membranes (membrane receptors, ligands etc.) or through various biochemical

substances found in the extracellular matrix (proteoglycans, inorganic substances and so on).

Literature information has shown that biomaterials with different chemical compositions trigger

different biological responses.

Results

The results present 18 images of the chorion and the treatment of the gingival mucosa epithelium

at the cellular level. It was observed that the biological habitat formed by wearing dentures, as

well as different prosthetic materials, can cause various clinical and histopathological changes on

gingival mucosa.

Conclusions

The cytohistological changes of the gingival mucosa are present in all types of prostheses, but

their intensity and variability depends on the biomaterial used in their production.

Subjects who used fixed dentures with metal components presented chronic inflammatory

reactions, as well as histological reactions depending on the type of metal used in making the

denture as follows: fixed dentures with metal part of gaudent revealed histological lesions such

as condyloma acuminata, while fixed dentures with metal parts made of wipla predominantly

showed papillomatous lesions.

ABSTRAIT

Contexte

La recherche se propose de présenter les résultats des testes microscopiques obtenues à

travers le prélèvement de la muqueuse gingivale des patients qui portent des prothèses dentaires

fixes. Dans cette recherche on a utilisé des techniques histologiques classiques pour l’examen

des tissus.

Méthode

La recherche cisto-pathologique a comme point de départ la connaissance de l’interaction

entre le bio-métal et le tissu, interaction qui se manifeste directement dans une zone très étroite

située près du bio matériel prothétique où on a à faire aussi avec des réactions secondaires et des

interactions indirectes à distance et au profondeur de ces zones. Les cellules peuvent

interactionner comme des regroupements cellulaires aux niveaux de leurs membranes (récepteurs

de membrane et éléments de liaisons) ou comme des substances biochimiques qui se trouvent

dans la matrice extracellulaire (substances anorganiques). Dans la littérature de spécialité on voit

que les bio matérielles ayant de différentes compositions chimiques donnent des réponses

biologiques différentes.

Résultats

Les résultats présentes 18 images du chorion et du traitement épithélial de la muqueuse

gingivale au niveau cellulaire. On a observé que l’habitat biologique formé à la suite de

l’utilisation des prothèses, comme aussi des divers matériaux prothétiques, peut porter à des

changements cliniques et histo-pathologiques au niveau de la muqueuse gingivale.

Conclusions

Pour toutes les prothèses on observe des changements cyto-histologiques de la muqueuse

gingivale, mais leur intensité et leur variabilité dépend du bio matériel utilisé dans leur

fabrication.

Ceux qui ont utilisé des prothèses dentaires fixes ayant du métal en composition, ont eu

des réactions d’inflammation chronique, mais aussi des réactions histologiques selon le type du

métal utilisé: les prothèses fixes ayant en composition du gaudent met en évidences des lésions

histologiques –type condylome acuminé, en tant que les prothèses fixes avec du métal de type

wipla ont eu surtout des lésions papillomateuses.

INTRODUCTION

The human gingival mucosa is a variety of oral mucosa that forms a unitary and

integrated morphofunctional complex in the stomatognathic system, the most exposed to external

influences.

The gingival mucosa changes caused by fixed partial dentures aggression can determine

clinical and histopathological manifestations and provide important data on the oral cavity tissue

tolerance to materials used in making dentures.

Different prosthetic parts can cause changes of gingival mucosa, some featuring

adjustment and disappearing after the adjustment process, while others can cause histological

changes with inflammatory character, both in the areas of direct contact with prosthetic material

and perilesional, in the proximity or small distance from it.

OBJECTIVE:

The study of microscopic preparations obtained by prelevating gingival mucosa from

patients with fixed dentures (who have agreed on that) was conducted using conventional

histological techniques for overall examination of tissues.

This is the first part of a more detailed study which covers a larger number of

preparations.

MATERIAL

Histological investigations were performed on a total of 83 fragments of human gingival

mucosa prelevated from patients wearing unidental and pluridental fixed dentures, during 2002-

2008. The dentures were made of Gaudent – copper-based alloys, Wipla, Remanium and

acrylate.

The study of microscopic preparations was conducted using common colorations to

examine the overall tissue architecture.

For the comparative analysis a control group was used, with normal clinical aspect,

belonging to the same subject.

Patients included in the study covered both genders, aged 31 - 74. The selection criteria

considered patients with no affections associated to oral mucosa or registered with general

affections. The patients came from the geographical environment of Dobrogea, both urban and

rural areas. Out of the 45 patients wearing fixed prostheses, 15 patients had pluridental fixed

partial dentures, while 30 patients had unidental fixed prostheses. The patients had worn fixed

dentures for at least 10 years, and the material used in making dental prosthesis consisted of

Wipla, Gaudent, Remanium and acrylate.

No.

crt.

Topography of prelevated edentulous ridge No.of fragments

1. Agrressed maxillary edentulous ridge 14

2. Maxillary edentulous ridge to fixed prostheses with normal

clinical aspect

10

3. Agrressed mandibular edentulous ridge 19

4. Mandibular edentulous ridge with normal clinical aspect 10

5. Agrressed marginal periodontium 30

6. Total of prelevated fragments of gingival mucosa 83

Table 1. Topographical distribution of prelevated fragments of gingival mucosa

The size of the prelevated fragments of gingival mucosa was 3-4 mm in surface, and in

depth the excision was done up to the periosteum of maxillary bone.

METHODS

The cyto-histological and histometrical study was based on the histologic technique of

paraffin inclusion used in the Laboratory of Cytohistology of the Faculty of Medicine Constanţa

(Professor. Dr. Teofil Mehedinţi). The histopathologic exam necessary for the speciality

diagnosis was performed in the Anatomical Pathology Laboratory, No.2 Clinic, Municipal

Hospital Constanta (dr.Gheju Anca).

The histologic sections were stained both in common dyes with hematoxylin and eosin,

as well as in Masson’s trichrom for overall structures.

RESULTS

During optical microscopy there were observed acute inflammatory lesions of the

perilesional tissue with vascular dilatation and inflammatory exudate (by plasma extravasation,

of figurative elements, etc.) (Fig.1)

Fig.1: Chorion with lymphocyte-plasmocyte-granulocyte polymorphous

inflammatory infiltrate, hematic hyperemiated and extravasated capillaries. Col. Masson’s

trichrome, ob X 40

In chronic inflammations of perilesional mucosa there were found proliferative lesions in

the lesional focal area. The infiltrated chorion appears to be thickened with a conjunctive-

vascular tissue of neoformation and limfoplasmocitar infiltrate with diffuse or nodular

appearance formed by reaction cells (lymphocytes, macrophages). (Fig.2)

Fig.2: chorion with granulomatous infiltration with nodular disposition. Col H.E., obx10

The congestion of chorion was found at the level of periprosthetic gingival cuff. In some

patients there was found a gingival mucosa with no inflammatory lesions, but there are

degenerative lesions both in the epithelium level (cytoplasmic vacuolation) and on the chorion

level. (Fig.3)

Fig.3: Epithelial cytoplasmatic vacuolation. Col.H.E., obX40

There were also observed the widening of the intercellular space and desmolysis lesions,

especially within the suprabasal layer. (Fig.4)

Fig.4: Desmolysis in the epithelial suprabasal layer. Col.H.E., obX40

The examination of the basal shell of periprosthetic gingival epithelium showed

significant changes at the level of the junctional systems, both intercellular systems and between

the basal pole of epithelial cells and the basal membrane, changes manifested by partial

dislocations of the epithelial stratum from the subjacent chorion. (Fig.5)

Fig.5: Partial dislocations of the epithelial stratum from the subjacent chorion. Col.H.E.,

obX40

The papillary chorion looks to be low in cells and richer in fibers of collagen type I, and

it also presents vasodilatation and edematous infiltrate. (Fig.6)

Fig.6: Fibered chorion witn numerous capillaries. Col.H.E., obX40

The loss of connections between epithelial cells leads to the formation of cleavages,

blisters or bubbles (acantholysis).

At the cellular level, the gingival mucosa epithelium showed both nuclear changes, such

as picnosis, cariorexis or karyolysis, and changes of the tinctorial reaction and the number of

nuclei or nuclear area. (Fig.7)

Fig.7: Diminishment of the epithelial nuclei number. Col.H.E., obX40

The microscopic examination of gingival mucosa epithelium also revealed modifications

of cell cytoplasm, manifested by vacuolation or replacement of these cells with koilocytes, cells

modified by prosthetic stress, changes accompanied by nuclear involutions. The presence of

these cells was found throughout the thickness of the epithelium. (Fig.8)

Fig.8: Koilocytes. Col.H.E., obX40

Cells showing small core, double or multiple, with chromatin in the form of islands of

different shape and size, without nuclear membrane. The core is surrounded by an optically clear

perinuclear cavity, bordered by an intense eosinophilic cell membrane.

The stratified squamous epithelium shows acantholysis type changes; increased epithelial

thickness by squamous cell hyperplasia with hypergranulosis of stratum corneum (Fig.9)

Fig.9: Squamous epithelial hyperplasia with acantholysis, Col.HE, obX10

Superficial chorion papillae appear elongated and epithelial ridges deepen toward the

profound layer of the chorion, having the appearance of papillomatosis.

Ob.20 analyzed sections and stained with Masson method highlights collagen fibers

arranged in thick fuxinophil bundles, targeted at different incidences.

In some regions, there are observed deposits of hyaline dystrophy.

In some cases there were noted, at the level of paraprosthetic gingival mucosa,

papillomatosis type lesions, in some areas the interruption of the continuity of junction

epithelium - lamina propria (basement membrane). These lesions have facilitated disruption of

selective biological control of basement membrane upon the tissue fluid content in the chorion

extracellular matrix. In this way, epithelial-mesenchymal interactions, established already in the

embryonary life, are changed, getting other meanings and other parameters.

On some sections of the same batch there were found acute inflammatory processes with

the predominance of polymorphonuclear neutrophils, phenomena of increased leukocyte

margination, as well as phenomena of angiogenesis, with risk of neoformation arranged as

islands.

There were also observed areas of necrobiosis, both at the level of papillary chorion and

at the level of the profound chorion. Fibers lose their individuality, fibril structure and

fundamental substance, creating a unique mass. Fibrinous degeneration appears to be

homogenous with eosinophilic appearance. The collagen fibrins break apart into fragments,

decompose into fibrils and finally dissolve with fibrinoid appearance.

In other areas, at the level of lamina propria, a disorganized structure of lax type, with an

edematous infiltration, was noted.

The microscopic examination of the lamina propria, together with reaction cells

characteristic for acute or chronic inflammatory processes, the hypertrophy of the nerve fascicles

is observed, some of them presenting phenomena of reactive neuritis adjacent to the necrobiotic

area.

On microscopic examination of histological sections obtained from fragments of human

gingival mucosa prelevated from patients with fixed dentures with metal part of Gaudent S, there

were detected condylomatous lesions with changes such as acanthosis and parakeratosis type,

with keratinization of both the superficial layer with microscopic appearance of squames and

corneous globes. Parakeratotic elements have rests of nuclei, no granular layer.

There were also been highlighted fibersclerosed areas with network disposition.

At the junction epithelium - lamina propria there are observed infiltrates with reaction

cells (monocytes, lymphocytes, plasma cells) scattered diffusely. In the distal areas of the

interpapillar epithelial ridges there are observed cell areas showing cariolisis and cariopicnosis.

Superficial areas of the gingival epithelium present numerous cells with vacuolated

cytoplasm. The cavitary alteration of the cell occurs as an early cellular edema within the

malpighiene epithelial cells. In some cases there are aspects of reticular degeneration by bursting

of the cells and creation of multinucleated blisters.

In the deep chorion, the connective fibers appear dissociated from edematous infiltration;

there is also noted the hyperplasia of nervous fillets and reactive neuritis.

The contact surfaces of the prosthesis with the gingival mucosa show small ulcerative

lesions and below them there is a fibersclerosed edematous connective tissue.

DISCUSSIONS

The citohistological study of gingival mucosa lesions belonging to the prosthetic field is

of great importance. It was observed that the biological habitat formed by wearing dentures, as

well as different prosthetic materials, can cause various clinical and histopathological changes on

gingival mucosa. During the first period of use of the prosthesis may occur citohistological

changes that are adaptive, they disappear after a period of adjustment, in other cases due to

distress (chronic stress) at the gingival mucosa level in contact with denture may occur changes

of the citohistological picture which may indicate the presence of inflammatory or proliferative

chronic lesions.

The human body is perfectly adapted to certain conditions such as gravity, but it is also

versatile, able to adapt to different conditions in order to maintain body homeostasis. Any change

in the external environment determines the human body to adapt to annihilate the harmful action

and lead to maintaining or restoring the homeostasis. The excessive action of some

environmental factors (pressure, postural demands, biological and chemical factors of the

environment etc.) can affect the homeostasis and create a pathogenic potential for various

diseases.

From the microscopic analysis of the histological preparations there was found that the

cytohistological changes of the periprosthetic gingival mucosa are present in all types of

prostheses (unidental or pluridental), but their intensity and variability depend on the biomaterial

used in their manufacture.

In 1994 Steflik published a detailed study regarding the necessary conditions for a

biomatwerial to ensure the two sequences of biocompatibility:

- Primary sequence, which considers the short-term reaction to achieve gingival mucosa-

prosthesis interface;

- Secondary sequence, on long term, where the direct interface is subject to functional

demands called "functional biocompatibility”.

There is experimental data in literature showing that biomaterials with different chemical

compositions trigger various biological responses. In this respect, the chemical composition of

the contact surfaces is mainly determined by the action of metal oxide (Kasemo, 1983). Metal

oxides influence the cellular connection type of the interface prosthesis-periodontium. There

were reported dynamic disorders of blood circulation, with altered vascular permeability and

inflammatory exudate formation, either diffuse or nodular. The gingival mucosa showed

inflammatory and degenerative lesions both in the epithelium and in the chorion.

At the cellular level changes occur in all cellular components (cytoplasmic membrane),

but especially at the nuclear level. These changes are detectable microscopically both directly

and indirectly through quantitative methods of biometric type.

It is known that, of all the constituent components of the cell, the nucleus is the one

having the most comprehensive participation, both in physiological processes, and in

pathological ones, although such participation is not always reflected by morphopathological

changes. That is why we have included in our study, in addition to the microscopic study

exploring the visible microcosm, a biometric study that explores the unseen interior of the

microcosm.

The nucleus is involved in almost every activity of response to the stress apllied upon the

cell. The altered function of the nucleus might be the cause of some cytoplasmic dystrophic

lesions. During the stage of eustress generated by the action of the dentures, some lesions that

appear may be transitional, since the cell lesion is reversible as long as the nucleus is still

unaffected.

Morphological lesions of the nucleus are relatively poor and nonspecific. The correlation

of the morphological data with the genetic, quantitative and cytological translates into

determining changes in the structure of the nucleus, so if the cell is chronically injuried, there is

an activation of the nuclear chromatin and the nucleus becomes heterochromatic, the basophilia

of the nucleus is erased. This process is described as pyknosis, a process that is largely due to the

permeability of cellular and nuclear membranes for calcium ions. It blocks the negative

phosphate groups of nucleic acids causing changes of nuclear basophilia and chromatin

aggregation.

The nucleus lysis - karyolysis - may not be preceded by the appearance of pyknosis if the

nuclear membrane is affected and lysosomal enzymes are activated early.

Hyperplasia occurs in response to excessive stimuli. It may precede benign or malignant

neoplasms.

Our study reveals results compared to those obtained in similar studies such as the ones

cited below.

A research conducted by Bergendal and Isacsson (1983) has shown, at the level of

periprosthetic gingival mucosa, the existence of nonspecific inflammatory reactions

characterized by the presence of a stratified malpighian epithelium with parakeratosis, marked

acanthosis and spongyosis, and a mononuclear inflammatory infiltrate and capillary

vasodilatation at the level of the subjacent connective tissue.

Schroder (1996) considered that chronic inflammation of the gingival mucosa might lead

to epithelial dysplasia or metaplastic changes.

Ursache (1996) cites histopathological changes of the gingival mucosa in acrylic denture

wearers, manifested by hyperplastic epithelium, with filiform or digitiform elongated ridges,

with ortokeratosis or parakeratosis with inflammatory phenomena, atrophic epithelium with

acanthosis and discheratosis, and at the chorion level with collagen bundles with edematous

reaction, the basement membrane with uneven thickness and trajectory, with interruption of

continuity.

Costin et al (1998) observed, in patients with gaudent prostheses, subacute and chronic

inflammatory changes. At the level of lamina propria, the authors noted the presence of dilated

vessels, the limfoplasmocitar perivascular inflammatory infiltrate, and hemosiderin granules.

A study by Minic et al (1991) on the oral mucosa necropsied from individuals who had

worn dentures showed epithelial calcification sources, chronic inflammatory infiltrate in the

gingival stroma, presence of granulation tissue, connective tissue myxo degeneration.

Tovaru (1999), Staniceanu (2001) emphasized the role of HPV (Human Papilloma Virus)

in condylomatous lesions in the oral area. This can cause both benign and malignant tumors.

The koilocytes described by Koss (1976) as a squamous cell of superficial or

intermediate type, with the nucleus (with HPV parasites) being single, double or multiple,

present chromatin of variable shapes and sizes, without apparent nuclear membrane, without

nucleoli. The nucleus is always surrounded by an optically clear perinuclear cavity, margined at

the periphery by a well-defined cytoplasmic membrane, with eosinophilic, cianofil or

policromatofil stain (Meisels et al, 1992). Perinuclear vacuole usually contains usually

cyanophilic remains.

In our study, in the mucosae with papillomatous lesions we have shown koilocytes in the

superficial layers accompanied by nuclear involutions of cariorexis or karyolysis type. We have

met the presence of koilocytes on the entire thickness of the epithelium.

Sajin et al (1999) stated that the citomorphologic diagnosis of the condylomatous lesions

is based on two pathognomonic (specific) elements: specific koilocytes and dyskeratosis.

Trusal et al (1975), Simirad et al (1986), Rihet et al (2000) have continued their studies in

this direction.

CONCLUSIONS

The biomaterial used in making unidental or pluridental fixed dentures can interact with

the components of periodontium and determine clinical and histopathological changes.

Some of these are adaptive changes which disappear after a period of time, others are

changes associated to physical or biochemical distress leading to inflammatory or

proliferative chronic lesions.

During the microscopic examination of histological preparations there was found that the

citohistological changes of the gingival mucosa are present in all types of prostheses (uni-

or pluridental), but their intensity and variability depends on the biomaterial used in their

production.

In subjects who used fixed dentures with metal part of gaudent there were present,

besides chronic inflammatory reactions, histological lesions such as condyloma

acuminata.

The microscopic study of gingival mucosa fragments obtained from patients with fixed

dentures with metal parts made of wipla predominantly showed papillomatous lesions.

On the paraprosthetic condylomatous gingival mucosa sections there was found, on the

entire thickness of the epithelium, the presence of koilocyte shape cells, cell vacuolation,

picnotic nucleus pushed to the periphery with involutive lesions involution, cariorexis

and karyolysis. The nuclear involutive lesions (the nucleus being the vital center of the

cell) induced changes of the whole cell metabolism, which favored dysmetabolic or

metaplastic processes.

The pattern of citohistological changes of gingival mucosal from patients who have worn

dentures with wipla type metal component had papillomatous changes.

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