Pulp Fibroblast Cell Apoptosis After Application of Hema Dentine Bonding Material with Ethanol and Water Solvent

Brazilian Dental Journal (2019) 30(3): 208-212 http://dx.doi.org/10.1590/0103-6440201902524 ISSN 0103-6440 Pulp Fibroblast Cell Apoptosis After Application of Hema Dentine Bonding Material with Ethanol and Water Solvent 1Department of Conservative Ira Widjiastuti1 , Ratih Elisa Nandarani2 , Latief Mooduto1 Correspondence: Ira Widjiastuti, Jl. Prof. Moestopo 47, Surabaya. e-mail: The most common main materials for dentin bonding for composite resin restoration is 2-hydroxyethyl methacrylate (HEMA). HEMA has beneficial physical and chemical properties, and stable, yet toxic. The addition of ethanol or water, may reduce the toxic effect of HEMA. Ethanol solvent has lower H-bonding capacity compared to water solvent, so it can bind less free radicals from the residual monomer. This study aimed to analyze apoptosis due to dentine bonding application with ethanol and water solvent. Fibroblast culture cells were obtained from extracted third molar, by means of tripsinasion method. The cells were divided into 4 groups as reached confluent: cell culture without treatment as control, cell culture with scaffold chitosan, cell culture with scaffold and polymerized dentin bonding with ethanol or water solvent. Apoptosis observation was conducted using immunohistochemistry method with ethidium bromide acridin orange staining, under fluorescent microscope with 40× magnification. There was a significant difference among groups (p=0.0001), yet no differences found between different solvent. Apoptosis rate in fibroblast cells culture exposed to HEMA bonding with ethanol solvent was 67%, while the cells exposed to HEMA bonding with water solvent was 44%. The effect of dentin bonding with ethanol solvent and water solvent towards apoptosis rate of pulp fibroblast cells is not different. Introduction Composite resin is restoration material that is commonly used in conservative dentistry for its esthetic value is better compared to other restoration material. Composite resin binds to teeth structure through adhesive material called dentin bonding. Most of various dentin bonding that is currently available, thus far, containing 2-hydroxyethyl methacrylate (HEMA) as basic material (1). HEMA is widely used since have several advantages, such as having good physical-chemical properties, that is stable, both as base material bonding and when mixed with other materials (1). HEMA is one of the derivatives of methacrylate resin, that can be hydrolyzed into two compounds namely methacrylic acid and ethylene glycol, which has a toxic effect (2). Based on in vitro study, HEMA can stimulate apoptosis after 24 h (3). HEMA can also induce the production of Reactive Oxygen Species (ROS) in fibroblast cells that cause DNA fragmentation, characterized by apoptosis due to oxidative stress (4). HEMA monomer is toxic to mammalian cells because it contains hydroxyl groups which are free radicals that can induce oxidative stress (5). According to Anusavice (1), free radicals are negatively charged ions that have no pairs in an atomic orbit, so to fill the void of the ion, it will attract other free ions which are nearby. According to Kitamura Dentistry, Faculty of Dental Medicine, Universitas Airlangga, Surabaya-Indonesia 2Residence of Conservative Dentistry, Faculty of Dental Medicine, Universitas Airlangga, Surabaya-Indonesia Key Words: dentine bonding, apoptosis, 2-hydroxyethyl methacrylate, ethanol solvent, water solvent. et al. (6), the diffusion of HEMA into dentin may lead to pulp irritation. An in vitro study discovered that HEMA can induce apoptosis after 24 h (7). Other study by Bakopolou et al (8) stated that HEMA could diffuse through dentin, toward pulp chamber in a significant high concentration, while other study stated that HEMA could affect human lungs epithelial cell, by inducing the formation of ROS, leading to DNA fragmentation, indicated by apoptosis (9). Cytotoxic activity of chemical compounds can be observed from its ability to stimulate cell death (8). According to Krifka (10), monomer-induced apoptosis is an active cell response to ROS levels that exceed the cells ability to maintain reduction and oxidation homeostasis. Cells activate different enzymatic cellular antioxidant tissue balance to control intracellular oxidative states after exposure to HEMA monomers. It has also been shown that HEMA may increase ROS production which further lead to inflammation and delay the cell cycle process (11). Biochemical materials as well as resin-based dentin bonding materials can produce ROS in polymerization process (1). The solvent contained in dentin bonding plays role in adequate bond formation between dentin and resin. Besides, bonding also act as the carrier of resin monomer into the dentin after etch. However, the residual solvent may also affect the polymerization process, impair the Braz Dent J 30(3) 2019 bond strength, and causing inteface degradation (12). The solvents that is frequently used in adhesive systems are water, ethanol and acetone (13). According to Bianci (14) in his study suggested that the ratio of cell death both apoptosis and necrosis in the dentin-self-etch group of dentine-saturated-ethanol ranged between 3% -10% and 12% -47%. Indirectly, transdentinal diffusion of monomers occurs in the presence of ethanol, so that the presence of monomers diffusing into the pulp can induce oxidative stress and cytotoxicity in the pulp (14). The induction of apoptosis associated with the production of ROS is a major incident of direct cell response to monomers exposure. Apoptosis is programmed cell death, active, requires energy and not accompanied by inflammation. Changes in cell morphology with apoptosis include cell shrinkage, chromatin segregation, cytoplasmic condensation and apoptotic bodies. The process of apoptosis through intrinsic and extrinsic pathways (15). Dentin bonding in this study will be tested on pulp fibroblast human fibrous cell culture because fibroblast is the largest component of the pulp, periodontal ligament and gingiva. Fibroblast is the main cell in pulp tissue that can produce collagen fibers and differentiate into odontoblast like cells. Pulp fibroblasts are capable of producing growth factors and cytokines that act as growth control and response to injury (16). The purpose of this study was to investigate the effect of apoptosis on fibroblast pulp cells after dentin bonding with HEMA based on ethanol and water. Apoptosis Examination Material and Methods Based on the observations using a fluorescence microscope, the cultured cells from all groups was showing the characteristics of pulp fibroblast cell (Fig.1) Based on the acquired data, the mean and standard deviation of apoptotic fibroblast cells are shown in Table 1. The acquired data were not normally distributed, therefore, Kruskal-Wallis was used to find the differences among groups. A significant difference was found among groups, with p value of 0.0001. Chitosan scaffold control (...truncated)