Gingiva and

Zsolt Lohinai DMD, PhD university adjunct Semmelweis University Dept. of Conservative [email protected]

 Gingiva  Periodontal ligaments  Root  Alveolar bone  Summary/integration

 Knowledge of normal morphology and structural biology is a prerequisite for understanding pathology and causal therapy!

 Summary/integration, clinical aspects, DMD vs. DDS

GINGIVA The gingiva is a protion of the . Covers the alveolar bone and the cervical part of the teeth. It is firm in consistency and not mobile. Specially adaptated to the masticatory functions.

 Free (gingiva marginalis)  Attached gingiva (gingiva propria)   Mucogingival line

GINGIVA

Pink and tight Interdental papilla has one vestibular and one oral portion separated by the concave col region Gingival sulcus or crevice

Narrow groove surrounding the !!! Gingival sulcus or crevice The dentogingival interface

 The gingival sulcus is an area of potencial space between a tooth and the surrounding gingival tissue

 Outer (lateral) wall: sulcular of the free gingiva  Inner wall: enamel of the tooth (cementum)  Bottom: of the free gingiva

 In clinically healthy gingiva it is only a virtual space or more frequently a less than 2 mm deep fissure around the teeth

!!! Significance of the gingival sulcus

 Discontinuity in the epithelial lining - breakage in barriers - medical aspects - locus minoris resistenciae - periodontal seal !

 Battle for the solid surface between host and bacteria

 The teeth are nonshedding, solid surfaces, which provide a solid substratum for bacterial colonization and spreading into the deeper tissues

 The modified lining epithelium is firmly attached to the tooth, forms a strutural barrier against plaque bacteria

 In periodontitis there is an apical migration of the epithelial cells and periodontal breakdown and consequent conversion of gingival sulcus into an infected periodontal pocket

The epithelial components of the gingiva Oral epithelium Multi-layer stratified squamous epithelium Stratum corneum: keratinized layer

Stratum granulosum: electrondense keratohyaline and glycogen containing granules, less and less nucleus

Stratum spinosum: simplier IC stucture, less mitochondria and Golgi, more desmosomes

Stratum basocellulare: basal layer, division, connection to basal membrane by hemidesmosomes, fibrills (VII-type coll.)

Non keratinocytes: melanocytes and Langerhans cells

Oral epithelium

Oral epithelium str. spinosum

tonofilaments

Epithelial cells

desmosomes Intercellular space Oral epithelium basic features

 When traversing the epithelium from the basal layer to the epithelial surface, the keratinicytes undergo continuous differentiation and specialization  Keratinized outer mechanical és microbiological defendig layer  Impermeability for water soluble materials  Strong connections to subepithelial connective tissue  Turnover (4 weeks)  Width of attached gingiva has a clinical significance: Protect the tooth/implant vs. gingiva interface! Minimum width of 2 mm is necessary to maintain perio health!  Height: thin or thick biotype, inflammatory response, impression taking

The epithelial components of the gingiva, Sulcular epithelium basic features

Lateral wall of the sulcus

 Non-keratinized  Relative impermeability to fluids and cells; good resistance to mechanical forces  Increased lysosomal activity The epithelial components of the gingiva, junctional epithelium Junctional epithelium basic features

A collar like modified epithelial ring around the neck of the teeth

 Epithelial attachment to the tooth, sealing  Non-keratinized, there are no separated layers, stratum basale (facing the CT) & suprabasale (fasing the tooth), the cell axis is parallel with axis of the tooth, from couple to appr. max. 30 layers  Two basal lamina (inner to tooth/outer to CT)  Increased permeability (wide intercellular spaces, few intercellular connections)  Main route of leukocytes toward the sulcus  Fast turn over (7 days) Junctional epithelium

Inner basal membrane

Outer basal membrane Epithelial attachment apparatus capillary plexus

Capillar pl. Inner and outer basal lamina

omatin

lagen fiber

ementum Epithelial attachment apparatus The hemidesmosome of DAT cell

 Tonofilament-bullous pemphigoid antigens (collagenous protein type VIII)-?  Tonofilament-integrin- laminin

 The components of IBL are synthetized by DAT cells in the absence of the immediate vicinity of CT DAT cell turnover

 The DAT cells divide and migrate

 The daughter cells replace the generating cells on the tooth surface

 Gradually migrate coronally, eventually break off into the sulcus

Junctional epithelium in the antimicrobial defense

 Quick cell exfoliation  Strong funelling effect  Basement membrane barrier  Production of antimicrobial substances (beta- defensins, lysosomal enzymes: proteinases, MMP)  Secretion of chemokines (IL, TNF, cytokines) attract and activate professional defense cells (PMN, lymphocytes)

!!! Importance and production of crevicular fluid (GCF)

 GCF is basicly a transudate of varying composition found in the sulcus/periodontal pocket between the tooth and the marginal gingiva

 GCF passing through the relatively wide intercellular spaces of SE and JE into the sulcus (by oncotic/capillary pressure gradient – leukocytes by chemotatic factors!) and provides nutrients for DAT cells to grow  At the gingival sulcus the GCF become contaminated so that agents from the oral cavity challenge the most coronal DAT cells Blood supply of the gingiva

From periodontal ligaments (mainly) From From subperiosteal plexus

!!! Function of crevicular fluid

 In healthy sulcus the amount of GCF is very small – normal maintenance of function of epithelium – composition: IS fluid- like, epithelial cells, leukocytes, bacteria

 During inflammation the GCF flow increases and resembles that of an inflammatory exudate – host defense by flushig bacterial colonies and their metabolites away from the sulcus - composition: 1) inflammatory exudate: plasma-like exudate, prostaglandins, cytokines, complement system, lysozyme, alkaline phosphatase, cathepsin, lactoferrin etc. 2) bacteria, bacterial metabolites/enzymes: endotoxins, hydrogen sulfide, butyric acid, collagenases, proteases, hyaluronidase etc. 3) products of tissue breakdown: lactate dehydrogenase, polyamines, collagen peptides etc.

 GCF amount and composition – rapid diagnostic test

!!!

Periotron

Metabolite: PGE2,

Inflammatory cytokines: IL, TNF Catabolic tissue enzymes: collagenase, protease,

Host vs. bacteria Battle for solid tooth surface!

 Colonization of GR- bacteria  Degeneration and detachment of DAT cells  Conversion of sulcus into pocket  Periodontal tissue breakdown

Bacterial infection

 Periodontitis is NOT a civilisation disease! Its incidence is smaller in developed contries  Endogenous infection (sugar, anaerobic environment, recolonization)  Depth of pocket determines the composition of bacterial flora – bacterial complexes  Vertical and horizontal bacterial transmissions

Bacteria vs. host defense lines

 1st: saliva  2nd: gingival sulcus  3rd: connective tissue of the gum  4th: systemic immune response

Immune cells traffic to the periodontium

 1. Plaque antigen diffuse through the JE  2. Langerhans cells capture and process the antigen  3. Antigen presenting cells leave the gingiva in the lymphatics  4. In the lymph node they stimulate the lymphocytes to produce a specific immune response  5. Antibodies travel back to the gingiva via blood vessels, leave the circulation in the exsudate and carried to the plaque in GCF  6. Periodontally specific B and T cells proliferated in the lymph nodes home back to the periodontium and begin there humoral and cell mediated immune activities

Clinical aspects

 The proper plaque-controll is the base of periodontal health: so the gingival massage alone no, just the plaque removal is effective (interdental brush, floss)

 In case of /periodontitis the volume of sulcular fluid (GCF) is increased, composition is altered - diagnostic possibility !!! Clinical aspect Probing of healthy gingiva

 0.2 N force - till junctional epithelium  Probing may under or overestimates the depth of sulcus

Clinical aspect Probing of inflammed gingiva

Bleeding? Till the resistant collagen fibers or till the alveolar bone Clinical aspect of probing

Visual exam with probing depths

Periodontal probing chart !!! Finishing line position Clinical aspects

 Finishing line position of the dental restaurations: 1) Supragingival (preferably)

2) Paragingival

3) Subgingival - intracrevicular (above JE) - temporary expanding the sulcus for impression material - the deeper the finishing line  the greater risk to iatrogen dental attachment loss! Never violate the biologic width!

!!! Biologic width Clinical aspect

 Biologic width (on average 2.04 mm) is the distance between the bottom of the sulcus and the top of the alveolar crest, including the JE (0.97 mm) and CT attachment (1.07 mm) to the root surface of a tooth.

 This distance is important to consider when fabricating dental restorations, because they must respect the natural architecture of the gingival attachment if harmful consequences are to be avoided.

 When dental restorations violate biologic width, chronic inflammation of the periodontium and unpredictable loss of alveolar bone will occur untill the biologic width is restored by tissue breakdown

 The ultimate goal is to maintain the insulation barrier! Clinical aspect

 Crown lengthening is a surgical procedure performed by a to expose a greater amount of tooth structure for the purpose of subsequently restoring the tooth prosthetically.

 This is done by incising the gingival tissue around a tooth and, after temporarily displacing the soft tissue, predictably removing a given height of alveolar bone from the circumference of the tooth being operated on. Own experimental human study Background - 1 •Dental biofilm extracts contain a heat-sensitive factor (160 kDa) that inhibits mammalian cell growth in culture. This factor was identified as lysine decarboxylase enzyme. •Lysine decarboxylase depletes the sulcus of essential amino acid lysine by converting it to carbon dioxide and cadaverine, a contributor to oral malodor and inhibitor of host response by decreasing the release of bacteriocide superoxide and peroxynitrite from activated leukocytes. •Eikenella corrodens is a major producer of lysine decarboxylase in the healthy oral cavity.

46 Background - 2

Initiation of the inflammatory pathomechanism ???

GCF transudate provides nutrients for healthy dentally-attached (DAT) cells that continuously proliferate (turnover) to maintain attachment.

At the gingival sulcus the GCF become contaminated so the bacterial agents from the oral cavity challenge the most coronal DAT cells.

Degeneration and detachment of DAT cells leads to pocket formation.

Our working hypothesis: E. corrodens lysine decarboxylase depletes coronal DAT cells of lysine.

DAT cells become stressed and release proinflammatory mediators which induce GCF.

Enhanced GCF exudation restores lysine, but on the other hand promotes colonization by the pathogenic, gram-negative (successor) microbiota and gingivitis.

Inhibiting lysine decarboxylase activity may retard the development of gingivitis. Aims

To determine the biofilm lysine and cadaverine contents under healthy condition in the oral cavity and their association with plaque index and GCF exudation after restriction for a week.

Methods -1

Participant selection and examination The human protocol was reviewed and approved by the Ethics Committee of the Hungarian Medical Research Council.

Subjects were entered into the study only after having given written consent.

Sixteen healthy volunteers, 7 men and 9 women were enrolled, aged 19 to 38 years.

Inclusion requirements were at least 26 teeth, no existing or prior medical conditions and no drug therapy or smoking tobacco.

Dental exclusion categories were partially erupted wisdom teeth, cavities requiring treatment, a pocket with a probing depth >2 mm, >10% of teeth with gingival sulci that bled on probing, and the presence of or .

Human Partial Experimental Gingivitis Protocol Methods - 3 The buccal and mesio-buccal surfaces of the upper second incisor, first premolar, and first molar from the bioguarded region were scored for plaque index according to Loe, 1967.

GCF exudation measured by Whatman No.1 paper strips placed into the gingival sulci at each of the three buccal surfaces from which plaque index was obtained. Their wetted length were measured with a caliper.

Dental biofilm was then collected from above mentioned areas with a curette.

Lysine and cadaverine were determined from saliva, and dental biofilm by laser-induced fluorescence after capillary electrophoresis (Anal Bioanal Chem 2008; 391:647-651). Results - 1 Results - 2 Results – 3

PI = 1.48 + 3.75*Lysine; R2 = 0.61, p <0.001

GCF = -18.5*Lysine2 + 4.5*Lysine + 0.05; R2 = 0.37 p <0.01; Coefficiens: Lysine2 ‘t’ statistic = -3.09, p <0.01; Lysine ‘t’ statistic = 3.27, p <0.01.

Relationships of plaque index and GCF exudation rate to plaque lysine content. Conclusions - 1

Biofilm lysine originates from GCF, not saliva or the diet.

Stopping oral hygiene increased bacterial lysine decarboxylase activity, reduced biofilm lysine content to below the blood plasma levels at baseline and increased Its cadaverine content.

The residual lysine content may control: 1) biofilm development, 2) by modifying epithelial attachment integrity the access of bacterial proinflammatory agents to subgingival tissues.

Conclusions - 2

Biofilm lysine content may be a new marker for biofilm accumulation and the associated inflammatory response.

Inhibiting E. corrodens lysine decarboxylase may preserve lysine in the gingival sulcus and strengthen the epithelial barrier to bacterial proinflammatory agents. Thank you for your attention!

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