Isolation and Characterization of Mouse Specificity Protein 6 Promoter

Specificity protein 6 (SP6) is a member of the SP/Krüppel-like transcription factor family and plays key roles in tooth development. To study its biological roles, it is important to understand the spatiotemporal regulation of Sp6 gene expression. For this purpose, we first identified two separate 5' ends of the Sp6 cDNA by 5' RACE analysis using mouse mandibular RNA. Next, we isolated mouse genomic DNA fragments covering the Sp6 gene including two putative mouse Sp6 promoter regions and generated a series of luciferase reporter constructs. We confirmed the activity of both promoters by a luciferase assay and found strong second promoter activity in dental epithelial cells. Unexpectedly, we also detected potential third promoter activity in the intron 2 of the Sp6 gene. Last, we also found that bone morphogenetic protein and wingless signals could enhance Sp6 promoter activity in dental epithelial cells, suggesting the regulatory roles of two cytokines in Sp6 gene expression during tooth development. Our findings may shed new light on the regulatory mechanisms of Sp6 gene expression and provide a possible linkage between cytokine regulation of Sp6 expression and inductive epithelial and mesenchymal interactions.

Remodeling Capacity of Femoral Bone Defect by POP-CHA Bone Substitute: A Study in Rats’ Osteoclast (First Series of POP-based Bone Graft Improvement)

Reconstruction of large bone defects caused by trauma, excision of tumors, and congenital malformations can be very difficult to perform. Bone engineering offers an option to improve bone reconstruction procedures. This interdisciplinary field applies the principles of biology and engineering to the development of functional substitutes for damaged bone. Our research aimed to find the ideal scaffold for bone regeneration, focusing on Calcium and Phosphate combination. In this study, Plaster of Paris (POP) was combined with CHA and implanted in femoral condyles of rats. According to the experimental result, it can be concluded that there was no significant difference in response to the implantation of POP and POP-CHA in Sprague Dawley rat femur condyle (p<0.05). It can be stated that both POP-CHA and POP shows similar trait in bone healing.

The Newly Bone Formation with Carbonate Apatite-Chitosan Bone Substitute in the Rat Tibia

Large bone defect still represent a major problem in orthopedics. A tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds and growth factors can play in a role to the bone repair. Bone consist a mineral phase such as carbonate apatite and an organic phase such as collagen. Synthetic carbonate apatite ceramics are considered as promising alloplastic materials for bone substitute. Chitin is the organic matrix of the hard parts of exoskeleton of insect, crustacean and present in a small amounts in mushrooms. It is an insoluble, similar to cellulose and composed of N-acetylglucosamine unit. Partial deacetylation from chitin result in the formation of chitosan. Chitin’s properties as a flexible and strong material make it favourable as surgical thread. It has novel properties such as biocompatibility, biodegradability, anti bacterial, wound healing activity, tissue regeneration and hemostatic activitities. The composit from carbonate apatite and chitosan may have a great impact on human health care system as bioresorbable bone substitute. The aim of the study was to evaluate the newly bone formation on the bone healing of defect tibia treated with carbonate apatite-chitosan bone substitute. Eighteen Sprague Dawley rats, male, 3 months, weighing 250-300g used in this study. Bilateral defect were created in each tibia rat. The defects were filled with carbonate-apatite chitosan bone substitute. The rats were sacrificed after respectively 1, 2 and 3 weeks. The result of this study showed that carbonate apatite-chitosan significantly increased a number of osteoblast (p<0.05). Carbonate apatite-chitosan group showed that matrix deposition faster than the other groups and have a good interface with the old bone. These data indicate that carbonate apatite-chitosan are potential candidate for bone substitute

Effect of Allicin for Re-epithelialization During Healing in Oral Ulcer Model

Allicin (diallyl thiosulfinate) is the main component of fresh garlic with multi-bioactivities as therapeutic agent. Recently, allicin is being proposed as alternative therapy for aphthous ulcer. The purpose of this study was to determine the effect of topical allicin in aqueous garlic extract on re-epithelialization during healing of acetic acid induced oral ulcer model. Thirty males SD rats aged 2 months were equally divided into two groups as experimental and control groups. After sedated with 0.2 ml intramuscular Ketamine injection, ulcer model was made by applying 99.5% glacial acetic acid-moistened paper disc on rat buccal mucosa. This would gene- rate an immediate necrotic tissue, which produced a single crateriform ulcer in each of the experimental rats for the next two days. In the experimental group, one drop of allicin was applied twice daily onto the lesion. Three rats in each group were sacrificed on days 2, 3, 6, 9 and 12 after induction and were recorded as H0, H1, H4,H7 dan H10 groups, respectively. The tissue samples from buccal mucosa were processed for HematoxylinEosin (HE) staining, and then evaluated histologically. Re-epithelialization was assessed by measuring the thickness of the epithelium using an ocular micrometer under a light microscope. Data were analyzed statistically by Independent t-test with significant p-values of <0.05 (95%). Based on histological findings and trend graphs, it can be concluded that the topical application of allicin can accelerate re-epithelialization of ulcer healing process. This study suggests that allicin in aqueous garlic extract could be developed as therapeuticagent for aphthous ulcer

Analysis of Patients Visiting Niigata University Medical and Dental Hospital with Chief Complaints of Metal Allergy and/or Focal Infection in the Previous 8 Years

Dental metal allergy and dental focal infection are possible causes of dermatological diseases, but have been the subjects of few reports to date. We have been treating such patients in our special clinic for more than 20 years.The purpose of the present study was to investigate the mouths of patients visiting our dental hospital over an 8-year period, with the aim of clarifying whether dental metal allergy and/or dental focal infection affects their dermatologic conditions.We surveyed all clinical records of the 185 patients who visited Niigata University Medical and Dental Hospital with chief complaints of dental metal allergy since 2002. Diagnostics of skin diseases, periodontal records, periapical lesions, dental caries, dental metal series patch test results and Electron Probed Micro-Analysis (EPMA) data were investigated. Ninety-two (49%) patients were suffering from pustulosis palmaris et plantaris and 20 (11%) patients had lichen planus. Eighty-two (49%) patients showed positive reactions on patch testing. Based on the result of patch tests, Ni showed the highest positivity rate (62%, 51 patients), but on EPMA, the number of patients with Ni as an allergen was 14 (27%). On the other hand, more than 98% of patients who showed positive reactions on patch test to Pd and Au had these metals in their dental prostheses. In addition, 112 (60%) patients showed the possibility of dental focal infections

The Effect of Calcium Hydroxide on Fibroblast Cells Viability

Calcium hydroxide [Ca(OH)2] is widely used as medicament in dental pulp and root canal therapy. Previous studies demonstrate the ability of calcium hydroxide to induce necrosis in dental pulp tissue. However, the mechanism of tissue destruction remains unknown. The aim of this study was to investigate fibroblast cell viability in response to calcium hydroxide exposure. In this study, Vero fibroblast cell line was treated with various concentrations of calcium hydroxide for 24 hours. Cell viability was measured by using MTT assay. Our results showed significant decrease in cell viability after exposed with calcium hydroxide at concentration 62.5 and 125 µg/ml. The result indicated that calcium hydroxide induced cell death in Vero cell line in a dosedependent manner. This study suggests that fibroblast cell death may involved in the mechanism of pulp tissue necrosis caused by calcium hydroxide

Oral Health Status Among Schoolchildren: Does Partnership With School of Dentistry Make A Difference?

The purpose of this study was to evaluate whether the partnership between 26 primary schools with the Faculty of Dentistry, Universitas Gadjah Mada, during the past 12 years improved oral health status of the schoolchildren. A sample survey was carried out, involving 106 fifth and sixth graders from 9 schools which have been participating in the partnership and 90 schoolchildren from non-participating schools. They were examined by trained dental students. Oral health status was represented by OHI-S and DMF-t measurement. Knowledge and attitude of oral health were measured using structured questionnaires. The study indicated that the means of OHI-S and DMF-t among schoolchildren participating in the partnership were 0.11 and 0.01 lower respectively compared to those of their counterparts, although the differences were not significant statistically. The knowledge and attitude among schoolchildren in the partnership were 2.49, 4.18, and 3.86 higher, all were highly significant (p < 0.001). Path analyses showed that the partnership reduced OHI-S and DMF-t with an overall path coefficients – 0.086 and -0.076 respectively. Although knowledge was associated with DMF-t and attitude with OHI-S, there were other unmeasured variables which were more strongly associated with oral health status of the schoolchildren.

Influence of Exposing Root Canal Dentin to Calcium Hydroxide on Its Flexural Strength

Calcium hydroxide has been used extensively in endodontic treatments, for instance as an intra-canal dressing; however, the exposure of root canal dentin to calcium hydroxide may affect its flexural strength and could have important clinical implications for endodontic treatment. The purpose of this in vitro study was to investigate the influence of calcium hydroxide on the flexural strength of root canal dentin.Seventy-two extracted single-rooted human mandibular premolars were used in this study. Each tooth was instrumented using crown-down technique and was irrigated using sterile saline. The teeth were assigned into three groups of 24 each. The prepared root canal system of each tooth was filled with calcium hydroxide mixed with sterile saline (group 1), a calcium hydroxide commercially available product (UltraCal®) (group 2) or saline solution (group 3, as control). The apices and access opening were sealed using composite resin, and the teeth were immersed in artificial saliva. After 7, 14 and 30 days of immersion, the inner root canal dentin of 8 teeth respectively from each group were sectioned to create dentin bars (1 X 1 mm, with 7 mm in length). Each dentin bar then was subjected to a three-point bending flexural test using MTS (Universal Testing Machine). Data gathered were then analyzed using two-way ANOVA, followed by Tukey’s test with the level of significance of 95%. The results showed that exposure to calcium hydroxide either using calcium hydroxide mixed with sterile saline or UltraCal® for 14 and 30 days can reduce flexural strength of root canal dentin compared to control group (p<0.05). In contrast, after 7 days exposure, there was no significantly different of flexural strength between three groups (p>0.05). It can be concluded that calcium hydroxide reduced the flexural strength of root canal dentin. The longer the exposure to calcium hydroxide would produce a greater effect on flexural strength of root canal dentin.

Dynamic Changes of Sp6 Transgene Expression in Dental Epithelial Cells during Long-term Culture

To investigate the function of specificity protein 6 (SP6) transcription factor by gain-of-function procedure, we established cytomegalovirus (CMV) promoter-driven Sp6 stable transformants, C9 cells, using dental epithelialderived cells. Initially, C9 cells produced a significant amount of SP6 protein. However, SP6 expression was reduced in these cells upon long-term culture. We could detect Sp6 transcripts in C9 cells by RT-PCR throughout the passages, although the CMV promoter is known to be epigenetically silenced. We recently found that SP6 was a short-lived protein that was degraded by a ubiquitin-independent proteasome pathway, although it is yet unclear how Sp6 expression was regulated during culture. Thus, we studied the possibility of epigenetic regulation of Sp6 expression. Comparative analysis of endogenous and exogenous Sp6 mRNA expressions demonstrated the specific down-regulation of exogenous Sp6 mRNA levels during culture passages. A DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5AC), and a histone deacetylase inhibitor, valproic acid (VPA), enhanced or induced SP6 protein expression up to passage 28 without enhancing the mRNA level. The dramatic up-regulation of exogenous Sp6 mRNA was uniquely observed only at passage 50 by 5AC or VPA treatment. These findings indicate that multiple epigenetic regulatory mechanisms operate to fine-tune Sp6 expression during long-term culture.

CDHA Ceramic Microspheres for Periodontitis Treatment: Synthesis, Characterization and Doxycycline Release Profiles

The present study is focused on the development of doxycycline loaded calcium–deficient hydroxyapatite (CDHA) microspheres for the treatment of periodontitis. The CDHA microspheres were formed by liquid immiscibility effect using gelatin and paraffin oil with varying Ca/P ratios using calcium hydroxide and diammonium hydrogen orthophosphate as precursors. The morphology of the microspheres as characterized by SEM was optimized by varying the gelatin content. The doxycycline incorporation and its release profiles were studied by UV-Visible spectroscopy in phosphate buffer at physiological conditions. The pH of the buffer solution was initially optimized to have maximum amount of drug loading. Doxycycline loading around the physiological pH of 7 has the highest amount of drug incorporation. All the microspheres exhibit similar release profiles with an initial gradual increase reaching a maximum value and then nearly constant release. The microspheres formed using 6% gelatin shows maximum amount of drug release of 80%.