Basic Informations

C.V

Dr. Moahmed Yehia Saad Abdelfattah

Assistant Lecturer Oral and Craniofacial Biology
Faculty Of Dentistry, Beni-Suef University

Master Title

Survey of Tenascin-C and Integrin ß1 during Odontogenesis and Eruption of Albino Rat Molar (A Histological and Immunohistochemical study)

Master Abstract

SUMMARY Tooth eruption and are complicated processes that involve tightly programmed events coordinating functions of osteoblasts, osteoclasts, dental follicle cells, and periodontal ligament cells. During pre-emergent tooth eruption, the rate of resorption of overlying structures is critical to direct eruption, and hence support the erupting tooth to move along the appropriate path. Tenascin is an extracellular matrix glycoprotein which interacts with other matrix molecules and with cells, and which appears to play important roles in development and growth. It regulates cell migration and organogenesis. Integrins are transmembrane receptors which promote both cellcell and cell-extracellular matrix adhesion. The a and ß subunits show a wide tissue distribution and play key roles in developmental, physiological, and pathological processes. Integrins are involved in many fundamental cellular functions, including proliferation, adhesion, motility, differentiation, survival, and apoptosis. It was established that tenascin-C and integrin ß1 have got a role in cell movement and migration and may be implicated in tooth movement. Also they are involved in regulation of development and growth. The aim of our study was to evaluate immunohistochemical localization of tenascin-C and integrin ß1 in the odontogenic tissues of Summary 101 lower first molar at different stages of its development and along its pathway of eruption. Materials and methods: To fulfill such aim, this study was carried on 40 adult male albino rats of average weight 200-250 gm. The animals were kept on a laboratory balanced diet supplied daily adlibitum. Three females to one male rat were caged together and left for mating. When pregnancy was determined by pregnancy test, the pregnant rats were caged separately waiting for delivery. The offsprings were sacrificed by cervical decapitation at the following age: At birth (Group I) At one week (Group II) At two weeks (Group III) At three weeks (Group IV) At four weeks (Group V) The lower jaws of offsprings sacrificed at birth were dissected out, fixed in neutral formol, dehydrated in ethanol, cleared in xylene and mounted in paraffin wax. Whereas the specimens of other age groups were subjected to decalcification. Decalcification was performed in EDTA solution prior to dehydration and clearing. Three to four micron-thick sections were subjected to routine (Haematoxylin and Eosin) staining and immunohistochemical Summary 102 investgation using primary antibodies for detection of tenascin-C and integrin ß1. Histological results: The tooth germ of lower first molar of albino rat at birth (group I) appeared at the beginning of late bell stage showing thin layer of dentin matrix deposited at the cusps. The dental papilla was moderately vascular and thin bone trabeculae were deposited at the fundus of the crypt and overlay the tooth germ. At one week (group II), there were a disintegrating dental lamina and thick layers of enamel and dentin matrices deposited. Thick interconnecting bone trabeculae surrounded the tooth germ. At two weeks (group III), the dental lamina was disintegrated, and there was a close proximity of the tooth germ to the oral epithelium with shrunken enamel organ. The tubular structure of dentin was evident. Dense, interconnected and thick bone trabeculae surrounded the tooth germ in a continuous manner and presented nutrient canals at the base of the socket. At three weeks (group IV), portions of the roots were developed and the middle cusp was piercing the overlying mucosa. Dense and wavy collagenous bundles of periodontal ligament were obliquely directed at the cervical region. Thin interradicular bone trabeculae separated the root portions. The lower first molar of albino rat was erupted at age of four weeks (group V). Most of the root length was developed and appeared Summary 103 associated with more organized interradicular and interdental bone. The outline of alveolar bone proper was irregular and the periodontal ligament displayed higher vascularity at the area facing the bone than that facing the root. The intermediate plexus was identified in the cervical portion of periodontal ligament and the alveolar crest, interdental , horizontal ,oblique and interradicular groups were distinguished. Immunohistochemical results: In immunohistochemical investigation of the present study, mild staining reactivity for tenascin-C appeared in most of the odontogenic tissues of lower first molar of albino rats at birth. The reaction increased markedly at age of one week by the differentiation of formative cells and deposition of protein matrix. The expression of tenascin increased in dentin, bone and bone marrow but was negative in the enamel matrix. At age of two weeks, the reactivity to tenascin-C decreased mostly in the odontogenic tissue but minimal traces of tenascin appeared in the enamel matrix that previously reacted negatively. Further decrease in tenascin was observed at the age of three weeks but the persisting layer of enamel matrix seen cervically presented remarkable increase in intensity. The expression increased again at the age of one month by the eruption of the tooth. The periodontal ligament fibers showed stronger reactivity in the gingival, transeptal, alveolar crest, horizontal and Summary 104 inter-radicular fibers as compared to the oblique group and follicular tissue apical to the developing roots. However, the greatest reactivity was manifested in the zone of intermediate plexus cervically. Integrin expression in the odontogenic tissues of lower first molar of albino rats at birth presented mostly weak to mild intensities with a slightly greater reactivity in developing bone trabeculae and associated bone marrow. The reactivity to integrin decreased as the tooth germ grew from birth to one and two weeks and then increased slightly at the age of three and four weeks particularly in the bone marrow, interstitial tissue of periodontal ligament and in the zone of intermediate plexus cervically.

PHD Title

Evaluation of the effect of botulinum toxin (Botox) on submandibular salivary gland of the rats (Histological and Ultrastructural study)

PHD Abstract

Summary 126 SUMMARY Botulinum toxin (Botox) is best known for its beneficial role in facial aesthetics but recently it was established that Botox has a role in treatment of multiple non-cosmetic medical and surgical conditions. Botox is an exotoxin produced from Clostridium botulinum, so it is considered as a bacterial toxin that could be used as a medicine and it is looked upon to be ‘the poison that heals”. It works by blocking the release of acetylcholine from the cholinergic nerve end plates leading to inactivity of the muscles or glands innervated. The aim of this study was to evaluate the effect of botulinum toxin (Botox) on submandibular salivary gland of the rats (Histological and Ultrastructural study). To fulfill such aim, this study was carried out on 60 adult male albino rats of average body weight 250-300 gm. Animals were divided into two main groups as follows: I-Control group; Group I: thirty rats were used as control and injected intraglandular with saline. II-Experimental groups; Group II: thirty rats were injected intraglandular with Botox (5 IU) at the beginning of the experiment then sacrificed to take the specimens as follow: - Group II (A): ten rats where the animals were left for 2 weeks. - Group II (B): ten rats where the animals were left for 4 weeks. - Group II (C): ten rats where the animals were left for 12 weeks. 5 U Botox in 0.1 ml saline was injected into the submandibular gland. The control group was injected by 0.1 ml saline in a similar manner. Summary 127 After two, four and twelve weeks, the rats were anaesthetized as mentioned above and sacrificed. The submandibular glands were dissected from the surrounding connective tissue and removed. Tissue samples were preserved for studying their histology by routine staining by hematoxylin and eosin (H&E) (sections of about 6-7µm) as well as for ultrastructural study using transmission electron microscope (sections of about 50 nm in thickness). Histological, Morphometric and ultrastructural results: Histological results: The results of the present work suggest that injection of Botulinum toxin intra-glandular into the submandibular salivary gland of albino rats resulted in structural and ultrastructural alterations in these glands, such alterations were more marked after BoNTA injection by four weeks more than two weeks but after 12 weeks the structures of the glands became close to normal glandular architecture. In the present study, the control SSGs showed normal histological structures of serous acini and duct system without significant histological differences at 2, 4 and 12 weeks, indicating that the age changes or growth effects on these structures are not significant. The light microscopic examinations revealed that there was regular appearance of serous acini and duct system. Acini were spherical in shape acini, granular convoluted tubules had cuboidal cells, striated ducts presented low columnar cells and characteristic basal striations. Most of blood vessels were patent and engorged with red blood cells. Well defined perineurium, wavy course of myelinated nerve fibers, along with regular arrangement of Schwan cells nuclei were presented during examination of histological structure of the nerve. Summary 128 Two weeks interval after BoNTA injection, light microscopic examination revealed loss of spherical fashion and cytoplasmic vacuoles of some serous acini. Also, GCT became distended with stagnated secretion in their lumen. In addition, degenerative changes of striated ducts such as loss of basal striations, cytoplasmic vacuolization and defective borders were observed. Moreover, shrinkage of some acinar cells and wide intercellular spaces were also presented. ILD presented signs of nuclear degeneration. A lot of blood vessels were obliterated by engorged RBCS. Nerve fibers presented loss of wavy course and irregular arrangement of Schwan cells nuclei and vacuolization. However, some nerve fascicles showed a normal wavy course of myelinated nerve fiber bundles and surrounded by well-defined perineurium. Changes in histological structure 4 weeks after BoNTA injection were presented in the form of atrophy and degeneration which was more marked than the previous group. Acini were completely degenerated, and atrophied, and only remnants of their nuclei appeared scattered. The degenerated parts were replaced by fatty droplet formation or hyalinization. In some lobules, the acini and ducts became shrunken and atrophic leaving very wide inter acinar spaces replaced by fibrous hyalinization. In atrophic part, ICDs were dilated with hyperchromatic nuclei and forming proliferating cords. GCTs lost their normal architecture and presented stagnated secretion. Striated ducts lost their basal striations in addition to cytoplasmic vacuolization and stagnated secretion. Blood vessels were completely occluded and obliterated with thrombus formation. Severe histological alterations were observed in the nerve fascicles examined at this interval. Nerve fascicles may be distended, severely degenerated, or lost their normal architecture. Cellular infiltration, axonal degeneration, and loss of wavy course of nerve fiber Summary 129 bundles and their Schwan cells. Severe histological changes in nerve fiber bundles were presented close to atrophied and degenerated acini. Twelve weeks interval revealed different histological patterns where some lobules demonstrated histological alterations while many other lobules demonstrated glandular architecture approximately close to normal. In atrophic lobules of the gland, acini revealed some vacuolization of the cytoplasm and abnormal shape of the nucleus. In addition, ICD exhibited tubular or ductal like structures with proliferating cords branching into terminal secretory Bulbs. Striated ducts in atrophic part of the gland, striated ducts surrounded by occluded blood vessels exhibited ill distinct cell membrane and ill-defined cell boundaries, loss of characteristic basal striations, nuclear changes, cytoplasmic vacuolization, and dilated lumen filled with stagnated secretion. In many other lobules, where the blood vessels became patent, the acini presented normal morphology of spherical fashion with pyramidal shaped cells and morphometric analysis results showed that the mean of the area percent occupied by acini of this group was very near to that of control group and the P-value was insignificant. The striated ducts appeared with normal like architecture with rounded well-defined borders, rounded and lined by a single layer of columnar cells with open vesicular nuclei and characteristic basal striations. Most of blood vessels were patent and some of them were obliterated. Histological structure of the nerve fascicles was close to normal morphology architecture of the control group. However, some lobules in atrophied part of the gland demonstrated obvious changes in nerve fascicles presented by loss of wavy course of nerve fiber bundles, vacuolization and irregular perineurium. Summary 130 Morphometric results: Statistical analysis results of morphometric evaluations of mean of surface area percentage of the acinar cells and mean of surface area percentage of the blood vessels were obtained by comparing between the mean percentage of both surface areas of acinar cells and blood vessels in the Control [Group I(GI)] and BoNTA injected groups [Botox 2weeks (GIIA), Botox 4weeks (GIIB) and Botox 3 month(GIIC)]. The results obtained from morphometric evaluations indicated that the mean of surface area percentage of the acinar cells was significantly smaller in rats treated with BoNTA (Group II) than the control group (GroupI) (P<0.001). The only group with no significant difference with the control group was GIIC (P>0.05). Statistically significant differences were also found between the BoNTA subgroups (A, B and C) (P<0.001). The mean of surface area percentage of the blood vessels as measured histomorphometrically was very similar and followed the same trend as the percentage area of the acinar percentage area. Ultrastructural results: In the current study, the ultrastructural findings of submandibular salivary gland confirmed the findings of the structural changes found in the different intervals. The ultrastructural changes of intraglandular botulinum toxin A treated submandibular salivary glands were marked and showed more prominent changes at four weeks interval more than that of two weeks and presented almost normal ultrastructural findings after three months. Ultrastructural examination of control group revealed that the acinar cells presented pyramidal cells with basally located rounded nuclei, rER and secretory granules. However, Golgi apparatus located apical and lateral to the nucleus. The cytoplasm was dispersed with numerous mitochondria. The Summary 131 granular convoluted tubules with electron lucent basally situated nucleus surrounded by electron dense secretory granules. The cells of the striated ducts showed characteristic basal infoldings of the plasma membrane and numerous mitochondria. Their nuclei surrounded by cisterna of rough endoplasmic reticulum, minute secretory granules and huge number of mitochondria which are scattered throughout the cytoplasm. Ultrastructural examination of treated rats two weeks after BoNTA injection presented acinar cells with irregular nuclear pattern, degenerated mitochondria, dilated rER and Golgi apparatus. There was lipid droplets and electron lucent secretory granules of different size and density in addition to cvaytoplasmic vaculisation. The striated ducts presented irregular nucleus, loss of basal striations and swollen or degenerated mitochondria. Four weeks after BoNTA injection, serous acinar cells showed disfigurement of nuclear pattern, ruptured and degenerated mitochondria, deformed Golgi apparatus and thickened rough endoplasmic reticulum. Myoepithelial cell with flattened cell body and elongated nucleus was observed between the basal lamina and the basal plasma membranes of the acinar cells. The intercalated ducts showed different nuclear pattern with peripheral chromatin, many secretory granules and some cytoplasmic vacuoles were noted. Stagnated secretion was also observed in their lumen. The granular convoluted tubules showed cytoplasmic vacuolization, electron dense secretory granules and degenerated mitochondria with loss of their cristae. The striated ducts showed signs of degeneration as loss of basal infolding, different nuclear pattern, cytoplasmic vacuolization, degenerated mitochondria with loss of cristae and some ruptured mitochondria although some normal mitochondria might have been noted. Twelve weeks interval, the serous acinar cells were pyramidal in shape with centrally located nucleus, mitochondria, and rough endoplasmic Summary 132 reticulum still thickened adjacent to the nucleus, secretory granules of variable electron density. Normal mitochondria were observed but some slightly swollen mitochondria and some cytoplasmic vacuolization were also observed. The intercalated ducts showed different nuclear pattern and surrounded by slightly widened Golgi apparatus and coalescent secretory granules. The granular convoluted tubules showed normal nuclear pattern, electron lucent basally located nucleus and electron dense secretory granules of variable sizes. The striated ducts showed infolding of the basal lamina and a lot of mitochondria that had normal internal cristae while some of them loose some of these cristae To summarize the findings of this study it was found that significant histological structural and ultrastructural alterations were observed in BoNTA-injected SSGs in 2 weeks, and these changes became more obvious and aggravated in 4 weeks. Nonetheless, these alterations were transient, and these atrophic changes were returned to almost normal histological structure in 12 weeks. These results indicated that BoNTA had a reversible biological effect on the histology and ultrastructure of SSGs. This may be due to proliferation of intercalated ducts (containing stem cells of salivary gland) and normal mitosis of the nuclei of serous acini to replace the atrophied and depleted part of the gland that started at four weeks interval. Moreover, sprouting of the nerve, transient effect of BoNTA on parasympathetic nerve supply in addition to nutrition supply from patent blood vessels may play a role in major recovery of the gland to normal function and normal glandular architecture.