الفهرس 
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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,
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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®).
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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.
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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.
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165
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
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number of inflammatory cells were evident in relation to
HA deposits in some sections.
The LP of most HAc sections presented large wellcircumscribed
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.
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167
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.
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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
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HAc section showed few positive CD68 immuno-reactive
cells in the LP.
Da sections showed numerous CD68 immunoreactive
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 (Cve,
D and HA groups) with time was statistically
insignificant.