Basic Informations

C.V

Master Title

The Efficacy and Comparison of Granulocyte Colony Stimulating Factor (G-CSF) between Induced Pulpal and Periodontal Inflammation in Neutropenic Rats

Master Abstract

Summary This study was performed to evaluate the effect of recombinant granulocyte colony-stimulating factor on the number of neutrophils and on the histopathological changes of inflamed pulpal and periodontal ligament in neutropenic rats. 64 healthy adult male albino rats of average body weight 200-250 gm were used in the present study. The animals were divided into 4 groups, 16 rats each. Group I (control group): The rats did not receive any injection and divided into : a- Negative control (subgroup Ia), b- Positive control (subgroupIb) Group II (Neupogen group): The rats were received an intraperitoneal injection of granulocyte colony stimulating factor (neupogen) before pulp exposure to left mandibular molars , before induced periodontitis to periodontal ligament of lower incisors and every day during experiment. Group III (Methotrexate group): The rats were received an intraperitoneal injection of 7.5mg/kg body weight of methotrexate. GroupIV (Methotrexate and Neupogen group):The rats were received an injection of methotrexate in the same manner as group III then they were received the G-CSF (neupogen) as in the same manner on group II. All animals were anaesthetized, and then the pulpal tissues of left mandibular molars were exposed to induce pulpal inflammation except in the negative control subgroup. Periodontal ligament was inflamed by placement silk ligature around cervix of mandibular incisors throughout the experiment except negative control sub group. Four animals from each group were sacrificed at 2, 4, 7 and 10 days from the beginning of the experiment respectively and before rat sacrification the peripheral blood was sampled. The light microscopic examination of group I (negative control subgroup Ia)revealed that the pulp appeared with moderately vascularized and well organized odontoblast cells forming odontoblastic layer under a thin layer of predentin. The blood vessels of the pulp were normally dispersed within fine oriented collagen fibers which seemed to be within normal size and its endothelial lining was intact. In positive control group (subgroup Ib) at 2 and 4 days, evidence of inflammation was seen by disrupted odontoblastic layer which was infiltrated with inflammatory cells, dilated blood vessels filled with RBCs and irregular collagen fibers orientation. At 7 days, edematous connective tissue led to formation of cytoplasmic vacuoles and necrotic areas were found. The inflammation in the residual pulpal and periapical tissues was detected as well as alveolar bone resorption was obvious. After 10 days, the necrosis spread to over half of the pulpal tissue. The inflammation in the residual pulpal and periapical tissues had become stronger, and signs of alveolar bone resorption were evident. During all experimental periods, the histological changes in group II were similar to those of group I (positive control subgroup Ib) with an increase in the spread of acute and chronic inflammatory cells and slightly decrease in the severity of the degeneration. In group III, the histological changes at 2 and 4 days demonstrated an increase in the severity of the inflammatory reaction in comparison to those of group I. However, After 7 days, most of the pulpal tissue had become necrotic. Inflammatory changes in the residual pulpal and periapical tissues and resorption of the alveolar bone were seen. After 10 day, the pulpal tissue was completely necrotic. Signs of severe periapical tissue inflammation and alveolar bone resorption were observed. In group IV, the histological changes were similar to those of groups I(positive control subgroup Ib) and groupII .Furthermore,The pulpal necrosis and periapical inflammation were less as compared with those of group III. The light microscopic examination of group I (negative control subgroupIa ) the periodontal ligament was divided into two distinct compartments one zone close to the alveolar bone (alveolar part), being rich in blood vessels, and an avascular zone located close to the tooth surface(tooth part).The periodontal ligament consists of cells and an extracellular compartment. Blood results revealed the normal count of total leukocytes and neutrophils of the rats. In positive control subgroup (Ib), the light microscopic examination after 2 days revealed that the periodontal ligament in the tooth part appeared with change of collagen fibers orientation while in the alveolar part there was dilatation of blood vessels which filled with RBCs. After 4 days in the tooth part, the irregularity of collagen fibers and edema in the connective tissue and areas of hyaline degeneration appeared. However, at the alveolar part blood vessels were dilatated with interrupted endothelial lining, so extravasated RBCs were present in the connective tissue and some of them were engorged with coagulated blood. Furthermore, there were spread of acute and chronic inflammatory cells in both parts where they clustered around blood vessels and between collagen fibers. After 7 days, there was degeneration of collagen fibers with few fibroblasts. At the alveolar part, the alveolar bone resorption with howship's lacunae which contained osteoclasts were detected. After 10 days, the tooth part of periodontal ligament showed formation of necrotic areas while, in the alveolar part the detachment of collagen fibers from alveolar bone was detected and signs of bone resorption were noticeably increased. During all experimental periods, the histological changes in group II were similar to those of group I (positive control subgroup Ib) with an increase in the spread of acute and chronic inflammatory cells in both parts of periodontal ligament and slightly decrease in the severity of the degeneration. In group III, the histological changes at 2 and 4 days were demonstrated an increase in the severity of the inflammatory reaction in comparison to those of groupI (positive control subgroup Ib). Furthermore, resorption of alveolar bone was detected at 4 days. However, At 7 days most of the peiodontal tissue had become necrotic and resorption of alveolar bone was markedly appeared. After 10 days the periodontal tissue was almost completely degenerated and collagen fibers were detached from alveolar bone. In group IV, the histological changes were similar to those of groups I (positive control subgroup Ib) and group II. The periodontal inflammation, degeneration and bone resorption were less as compared with those of group III. Blood results were demonstrated that in group I (subgroup I b), the numbers of total leukocytes and neutrophils did not show any changes during the entire experimental period but increased than normal (negative control subgroup Ia) due to inflammation. In G-CSF-treated group II, the numbers of total leukocytes and neutrophils increased and were statistically significant (P < 0.05) higher than those of groupI (positive control subgroup) throughout the experimental period. In methotrexate-treated group III, the numbers of total leukocytes and neutrophils decreased and were statistically significant (P < 0.05) less than those of group I from 2 to 7 days. However, there was no significant difference at 10 days, between those of groups I (positive control subgroup) and III. In group IV, the numbers of total leukocytes and neutrophils decreased and were statistically significant (P < 0.05) less than those of group I at 2 and 4 days. At 7 days, there was no significant difference between those of groups I (positive control subgroup)andIV.At 10 days, the number of total leukocytes and neutrophils increased and as statistically significant (P<0.05) higher than that of groupI (positive control subgroup Ib).

PHD Title

Study of the Effect of Ovariectomy on Cementum, Periodontal Ligament and Alveolar Bone of Albino Rats (Histological, Immunohistochemical and Scanning Electron Microscopic Study)

PHD Abstract

Summary 206 Summary Ovariectomy is the surgical removal of woman's ovaries and it is established as the most prevalent and pertinent protocol to induce estrogen deficiency, bringing about hormonal deficiency similar to that of menopause. Post-menopause estrogen deficiency leads to increased and imbalanced bone turnover. The resultant altered bone turnover activity results in the net bone loss and reduction of bone quality (Osteoporosis) (Pacifici, 2006; Giro et al., 2011). Osteoporosis is a progressive systemic disease that reduces, mineralized bone per unit volume, and degrades its microstructure. These factors increase significantly the bone fragility, leading to increased susceptibility to fractures. The disease is a public health problem because fractures lead to high costs for the healthcare service due to hospitalizations and associated drugs (Sandhu and Hampson 2011). Several studies have reported a positive correlation between systemic osteoporosis and jaw conditions such as low mineral density in the mandible ,a large number of missing teeth, (Takaishi et al., 2005), changes in trabecular microarchitecture, and severity of residual ridge resorption (Yang et al., 2005; Liu et al., 2015). Summary 207 There is debate as to the magnitude of osteoporosis effects on the bone quality of the jaws. Not all skeletal sites are equally susceptible to estrogen deficiency induced bone loss. For this purpose, the aim of the present study was to assess the effect of ovariectmy on cementum, periodontal ligament and alveolar bone of albino rats (histologically, immunohistochemically and scanning electron microscopic study). MATERIALS AND METHODS: Thirty adult female albino rats (12-week old) were used in this study weighing between 200-250 grams. The rats were equally divided into the two groups (control and experimental) and study period was (4 months): Group I (control group): It consisted of 15 rats that was subjected to sham operation. Group II (experimental group): It consisted of 15 rats that was subjected to ovariectomy operation. By the end of the experiment period (4 months), blood samples were collected from both groups and the levels of estrogen in the sera was measured using the Enzyme Linked Immunosorbent Assay (ELISA). Summary 208 And also, the rats of all groups were killed separately by cervical dislocation and their heads were immediately dissected to obtain the mandibles from both groups, the molar areas of the mandibles were dissected. Thus, 2 samples (right and left molar areas) from each rat were obtained. One side of the molar area of each rat was identified and prepared for light microscopic examination by routine histologic examination (hematoxyline and eosin stain) as well as the immunohistochemical assay using antiosteonectin antibody to assess the histology of cementum, PDL and alveolar bone. The other side was prepared for scanning electron microscopic examination (15 samples from each group). Results: Light microscopic results: (I) Hematoxylin and Eosin stain: 1-Control group: Cementum examination revealed that both types of cementum appeared with normal histological structure of cells and matrix. PDL examination revealed that the principle fibers were arranged in regular manner and the fibers are attached to cementum and alveolar bone by Sharpy's fibers. And also, alveolar process examination revealed that the alveolar process Summary 209 appeared with normal architecture and thickness with entrapped osteocytes. 2- Experimental group Cementum examination of this group revealed that acellular cementum appeared in irregular manner due to resorption and remarkable variations of cementocytes within their lacunae. And, PDL examination revealed that disarrangement of the fibers orientation in all the fiber groups and some fiber bundles were detached from cementum and alveolar bone surfaces, in addition complete loss of fibers was detected in some regions. Alveolar bone examination revealed that remarkable thinning of bone trabeculae and widening of bone marrow spaces. Multiple resorption bays giving the border of the alveolar process irregular. Moreover, some osteocytes were completely degenerated leaving empty lacunae, while others showed signs of degenerations. II- Immunohistochemical results: 1-Control group: Examination of sections stained with anti-osteonectin antibody showed weak immunoreactivity in cementoblast and moderate to weak reactivity in cellular cementum, cementocytes and extracellular matrix (ECM).And also, PDL showed positive immunoreactivity in fibroblast cells of collagen fibers and ECM.Alveolar bone examination showed that the ECM of bone Summary 210 and osteocytes are positively stained. The endosteum was intensely stained. 2- Experimental group: Examination of sections stained with anti-osteonectin antibody showed negative immunoreactivity for osteonectin in cementoblasts and very mild to negative reactivity for in cementocytes and ECM. PDL sections showed a decrease in the staining intensity for osteonectin compared with the control group.Moreover, alveolar process sections showed ECM and osteocytes are very weak immunoreactivity for osteonectin. III-SEM results: 1-Control group: Cemrntum examination demonstrated the root surface that showed regular cracks with no signs of resorption and showed external cuts from Sharpey's fibers inserted into the cementum. PDL and alveolar bone examination confirmed results of histological study. 2-experimental group: Cementum examination revealed that cracks on the surface of the cementum relatively wider than those on the control group and irregularities and multiple resorptive areas infiltrating the cementum surfaces.PDL and alveolar bone examination showed the same results obtained by the histological study.