1. ULTRASTRUCTURE OF THE GINGIVA
Presented by: Supervised by: Gabriela Jude Fernandes Dr. Abhiram Maddi [PGY2]

2. OUTLINE Introduction Development of Gingiva
Clinical Features of Gingiva
Normal Microscopic Features
Functions of Microscopic Structures
 Age Changes in Gingiva
Correlation of Clinical and Histological Aspects of Healthy and Diseased Gingiva
Clinical Considerations

3. INTRODUCTION
Gingiva is part of oral mucosa that covers the alveolar processes of jaws and surrounds the neck of the teeth .
Composed of thin outer epithelium and underlying connective tissue .
Consist of four anatomical portions –
Gingival sulcus , Free gingiva, Interdental gingiva and attached gingiva .
Texture of Gingiva – healthy gums have stippled ,translucent appearance.orange peel appearance .

4. Colour of Gingiva-coral pink, depends on the thickness and degree of keratinization of epithelium, blood flow to gingiva ,disease and medication ,natural pigmentation.
Contour of Gingiva-scalloped and knife edge margin .
Size of Gingiva-corresponding with the sum total of the bulk of cellular and intercellular elements and their vascular supply.

5. Gingival sulcus: It’s a V shaped notch. Bounded by surface of tooth
on one side and epithelium lining the free margin of gingiva on the
other side. Probing or clinical probing depth is depth of penetration
of probe and it is 2-3mm in normal gingival sulcus in humans.
In histological section depth is 1.8mm with variation from 0-6mm.
Marginal Gingiva: Also called as free or unattached gingiva. Surrounds the tooth in collarlike/cufflike manner. Characteristic – fits closely around but not directly
attached to the tooth-also form soft tissue wall of the gingival sulcus. It is 1mm wide. Free Gingiva is demarcated from adjacent attached gingiva by shallow linear depression that follows the Contour of the tooth called ‘ marginal groove ’.
It can be separated from tooth surface by peridontal probe.

6. Attached gingiva:Part of gingiva tightly connected to periosteum of alveolar bone. Lies between the two movable tissue.Colour- pale and pigmented in dark skinned individuals. Width- widest in incisor and molar region and narrowest in premolar region . Premolar= maxilla-1.9mm.Mandible-1.8mm.
Interdental gingiva:It occupies the gingival embrasure. Gingival embrasure is the interproximal space beneath the area of tooth contact.

7. Gingival fibres:

8. Kobayashi, K., et al.: Ultrastructural histochemistry of the dento-epithelial junction. I. J. Perio. Res.12: , 1977.[Fernandes]
Aim : To study the primary component of the dento-enamel junction. Materials and methods: Three young rhesus monkeys.Tissues obtained from the Dej.Various staining procedures performed on the tissues [PTA,PA Silver,PA-TSC-SP] Results: Various staining reactions differentiated the basal lamina as a reaction positive layer and the dental cuticle as a reaction negative layer. Conclusion: The dental lamina is a key participant in the attachment at the DEJ whearas the dental cuticle just plays a transitory role.
Clinical significance:dental lamina  if still persists and does not get resorbed becomes eruption cyst.hence important to know if dental lamina or dental cuticle is responsible for eruption cyst.

9. Saglie, R. , Sabag, N. and Mery, C
Saglie, R., Sabag, N. and Mery, C.: Ultrastructure of the Normal Human Epithelial Attachment. J. Periodontol. 50: , 1979.[Fernandes]
Aim: To investigate new findings in the structure of the epithelial attachment in clinically normal periodontium. Materials and methods: Biopsies were obtained from 12 recently erupted human permanent teeth with their corresponding clinically normal free and attached buccal gingiva and examined in the Transmission Electron Microscope. Results: Three zones were observed: an apical, a middle and a coronal zone. The division  quantification of the hemidesmosomes, their thickness and extension, the morphology of the cellular membrane adjacent to the tooth surface, and the cytological aspect of the epithelial cells Conclusion: There is no actual barrier between the base of the sulcus and the gingival tissue and that there may exist zones which could be more appropriate than others in achieving their protective and adhering functions.
No barrier,hence more susceptible to pocket formation

10. Shroeder HE, Listgarten MA
Shroeder HE, Listgarten MA. The gingival tissues: the architecture of periodontal protection. Periodontology :91-120, 1997 Feb.[Fernandes]
Gingival epithelium acts in defense via its degree of keratinization & turn over rate.
Principle cells of stratified epithelium are keratinocytes, the others being non- keratinocytes which include Langerhans cells,merkel cells & melanocytes.
The main function of the epithelium is to protect the deep structure while allowing selective interchange with the oral environment. This is achieved through the proliferation & differentiation of the keratinocytes.
The upper most cells of the stratum spinosum contains dense granules known as keratinosomes or odland bodies which are modified lysosomes. They contain large amount of acid phosphatase, which is related to the degree of keratinization.
Langerhans cells which are located among keratinocytes at supra basal levels, belong to the mononuclear phagocyte system and play a role in immune reaction as antigen presenting cells for lymphocytes.

11. Scully C, Bagan JV, Black M, Carrozzo M, Eisen D, Escudier M, Farthing P, Kuffer R, Lo Muzio L, Mignogna M, Porter SR.Epithelial biology. Oral Dis Mar;11(2): Review.[Fernandes]
ORAL EPITHELIUM :
Stratum basale:- cuboidal cells.
Stratum spinosum:- large polyhedral cells →Desmosomes
Stratum corneum:- superficial most layer Large, wide, flat and lacking nucleus.
SULCULAR EPITHELIUM:
Extends from the coronal area of the junctional epithelium to the free margin of the gingival.
Epithelium is nonkeratinized due to constant irritation of plaque.
Epithelium lacks heavy ridges and papillae.
JUNCTIONAL EPITHELIUM:
It consist of stratified squamous nonkeratinizing epithelium of 3-4 cell thickness having length of mm.
The adjacent teeth & are also referred to as transseptal fibers. These fibers make the interdental ligament.

12. DEVELOPMENT OF GINGIVA

13. Listgarten, M.A.: Phase contrast and electron microscopic study of the junction between reduced enamel epithelium and enamel in unerupted human teeth. Arch Oral Biol. 11: 999, 1966.[Fageeh, Hytham]
Aim:
To compare cuticle “A” , found exclusively over enamel, to the so called primary cuticle.
Method:
Phase contrast and EMS : unerupted vs. fully erupted premolars removed for Othro res. (n=??)
Immediate fixation and demineralization
Results:
Reduced enamel epi and basal lamina are attached through hemidesmosomes and are 400å wide.
EMS did not reveal cuticle structure on enamel side of Ameloblasts, suggesting that the primary enamel cuticle is an optical phenomenon observed only in Phase contrast microscopy.
Cuticle “A” was found to be an extension of cemental layer over enamel that was devoid of collagen fibrils.
Conclusion:
First published evidence of a well defined cementum layer found on the enamel surfaces of unerupted human teeth.

14. NORMAL MICROSCOPIC FEATURES

15. Ramírez K, García-Rodríguez O, Murillo-Arocho M, Fernández-López O, Elías-Boneta AR. Dentogingival complex: dimension based on biotypes. P R Health Sci J Dec;32(4): [Fageeh, Hytham]
Aim: To estimate the dimensions of the DGC dimensions and determine whether these dimensions varies by gingival biotype.
Methods:
(n=53) participants who needed restorative crowns were recruited.
Tooth gingiva was classified as either thin, thick or mixed according to transparency of periodontal probe. (1= probe can be seen, 2= not visible, 3= probe can be seen but not clearly.
DGC dimension by bone sounding on mesial, medial and distal of tooth surface from tip of marginal gingiva to bony crest.
Results:
Mean DGC dimensions ( Mesial= 3.09mm ,Medial = 3.40m, Distal= 2.70mm)
In all biotype groups , mesial sites showed the greatest DGC dimensions
Mean DGC was greater for the THIN biotype compared to mixed and thick biotypes.
Conclusion: DGC dimensions are different for thin, thick and mixed gingival biotypes.
Standard not right!thick thin mixed??what about investigators and agreement?

16. Clinical features of gingiva

17. Baume, L.J.: The structure of the epithelial attachment revealed by phase contrast microscopy. J. Perio.24: 999, April 1953.[Fageeh, Hytham]
Aim:
To study the structural elements which procure the mechanical connection between eipthelium and tooth surface.
Methods:
Gingval specimens from human donors (n=6) and (n=20) Rhesus monkeys.
Samples had been preserved in formalin from two weeks up to two years.
The adherence of gingiva over enamel was checked by force to remove tissue.
Results:
No force was required to remove tissue since samples were stored for long periods of time
Observation of tonofilaments from basement membrane and extension into epithelium
(Fibrillar structure and attachment remains concealed to conventional histo methods)
Conclusion:
Mechanical connection between crown and gingiva was not demonstrable.

18. Saito, I. et al. Intercellular junctions and the permeability barrier in the junctional epithelium: a study with freeze-fracture and thin sectioning. J. Perio. Res. 1981, 16: [Fageeh ,Hytham]
Aim:
To study the morphology and distribution of intercellular junctions of normal junctional epithelium of dogs
Methods:
(n=6 ) adult cross breed dogs with minimal plaque and normal gingiva (Maxillary ant. teeth )
Random sections of tissue were highly magnified x30,000 and printed, then distribution and incidnece of desmosomes in the cellular layers was calculated by percentage.
Results:
Desmosomes observed in coronal and apical portions of J.E
Gap junctions with diameters varying between um, larger ones in the coronal portion
Conclusion
J.E is a permeable physiological barrier due to the presence of dense granules.

19. Pitaru, S. and Melcher, A. Organization of an oriented fiber system in vitro by human gingival fibroblasts attached to dental tissue: Relationship between cells and mineralized and demineralized tissue. J. Perio. Res. 1987, 22:6-13.[Al-marghlani]
Aim:
Examine the effect of partial demineralization of the cementum on the morphology of the interface between gingival fibroblast and tooth (in-vitro).
Materials & Methods:
32 pairs of transversally root slices of procine premolars attached to the floor of culture dish, half of the root slices demineralized with 12% EDTA.
Results:
Rate of development of refractile material was greater in the demineralized site. Light microscope: Demineralized root slices were more cellular, higher degree of fibroblast orientation. Electron microscope: Gingival fibroblasts and collagen were oriented perpendicular to demineralized root slices, for the non-demineralized root surfaces the orientation was parallel to the cementum.
Conclusion:
The nature of the tooth surface that provide substrate for gingival fibroblast may influence the morphology of that they express.

20. Chavrier, C. , Hartmann, D. , Couble, M. , and Herbage, D
Chavrier, C., Hartmann, D., Couble, M., and Herbage, D. Distribution and organization of the elastic system fibres in healthy human gingiva. Ultrastructural and immunohistochemical study. Histochemistry. 1988, 89: [Al-marghlani]
Aim:
To report the ultrastructural distribution and organization of the different components of the elastic fibres in healthy human gingival connective tissue.
Materials & Methods:
Five 23-year-old students in dental surgery, 9 mm of attached gingiva were removed from the superior incisive area of each patient. two groups of blocks: for standard electron microscopy and indirect immunolabelling procedure.
Results:
In the subepithelial layer, bundles of parallel microfibrils characteristic of oxytalan fibres were observed, in longitudinal section microfibrils had a rail-road appearance, In transverse section the mean diameter ranged from 10 to 15 nm.
Conclusion:
The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with micro fibrils and amorphous material of the elastic system fibers throughout the gingival connective tissue.

21. Graner, E. , Line, SRP. , Jorge, J. , Lopes, MA. , and Almeida, OP
Graner, E., Line, SRP., Jorge, J., Lopes, MA., and Almeida, OP. Laminin and collagen IV distribution and ultrastructure of the basement membrane of the gingiva of the rat incisor. J. Perio Res. 1995, 30: [Al-marghlani]
Aim:
Investigates the presence of laminin and collagen IV as well as the ultrastructure of the basement membrane.
Materials and Methods:
Twelve lower incisors of 6 rats were used for optical microscopic studies. For electron microscopy the lower incisors of two rats were removed with the juxtaposed gingiva and fixed in 2S/'0 glutaraldehyde. The observations were made in a Zeiss EM -10 transmission electron microscope.
Results:
The epithelium comprises the gingival oral epithelium, junctional epithelium, sulcular epithelium and circumdental papilla.
The junctional epithelium, attached to the cementum surface, is non-keratinized and consists of squamous epithelial cells parallel to the tooth surface.
The pattern of the immunochemical reactions for laminin and collagen IV was the same.
Conclusion:
In summary, the data presented here indicate that laminin and collagen IV are not components of the dento-epithelial junction of the rat incisor. It was confirmed that basal lamina is absent or fragmented on the cell surface of the circumdental papilla, adjacent to the collagen resorption area.

22. Ogata, Y. , Niisato, N. , Sakurai, T. , Furuyama, S. , and Sugiya, H
Ogata, Y., Niisato, N., Sakurai, T., Furuyama, S., and Sugiya, H. Comparison of the characteristics of human gingival fibroblasts and periodontal ligament cells. J. Periodontol. 1995, 66: [Al-marghlani]
Aim:
To elucidate the characteristics of human periodontal ligament cells.
Materials and Methods:
Human gingival fibroblasts (HGF) were derived from the expiants of gingival connective tissues obtained from a female patient. Human periodontal ligament (HPDL) cells were derived from the expiants of a healthy periodontal ligament obtained from a female patient from the fully erupted third molar. Human alveolar bone (HAB) cells were derived from expiants of maxillary alveolar bone obtained from a female patient during periodontal surgery.
Results:
Human periodontal ligament (HPDL) cells had a sharper spindle shape and exhibited a higher growth rate than human gingival fibroblasts (HGF). HPDL cells had a high level of alkaline Phosphatase (ALPase) activity, whereas HGF had a low level of such activity.
Conclusion:
These results demonstrate that HPDL cells have quite different characteristics from HGF. HPDL cells proliferate at a higher rate than HGF, show higher levels of cAMP production and greater ALPase activity, and respond in a different fashion to chemical mediators (BK and HIS) compared with HGE

23. Kuru L. Parkar MH, Griffithi GS, Newman HN. Olsen I
Kuru L. Parkar MH, Griffithi GS, Newman HN. Olsen I. Flow cytometry analysis of gingival and periodontal blood mononuclear cells. Journal of Dental Res. 77(4):555-64, 1998 Apr.[Valente]
Aim:
To examine the phenotypic and functional features of the cells obtained from gingival and PDL biopsy samples using flow cytometry.
Materials & Methods:
Gingival tissues obtained from 8 healthy subjects. Flow cytometry was used to measure the size and granularity of the cultured cells.
Results:
The gingival fibroblasts were smaller and less granular compared with the PDL cells but this difference was not statistically significant.
Fibronectin fluorescence intensity was higher in the PDL cells than that in the gingival fibroblasts.
Collagen type I was also found to be up-regulated in the PDL compared with the gingival cells
Tenascin was expressed at similar levels
PDL cells had higher activity of Alkaline phosphatase than gingival cells
Conclusion:
Despite their similar spindle-shaped appearance, fibroblasts derived from gingival and PDL tissues appear to display distinct functional activities

24. Holly McKnight,W. Patrick Kelsey,Deborah A. Hooper, Thomas C
Holly McKnight,W. Patrick Kelsey,Deborah A. Hooper, Thomas C. Hart, and Angelo Mariotti Proteomic Analyses of Human Gingival and Periodontal Ligament Fibroblasts Journal of Periodontology June 2014,85: [Valente]
Aim:
To evaluate the putative differences in the membrane-bound and -associated proteome of hGFs and hPDLFs.
Materials & Methods:
Four matched pairs of hGFs and hPDLFs were cultured. Membrane-bound and -associated proteins from cells of the fourth passage were extracted and evaluated using capillary-liquid chromatography-nanospray tandem mass spectrometry.
Results:
450 proteins were common to both hGFs and hPDLFs.
214 were known membrane-bound or -associated proteins, 165 proteins were known nuclear-associated proteins.
27 proteins were detected in statistically significant greater quantities in either (13) hGFs or (14)hPDLFs.
Conclusion:
Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGFs in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDLFs in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization.

25. DeCarlo AA, Cohen JA, Aguado A, Glenn B
DeCarlo AA, Cohen JA, Aguado A, Glenn B. Isolation and characterization of human gingival microvascular endothelial cells. J Periodontal Res Apr;43(2): Epub 2008 Jan 23. [Valente]
Aim: To establish a replicable protocol for isolating microvascular endothelial cells from the gingiva. Materials & Methods: Gingival microvascular endothelial cells were isolated from six different nondiabetic patients and characterized by morphology, the expression of factor VIII-related antigen, the expression of UEA-1 ligand, the uptake of acetylated low-density lipoprotein, network formation on Matrigel, and by the expression levels of urokinase plasminogen activator, tissue plasminogen activator, plasminogen activator inhibitor-1 and collagen IV. Dermal fibroblasts, dermal microvascular cells and human umbilical vein endothelial cells were used as controls. Results: These gingival endothelial cells, when isolated by the protocol established herein, demonstrated endothelial characteristics and constitutively secreted plasminogen activators and plasminogen activator inhibitor-1 in culture. Conclusion: The method herein described has demonstrated to be relatively simple and reliable approach to isolate and enrich gingival endothelial cell populations.

26. Functions of the microscopic structures

27. Wiebkin, O. W. and Thonard, J. c
Wiebkin, O.W. and Thonard, J.c. Mucopolysaccharide localization in the gingival epithelium.: an autoradiographic demonstration. J. Perio. Res. 1981, 16: [Valente]
Aim: To describe an autoradiographic method for localizing [35S]-sulphate (a specific precursor of glycosaminoglycan) or [3H]-acetate in macromolecular intercellular material, the synthesis of which can be traced to the gingival epithelium itself. Materials & Methods: Small pieces of human gingival tissues were obtained and shortly incubated at 37°C with either[35S]-sulphate or [3H]-acetate, the incorporation of which was observed following histological sectioning, by autoradiography. In vivo incorporation and localization was also accomplished by intraperitoneal injection in rats. Results: Critical electrolyte salt concentration elution indicated that most of the intercellular material was soluble in 0.3M-MgCl2, and any material which remained was intercellular. Conclusion: Proteoglycans (mucopolysaccharides) are a major intercellular component of human gingival epithelium. The method described, together with in vivo incorporation studies, provides a useful technique for studying direct effects of some microenvironmental influences on gingival epithelium.

28. Cho, M. ; Lee, Y. ; and Garant, P
Cho, M.; Lee, Y.; and Garant, P. Imunocytochemical localization of extracellular matrix components in beagle dog periodontium: Collagen types I and III in healthy gingival connective tissue. J. Perio. Res. 1987, 22: [Ibraheem]
Aim:
To identify type I and III collagen in healthy gingival connective tissue of a beagle dog.
Materials and methods:
using indirect immunofluorescent and immunoferritin electron microscope to localize and identify collagen I and III in two 6 week old beagles.
Results:
Type I collagen:
indirect immunofluorescent : evenly distributed throughout the lamina propria and the underlying CT in form of thick collagen fibers.
immunoferritin electron microscope: diameter ranging from 40 to 100 nm.
Type III collagen:
indirect immunofluorescent: restricted to the lamina propria and the CT around the blood vessels.
immunoferritin electron microscope: diameter ranging from 18 to 32 nm.

29. Del Fabbro M, Galardi E, Weinstein R, Bulfamante G, Miserocchi G
Del Fabbro M, Galardi E, Weinstein R, Bulfamante G, Miserocchi G. Fluid dynamics of gingival tissues. Journal of Per REs 33(6):328-34,1998 Aug. [Ibraheem]
Aim: To study fluid dynamics of gingival tissue (free gingiva, attached gingiva, and oral mucosa). Materials and methods: The experiment was performed in 18 rabbits using glass micropipettes to measure gingival hydraulic interstitial pressure. Results: Conclusion: Across sulcular epithelium, the pressure gradient sustains fluid absorption from the sulcus into the gingival inerstitium.
Plasma proteins may leak from microvessels into plasma proteins.

30. Tuomas P., Ismo V. Jaana O. Function of Laminins and Laminin-Binding integrins in gingival epithelial cell adhesion. Journal of Periodontology. 73(7) : , 2002 July [Ibraheem]
Aim: To study how cultured immortalized gingival epithelial cells produce laminins and express laminin-binding integrins. Materials and methods: Culturing gingival keratinocytes and studied their production of laminin using immunofluorescence microscopy, immunoprecipitation, and immunoblotting method. Results: The cells were seen to organize chains of laminin 5 to extracellular patches while α5 chain of laminin-1 0could only be seen intracellulary. Of the laminin binding integrin subunits, integrin α6 subunit was organized to dotted arrays typical to prehemidesmosomal adhesions, whereas integrin α3 subunits located at cell-cell junction.

31. AGE CHANGES IN GINGIVA

32. Matheny, J. L. , Johnson, D. T. , Roth, G. I
Matheny, J.L., Johnson, D.T., Roth, G.I. Aging microcirculatory dynamics in human gingiva. J. Clin Perio, 1993, 20: [Ibraheem]
Aim:
To study gingival vascular function in humans of different ages.
Materials and methods:
60 health male humans assigned to 3 groups. Each group has 20 subjects (Y: young, M: middle, and O: old). Videomicroscopy and laser droppler flowmetry were used to assess marginal gingival circulation.
Results:
Blood pressure increased with age.
Volume of flow in the marginal gingiva increased slightly with age while blood velocity decreased with age.
The numbers of visible vessels per microscopic field increased with age.
Red blood cells velocity showed no statistical difference among the age of groups.
A significance decrease was seen in peripheral oxygen saturation in O compared to Y and M.
Conclusion:
The differences in numbers of visible vessels and the number of vessels with active blood flow may reflect how the gingiva adapts with age to changing nutritional needs, microbial, or mechanical challenge.
Systemic cardiopulmunary parameters were monitored as controls
2nd point is not statistically significant.

33. ANGELO MARIOTTI, THOMAS HASSELL, PATRICK KAMINKER THE INFLUENCE OF AGE ON COLLAGEN AND NON-COLLAGEN PROTEIN PRODUCTION BY HUMAN GINGIVAL EPITHELIAL CELLS Arch, oral Bio. Vol. 38, No.8, pp , 1993 [Annaba]
Aim: To investigate the effects of donor age on the proliferation and secretory phenotype of cultured human gingival epithelial cells. Materials & Methods: Pure cultures of epithelial cells were isolated from human gingiva of old (61-75 yr) and young (18-30 yr) adults and serially cultivated in a serum-free medium at 37°C in humidified air containing 5% CO2, For each experiment, cells were seeded at 150/mm2 and the medium changed every other day. Cell number, collagen and non-collagen protein production and relative collagen synthesis (percentage collagen synthesized) were determined at days 2, 4, 6 and 8. Results: there were no differences in their rates of proliferation nor in collagen production between the two groups; however, cells from elderly individuals produced significantly less non-collagen protein. Conclusion: The reduction of non-collagen proteins may play an important part not only in determining the composition of the extracellular matrix but also the gene expression of the ageing epithelial cell.

34. Saario M, Ainamo A, Amttila K, Ainamo J The width of radiologically-defined attached gingiva over permanent teeth in children. J Clin Periodontol 1994; 21: 666–669 [Annaba]
Aim: To assess the width of radiologically-defined attached gingiva (RAG) over the permanent teeth of age 6, 10, 12 yr old. Materials & Methods: 123 children, 60 males and 63 females were recruited, the MGJ was marked over each tooth with a piece of metal wire before taking a panoramic radiograph. The width of RAG was measured on the radiograph from the midfacial of the CEJ to MGJ marker. Results: There was statistically significant difference between the groups when comparing the RAG over the 1st molars and the 1st and 2nd incisor; but the difference was not statistically significant on the 1st maxillary incisor between age group 10 and 12 years. Conclusion: Inadequate width of AG will correct itself from 6 to 12 years of age without interference by means of periodontal surgery.

35. Muller HP, Eger T, Gingival phenotypes in young male adults
Muller HP, Eger T, Gingival phenotypes in young male adults. J Clin Periodontol 1997; 24: 65–71 [Annaba]
Aim: To extend a previous study analyses by studying the relation crown shape and its relationship with gingival thickness (GTH). Materials & Methods: 42 healthy young males, without any attrition, abrasion, or crown restoration were recruited. (GTH) was determined at the facial aspects of premolars, canines, and incisors by an ultrasonic device, PPD, recession , width of gingiva (WG), (CW/CL) was recorded. Results: Three groups were identified, group A comprised of 2/3 of subjects with normal GTH, WG, and CW/CL; group B had a significantly thicker and wider gingiva and more quadratic form of upper front teeth, group C was showing normal GTH and high CW/CL but narrow zone of keratinized tissue. Conclusion: Present results clearly indicate evidence of different gingival phenotypes.

36. Abiko Y, Shimizu N, Yammaguchi M, Suzuki H, Takiguchi H
Abiko Y, Shimizu N, Yammaguchi M, Suzuki H, Takiguchi H. Effect of aging on functional changes of periodontal tissue cells. Annals of Periodontology. 3(t):350-69, 1998 Jul. [Fageeh]
Aim: To define how cellular aging affects the progression of periodontal diseases associated with the aging process. Materials and methods: Bacterial Culture of C. Rectus and AA, followed by extraction of LPS. Also, HGF and HPLF cells from six pts. with different ages were cultured. Then, HGF and HPLF cells were treated with (LPS) and cyclic tension force, respectively. Amounts of PGE2, interleukin (IL)-lß, IL-6, and Plasminogen activator (PA) were measured Results: The LPS-stimulated PGE2, IL-lß, IL-6, and PA production was increased in "old" HGF compared to younger cells. Also, the cyclic tension force to HPLF stimulated phenotypic and gene expression of IL-lß, PGE2 and PA with higher rates in old Pt. Conclusion: Aging in both HGF and HPLF may be an important factor in the severity of periodontal disease through higher production of inflammatory mediators in response to both LPS and mechanical stress.

37. Andreescu CF, Mihai LL, Răescu M, Tuculină MJ, Cumpătă CN, Ghergic DL
Andreescu CF, Mihai LL, Răescu M, Tuculină MJ, Cumpătă CN, Ghergic DL. Age influence on periodontal tissues: a histological study. Rom J Morphol Embryol. 2013;54(3 Suppl): [pubmed ] [Fageeh]
Aim: To investigate the morphopathological changes that occur in gingival tissue of senior pts. in relationship with histological aspect of gingival tissue of young pts. Materials and methods: Gingival tissues were collected from two groups of patients. The first groupcontrol, [age:20-29 ys. seniors age: >65 ys]. Samples observed under microscope. Results: modification in both epithelium & CT. At epithelial level the study group shows acanthosis, atrophy, dystrophy of epithelial cells & necrosis. At CT level the study shows increased number of fibers and decreased number of cells and Degradation of connective tissue. Conclusion: Aging is a complex, continuous, and slow process that adversely affect the organs functionally and morphologically.

38. CORRELATION OF CLINICAL AND HISTOLOGICAL HEALTHY AND DISEASED TISSUE

39. Matsuura, M. , Herr, Y. Han, K. , Lin, W. , Genco, R. , and Cho, M
Matsuura, M., Herr, Y. Han, K., Lin, W., Genco, R., and Cho, M. Immunohistochemical expression of extracellular matrix components of normal and healing periodontal tissue in the Beagle dog. J. Periodontol. 1995, 66: [Fageeh]
Aim: To understand the formation of new periodontal tissues during periodontal repair and regeneration. Materials and methods: furcation defects were created around mandibular P2 and P4 of 6 dogs. The root surfaces were conditioned with citric acid and (ePTFE) membranes were placed. flaps were sutured. Results: Morphological: at 2 weeks defects were filled with granulation tissue which was replaced by fibrous CT, PDL , cementum, and bone bet. 4 and 8 weeks. Immunohistochemical: at 2 weeks the granulation tissue was stained for FN and VN. At 4 and 8 weeks, staining for CI, FN, and VN was found in fibrous connective tissue, the newly-formed PDL, cementum, and osteoid. Conclusion: This study indicate that FN and VN are involved in the early stages of periodontal repair, and periodontal regeneration.

40. Bissell J, Joly S, Johnson GK, Organ CC, Dawson D, McCray PB Jr, Guthmiller JM Expression of beta-defensins in gingival health and in periodontal disease. J Oral Pathol Med May;33(5): [Al Oraini]
Aim:
To evaluate and compare the expression of HBD-1, HBD-2, and HBD-3(the β- defensins) in healthy gingiva and in periodontal disease.
Material and Methods:
49 gingival samples collected from patients ( 20 healthy, 29 with periodontitis). RNA was isolated and cDNA was synthesized to express the β- defensins, which was analyzed using electrophoresis.
Results:
All three β- defensins are present in the tissues regardless of its status. However, higher level of HBD-2 and HBD-3 are present in healthy tissues.
Conclusions:
the finding suggests that the β- defensins are protective in periodontal disease. However, further studies are needed to understand their role.

41. Gilowski L, Wiench R, Płocica I, Krzemiński TF
Gilowski L, Wiench R, Płocica I, Krzemiński TF. Amount of interleukin-1β and interleukin-1 receptor antagonist in periodontitis and healthy patientsArch Oral Biol Jul;59(7): [AlOraini]
Aim : Study the level of IL-1β and IL-1ra in GCF samples obtained from healthy patient and patient with periodontitis. Materials and methods: 80 CGF samples was collected from non-smoking patients-50 with moderate/severe periodontitis, 30 healthy patients (control group). the samples were centrifuged then IL-1β and IL-1ra was analyzed using ELISA technique. Results: Both IL-1β and IL-1ra were found to be in high concentration in periodontitis group- IL-1β higher. The molecular mass of IL-1ra was 800 fold more moles than IL-1β in the control group but only 300 fold more in periodontitis group. Conclusion: IL-1ra has a defense role in periodontitis but no enough concentration to control the release of IL-1β,further studies required to identify the exact level needed.

42. Li M, Firth JD, Putnins EE. Keratinocyte growth factor-1 expression in healthy and diseased human periodontal tissues. J Periodontal Res Apr;40(2): [AlOraini]
Aim : To examine if KGF-1 and KGFR proteins were expressed in the sample collected from health patients and patients with periodontal disease. also., compare the protein expression in health and in disease. Materials and methods: Tissue samples were collected ( 15 healthy, 13 from patients with advanced periodontitis). Tissues were forzen and stained. Laser capture micro dissection preformed. The samples were incubated with suramin to examine the interaction between KGF-1 and KFGR. Results In healthy tissues, KGF-1 was located in the junctional and basal cell of oral epithelium. KGFR was found in junctional and parabasal cells. In diseased tissues, KGF-1 concentration was high in periodontal pocket epithelium and KGFR was intensely stained in the periodontal pocket epithelium. Conclusion KGF-1 protein expression periodontitis disease tissues may play a role in regulating onset and progression of periodontal pockets.

43. CLINICAL CONSIDERATIONS

44. Keagle, J. Garnick, J. Searle, J. , and Thompson, W
Keagle, J. Garnick, J. Searle, J., and Thompson, W. Effect of gingival wall on resistance to probing forces. J. Clin. Periodontol. 1995, 22: [Fernandes]  
Aim: To study the effect of the gingival wall on resistance to different degrees of probing forces. Materials and methods: An electromechanical device was used to advance a probe tip into the facial sulcus at a constant speed until resisting forces of 0.70 N were encountered.The gingiva of all 2nd incisors, 2nd premolars, and 1st molars of 4 young adult male beagle dogs were tested. Results: Less variability of probing distance in different animals occurred at higher forces. Conclusion: The pressure at which probing distance had less variability among animals and least affected when the gingival sulcular wall was incised was estimated to be 106 N/cm2.

45. Hormia M, Willberg J, Ruokonen H, Syrjanen S
Hormia M, Willberg J, Ruokonen H, Syrjanen S. Marginal periodontium as a potential reservoir of human papillomavirus in oral mucosa. JP. 2005;76(3): [Alkudmani]
Aim:
To investigate whether HPV DNA could be detected in gingival biopsies from patients with periodontal disease.
Materials and methods:
Gingival biopsy from 38 patients diagnosed with periodontitis, . Examination of 12/38 under dissecting microscope, 7/38 under in situ hybridization (ISH), 31/38 using PCR
Results:
HPV DNA was detected in 8/31 gingival samples analyzed with PCR (26%)
In all samples HPV was present only in outer gingival epithelium, except one patient (sulcular and outer gingival epithelium)
In 2/7 samples, ISH revealed viral DNA in the nuclei in the coronal parts of junctional and sulcular epithelium
Conclusion:
periodontal pocket might serve as a reservoir of HPVs in oral mucosa

46. Keum Jin Baek, Youngnim Choi, and Suk Ji
Keum Jin Baek, Youngnim Choi, and Suk Ji. Gingival fibroblasts from periodontitis patients exhibit inflammatory characteristics in vitro. Archives of Oral Biology. 2013;58(10): [Alkudmani]
Aim:
Compare gene expression in GFs isolates from healthy and periodontitis patients in the presence and absence of P.gingivalis
Materials and Methods:
GFs samples were collected from 10 healthy (H) and 10 periodontitis (P) pts, real-time PCR used to measure gene expression of IL-1B, IL-6, IL-8, IL-4, IL-10, MMP-1 and 2, TIMP-3 and OPG. Enzyme-linked immunosorbent assay was used to measure IL-1B, IL-6 and TIMP-3 protein levels
Results:
mRNA expression of IL-6 and IL-1B was higher in the P group whereas IL-4 was higher in H group, in the absence and presence of some of the P. gingivalis strains.
mRNA expression of TIMP-3 was higher in GFs from the P group in the absence and presence of some of the P. gingivalis strains.
Levels of IL-6, IL-1B and TIMP-3 protein were also higher in the periodontitis group in the absence and presence of the P. gingivalis strains.
Conclusion:
High expression of pro-inflammatory cytokines and TIMP-3, and low expression of IL-4 can be a signature of GFs associated with periodontitis.
As there should be a balance between proinflammatory cytokines and anti-inflammatory cytokines then IL-1 and 6 higher in periodontitis and IL-4 in healthy
They check the difference between the gene expression and the protein to check if p.gingivalis could degrade the protein by the protease

47. Lorenzi T et al. The Novel Role of HtrA1 in Gingivitis, Chronic and Aggressive Periodontitis. PLOS one. 2014;9(6):e96978 [Alkudmani]
Aim:
Examine the profile of HtrA1 (High temperature requirement A1) protein and mRNA expression in diseased compared to clinically healthy gingiva
Materials and methods:
2 gingival samples were collected from 16 healthy, 16 gingivitis, 14 CP and 10 GAP pts and tested with immunostaining and PCR for the levels of HtrA1
Results:
Presence of epithelial HtrA1 in all samples, with gradual increase from basal to superficial layer of epithelium
Significant increase specifically in the superficial layer with the increase of severity of disease
Increase of plasma cell HtrA1 immunostaining from gingivitis to chronic and aggressive periodontitis, with the higher intensity in aggressive disease.
Conclusion:
HtrA1 gene expression is not a marker for a particular type of periodontitis but more probably for periodontal inflammation in general.

48. Ben Syndergaard, Mohanad Al-Sabbagh, Richard J
Ben Syndergaard, Mohanad Al-Sabbagh, Richard J. Kryscio, Jing Xi, Xiuhua Ding, Jeffrey L. Ebersole, Craig S. Miller. Salivary Biomarkers Associated with Gingivitis and Response to therapy. JP. 2014;85(8): [Alkudmani]
Aim:
1- Compare levels of IL-1B, 6, PGE2, MMP-8, MIP-1a in whole saliva between gingivitis and healthy pts. 2- Determine if these levels would change after perio tx for gingivitis pts
Material and methods:
Whole saliva was collected from 40 gingivitis and 40 healthy patients at baseline, 7-30 days later and 7-30 days after perio tx for gingivitis pts. Salivary IL-1B, IL-6, MMP-8, MIP-1a and PGE2 were measured
Results:
At baseline, clinical parameters, MIP-1a and PGE2 were higher in gingivitis pts
After perio tx, concentrations of IL-1B, IL-6, and MMP-8 approached healthy levels, whereas MIP-1a and PGE2 concentrations remained significantly higher than in the healthy group
Odds ratio analyses showed that PGE2 concentrations, alone and in combination with MIP-1a, readily discriminated gingivitis from health.
Conclusions:
Salivary PGE2 and MIP-1a discriminate gingivitis from health, and patients with gingivitis who return to clinical health continue to produce inflammatory mediators for weeks after dental prophylaxis.

49. Yu X, Ge S, Chen S, Xu Q, Zhang J, Guo H, Yang P Human gingiva-derived mesenchymal stromal cells contribute to periodontal regeneration in beagle dogs. Cells Tissues Organs. 2013;198(6): [Fernandes]
Aim: To establish that gingiva-derived mesenchymal stromal cells (GMSCs) is a good alternative for periodontal regeneration. Materials and methods: Enhanced green fluorescent protein-labeled GMSCs were transplanted into class III furcation defects created in beagle dogs. Results: Transplanted GMSCs significantly enhanced the regeneration of the damaged periodontal tissue, including the alveolar bone, cementum and functional periodontal ligament (PDL). Conclusion: GMSCs represent a novel cell source for periodontal tissue reconstruction.

51. THANK YOU