Association between Shammah Use with Periodontal Disease and Shammah -induced Leukoplakia-like Lesion among Adult Males in Dawn Valley, Yemen

s | Abstracts of Theses Approved for the MSc and PhD at the School of Dental Sciences 157 www.mjms.usm.my Sex differences were assessed, and interrelationships between different variables were explored within the study group. For the data obtained by SM techniques, the men had statistically significant larger arch dimensions and geomorphometrics norms of tooth size than the women (P<0.05). For the Bolton‘ tooth size ratio (intermaxillary tooth size discrepancy), the sum of anterior tooth size and overall tooth size via SM methods showed statistically significant result in relation sexual disparities (P<0.05). No significant sexual disparities for Bolton‘s anterior ratios (BAR) and Bolton‘s overall ratios (BOR) were observed. This study has established a new reference database of tooth size and arch dimensions via SM for first time on Pakistani population. These norms for tooth size and tooth size ratio will be helpful for clinical treatment planning in dentistry and forensic dentistry. Supervisor: Dr Mohammad Khursheed Alam Co-Supervisor: Dr Mohd Fadhli Khamis EFFECT OF VEGF AND BCP ON WOUND HEALING IN CRITICAL SIZED MANDIBULAR DEFECTIN VITRO AND IN VIVO STUDIES Hamid Hammad Enezei PhD (Dentistry) School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia One of the most challenging problems in Oral and Maxillofacial Surgery is the repair of critical-sized bone defects in the mandible and craniofacial region that needs a solution. The aim of this study was to produce a clinically applicable model of bone tissue engineering in composite bone graft that has osteoconductive and osteoinductive properties capable of treating critical-sized bone defects in a single step surgical procedure in the rabbit’s mandible, beginning with in vitro molecular phase and followed by in vivo clinical phase. Part I and II in vitro phase was conducted to evaluate the effectiveness and the potential influence of VEGF-added-BCP on both angiogenic and osteogenic gene expressions of endothelial cells (ECS) and dental stem cells (DSCs) treated with BCP extract with and without addition of VEGF respectively. The results provided critical insights into the use of BCP granules with and without addition of VEGF which is important for in vitro test before evaluating the efficacy in vivo. This was followed by performing reverse transcriptase-PCR (RT-PCR) to amplify the osteogenesis and angiogenesis-regulated genes. In part III, the in vivo study, the optimal effective concentration of VEGF in bone regeneration was established using Fibrin sealant delivery system. The in vivo clinical phase included a total of 42 male New Zealand White rabbits, aged between 5 and 6 months; giving a total of 84 mandible sides for experiment. The mandible sides were divided into 4 groups subjected to the treatment given and analysis done. 24 rabbits (48 mandible sides) were subjected to micro-CT and histological studies, while 18 rabbits (36 mandible sides) were subjected to molecular studies. Nine untreated mandible sides (designated as A) versus BCP/FS treated mandible sides (designated as B), control treatments comprising n= 9 rabbits; and the experiment group comprising BCP/FS treated mandible sides (designated as C) versus BCP/ FS + VEGF treated mandible sides (designated as D). These rabbits were sacrificed at day 14, 30, 45, and 60 after surgery for micro-computed tomography and histological analyses. For molecular analysis, same designated treatment groups were used and the rabbits were sacrificed at day 7, 14, and 21 after surgery. Results: In part I and part II in vitro studies showed angiogenesis gene VEGF was highly expressed in ECS and DSCs by VEGF only treatment but showed some changes in combination of VEGF/BCP treatment in both cell lines. Osteogenesis genes; Bone morphogenetic proteins-2 (BMP2), Alkaline phosphatase (ALP), Osteocalcin (OC) and Osteopontin (OPN) were shown to be positively affected by both BCP and VEGF. The BCP only treatment group induced high expression of early regulating osteogenesis genes (BMP2 and OPN). Mineralized gene markers (ALP and OC) were however, highly expressed with VEGF/BCP treatment. Besides that, osteogenesis genes (BMP-2 and OPN) for DSCs were shown to be positively affected by both BCP only and VEGF only groups and slightly effected in VEGF/BCP group. Some genes were expressed at an earlier time interval compared to the other genes depending on the type of treatment. BCP only treatment induced high expression of initial-regulated osteogenesis genes (BMP-2 and OPN). Results in part III study shows micro-computed tomography images at week 8 after surgery demonstrating complete resorption of BCP granules and young bone formation in group D implanted with VEGF/BCP compared with other groups of study (p<0.05). Histological and histomorphometrical findings at week 8 after surgery (group D) showed marked new bone formation. Group D showed up-regulation of VEGF gene expression at week 1. Osteogenesis markers in group D showed higher expression than other groups at early stage of bone healing. The results suggest that the use of BCP/FS composite bone graft loaded with VEGF is ideal for local controlled release of VEGF resulting in accelerated bone formation and healing process of mandibular critical-sized bone defects and this may contribute to the scope of modern reconstructive surgery. Supervisor: Associate Professor Dr Noor Hayati Abdul Razak Co-Supervisor: Associate Professor Mutum Samarendra Singh Dr Azlina Ahmad Dr Mohd Fadhli Khamis Dr Roselinda Ab. Rahman CHEMICAL ANALYSIS, BIOCOMPATIBILITY AND DENTINOGENIC DIFFERENTIATION POTENTIAL OF TWO FORMULATIONS OF WHITE PORTLAND CEMENT OF DIFFERENT ORIGIN Hany Mohamed Aly Ahmed PhD (Dentistry) Malays J Med Sci. Sep-Oct 2016; 23(5): 156-163 158 www.mjms.usm.my School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Advances in endodontic bio-materials are at the forefront of endodontic research. The aim of the current study was to determine and compare the chemical composition, biocompatibility and dentinogenic differentiation potential of two formulations of white Portland cement (WPC) of different origin and white mineral trioxide aggregate (WMTA). Normal and fast (addition of calcium chloride dihydrate) set formulations of WMTA, Malaysian and Egyptian WPCs (MAWPC and EGWPC) were prepared for chemical analysis. Scanning electron microscope (SEM), energy dispersive X-ray (EDX) micro-analysis and X-ray diffraction (XRD) were used for surface morphology evaluation, elemental and phase analysis, respectively. After the preparation of optimized serial dilutions, the cytotoxicity was evaluated on human periodontal ligament fibroblasts (HPLFs) and dental pulp stem cells (DPSCs) using methyl-thiazol diphenyltetrazolium (MTT) assay after 24 and 72 hours. Statistical analysis was performed using Kruskal-Wallis test (P=0.05) followed by pairwise comparisons using Mann-Whitney test. Cell attachment properties were examined on HPLFs and DPSCs under SEM using a novel technique after 24 and 72 hours. The dentinogenic differentiation of DPSCs was assessed based on the expression of BGLAP, DSPP, RUNX-2 and SPP1. After one, three, seven and 14 days of incubation, the expression was examined using real-time PCR. One way ANOVA followed by post-hoc comparisons was used for statistical analysis (P=0.05). The results showed that the surface morphology and chemical composition of both formulations demonstrated considerable variations. The elemental composition of WMTA differed from both WPCs by the presence of bismuth and absence of sulphur. Potassium was merely observed in MAWPC. Phase analysis demonstrated the presence of various chemical compounds. The cytotoxicity evaluation showed different cellular responses of HPLFs compared to DSPCs. Generally, both formulations favoured the viability of HPLFs. However, the fast set formulations demonstrated severe to moderate cytotoxicity on DPSCs at three successive concentrations. Significant differences between EGWPC and MAWPC were identified (P<0.05). The cell attachment properties of all materials were favourable, however, HPLFs attached and spread over the samples better than DPSCs. The dentinogenic differentiation potential showed fluctuating expressions at days 1, 3 and 7. However at day 14, all genes were up-regulated. Generally, fast set formulations showed higher expressions than normal set counterparts (P<0.05). In conclusion, WPC of different origin shows differences in chemical composition and biological properties. However, the biological profile of both WPC is comparable to that of WMTA. This holds promise for potential use of MAWPC and EGWPC as cost effective substitutes in clinical dentistry. Supervisor: Associate Professor Dr Norhayati Luddin Co-Supervisor: Associate Professor Dr T. P. Kannan Associate Professor Dr Khairani Idah Mokhtar @ Makhtar Dr Azlina Ahmad HUMAN HERTWIG’S EPITHELIAL ROOT SHEATH CELLS IN ASSOCIATION WITH CHITOSAN SCAFFOLD AND TGFβ1 MODULATE THE CEMENTOBLASTIC DIFFERENTIATION OF STEM CELLS FROM HUMAN EXTRACTED DECIDUOUS TEETH Manal Abdulrahman Farea Ali PhD (Dentistry) School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Destruction of the periodontium as a result of furcal perforation may lead to loss of the tooth. egeneration of cementum is essential to allow the reconstruction of the lost attachment. Multipotent dental pulp stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue engineering. Hertwig’s epithelial root sheath (HERS) cells have the ability to form cementum-like tissue and differentiate into cementoblastlike cells. On the other hand, transforming growth factor-β1 (TGFβ1) plays a key role in controlling cell proliferation and differentiation. Chitosan has been chosen as a scaffold in the current study because it supports cells attachment, proliferation, osteoblastic differentiation and hard tissue regeneration. To the best of our knowledge, the ability of SHED to differentiate into cementoblast-like cells has not been investigated in the dental field yet. The aim of this study was to evaluate the the ability of SHED co-cultured with HERS cells in presence of chitosan-TGFβ1 to differentiate into cementoblastlike cells. To this end, HERS cells were isolated using selective digestion method and characterized by immunofluorescence staining, flow cytometry and sqRT-PCR. Thereafter, SHED seeded on chitosan scaffold and co-cultured with HERS cells in the presence of TGFβ1. Eight groups were assigned for the downstream analysis: SHED (S), SHED+chitosan (SC), SHED+TGFβ1 (ST), SHED+chitosan+TGFβ1 (SCT), SHED+HERS (SH), SHED+HERS+chitosan (SHC), SHED+HERS+TGFβ1 (SHT), SHED+HERS+chitosan+TGFβ1 (SHCT). SHED proliferation was assessed by PrestoBlue assay. Live/dead assay was then performed and SHED attachment to chitosan scaffold was examined by scanning electron microscope (SEM). For cemento/osteogenic differentiation analysis, morphological appearance, alkaline phosphatase (ALP) activity, mineralization behaviour and gene/protein expression of cemento/osteoblast phenotype of SHED were evaluated. In addition, the inflammatory response of SHED was analysed. Results of the present study showed that HERS cells had typical epithelial-like cells morphology and expressed epithelial-like markers. SHED remained viable and attached well to the chitosan structure. HERS cells in association with chitosan-TGFβ1 significantly enhanced the proliferation and cemento/osteogenic differentiation of SHED, which was demonstrated by high ALP activity, strong mineral deposition and up-regulation of cementum/bone-related gene and protein expressions (i.e. ALP, collagen type I, bone sialoprotein, osteocalcin and cementum attachment protein). Low levels of inflammatory genes expression were detected. In conclusion, HERS cells have been successfully isolated using selective digestion method. Our co-culture system Abstracts | Abstracts of Theses Approved for the MSc and PhD at the School of Dental Sciencess | Abstracts of Theses Approved for the MSc and PhD at the School of Dental Sciences 159 www.mjms.usm.my confirmed the synergistic effect of HERS cells in a combination with chitosanTGFβ1 to induce SHED differentiation along the cemento/osteoblastic lineage; which possesses a novel therapeutic strategy for endodontic furcation perforation repair and periodontal tissue engineering. Supervisor: Professor Dr Adam Husein Co-Supervisor: Dr Zurairah Berahim Professor Dr Ahmad Sukari Halim Dr Nurul Asma Abdullah Dr Kasmawati @Norhidayati Mokhtar Associate Professor Dr Khairani Idah Mokhtar @ Makhtar EFFECTS OF NON-INVASIVE LOW LEVEL LASER THERAPY, LOW INTENSITY PULSED ULTRASOUND TREATMENT AND THEIR COMBINATION ON ORTHODONTIC TOOTH MOVEMENT Mohammed Mahmood Jawad Al-Azzawi PhD (Dentistry) School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Orthodontic treatment is a process of achieving appropriate functions and esthetics by the movement of a tooth through the application of an external physical force in order to obtain physiological tissue reactions around a tooth, while avoiding the side effects of an external force. However, longterm therapy for several years is required. Accordingly, faster tooth movement without harmful effect on periodontal tissue and alveolar bone has been an important issue of interest to orthodontists and patients. Recently with the development of science, there is high attention among orthodontists to investigate the non-invasive stimulating techniques to improve orthodontic treatment. Many researchers have shown that proper use of low level laser treatment (LLLT) in orthodontic clinic can accelerate treatment, reduce appointments, and provide superior results, but there is no established regimen that all LLLT users within orthodontics are agreeing to follow. The non-invasive low intensity pulsed ultrasound (LIPUS) has also been used to stimulate bone fracture healing by improving osteogenesis, remodeling and angiogenesis. However, there are limited numbers of studies on LIPUS stimulation for orthodontic tooth movement. To the best of our knowledge, there is no other study comparing the effect of LLLT with LIPUS for orthodontic tooth movement, or comparing the stimulatory effect of the combination of LLLT with LIPUS for orthodontics. This research was aimed to study in vitro and in vivo effect of different low level laser regimes, low intensity pulsed ultrasound and the combination of both techniques on orthodontic tooth movement. For in vitro, Human fetal osteoblast cell line (hFOB) was cultured and divided into different groups; 1st group was treated with 940 nm LLLT (with power ranging between 100-300mW), 2nd group was treated with LIPUS, 3rd group was treated with combination of LLLT and LIPUS. The application of LLLT or LIPUS or combination of them was once a day for 7 days. For cell proliferation, MTT assay was used. Both alkaline phosphatase and osteocalcin activity assays were assessed for cell differentiation. RT-PCR was also performed to elucidate the osteoblasts gene expression for COL1A1, RUNX-2 and BSP. For in vivo, 6-week-old Sprague Dawley male rats were used. Orthodontic appliances were inserted. A force of 10g was applied to the molars to induce tooth movement. The rats were grouped into four groups. The 1st group was irradiated with LLLT, 2nd group was treated with LIPUS and a 3rd group was treated with combination of both LLLT and LIPUS. A 4th group was a control group. The LLLT and LIPUS were used to treat the area around the moving tooth once a day till 1, 3, 7, 14 and 21 days. To determine the amount of tooth movement, plaster models of the maxillae were made. The models were imaged and analysed. Histological examination was performed after staining with (haematoxylin and eosin) and (Alizarin red and Alcian blue) stain. RT-PCR was also performed to elucidate the gene expression of RANK, RANKL, OPG and RUNX-2 in the area of treatment. The results of in vitro showed that all treatment groups significantly increased rate in cell proliferation and differentiation compared to the control group. The LIPUS group and the 300 mW LLLT group significantly increased the amount of cell proliferation. By contrast, the combination groups showed significantly greater amount of cell differentiation and gene expression. The results of in vivo showed that the amount of tooth movement, the histological bone remodelling and the RT-PCR was significantly greater in the treatment groups than that in the control group. Among the treatment groups, the combination group was the highest and the LIPUS group was the lowest. The findings of this study suggest that LLLT and LIPUS can stimulate osteoblast cells for bone formation. Additionally, they facilitate the velocity of tooth movement and improve the quality of bone remodelling during orthodontic tooth movement especially when they are combined together. Supervisor: Professor Dr Adam Husein Co-Supervisor: Professor Dr Rozita Hassan Dr Rumaizi Shaari Dr Azlina Ahmad Dr Mohammad Khursheed Alam MORPHOMETRIC ANALYSIS AND FABRICATION OF PROSTHETIC EAR USING CAD/CAM AND ADDITIVE MANUFACTURING TECHNOLOGIES Nor Azura Mohamed MSc (Dentistry) School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Prosthetic ear rehabilitation is one of the treatments for auricular defect. Current practice by surgeon require patient to go for osseointegrated craniofacial implant surgery Malays J Med Sci. Sep-Oct 2016; 23(5): 156-163 160 www.mjms.usm.my for retention of the prosthetic ear. Traditionally, the wax pattern was created from the impression taken from patient and the final prosthesis is processed with silicone material. This conventional method has always been time consuming, massive work and caused discomfort to patient. Moreover the accuracy of the final prosthetic sometimes was not satisfied. Impression technique play a vital role in determining accurate reproduction of affected and unaffected ears, orientation of the ear during wax try in and fabrication of ear prostheses. Hence, the ear anthropometric data is important to determine the correct orientation and position of the prosthetic ear. This paper aims to provide morphometric data of a few standard ear parameters for Kelantanese and also describes a novel method of design and fabricating the prosthetic ear applying CAD/CAM and additive manufacturing technologies. A clinical study is done onto a patient in HUSM and comparison is made between traditional method and the new approach using computer aided technology. Study also validates the prosthetic ear obtained from both techniques with the morphometric data. The measurement technique of the final prosthetic ear to validate the result with regards to the morphometric data was done digitally using software. Morphometric study was conducted on 68 samples of normal ear for both left and right with 15 parameters measured. Data was retrieved from CT scan and convert to 3D image using soft tissue development. Mirror image technique was applied to reconstruct the missing ear, and then fabricate the 3D model of the prosthetic ear using Stereolitography (SLA) technology. The 3D model will become the master mold to produce the final prosthetic ear using vacuum casting technology. Morphometric analysis gave the mean and standard deviation values for auricular length and width, length and height of tragus, insertion length of auricle, length and width of lobular and conchal, protusion at superaurale and tragal level as well as the inclination and symmetrical angle. While study also illustrates that there is significant different between traditional and computer aided approach. The new method shows time reduction during design and fabrication stage and also show improvement in accuracy and aesthetic appearance. Supervisor: Associate Professor Dr Zainul Ahmad Rajion Field Supervisor: Associate Professor Dr Md Saidin Wahab SYNTHESIS AND EVALUATION OF SILICA PARTICLES FROM RICE HUSK FOR FABRICATION OF DENTAL NANOHYBRID COMPOSITES Mohammed Noushad PhD (Dentistry) School of Dental Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia The objective of this study was to synthesize silica particles from rice husk with ideal characteristics, like spherical shape, low surface area and wide particle size distribution, for use as fillers in dental composites. The silica was leached in to a solution of NaOH to form sodium silicate. The silica was synthesized from sodium silicate solution using a simple acid precipitation technique. Several parameters were tested to obtain silica particles with ideal characteristics for use as fillers in dental composites. These included the effect of various precipitating acids and solvents, effect of dilution of sodium silicate solution, mixing speed, feed rate and various concentrations of solvents. It was possible to obtain spherical silica particles with a BET surface area of 30 m2/g and a mean diameter of 261nm. The silica particles were modified using a coupling agent, 3–(Trimethoxysilyl) propyl methacrylate. The silica surface was successfully modified using a very simple technique and was characterized using FTIR and NMR studies. Dental composites with two different filler/matrix ratios (EC1: 40/60 and EC2: 50/50) were fabricated from the surface modified nanohybrid silica, followed by mechanical properties testing, which included flexural strength, flexural modulus, compressive strength, Vickers’ hardness and surface roughness tests. Although both experimental dental composites showed promising results, EC2 exhibited better test values. EC2 showed a flexural strength of 106.6 MPa, flexural modulus of 6.2 GPA, compressive strength of 190.6 MPa, Vickers’ hardness of 38.66 HV1 and surface roughness of 0.057 Ra which are comparable with a commercial dental composite. Biocompatibility of the silica particles and EC2 were tested using cytotoxicity studies on human periodontal ligament fibroblast cells using the MTT assay. The silica particles were non-cytotoxic at all the concentrations tested. The results for experimental dental composite showed that it was moderately cytotoxic only at the highest tested concentrations, which is in agreement with several other studies. The results indicate that rice husk, which is an agricultural waste, is a good and inexpensive source of silica for potential use as fillers in dental