Evaluation of gypsum-chitosan as a biomaterial in dental pulp protection

In dentistry, preservation of vital pulp tissue through pulp therapy is a challenging clinical approach. In the procedure of direct pulp capping, the exposure site is sealed by dental material to maintain the vitality of the pulp and induce reparative dentin formation. Several materials such as calcium hydroxide and mineral trioxide aggregate have been used and investigated for dental pulp capping with limitations. Thus, chitosan (CHT) and calcium sulfate (pure gypsum - Gyp) incorporated with bone morphogenetic protein 2 (BMP-2) were chosen in this study to exploit their advantages and develop a biomaterial with high biological and mechanical characteristics. Gyp is highly biocompatible, moldable and used as carrier of antibiotic. CHT is biocompatible, has antibacterial activity and enhances tissue growth. BMP-2 induces differentiation of pulp cells into odontoblasts and reparative dentin formation. The aims of this study were to prepare an experimental gypsum-based biomaterial and to study the effects of the biomaterial with several concentrations of CHT (10%, 5%, 2.5%, 1%, and 0% chitosan solution) on setting time (min), pH value, compressive strength (MPa), solubility (%), antibacterial activity against cariogenic bacteria, and the proliferative activity of SHED. In addition, BMP-2 was incorporated with Gyp-CHT and its effect was evaluated on alkaline phosphatase activity (ALP) of SHED. The adhesion and proliferation of SHED on the experimental biomaterial was also observed by scanning electron microscope (SEM). Commercial dental liner Dycal and Fuji IX were prepared according to manufacturer’s instructions and selective properties were evaluated for comparison purpose. For the experimental biomaterials the results of setting time were ranged between 4.1 and 6.6 min which is considered acceptable for clinical application. The pH values after 24 hours were ranged 5.7 and 6.4 which is suitable for application as pulp capping. The compressive strength increased with higher CHT concentration in the biomaterial which ranged between 2.63 and 5.83 MPa. The solubility rate decreased with CHT incorporation compared to that without CHT except for Gyp-CHT 10% which showed the highest solubility. The experimental biomaterial showed potent antibacterial activity against S. mutans and S. sobrinus, which was more evident with greater CHT concentration and comparable with other lining materials. The biomaterial showed good cell compatibility to SHED in both direct and indirect MTS tests. The biomaterial tended to increase the release of ALP outside the cells. The experimental biomaterial induced the ALP activity of SHED with higher activity in biomaterial incorporated with BMP-2. SEM observations showed that the seeded SHED were apparently healthy, well adhered and were spread on the surface through proliferative activity. The in vitro results of this study suggest that the physical and mechanical properties of gypsum based chitosan biomaterials were acceptable in relation of pulp capping biomaterials. The ALP activity of SHED which is an indicator of mineralization was significantly higher in Gyp-CHT-BMP-2 biomaterials compared to Dycal. Antibacterial properties of experimental biomaterials were found comparable with the commercial materials. Thus, the crystalline gypsum impregnated with CHT and BMP-2 organic matrices may have the potential for application in vital pulp therapy

Physico-chemical properties and effects of chitosan-based accelerated portland cement on stem cells from human exfoliated deciduous teeth

Advancement in the field of endodontic such as techniques, instrumentations and materials have considerably improved the oral health care and have made the dental treatment more efficient, as well as cost and time effective. Accelerated Portland cement (APC) is a potential material with favourable chemical, physical and biological properties. It was studied as an alternative material to overcome the major limitations of mineral trioxide aggregate (MTA) and portland cement (PC) such as delayed setting time and high cost of MTA. Chitosan (CT) has also been used in numerous medical applications due to its various biological properties. In this study, APC was prepared in combination with CT and designated as APC-CT. This study aimed to evaluate the chemical, physical and mechanical properties of APC-CT and to evaluate its biocompatibility, mineralization activity and dentinogenic/osteogenic differentiation potential on stem cells from human exfoliated deciduous teeth (SHED). APC-CT was prepared with various CT concentrations of 0.625%-, 1.25%- and 2.5%-CT solutions, and APC was used as control. The chemical characterizations by FTIR and FESEM/EDX were evaluated, in addition to the physical and mechanical properties such as setting time, compressive strength, surface microhardness, pH and solubility. Then, the effect of APC-CT on cell viability, attachment and apoptosis were assessed. The mineralization activity of SHED was evaluated by Alizarin Red staining and Von Kossa stain. Finally, the dentinogenic/osteogenic differentiation of SHED was analysed by evaluating the gene expression of selected dentinogenic/osteogenic markers i.e. DSPP, MEPE, DMP-1, OPN, OCN, OPG, RANKL, RUNX2, ALP and COL1A1 by real-time PCR. The results confirmed the interaction of CT with APC by FTIR spectra. The surface morphology of APC-CT was characterized by the presence of CT crystallites which spread and filled the spaces in APC structure that resulted in more homogeneous phases. The chemical compositions of APC and APC-CT were almost identical with intensified O, C and Si in APC-CT. The setting time, compressive strength, microhardness, pH and solubility obtained ranged between 46.6-48.5 min, 51.3-39.1 MPa, 44.89-38.57 HV, 11.04-11.02 (24 hrs) and 3.23-2.44%, respectively. CT improved the pH and solubility of APC and extended its setting times. However, compressive strengths were reduced and minimum effect on microhardness was observed. Cytotoxicity assays demonstrated that APC-CT supported the cell proliferation and interaction of SHED to the materials; as well as no apoptotic effect was observed. Alizarin Red and Von Kossa stainings demonstrated increased mineralization activity of SHED when treated with APC-CT. The expressions of DSPP, MEPE, DMP-1, OPN, OCN, OPG and RANKL markers were up-regulated in APC-CT-treated SHED. While, the expressions of RUNX2, ALP and COL1A1 markers were down-regulated. These findings demonstrate that APC-CT exhibits good chemical, physical and mechanical properties. APC-CT is non-toxic and promotes dentinogenic/osteogenic differentiation and mineralization activity; which provides potential applications of APC-CT in tooth/bone tissue engineering