Chapter 17. Oral mucosa and gingival crevicular fluid
structure and composition
Functions 235
Classification 235
Epithelium 236
Cytokeratins 237
Non-keratinocytes 237
Lamina propria 237
Overview
The oral mucosa shows regional variations relating to different functions in different regions. Masticatory mucosa is found in areas subjected to significant loading such as the hard palate and gingiva. Lining mucosa is subjected to far less stress and is found in regions such as the lip, cheek and floor of mouth. The anterior two-thirds of the dorsum of the tongue is lined by a specialized gustatory mucosa. Where the teeth are exposed in the mouth, the site is sealed and protected by the specialized junctional epithelium. Gingival crevicular fluid (GCF) is an exudate of the periodontal tissues and collects in the gingival sulcus. It is thought to have protective properties towards the tooth and gingival tissues, as it may flush away bacterial cells and host inflammatory molecules from the gingival sulcus, and may have antibacterial properties. The health of the oral mucosa (and teeth) is dependent on the quantity and quality of saliva produced by glands lying in the submucosa.
Learning objectives
You should:
• know the basic structure of the different types of oral mucosa
• understand the reasons for the regional variation of oral mucosa and its significance in different clinical situations; a full appreciation of the normal appearance of the oral mucosa is essential in obtaining a diagnosis for the many pathological conditions seen within the oral cavity
• have a detailed knowledge of the gingiva, as this is essential for an appreciation of periodontal disease
• know the composition, formation and function of GCF
• understand how inflammation influences GCF composition and production
• know that GCF contains proteins which may act as biomarkers of disease progression and appreciate its usefulness as a possible diagnostic and prognostic fluid.
The oral mucosa lines the oral cavity. It consists of two basic layers separated by a basement membrane:
• An outer stratified squamous epithelium
• An underlying connective tissue layer, the lamina propria.
In many regions, a third layer (the submucosa) is found between the lamina propria and the underlying bone (palate) or muscle (cheeks and lips). The submucosa consists of a looser connective tissue containing the main nerves and blood vessels, as well as glands.
Functions
The oral mucosa has a number of important functions:
• It is protective mechanically against both compressive and shearing forces associated with mastication.
• It provides a barrier to microorganisms, toxins and various antigens.
• It has a role in immunological defence, both humoral and cell-mediated.
• Salivary glands within the oral mucosa secrete saliva which has many functions, including lubrication and buffering activity, as well as providing some antibodies.
• The viscoelastic mucous film also acts as a barrier, helping to retain water and electrolytes.
• The oral mucosa is richly innervated, providing sensory inputs associated with the modalities of touch, proprioception, pain and taste.
Classification
The oral mucosa may be classified into three types – masticatory, lining and specialized mucosa:
• Masticatory mucosa is found in regions where there is high compression and friction, and is characterized by a keratinized epithelium and a thick lamina propria, which is usually bound down directly to underlying bone (mucoperiosteum).
• In lining mucosa, the epithelium is non-keratinized, as it is subjected to less stress.
• The tongue is regarded as a specialized mucosa, as it contains taste buds and is papillated. The vermilion (red) zone of the lip may also be classified as specialized.
Within the oral cavity about 60% of the mucosa is lining mucosa, about 25% of the mucosa is masticatory mucosa and the remaining 15% is specialized mucosa.
Epithelium
Oral epithelium is classified as a stratified squamous epithelium, as it has several layers of cells with distinct morphologies.
masticatory epithelium
For masticatory epithelium, four layers are present.
Basal layer
The basal layer (stratum germinativum or stratum basale) is the single cell layer adjacent to the lamina propria and is demarcated from it by a basement membrane. It consists of low columnar/cuboidal cells, among which is a population of stem cells. On mitosis, stem cells give rise to two daughter cells, one of which remains a stem cell. Stem cells generate transit-amplifying cells that will undergo a number of further cell divisions, migrate from the basal cell layer and differentiate to give rise to replacement keratinocytes in the epithelial layers above. The cells of the basal layer are the least differentiated within the oral epithelium. Cell contacts in the form of desmosomes, hemidesmosomes, intermediate and gap junctions are present, allowing for adhesion and cell signalling.
Prickle cell layer
Above the basal layer lies the prickle cell layer (stratum spinosum). The cells of this region show the first stages of maturation, being larger and rounder than those in the basal layer. The transition from basal to prickle cell layer is characterized by the appearance of new cytokeratin types. They contribute to the formation of the tonofilaments, which become thicker and more conspicuous towards the surface. In the upper part of the prickle cell layer, small, intracellular membrane-coating granules appear. These granules are rich in phospholipids and, in the more superficial layers of the stratum spinosum, come to lie close to the cell membrane. Within the prickle cell layer, desmosomes increase in number and eventually occupy about 50% of the intercellular space. The term ‘parabasal’ is used to refer to the deepest layer of cells of the prickle cell layer that lie next to the basal layer. They may show features similar to that of the basal layer and may undergo cell proliferation.
Granular layer
Above the prickle cell layer lies the granular layer (stratum granulosum). The cells of the granular layer show a further increase in maturation compared with those of the basal and prickle cell layers. Many organelles are reduced or lost, such that the cytoplasm is predominantly occupied by the cytokeratin tonofilaments and tonofibrils. The cells are larger and flatter, and contain numerous small granules called keratohyaline granules. These contain profilaggrin, the precursor to the protein filaggrin that eventually binds the cytokeratin filaments together into a stable network. The membrane-coating granules first seen in the prickle cell layer move towards the superficial surface of the keratinocyte and discharge their lipid-rich contents into the extracellular space. This intercellular ‘cement’, together with the cell contacts (especially tight junctions in the upper region of the granular layer), helps limit the permeability of the layer and prevents water loss. Synthesis increases of the additional proteins, loricrin and involucrin, first apparent in the prickle cell layer. These proteins will help form a more resistant cell wall (envelope).
Keratinized layer
The most superficial layer in masticatory epithelium is the keratinized layer (cornified layer, stratum corneum). In this final stage in the maturation of the epithelial cells, there is loss of all organelles including the nucleus. The cells of the keratinized layer become filled entirely with closely packed tonofilaments surrounded by the matrix protein filaggrin. This mixture of proteins is collectively called keratin; it contributes to the mechanical and chemical resistance of the layer. In the cornified layer, involucrin becomes cross-linked (by the enzyme transglutaminase) to form a thin (10 nm), highly resistant, electron-dense, cornified envelope just beneath the plasma membrane. The cells of the keratinized layer are shed (squames), necessitating the constant turnover of epithelial cells. Desmosomes weaken and disappear to allow for this desquamation.
In some areas such as the gingiva, the nuclei may be retained in the cornified layer. These cells are described as parakeratinized (in contrast to the more usual orthokeratinized cells without nuclei).
Lining epithelium
In lining epithelium, the cells are non-keratinized at the surface. Like the cells in keratinized epithelia, cells from the basal layer enlarge and flatten as they shift towards the surface. The surface layers differ from the cells of keratinized epithelia in that they lack keratohyaline granules. This accounts for the less developed and dispersed tonofilaments present in lining epithelium. There are also more organelles in the surface layers compared with those in keratinized cells, although there are still considerably fewer than in the basal layer. Nuclei persist within the surface layers. Membrane-coating granules are smaller and lack the lipid-rich lamellar structure of those in keratinizing epithelia. This is thought to account for the greater permeability of lining epithelium compared to keratinized epithelium. Lining epithelium generally lacks the proteins filaggrin and loricrin, but contains involucrin.
Turnover time of the epithelium is fastest in the region of the junctional and sulcular epithelia (about 5 days), which are located immediately adjacent to the tooth surface. This is probably about twice as fast as that seen in lining mucosa, such as the cheek. Turnover time in masticatory mucosa is a little slower than that in non-masticatory (lining) mucosa.
Cytokeratins
Within epithelial cells, cytokeratin intermediate filaments function as components of the cytoskeleton and cell contacts (desmosomes and hemidesmosomes). The products of each cytokeratin gene family are divided into the neutral or basic type II cytokeratins (numbered 1–8) and the acidic type I cytokeratins (numbered 9–20). They occur in pairs; the type I cytokeratin is the smaller of each pair. There is a specific distribution of cytokeratins within epithelia:
• Cytokeratins 5 and 14 are usually restricted to the basal and parabasal layers (although cytokeratin 14 may also be expressed by suprabasal keratinocytes).
• Cytokeratins 1 and 10 (or CK 2 and 11) are characteristically found in the suprabasal layers of masticatory mucosa.
• In lining mucosa, the suprabasal keratinocytes stain primarily for cytokeratins 4 and 13.
• In the epithelium covering the soft palate, cytokeratins 7, 8 and 18 (normally associated with simple epithelia, such as ductal luminal cells) are present.
Non-keratinocytes
As many as 10% of the cells in the oral epithelium are non-keratinocytes, and include melanocytes, Langerhans cells and Merkel cells.
Melanocytes
Melanocytes are pigment-producing cells located in the basal layer. They are derived from the neural crest. They are dendritic cells, having long processes that extend in different directions and across several epithelial layers. Melanocytes characteristically contain pigment that is packaged in small granules termed melanosomes. The pigment is passed to adjacent keratinocytes when the tips of the dendrites are actively phagocytosed. In dark-skinned patients, patches of melanin pigment may be seen in the mouth, particularly in the gingiva.
Langerhans cells
Langerhans cells are dendritic cells situated in the layers above the basal layer. They are derived from bone marrow precursors. They act as antigen-presenting cells. Ultrastructurally, the Langerhans cell contains characteristic trilaminar, rod-shaped granules called Birbeck granules.
Merkel cells
Merkel cells are found in the basal layer, often closely apposed to nerve fibres. They are thought to act as receptors and are derived from the neural crest. Merkel cells can be identified using antibodies for cytokeratins 8/18 and 20. Merkel cells are common in masticatory epithelia but less frequently found in lining mucosa. Ultrastructurally, the nucleus of the Merkel cell is often deeply invaginated and may contain a characteristic rodlet. The cytoplasm contains a collection of electron-dense granules, which may liberate a transmitter towards the adjacent nerve terminal, giving the cell a sensory function. Desmosomes are associated with the cell membrane. Free nerve endings not associated with a Merkel cell are also found within the epithelium. These are nociceptors.
Inflammatory cells
Some inflammatory cells may also be found in the epithelium, having migrated through it from the underlying lamina propria. Lymphocytes are the most common type of inflammatory cell, though polymorphonuclear leukocytes and plasma cells may also be encountered. The greater degree of permeability of non-keratinized epithelium may account for the larger number of inflammatory cells said to occur there compared with masticatory epithelium.
Lamina propria
The lamina propria underlying the oral epithelium provides mechanical support for the epithelium, as well as nutrition. Its ridges, the dermal papillae, interdigitate with the epithelial folds or rete; the folding in masticatory mucosa is more pronounced than in lining mucosa. Its nerves have an important sensory function, while its blood cells and salivary glands have important defensive roles. The principal cells of the lamina propria are fibroblasts, responsible for the production and maintenance of extracellular matrix. The collagen fibres are mainly type I (about 90%), with about 8% type III. Elastin fibres are also present, their number varying according to site. As with all general connective tissues, the usual defence cells are present, such as macrophages, mast cells and lymphocytes. Inflammatory cells will increase dramatically in inflammation, such as following gingivitis.
Regional variation
There is regional variation in the structure of the oral mucosa related to different degrees and types of stress during mastication, speech and facial expression. As a consequence, the structure of the oral mucosa varies in terms of the thickness of the epithelium, the degree of keratinization, the complexity of the connective tissue-epithelium interface, the composition of the lamina propria, and the presence or absence of the submucosa.
Masticatory mucosa
Masticatory mucosa is found where there is high compression and friction, and is characterized by a keratinized epithelium and a thick lamina propria, with a highly folded interface. The mucosa of the gingiva and palate is masticatory, the bulk of which is firmly bound down to underlying bone by dense collagen bundles forming a mucoperiosteum. In the roof of the hard palate, however, a submucosa is present, within which is found the main neurovascular bundles. There are also minor mucous glands (predominantly posteriorly) that open on to the surface by ducts, and adipose tissue (predominantly anteriorly). The nasal surface of the hard palate is lined by a respiratory mucosa.
Gingiva
The majority of the gingiva surrounding the neck of the tooth is attached to the tooth and alveolar bone, with no submucosa. Its external surface (oral gingival epithelium) is a masticatory mucosa that may show orthokeratinization or parakeratinization. Its margin (1 mm) is the free gingiva, which may be demarcated from the attached gingiva by the free gingival groove. The gingival margin marks the floor of the gingival crevice or sulcus.
The internal surface of the gingiva adjacent to the tooth shows two zones of epithelium:
• The crevicular (sulcular) epithelium that faces the gingival crevice
• The junctional epithelium that is in direct contact with the enamel surface at the base of the crevice.
These two epithelia comprise the dentogingival junction; both are non-keratinized.
Crevicular (sulcular) epithelium
The crevicular epithelium has a more folded interface with the underlying connective tissue. In addition, the two epithelia can also be distinguished by their different cytokeratin profi les. The superfi cial layers of the crevicular epithelium stain positive for the cytokeratins typical of lining epithelium (e.g. cytokeratins 4 and 13). However, junctional epithelium not only lacks the cytokeratins typical of lining epithelium, but expresses the basal keratinocyte cytokeratins 5, 14 and 19 throughout all its layers, typical of the cytokeratin profile of the odontogenic epithelium from which it is derived.
Junctional epithelium
The junctional epithelium shows a number of additional specialized features that distinguish it from other oral epithelia:
• In addition to the normal (external) basal lamina at its junction with the adjacent lamina propria, it has a second (internal) basal lamina uniting it to the enamel surface.
• It has fewer desmosomes and this is correlated with larger intercellular spaces that may comprise up to 5% of the volume of the tissue. This can be correlated in turn with its increased permeability, which allows crevicular fluid and defence cells to pass into the crevicular space.
• It has the highest turnover rate.
• The peripheral cells adjacent to the enamel surface have organelles, such as Golgi material and roughened endoplasmic reticulum, related to the synthesis and secretion of the (internal) basal lamina.
• The composition of its integrins is different, implying a difference in the adhesive mechanisms within its cells.
The lamina propria associated with the junctional epithelium has a rich blood supply arranged as a complex anastomosing network. This crevicular plexus is the obvious source of gingival crevicular fluid. The vessels of the plexus are very sensitive to stimulation and are likely to vasodilate under the slightest of insults. In response to plaque, they may become more permeable, increasing the production of crevicular fluid.
Principal gingival collagen fibres
The dentogingival junction seals the underlying connective tissue of the periodontium from the oral environment. The strength of the seal is thought to be dependent not only upon the properties of the junctional epithelium, but also upon the groups of principal gingival collagen fibres. Among these groups are:
• dentogingival fibres (arising from the root surface above the alveolar crest and inserting into the lamina propria of the gingiva)
• longitudinal fibres (extending along the free gingiva, some possibly for the whole length of the dental arch)
• circular fibres (encircling each tooth within the marginal and interdental gingiva)
• alveologingival fibres (running from the crest of the alveolar bone and into the overlying lamina propria of the gingiva)
• dentoperiosteal fibres (passing from cementum over the alveolar crest to insert into the periosteum)
• trans-septal fibres (passing horizontally from the root of one tooth, above the alveolar crest, to be inserted into the root of the adjacent tooth).
Interdental gingiva
The interdental gingiva is the part of the gingiva lying between adjacent teeth. The shape and arrangement of the gingival tissues between the teeth depends on the shape of the contact between the teeth:
• From the buccal or lingual aspects, the interdental gingiva has a wedge-shaped appearance.
• Between the anterior teeth (which contact only at a small point), it would appear similarly ‘pointed’ when viewed in a buccolingual plane.
• In the posterior cheek teeth, which have a broader area of contact, the appearance from the buccal or lingual side would show the typical wedge shape but, across its buccolingual plane, there are two peaks on the buccal and lingual aspects with a curved depression between them (the interdental col), which fills the contour around the contact point.
Gingival crevicular fluid
Gingival crevicular fluid (GCF) may be regarded as a transudate (or exudate) of the periodontal tissues. It gathers in the gingival sulcus and may be sampled by non-invasive means at the gingival margin. GCF is thought to be protective towards the tooth and gingival tissues, as it washes away potentially harmful cells (bacterial and host) and molecules from the gingival sulcus. It also contains antibacterial substances along with high calcium and phosphate concentrations. The major constituents of GCF are derived from plasma, interstitial fluid, microbial sources, dental plaque matrix, host inflammatory cells and host tissues. Flow rate of the GCF can also be directly related to the degree of gingival inflammation. In healthy tissues, low levels of flow rates are observed (typically 0.05–0.2 μl/min) and the fluid has a composition similar to interstitial fluid.
Osmosis
The GCF reaches the sulcus by intercellular routes across the epithelial wall, and this passage of fluid into the sulcus is thought to be mediated by osmosis. In the healthy patient, small amounts of subgingival plaque give rise to macromolecular plaque, which is usually removed by desquamating epithelial cells or phagocytosis. However, macromolecules can diffuse intercellularly towards the basement membrane, which is a limiting barrier. This creates an osmotic gradient and interstitial fluid flows into junctional and sulcular epithelium and sulcus.
Flow of GCF is influenced by several factors, the major one being the passage of fluid from capillaries into the gingival tissues. Flow rate is also directly affected by:
• removal of the fluid by the lymphatic system of the gingival tissues
• the filtration coefficient of the junctional and sulcular epithelia
• differences in oncotic pressure of the interstitial fluid and sulcular fluid.
The basement membrane filters out large components.
Inflammation
Inflammation can have direct influences on GCF production. In inflamed periodontal and gingival tissues, GCF flow rate is increased due to a greater osmotic gradient being created and fluid flows across a weakened basement membrane. There is a loose organization of the junctional and sulcular epithelium, which serves to increase fluid flow from the capillaries into the connective tissues as a consequence of the host response. The production of an inflammatory exudate allows the passage of cells and large proteins into the fluid, and pressure sources such as mastication and tooth brushing may cause an increase in GCF flow. These forces are thought to produce transient elevations in the pressure of inters/>
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