Positive control for cytotoxicity evaluation of dental vinyl polysiloxane impression materials using sodium lauryl sulfate.

OBJECTIVES: Vinyl polysiloxane (VPS) is elastomeric dental impression material which, despite having very few reports of adverse reactions, has shown high levels of cytotoxicity that is difficult to be interpreted without referencing to the positive control material. Therefore, in this study, positive control VPS was developed using sodium lauryl sulfate (SLS) for the reference of cytotoxicity test. MATERIALS AND METHODS: The positive control VPS with SLS was formed with a different proportion of SLS (0, 1, 2, 4, 8 and 16 wt%) added to the base. The cytotoxicity test was then carried out using the extractions or dilutions of the extractions from each of the test samples using murine fibroblast cells (L929). RESULTS: The final product of positive control VPS behaved similar to commercially available VPS; being initially liquid-like and then becoming rubber-like. Ion chromatography showed that the level of SLS released from the product increased as the proportion of added SLS increased, consequently resulting in an increased level of cytotoxicity. Also, the commercially available VPS was less cytotoxic than the positive control VPS with more or equal to 2 wt% of SLS. However, even the VPS with the highest SLS (16 wt%) did not cause oral mucosa irritation during the animal study. CONCLUSIONS: The positive control VPS was successfully produced using SLS, which will be useful in terms of providing references during in vitro cytotoxicity testing.

Cytotoxicity of dental alginates / Citotoxicidad de alginatos dentales

Alginate, or irreversible hydrocolloid, is one of the most accepted impression materials used in dentistry. However, some substances existing in these materials can be toxic. The aim of this study was to assess the cytotoxicity of alginates for dental applications. Fourteen different alginates were assessed: Jeltrate, Jeltrate Plus, Jeltrate Chromatic, Alga Gel, Printer Gel, Ava Gel, New Print, Kromopan 100, Tropicalgin, Cavex Orthotrace, Hydrogum, Orthoprint, Cavex Color Change, and Qualitygel. Three control groups were also used in this study: positive control group (C+) consisting of cell detergent Tween 80, negative control group (C-) consisting of PBS, and cell control group (CC) consisting of non-exposed cells. After manipulating the materials according to the manufacturer’s instructions, samples were made by using silicone rings. Next, the samples were immersed into Eagle’s minimum essential medium (MEM) for 2 minutes followed by removal of supernatants and contact with L929 fibroblasts. After contact with the medium, the cells were incubated for further 24 hours in which 100µl of 0.01 por ciento neutral red stain were added. Cells were incubated again for 3 hours so that the stain could be absorbed. After this period, the cells were fixed and viable cell counting was performed by using a spectrophotometer (BioTek, Winooski, Vermont, USA) at wavelength of 492 nm. The results demonstrated statistical differences between CC and C- groups in relation to other ones (p<0.05). No statistical differences were observed between Jeltrate Plus and Hydrogum groups, between Jeltrate and Jeltrate Chromatic, Printer Gel, Tropicalgin, and Qualitygel groups, and between Jeltrate Chromatic and Alga Gel, Ava Gel, New Print, Kromopan 100, Cavex Orthotrace, Hydrogum, Orhtoprint, and Cavex Color Change groups. One can conclude, based on the results of this study, that all alginate materials were found to be cytotoxic.

El alginato o hidrocoloide irreversible, es uno de los materiales de impresión más aceptados y utilizados en odontología. Sin embargo, algunas substancias existentes en estos materiales pueden ser tóxicas. El objetivo de este estudio fue evaluar la citotoxicidad de los alginatos para aplicaciones dentales. Fueron evaluados 14 alginatos diferentes: Jeltrate, Jeltrate Plus, Jeltrate Chromatic, Alga Gel, Printer Gel, Ava Gel, New Print, Kromopan 100, Tropicalgin, Cavex Orthotrace, Hydrogum, Orthoprint, Cavex Color Change y Qualitygel. También se utilizaron tres grupos de control también se utilizaron en este estudio: grupo control positivo (C+) que consiste en células de detergente Tween 80, el grupo de control negativo (C-) que consiste en PBS, y el grupo de células de control (CC) que consiste de las células no expuestas. Después de la manipulación de los materiales de acuerdo a las instrucciones del fabricante, las muestras fueron hechas mediante el uso de anillos de silicona. A continuación, las muestras se sumergieron en medio mínimo esencial de Eagle (MEM) durante 2 minutos, seguido de la eliminación de los sobrenadantes y el contacto con los fibroblastos L929. En caso de contacto con el medio, las células fueron incubadas durante 24 horas más en 100ml de tinción roja neutra al 0,01 por ciento. Las células se incubaron nuevamente durante 3 horas para que la tinción pueda ser absorbida. Después de este período, las células fueron fijadas y el recuento de células viables se realizó mediante un espectrofotómetro (BioTek, Winooski, Vermont, EE.UU.) a la longitud de onda de 492 nm. Los resultados demostraron diferencias estadísticamente significativas entre los grupos de CC y C- en relación con los demás (p<0,05). No se observaron diferencias estadísticas entre los grupos Jeltrate Plus y Hydrogum, entre Jeltrate y los grupos Jeltrate Chromatic, Printer Gel, Tropicalgin y Qualitygel, y entre Jeltrate Chromatic y los grupos Alga Gel, Ava Gel, New Print, ...

Avaliação da citotoxicidade de quatro alginatos utilizados na Odontologia / Evaluation of the cytotoxicity of four alginate used in Dentistry

O objetivo do presente trabalho foi avaliar a citotoxicidade de quatro diferentes alginatos de uso odontológico. Foram avaliados quatro alginatos divididos em quatro grupos: Jeltrate, Tropicalgin, Cavex Color change e Qualitygel. Corpos de prova foram imersos em meio Eagle por 2 min, onde então procedeu-se à remoção do sobrenadante e colocação em contato com fibroblastos L929. Após coloração, realizou-se contagem de células viáveis. Os resultados demonstraram diferenças estatísticas entre os grupos CC e C- e os demais. Todos os grupos experimentais apresentaram citotoxicidade.

Assessing the effects of three dental impression materials on the isolated sciatic nerve of rat and frog.

The effects on nerve tissue of three dental impression pastes were compared in this study. Two of the impression pastes, Examix and Express 3M, contained vinyl polysiloxane while the other, Xanthopren, did not. An in vitro model based on the isolated sciatic nerve of the frog and rat was used. As an indication of the proper functioning of the fibres in the nerve, the amplitude of evoked compound action potential (CAP) was monitored continuously. The results clearly showed that the number of active nerve fibres in the isolated sciatic nerves of either rat or frog exposed directly to impression pastes containing vinyl polysiloxane, decreased much faster than those of the nerves in contact to impression material without vinyl polysiloxane. When the nerve of the frog was exposed to Xanthopren there was a decrease in the CAP to 50% of the control values within 56.87+/-2.42 h (n=6). This value was called inhibition time to 50%, IT(50) and for Examix it was found to be 9.97+/-1.53 h. When the nerve of the rat was exposed to Xanthopren, the IT(50) was 15.34+/-2.97 h (n=6) for the Xanthopren and only 2.86+/-1.20 h for Examix and 2.76+/-0.48 h for Express 3M (n=6). There was no significant difference between the action of the last two compounds (P=0.85). This fast nerve fibre inactivation could be caused either by the chemical used for the synthesis of the two impression pastes, Examix and Express 3M, or by the unusual constriction of the nerve when it is embedded in the materials with vinyl polysiloxane. There is strong evidence to support the first case, since the incubation of the nerve in the presence of Examix, Express 3M and Xantopren in a way so the nerve was not in contact with the impression pastes, shows a much faster decrease of the CAP in the presence of the first two pastes. The decrease is caused by the death of nerve fibres, since there is no recovery in the CAP after the removal of Examix from the incubating saline.

A new generation of sterile and radiopaque impression materials: an in vitro cytotoxicity study.

Impression materials are largely used to record the geometry of dental tissue. Hence, the assessment of their possible cytotoxicity is a necessary step in the evaluation of their biocompatibility. The present study is carried out to evaluate the cytotoxicity of a new elastomeric sterile and radiopaque impression material. Human gingival fibroblasts, cultured in vitro are exposed directly to Elite Implant in three different viscosities, heavy, medium, and light. At 3, 9, 24, 48, and 72 h, the cellular proliferation is evaluated. In parallel, human gingival fibroblasts are exposed indirectly by means of fluid extracts of Elite Implant. The cellular viability is evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, (MTT) assay (Sigma, St Louis, Mo). The gingival fibroblasts proliferation and viability are unaffected by the presence of Elite Implant. This new impression material may represent a safe medical device for clinical and surgical applications. In addition, this material is radiopaque and, thus, can be identified radiographically.

Potencial tóxico dos alginatos para uso odontológico / Potencial toxicity of dental alginates

O alginato ou hidrocolóide irreversível é um dos materiais de moldagem mais aceitos e utilizados na Odontologia. Muitas substâncias como zinco, cádmio, silicato de chumbo e fluoretos foram adicionadas em algumas marcas de alginatos, com o objetivo de melhorar suas propriedades físicas, químicas, mecânicas e se tornaram causa de preocupação no que se refere à toxicidade desses materiais. Em algumas marcas de alginatos relatou-se a presença de fluoretos, cádmio, silicatos de chumbo e zinco potencialmente tóxicos, isoladamente ou em conjunto, consequentemente, cuidados especiais devem ser tomados na preparação desses materiais. É necessário que haja um controle contínuo de metais e substâncias potencialmente tóxicas nos alginatos para se evitar a contaminação dos profissionais da área odontológica e pacientes. Nesta revisão analisou-se o potencial tóxico de alginatos usados em odontologia.

Study of the potential cytotoxicity of dental impression materials.

The aim of this study was to assess the cytotoxicity of tow types of impression dental materials: polyethers (Impregum Penta, Permadyne Penta Heavy and Light) and vinyl polysiloxanes (Elite Mono Tray, Medium, Low viscosity and Elite H-D Putty). Their cytotoxic effects were studied by indirect and direct tests. The indirect tests were performed by incubating impression materials in serum free cell culture medium to prepare the soluble extracts. Balb/c 3T3 cells were incubated with extract dilutions (25, 50, 75 and 100%) for 24 h. The extracts of polyether materials caused a decrease of cellular viability, evaluated by light microscopy, by cell counting and by MTT test. The extracts of vinyl polysiloxanes materials induced a slight effect on cellular number and viability. The direct tests were performed by placing the impression materials in the centre of Petri dishes while Balb/c 3T3 were settling. The cellular proliferation was drastically reduced by polyethers and it was unaffected by the presence of vinyl polysiloxanes. These results show that: (a) the polyether materials are more toxic than vinyl polysiloxanes in our experimental conditions, (b) the impression materials are cytotoxic to the same degree in all assay methods.

An in vitro pulp chamber with three-dimensional cell cultures.

To better simulate the in vivo situation, a three-dimensional fibroblast cell culture was introduced into an in vitro pulp chamber model. The system was evaluated by testing a series of dental filling materials. After a 24-h exposure with (0.3 or 5 ml/h) and without perfusion of the pulp chamber, the tissues were subjected to a routine MTT assay. Zinc phosphate cement, conventional glass ionomer cements, a silicone impression material, and zinc oxide-eugenol did not influence cell viability, compared with untreated controls; but, a light-curing glass ionomer cement significantly reduced cell survival. Perfusion of the chambers did not significantly influence the results, but perfusion conditions of 5 ml/h lead to a general decrease of cell vitality. The three-dimensional cell culture system in an in vitro pulp chamber seems to be a substantial improvement, because zinc oxide-eugenol does not evoke a cellular reaction (as is the case in vivo), and the test system is sensitive enough to detect other toxicants.

[Comparative analysis of tissue reaction to acrylic resin materials in studies on Wistar strain rats]. / Analiza porównawcza reakcji tkankowej na tworzywa akrylowe w badaniach na szczurach szczepu Wistar.

The study takes up the issue of assessing rat tissue reaction to operatively inserted implants of different acrylic resin materials used in prosthetic dentistry. The materials subjected to analysis were polyacrylics: Vertex Soft, Vertex R.S., Vertex S.C., Superacryl and silicone material Molloplast B. The prolongation of life and the dynamic development of prosthetic treatment have caused removable dentures to be used longer and among more people. Polymerised acrylic resin material of these dentures is a potential pathogenic factor to the oral cavity mucosa which is in contact with it. As many as 20 to 70% of patients using removable acrylic dentures suffer from prosthetic stomatopathy. It is considered that the mucosa irritation may be caused by denture trauma, a mycotic infection or toxic action of some components of acrylic materials. Therefore the use of new generation acrylic materials in producing prosthetic dentures needs a precise assessment of undesirable local and systemic effects. A comparative analysis of the effect of correctly polymerised acrylic material on rat mucosa, parotid glands and lymphatic nodes was carried out. Systemic toxicity of these materials was assessed. Acrylic plates were prepared from the most often used acrylic resin materials in the Department of Prosthetic Dentistry PAM and a silicone material (these materials were polymerised precisely according to the producers instruction). Before implantation the plates underwent a thermodynamic analysis in order to ensure that the polymerisation process was carried out correctly and to determine thermal resistance of particular materials. Next sterile acrylic plates were implanted in rats under general anaesthesia. The animals were divided into 6 groups, 10 rats each. In four groups acrylic plates were implanted, in one group silicone material plates were implanted and it represented the comparative group, in one control group an incision of the buccal mucosa was made. The rats were observed during a period of 6 weeks, they were weighed every two weeks and no loss in body mass was noted (Tab. 1). After 6 weeks the rats were anaesthetised with ether and dissectioned. Biopsy specimens were taken from the buccal mucosa, porotid gland and lymphatic cervical nodes around the plates in order to make histological specimens. Blood samples were also taken to carry out blood cell counts and liver tests to determine eventual systemic toxicity of the studied acrylics. Histological specimens were stained with hematoxylin and eosin. In borne cases in order to precisely assess the intercellular substance other staining methods were used such as van Gieson, PAS and silvering of precollagen fibres on reticulum. Prepared specimens were assessed in a light microscope in magnification of 80 to 400. Basing on specimens of the control group an analysis of tissue reaction to the particular tested acrylic resin material was carried out. It was ascertained that the most irritative properties to the rat buccal mucosa were caused by self-cure acrylic material--Vertex S.C. This polymer caused in all rats in the tested group a reactive hypertrophy of cervical lymphatic nodes (Tab. 2 and Fig. 3). The least damaging effect on the surrounding tissues was caused by heat-cured acrylic resin material Superacryl (Fig. 4). The tested materials had no damaging effect on the rat parotid gland and did not have a toxic action on the internal organs.

Cytotoxicity testing of materials with limited in vivo exposure is affected by the duration of cell-material contact.

Silicones for dental impression largely are used to record the geometry of hard and soft dental tissues. They are considered to be medical devices, and the assessment of cytotoxicity is a necessary step in the evaluation of their biocompatibility. Extracts of six addition-type and six condensation-type silicones have been tested with L929 cells according to the ISO 10993-Part 5 standard. The cytotoxicity was evaluated by three different methods: neutral red uptake, propidium iodide (PI) staining, and amido black staining. According to the selected specific assay, contact between cells and material extracts was maintained for 24 h in the first series of experiments; then, considering that in vivo application of these materials is restricted to a few minutes, additional experiments were performed after 1 h of cell/extract contact. Analysis of the results showed that the addition-type silicones are nontoxic even when tested after prolonged exposure of the cells to the materials while the condensation-type silicones were cytotoxic at 24 h of incubation. Nevertheless, harm to the patient actually could be negligible, considering its very short time of exposure in vivo. This is supported by our finding that most are not toxic after 1 h. We suggest that the experimental conditions of cytotoxicity testing have to be relevant to the in vivo situation; accordingly, the time of exposure should be designed carefully.

Cytotoxicity of impression materials.

The purpose of this study was to assess the potential cytotoxicity of impression materials. The impression material brands tested included Impregum, Reprosil, Surgident, Permlastic, Jeltrate Regular and Jeltrate Fast Set. These impression materials and their components were tested for their possible cytotoxic effects by three different methods. The results showed that all impression materials were cytotoxic to some degree by all three assay methods and some of the components were also cytotoxic. These results support the literature showing in vivo adverse effects of certain impression materials and/or their components in patients and practitioners.