Cytotoxicity testing biomaterials

As to the biomaterial for human tissue replacement, it is necessary to demonstrate if the material has any effect on the biological properties of the tissue. Bioceramics exhibit some possible toxic reactions due to metal ions leaching from the ceramics, resulting in the tissue dying or heavy reactions. In this experiment, cytotoxicity test, hemolysis test as well as skin irritation were conducted to value the biocompatibility of the porous AI2O3 ceramics. 

Fischer D, Li Y, Ahlemeyer B et al (2003) In vitro cytotoxicity testing of polycations influence of polymer structure on cell viability and hemolysis. Biomaterials 24 1121-1131... 

Although the initially reported tissue compatibility tests for subcutaneous implants of poly(BPA-iminocarbonate) were encouraging (41,42), it is doubtful whether this polymer will pass more stringent biocompatibility tests. In correspondence with the properties of most synthetic phenols, BPA is a known irritant and most recent results indicate that BPA is cytotoxic toward chick embryo fibroblasts in vitro (43). Thus, initial results indicate that poly(BPA-iminocarbonate) is a polymer with highly promising material properties, whose ultimate applicability as a biomaterial is questionable due to the possible toxicity of its monomeric building blocks. 

In the following, we will focus on in vitro tests for cell compatibility and blood compatibility. In this context, we will also discuss ISO 10993-5 (tests for in vitro cytotoxicity) and ISO 10993-4 (selection of tests for interactions with blood). Finally, the risk of pyrogens in the biomaterial context, especially of bacterial toxins (endotoxins), will be briefly highhghted and selected methods for determining pyrogens/endotoxins (cf. also ISO 10993-11) will be presented. 

The extract dilution type of cell culture assay requires a solvent extraction of the biomaterial under consideration and testing of this extract, most commonly at various dilutions, for evidence of cytotoxicity and cellular interaction. This type of cell culture assay finds its most common use in providing information for regulatory compliance. As identified in the preceding Materials for Medical Devices section and in Table 1, low-molecular-weight extractables are of concern regarding biocompatibility. The extraction assay, carried out with a series of solvents that are hydrophilic and hydrophobic, permits examination of the potential cytotoxicity of extracts and the identification of materials within a biomaterial that may be cytotoxic. These types of assays ultimately permit identification and characterization of cytotoxic materials within biomaterials or the lack of cytotoxicity, as well as providing correlation with in vivo assays such as sensitization, irritation, intracutaneous (intradermal) reactivity, and other tests where the in vivo injection of extracts is required. 

In addition to biodegradabUity, another important prerequisite for an ideal biomaterial is cytotoxicity. This is also vital in biomedical applications. Two methods are generally employed for estimating the cytotoxicity of polymers, the direct contact method and the M lT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. However, an anti-haemolytic test can provide direct evidence of non-cytocompatibiUty and the power of the polymer to protect the cells from harmful free radicals. Red blood cells (RBC) are very susceptible to attack from free radicals which damage the cell membrane, permitting the leakage of haem protein which can then be estimated. This may be done easily under normal laboratory conditions and the method is briefly discussed below. 

The biocompatibility of two different blends of corn-starch, SEVA and starch/cellulose acetate (SCA), and their respective composites with HAp, were studied by Marques et al. [274]. Researchers found that both types of starch-based polymers exhibit a cytocompatibility that might allow for their use as biomaterials Eurthermore SEVA blends were found to be less cytotoxic for the tested cell line, although cells adhere better to SCA surface. Considering the overall behaviour of SEVA, SCA and their composites with HAp, it can be expected that their cytocompatibility will allow for their use in the future in applications such as bone replacement/fixation and/or tissue engineering scaffolding. 

Biomaterials/devices are generally composed of relatively inert, high-molecular-weight materials and it is their teachable components (eg, contaminants, additives, fillers, and residual compounds derived from manufacturing) that usually present cytotoxicity. As a result, the rationale behind the in vitro testing of biomaterials is that cytotoxicity can be assessed by evaluating the impact of constituents that can be released from the biomaterial/device in in vivo conditions. 

This test determines if a material itself has potential to release agents that may be cytotoxic and is suitable for biomaterials with large toxicity. The indirect contact test includes the agar overlay assay and filter diffusion, giving a qualitative assessment of cytotoxicity (Table 7.9). 

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