الفهرس 
• Histology of GingivaOnly 14 pages are availabe for public view
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Abstract
Nowadays, modern restorative dentistry aims to achieve “white” and “pink” esthetics in the esthetically important zone. “White esthetics” refers to the natural dentition or the restoration of dental hard tissues with suitable materials. “Pink esthetics” refers to the surrounding soft tissue, including the interdental papilla (IDP) and gingiva that can enhance or impair the esthetic results.
Loss of the IDP after crown and bridge restoration and implants causes food accumulation in this space, phonetic problems and interferes with good esthetics leading to the dreaded “black triangles” which is one of the most challenging aesthetic problems, for which there are limited successful treatment options.
The use of soft tissue fillers (STF) like Hyaluronic acid (HA) filler either alone or in conjugation with surgical treatment for gingival augmentation has been introduced and proved its positive augmentation effect. However it is still controversial and its results are unpredictable. This is, in part, due to lack of efficient preclinical experimental models.
Cross-linked dextran in hydroxyl propyl methylcellulose (DHPMC) is a novel Korean STF whose filling effect is long lasting in comparison to the other biodegradable STF including HA fillers. Its filling effect can persist for about five years. For the IDP regeneration, single intragingival injection may be sufficient for significant papilla fill.
Thus this research aimed to evaluate the histological alterations that can result from injection of DHPMC versus HA fillers into gingiva of rats.
Materials and Methods:
Sixty-three male Weister Albino rats weighing between 200-250 grams were used for this study. They were equally divided into three groups, each consisting of twenty one rats as follows:
Control Group: (C group)
Gingivae of rats of this group were intragingivally injected distal to the mandibular left incisor with 0.02 ml physiologic saline. Rats of this group were used as:
1- Control negative group (C–ve): represented by the right uninjected side of the rat gingiva distal to the mandibular right incisor.
2- Control positive group (C+ve): represented by the left side of the rat gingiva distal to the mandibular left incisor intragingivally injected with 0.02 ml saline.
Experimental groups:
• Hyaluronic Acid group (HA group):
where gingiva of rats distal to the mandibular left incisor was intragingivally injected with 0.02 ml HA (Restylane®).
• Dextran group (D group):
where gingiva of rats distal to the mandibular left incisor was intragingivally injected with 0.02 ml DHPMC (Licol Gold®).
Both control and experimental groups were equally subdivided into three subgroups according to the date of sacrifice. Each subgroup consisted of seven rats:
• Subgroup a (4 days PI): sacrificed four days after injection.
• Subgroup b (14 days PI): sacrificed 14 days after injection.
• Subgroup c (2 months PI): sacrificed two months after injection.
At the end of the experimental period of each subgroup, rats were sacrificed and the augmented gingiva with the surrounding alveolar mucosa and opposing portion of lip was excised.
The collected samples were fixed and embedded in paraffin blocks to be sectioned. Tissue sections were stained by:
1. H&E Stain: for histological examination.
2. Picro Sirius red (PSR) Staining: for visualization of collagen types I and III fibers.
3. CD68 Immuno-histochemical Staining: labeling macrophages and other members of the mononuclear phagocyte lineage.
Morphometric Study:
Seven H&E stained sections of each subgroup were analysed using a computerized image analyser to measure:
1. Epithelial thickness
2. Area % of PSR reaction
Statistical Analysis:
The collected data was tabulated and statistically analyzed using a computerized statistical analyzer using ANOVA and Post Hoc tests.
Results:
1. H&E Results:
• H&E stained sections of C-ve group at the three experimental periods (C-a, C-b and C-c) showed gingiva with ortho-keratinized epithelium (Ep) of normal architecture with well-defined basement membrane (BM), multiple long slender connective tissue papillae (CTP) and few intraepithelial clear cells. The lamina propria (LP) of C-ve group appeared as a moderately dense vascular connective tissue (CT). Some sections showed some dilated blood vessels.
• Regarding the C+ve group, H&E stained sections at the three experimental periods (C+a, C+b and C+c) featured an ortho-keratinized stratified squamous Ep with few and irregular CTP.
The LP of C+a subgroup appeared either dense and infiltrated by inflammatory cells and multiple small BVs or loose and disorganized with large degenerative areas, large intercellular spaces and dilated BVs. On the other hand, the LP of C+b and C+c subgroups appeared of moderate density and vascularity with less degenerative areas.
• Histologic examination of HA subgroup revealed variable tissue reactions to HA injection. The Ep was apparently thin with ill-distinct basement membrane (BM) and few short CTP in HAa and HAb subgroups sections. While HAc sections showed normal gingival epithelial thickness with few long and irregular CTP.
The LP of HA group at the three experimental periods presented HA droplets in the form of dark basophilic deposits. Some vacuoles appeared empty devoid of basophilic deposits but limited by an inflammatory infiltrate. HA droplets were observed in the injected gingivae as well as other remote areas of labial, vestibular and alveolar mucosae.
In HAa sections, HA deposits were surrounded by an inflammatory cell infiltrate including few multinucleated cells. On the other hand, HAb sections showed well circumscribed HA deposits appeared limited by dense collagen bundles with or without an inflammatory cell infiltrate. Granulation tissue and aggregation of a large number of inflammatory cells were evident in relation to HA deposits in some sections.
The LP of most HAc sections presented large well-circumscribed vacuoles containing very few amounts of HA deposits and large areas of degeneration, limited by dense collagen bundles with some collagen bundles running in between the HA remnants forming a fibrous meshwork appearance. On the other hand, few HAc sections showed large HA deposits, surrounded by thick dense collagen bundles.
• H&E sections of Da subgroup featured an apparently thickened gingival Ep with broad rete pegs and multiple intraepithelial clear cells. Db and Dc sections featured gingival Ep of normal architecture. Few Db sections featured some sort of basilar hyperplasia and hyperchromatism of nuclei of basal and prickle cells. Karyorrhexis of some prickle cells nuclei was evident in some Dc sections.
Regarding the LP of D group, it appeared very dense infiltrated by various amounts of D particles as well as heavy inflammatory cell infiltration and multiple small BVs at the three experimental periods (Da, Db and Dc subgroups). D particles appeared at the three experimental periods in the form of homogenous well-circumscribed acidophilic spheres. A small number of these spheres could be identified at 4 days PI (Da subgroup). However, the LP of Db and Dc contained a large number of these D particles. Besides, some D particles in Dc subgroup showed partial degradation, while others appeared as intact spheres with lightly stained core and darkly stained peripheries.
In Da sections, inflammatory cells including few multinucleated cells were observed in the LP. These multinucleated cells were not deposited on D particles surface. On the other hand, at 14 days and 2 months PI (Db and Dc), the inflammatory infiltrate appeared in the form of epithelioid cells and multinucleated cells that were deposited on D surface. Some D particles in Dc subgroup were surrounded by degenerative areas without epithelioid or multinucleated cells deposited on D particles surface.
The area of D injection appeared well circumscribed from the surrounding tissue by dense collagen encapsulation especially in Dc subgroup. Most of Dc subgroup sections featured a dense LP in between D particles with collagen bundles, multiple small blood vessels and inflammatory cells (granulation tissue). However these collagen bundles in between D particles were absent in some areas of Dc sections.
2. PSR Results:
• All C-ve PSR stained sections at the three experimental periods showed densely packed red stained collagen bundles in dense LP.
• The LP of C+a PSR stained sections appeared loose with large spaces in between red stained collagen bundles. Then, LP of C+b sections appeared moderately dense with some spaces in between red stained collagen bundles. At 2 months PI, C+c sections showed dense LP with densely packed red stained collagen bundles.
• HAa, HAb and HAc PSR stained sections showed moderately dense LP with red stained collagen bundles surrounding vacuoles of HA. Besides, HAc sections showed thin collagen bundles within the HA filler running in between HA deposits.
• Da sections showed loose LP with very few red stained collagen bundles in between D particles. Density of collagen bundles in between D particles apparently increased with time in Db and Dc sections compared to Da. However, LP in between D particles in some Dc section showed totally negative PSR reaction.
In Db and Dc PSR sections, the area of D injection appeared well encapsulated by a thick red stained collagen capsule even in the Dc sections with negative PSR reaction in between D particles.
3. CD68 Immuno-Staining Results:
The CD68 immuno-reactive cells showed cytoplasmic brown staining which appeared diffuse or granular.
• Most of C-ve and C+ve CD68 stained sections showed few immuno-reactive cells scattered in LP. However some C-ve sections showed almost negative CD68 immuno-reaction in the LP.
• HAa CD68 stained sections showed some CD68 immuno-reactive cells scattered in LP in relation to HA vacuoles. Then, HAb sections showed multiple CD68 immuno-reactive cells limiting the empty vacuoles, extended within the vacuoles and scattered in LP. However HAc section showed few positive CD68 immuno-reactive cells in the LP.
• Da sections showed numerous CD68 immuno-reactive cells within LP in relation to D particles. The number of CD68 immuno-reactive cells in close relation to D particles apparently decreased in Db and Dc sections. The epithelioid and multinucleated cells presented negative CD68 immmuno-reaction in Dc sections.
4. Statistical Results:
I- Epithelial Thickness:
The epithelial thickness was found to be significantly increased in only Da subgroup compared to the control and HA groups. Then, it significantly decreased with time at 14 days and 2 months PI (Db and Dc subgroups). On the other hand, the other groups showed insignificant changes in epithelial thickness with time over the three experimental periods.
II- Area % of PSR Reaction:
The area % of PSR reaction was significantly decreased in HA and D groups compared to C-ve group at the three experimental periods. Meanwhile, C+ve group showed a significant decrease in area % of PSR reaction from C-ve group at 4 and 14 days only.
There was a significant increase in area % of PSR reaction with time in the C+ve group at 2 month PI. While the increase in PSR reaction area % of the other groups (C-ve, D and HA groups) with time was statistically insignificant.