الفهرس 
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Abstract
Nanotechnology is a science that deals with the research and development of
materials and devices at the atomic or molecular level. Almost every aspect of
human life is influenced by future nanotechnologies. With the advancement in the
technology, it is also getting incorporated in various medical fields including
dentistry (nanodentistry). The oral cavity possesses a number of features which make
it a distinct habitat for many of microorganisms as Porphyromonas gingivalis. There
is strong evidence that Porphyromonas gingivalis, a Gram-negative anaerobes, is the
keystone species in the development of chronic periodontitis. Tetracyclines were
attributed as one of the most effective broad spectrum antibiotics but their
widespread use rendered generations of bacteria resistant, which enhance a lots of
study to develop a new consideration between the nanotechnology and antibiotics
forming a protocol in treating bacterial diseases.
The present investigation was concluded to compare the effect of local
administration of silver nanoparticles versus tetracycline manufactured in nanosizing
in treatment of induced infection of the buccal mucosa of the rat with
Porphyromonas gingivalis.
Thirty six adult male albino rats 150 - 180 gram body weight were used in this
investigation. They were divided randomly into six groups as follows:
▪ group I consisted of 6 rats, they served as normal negative control group.
▪ group II (positive control) consisted of 6 rats, they were infected once by
injecting the buccal mucosa of the vestibule opposite to upper first molar with 1×108
colony forming unit (CFU)/1mL of Porphyromonas gingivalis that was cultured at
National Research Center in Egypt and then left for fourteen days untreated.
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▪ group III consisted of 6 rats, they were subjected to the same procedure as
group II then to daily injection at the site of infection with diluted tetracycline
powder with concentration of 50mg/1ml of distilled water for fourteen days.
▪ group IV consisted of 6 rats, they were subjected to the same procedure as
group II then to daily injection at the site of infection with nanosilver 20nm with a
concentration of 15𝜇g/1ml of distilled water for fourteen days.
▪ group V consisted of 6 rats they were subjected to the same procedure as
group II then to daily injection of tetracycline loaded on chitosan 50 nm at the site
of infection with a concentration of 140 𝜇g/1ml of distilled water for fourteen days.
▪ group VI consisted of 6 rats they were subjected to the same procedure as
group II then to daily injection at the site of infection with 5𝜇g/1ml of nanosilver
20nm loaded by nanotetracycline with a concentration of 30 𝜇g/1ml of distilled
water for fourteen days.
At the end of experiment, all rats were sacrificed by cervical dislocation. Samples
were taken from the buccal mucosa at the site of infection of the rats of different
groups and prepared to be examined through:
• Microscopic and submicroscopic examination to detect any structural and
ultrastuctural changes in the specimens
• Immunohistochemical localization of Proliferating Cell Nuclear Antigen
(PCNA) to assess the proliferating capacity of the cells at the site of infection.
• Immunohistochemical localization of interleukin-1beta as a pro- inflammotery
cytokine.
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The results of this study revealed the following:
A- Microscopic and submicroscopic results:
Buccal mucosa of group II infected with P. gingivalis showed a dramatic
change in histological appearance compared to uninfected controls, with increase in
thickness of epithelium. Epithelium showed cytoplasmic vacuolization and areas of
fusion of these vacuoles forming larger ones. A lot of clear cells appeared in different
levels in epithelial layers. Migration of basal cell layer into papillary layer of lamina
propria with discontinuation of basement membrane was detected. Lamina propria
showed destruction in the connective tissue fibers with localized area of
inflammatory cell infiltrations. Submucosal layer showed areas of collagen
dissociation with ill-defined bundles of muscles while minor buccal salivary glands
showed cytoplasmic vacuolization of variable size.
Transmission electron microscope imaging of group II (infected
group) showed P. gingivalis strain with its ultrastructural component and marked
ultrastructural deviation from normal picture where most of epithelial cells were
presented with variable sized cytoplasmic vacuoles. Marked degeneration of cell
organelles and decrease in the size and number of keratohyaline granules was
demonstrated. Decrease in the size and irregularity in shape of the nuclei and
homogenecity of chromatin was found in few of epithelial cells and basal lamina
showed areas of intruption. In lamina propria, fibroblast, mast and macrophages
presented with signs of degeneration manifested as. cytoplasmic vacuolization. The
collagen fibers were markedly dissociated with multiple areas of connective tissue
were completely devoid of collagen fibers. Submucosal layer presented in muscular
cells with obvious vacuolization with shrinked fibroblast and dilated blood vessel.
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The histological results of group III adminsterated with diluted
tetracycline powder as a traditional drug for bacterial infections as P.
gingivalis in group III didn’t approach the satisfied recovery for buccal
mucosa as hyperkeratinization and clear cells.were found. Inflammatory
cells infiltration in between the muscular layer underneath the submucosa
and partial regeneration of collagen fibers in connective tissue and dilated
blood vessel with persist focal hemorrhage. Minor buccal salivary glands
showed multiple cytoplasmic vacuolization in mucous acini.
The ultrastructural results obtained from TEM reveals no marked
signs of recovery as different sized cytoplasmic vacuoles in the epithelial
layers with marked degeneration of cell organelles particularly,
mitochondria. Marked decrease in the size and number of keratohyalin
granules was noticed. Widening of intercellular spaces was demonstrated
with presence of inflammatory cells particularly lymphocytes. Fibroblast
and muscle cell observed with vacuolization.
Inspite of the ultrastructural resembling of the epithelium and
decrease of clear cells as well as the inflammatory cells, and presence of
fibroblast cell in TEM images ,major findings decline the opportunity for
AgNPs treatment to reach the best recovery from the P. gingivalis infection
as disturbance in the epithelium and connective tissue interference which
was coincident with our ultrastructural findings where focused distortion
of basal lamina was detected as well as pleomorphic changes occurred in
multiple epithelial nuclei. Leucocytes were found as macrophage with few
phagosomes in cytoplasm
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group V was treated with nanotetracycline loaded on chitosan
revealed decreased in thickness of epithelium with clear cells seen through
different strata of epithelium. There was less elongation of the epithelial
ridges into lamina propria was frequently encountered. The results were
confirmed by examining the TEM images where thinner epithelium resting
on an intact basal lamina was found with few prickle cells layers that have
lost their characteristic polyhedral shape with more cytoplasmic
vacuolization were detected and less keratohyalin granules. The underlying
lamina propria showed fibrosis of collagen fibers in addition to
accumulation of many inflammatory cells and dilated blood vessels.
Best recovery signs were obtained by examining the epithelium
histologically of those specimens of group VI treated with nanosilver
particles loaded with tetracycline that showed almost normal structure of
its different strata as identified basal, prickle, granular and cornified layers
with less clear cells were found. Basal cells were rested on intact basal
lamina with new parabasal cells. No discontinuation of basement
membrane as well as basal lamina under TEM. Less cytoplasmic
vacuolization was observed and very limited to stratum granulosum. Non
keratinocytes were also detected as melanocyte and macrophage which
indicates reassembling structure of normal epithelium.
Regeneration of the under laying connective tissue were detected
were lots of collagen fibers formed. No sign of destruction in musculature
with well-organized bundles of muscles. Minor buccal salivary gland
showed almost normal appearance with less vacuolization in mucous acini.
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B. lmmunohistochemical results:
1- Proliferating Cell Nuclear Antigen (PCNA)
- PCNA labeling index
There was significant decrease in PCNA labeling index of the
surface epithelium of the buccal mucosa of group II animals compared with
normal group I as well as significant increase in PCNA labeling index of
group VI treated with tetracycline loaded on AgNPs with best results
considering proliferation and regeneration in basal and parabasl layers.
- PCNA staining reactivity
There was statistical significant increase in PCNA immunoreactivity
of all treated groups compared to control group I. Also, there was statistical
significant increase in PCNA immunoreactivity of groups V and VI
specimens treated with tetracycline loaded on chitisan and tetracycline
loaded on AgNPs respectively, showed better cellular proliferation of
epithelial cells of buccal mucosa, followed by group IV treated with AgNPs
which showed moderate positive reactivity compared to groups III treated
with tetracycline powder which showed weak positive reactivity with the
least cellular proliferation
2-Immunohistochemical results using interleukin-1 beta.
- IL-1β labeling index
There was significant increaese in IL-1β labeling index of the
epithelium and underlying lamina propria of the buccal mucosa of group II
animals compared with normal group I as well as significant decreaese in
IL-1β labeling index of group VI treated with tetracycline loaded on
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AgNPs with best results considering the host inflammatory response to
bacterial infection which leads to less to tissue damage.
- IL-1β staining reactivity
Normal group I sections examined under light microscope showed
weakly reaction to IL-1β all through different epithelial layers, lamina
propria as well as submucosa. While positive group II sections showed
strongly positive reaction at all strata of epithelium and more intense at
superfacial layers besides basal ones.
Examination of sections of group V and VI treated with local
administration of tetracycline loaded on chitosan and loaded on AgNPs
respectively, revealed weakly positive reaction to IL-1β as localized
patches of staining through epithelium and lamina propria, while sections
of group IV treated with AgNPs showed moderately positive reaction to
IL-1β at wider patches through epithelium. Similar results were obtained
within the lamina propria. group III sections showed strongly positive
reaction to IL-1β was found in the epithelium and lamina propria showing
their ability to produce inflammatory cytokines such IL-1β which is known
to cause degenerative changes in epithelium and connective tissues.