Clinical and Histological Analysis of Human Gingival Phenotypes

Study Overview
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Brief Summary
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The goal of this observational study is to compare the composition of the human gingiva in different gingival phenotypes. The main questions to answer are: Is there any difference in the cellular composition of the gingiva between thin and thick gingival phenotype? Is there any difference in the molecular composition of the gingiva between thin and thick gingival phenotype? The participants were divided in two groups (thin and thick phenotype) and a biopsy of healthy gingiva was obtained from each one them. The biopsies were analyzed histologically and the collected data will be analyzed statistically in order to identify possible differences between the gingival phenotypes.

Detailed Description
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Healthy volunteers were assigned in one of two groups: Group 1: Thin gingiva, when the free gingiva was evaluated as transparent, after the insertion of a periodontal probe (Hu-Friedy XP-23/QW, Hu-Friedy,Chicago,IL,USA) in the middle of the facial dentogingival sulcus of a maxillary central incisor Group 2: Thick gingiva: when the free gingiva was evaluated as non-transparent, using the same methodology. A full thickness sample of the oral mucosa of each one of the participants was collected under local anaesthesia. The sample had a rectangular shape, with a length of 4 millimeters and a width of 1 millimeter. It had a vertical orientation and it was expanding at the both sides of the mucogingival junction. The oral mucosa samples were processed for histological and immunohistochemical analysis. Staining with haematoxylin-eosin was performed in order to describe the tissue histologically and also calculate the total number of the cells it contained. Immunohistochemical staining with anti-Vimentin,anti-Cluster of Differentiation 68, anti-Ki-67 and anti-Smooth Muscle Actin antibodies was applied for the calculation of the numbers of fibroblasts, macrophages, Ki-67-positive cells and Smooth muscle actin positive cells respectively. Immunohistochemical staining was also performed in order to determine the levels of expression of Collagen I, Collagen V, Elastin and Hyaluronic acid. Whole slide images of the specimens were acquired with the use of the NanoZoomer 2.0HT (Hamamatsu Pho-tonics K.K., Hamamatsu, Japan). Cell counting will be performed automatically using an image analysis software (QuPath 3.0). The levels of molecular expression will be assessed with the use of the same software and will be expressed as a percentage of the area of the connective tissue that is occupied by the investigated molecules.

Official Title
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Clinical, Histological and Immunohistochemical Characterization of Human Gingival Phenotypes

Conditions
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Healthy

Intervention / Treatment
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* Diagnostic Test: Histological analysis
* Diagnostic Test: Immunohistochemical analysis


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Healthy adults Exclusion Criteria: Attachment loss or Probing depths greater than 2 millimeters at the maxillary central incisors Altered passive eruption of the maxillary incisors Previous surgery or orthodontic treatment in the anterior maxilla Medical conditions or medication affecting soft tissue metabolism Tooth crowding or abnormal angulation or abrasion of the maxillary incisors Restorations at the buccal surface of the maxillary central incisors Smoking Pregnancy or lactation

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Thin Phenotype<br>Participants with transparent free gingiva of the maxillary central incisor | Diagnostic Test: Histological analysis<br>* Histological staining of gingival biopsies with haematoxylin-eosin.<br>Diagnostic Test: Immunohistochemical analysis<br>* Immunohistochemical staining of gingival biopsies using anti-Vimentin, anti-Collagen I, anti-Collagen V, anti-Elastin, anti-Hyaluronic acid, anti-Smooth muscle actin, anti-Cluster of Differentiation 68 and anti-ki67 antibodies.<br>|
| Thick Phenotype<br>Participants with non-transparent free gingiva of the maxillary central incisor | Diagnostic Test: Histological analysis<br>* Histological staining of gingival biopsies with haematoxylin-eosin.<br>Diagnostic Test: Immunohistochemical analysis<br>* Immunohistochemical staining of gingival biopsies using anti-Vimentin, anti-Collagen I, anti-Collagen V, anti-Elastin, anti-Hyaluronic acid, anti-Smooth muscle actin, anti-Cluster of Differentiation 68 and anti-ki67 antibodies.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Cell density | The number of all types of cells per square millimeter of the connective tissue | Baseline | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Fibroblast Density | The number of fibroblasts per square millimeter of the connective tissue | Baseline | 
| Macrophage Density | The number of macrophages per square millimeter of the connective tissue | Baseline | 
| Density of Ki-67-positive cells | The number of Ki-67-positive cells per square millimeter of the connective tissue | Baseline | 
| Density of Smooth Muscle Actin positive cells | The number of Smooth Muscle Actin positive cells per square millimeter of the connective tissue | Baseline | 
| Collagen I area ratio | The percentage of the area of the connective tissue which is occupied by Collagen I | Baseline | 
| Collagen V area ratio | The percentage of the area of the connective tissue which is occupied by Collagen V | Baseline | 
| Elastin area ratio | The percentage of the area of the connective tissue which is occupied by Elastin | Baseline | 
| Hyaluronic acid area ratio | The percentage of the area of the connective tissue which is occupied by Hyaluronic acid | Baseline | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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gingiva, histological, immunohistochemical, periodontal, biotype, phenotype