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Research that matters – biocompatibility and cytotoxicity screening

2012; Wiley; Volume: 46; Issue: 3 Linguagem: Inglês

10.1111/iej.12047

1365-2591

Ove A. Peters,

Dental materials and restorations

In an attempt to enhance the quality of submitted manuscripts, the International Endodontic Journal has recently embarked on a series of editorials. The first two of these editorials dealt with investigations into irrigating solutions and root canal fillings. The recurring theme that connects these two areas with the question of biocompatibility is the recently increased number of submissions, unfortunately often of limited value for clinicians and researchers alike. In our opinion, the time is right for a renewed appraisal of areas that can be identified as relevant in the assessment of biocompatibility of endodontic materials. Moreover, the assays and experiments employed should also be reviewed and put into context. It is important to note that certain international standards exist in this area (ISO 10993 series, especially 10993-5, 10993-12 and ISO 7405) but researchers often fail to take these standards into consideration. To the contrary, many laboratories seem to have identified their particular strategy in biocompatibility testing and this makes any comparison of data problematic. In the following, we attempt to characterize relevant issues surrounding the biological testing of materials in endodontics that will be in contact with living cells and tissues. The first step is to define the properties that are addressed in this line of research, fully recognizing that there may be disagreement over some aspects of these definitions: Biocompatibility traditionally describes the ability of a material or a substance to perform with an appropriate host response when applied as intended. The term ‘appropriate’ means in this context that a biocompatible material is not necessarily inert, but the reaction to the test material must be an acceptable risk, compared to a clinically accepted material. The term ‘host response’ comprises a large array of different biological reactions including inter alia, systemic acute, subchronic and chronic toxicity, sensitization/allergenicity, local irritation/toxicity, and mutagenicity/carcinogenicity. Furthermore, bioactivity, that is, the stimulation of a desired tissue response by biomimetic approaches, is currently frequently connected to the term biocompatibility. In other words, biocompatibility = not harmful + efficient. For each of these biological reaction areas, a multitude of different in vitro and in vivo tests have been described in the literature. Well-known and extensively used tests are included in the above-mentioned ISO standards. Other test methods, for example using a variety of molecular biology approaches, may be useful for elucidating biological mechanisms behind an observed clinical reaction. This incomplete listing of the many different areas of biocompatibility together with the large number of different test methods confirms that biocompatibility can only be evaluated by using a battery of test, such as primary, secondary, usage and clinical tests. Furthermore, the rationale for the selection of the test method for each of these areas and for a specific scientific objective is an important aspect of the study design and must be justified in the manuscript. Cytotoxicity is defined as the capacity of a material to impact on cellular viability. Therefore, cytotoxicity tests are primary biocompatibility tests that determine the lysis of cells, the inhibition of cell growth and other effects on cells caused by test substances. This means that cytotoxicity only describes one single aspect of biocompatibility. Moreover, cytotoxicity itself has several aspects, depending on the cells, the growth medium and the endpoint used for measuring the cell response. Indeed, noxious effects are induced by cytotoxic molecules: intracellular adverse molecules are synthetized leading to glutathione depletion, production of reactive oxygen species, under the control of glutathione transferase P1, all these changes being involved in mitochondrial alterations. A new and crucial notion is that cytotoxicity is not only a problem of a toxic dose. With respect to bisphenol-A (BPA), very low doses are producing more defects or alterations than higher concentration. Interaction of some molecules with hormones may also be involved in BPA toxicity. In dye inclusion/exclusion tests, cells may produce different results when exposed to a test material, compared to tests, in which enzyme activity of cells is determined. The same is true when using different cell types, like primary, permanent or transfected cell lines. Again, it is important to justify the use of the cells, the endpoint and the test method, such as direct or indirect cell–material contact. Cytotoxicity tests can be employed to screen a new material comparing it to a large number of known materials (relative cytotoxicity analysis). The main question is, if there are substances being released from the material, which may be harmful to cells in general. For this very general approach, mainly ISO tests are recommended using permanent cell lines. These tests do not take the specific mode of application of the material into account, and the results are only a basis for further tests (screening tests). Data from such tests are mainly of interest for pre-market clearance but of limited scientific value. In a further step, cytotoxicity tests can be employed to evaluate more site-specific reactions. In this case, site derived cells should be employed, and the cell contact can be either direct or it can be indirect. Such approaches normally follow the above-mentioned screening tests and are of more interest for the reader of a scientific journal. Subsequently, mechanisms of interaction (e.g. inhibition of biomineralization or the stimulation of biomineralization or influences on the immune system) can be evaluated in very special tests using specific cells (e.g. odontoblast-like cells or macrophages). Here, normally very specific endpoints are measured by molecular biological methods. Data from such test are of high scientific interest. Under these general approaches, the scientist/author must justify, as mentioned above, the choice of the test method. Biocompatibility is by law an aspect, which must be evaluated before a material is allowed to be released to the market. It may be an issue worth investigating in endodontics for the following situations: Clinical relevance of data from cytotoxicity tests depends on the aim of the study and the methods used. For a simple screening test, clinical relevance is not achieved; rather basic biological information is aimed at in a relative sense comparing a set of materials. Clinical relevance of more specific tests depends on the degree of simulation (e.g. the cell–material contact, the choice of site-specific cells). However, it should be kept in mind that even when simulating the in vivo situation, in vitro test are only models and interpretation of results should take this into account. Finally, when cytotoxicity tests are used to elucidate the mechanism of a biological reaction, clinical relevance is of reduced interest: here, the test must be designed according to the specific question (e.g. measuring DNA double-strand breaks or the generation of reactive oxygen species). This means that clinical relevance must be discussed separately for each of the test levels used. Generally, direct and noncontemplated extrapolation of data from cytotoxicity tests to the patient situation is not possible. For example, capping of sterile exposed pulps has very limited predictive value for the applicability of a product. In summary, the two items biocompatibility and cytotoxicity, while in the same domain, are distinctly different. In addition to consulting pertinent literature (e.g. Hanks et al. 1996, Schmalz 1997, Murray et al. 2007, Goldberg 2008, Ribeiro 2008, Wataha 2012), I recommend the following general guidelines to authors planning to execute studies or submit manuscript regarding biocompatibility and cytotoxicity: It is my hope that this editorial, similar to the others in the series, will help to guide authors, as well as reviewers, for projects and manuscripts considered for submission to the International Endodontic Journal. The ultimate goal is to enhance the quality of research and, even more, to facilitate better clinical outcomes, a goal I am sure we all can agree on. I would like to express my sincere gratitude to Drs. Gottfried Schmalz, Michel Goldberg, Peter Murray, Jean Camps and Daniel Ribeiro for their valuable contribution to this editorial.