Cytotoxicity testing direct contact tests

Medical grade silicone elastomers became available in the early 1960 s. "Medical grade" refers to silicone elastomers specifically formulated, manufactured and qualified for implant uses. The formulations contain no materials with potential for biodegradation or adverse biocompatibility. Manufacturing and processing are done under carefully controlled, clean conditions to assure batch-to-batch duplication, and freedom from adulteration, contamination, and cross contamination. Batch-to-batch tests include assessment of chemical, physical, and biological properties. The materials must elicit no cytotoxic reaction by direct contact tissue-cell culture testing (5,6). Qualification of a controlled formulation for implant use typically requires 2-year minimum biocompatibility (host and tissue reaction) ( 7) and... 

The actions of chemicals that cause disease or death occur at the cellular level therefore, it is necessary to conduct an array of in vitro cell culture tests to determine cytotoxicity. The bulk absorbable material is tested also, hydrolyzed products as well as extracts are assessed. The most obvious marker of toxicity is cell death. Necrosis, as compared with normal cellular apoptosis, is characterized by cellular and organelle swelling followed by rupture. Apoptosis, in contrast, is programmed cell death, which is characterized by a reduction in cell volume and basophilic staining of the chromatin. There are three common in vitro screens for toxicity — agar, direct contact, and elution assays. 

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 cytotoxicity tests were performed with the use of the direct contact method on the extracts obtained by an 8-d incubation of the polymer PSU and PSU/Ag composite samples, placed at the bottom of the well of a 24-well culture plate, in 2 ml of culture mediiun. The incubation was conducted at 37°C in air atmosphere with a 5% content of C02and 100% of relative air humidity. Next, 0.2 ml of ihe obtained extract and its fourfold dilution in a proper culture medium was dosed for the cultures of human osteoblasts (HTB-85 cell line, ATCC, USA) and human fibroblasts (CRL-7422 cell line, ATCC, USA) adhered to the bottom of the well of a 96-well culture plate. In the case of the control test, the extract of the examined samples was replaced by the proper volume of culture medium. The plates were incubated at 37°C in air atmosphere with a 5% content of CO and 100% of relative air humidity. The incubation time for two parallelly conducted experiments was 24 h and 48 h. The cytotoxicity of PSU and the PSU/Ag composites was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay [9, 10] and by lactate dehydrogenase (LDH) activity measured with the use of a commercial cytotoxicity assay kit (Roche Diagnostics GmbH, Mannheim, Germany), [11]. Each of the indications was repeated three times. 

The medical grade silicone rubber Sylgard 184 consists of a crosslinked poly-dimethylsiloxane. In vitro and in vivo testing has proven the non-cytotoxicity in direct and indirect contact. There is no ocular toxicity and no acute foreign body reaction in contact with soft tissues. 

Cytotoxicity tests, such as agarose overlay, minimum essential medium elution, direct contact, and MTT assay. 

Ciapetti, G., Stea, S., Cenni, E., Sudanese, A., Marraro, D., Toni, A. and Pizzoferrato, A. (1994) Cytotoxicity testing of cyanoacrylates using direct contact... 

For the direct contact tests, materials with at least one flat surface need to be nsed, although the materials can have various shapes, sizes, or physical states (eg, liquid, solid, and gel). An important factor to consider is that the sterility of the sample mnst be maintained throughout the test procedure as, for example, bacterial contamination can lead to false evaluation of cytotoxicity. In this regard, the effect of sterilization on... 

For direct contact tests, HSFB were seeded onto polyurethane disks. After 3-5-72 hours incubation, cell survival was evaluated as described for cytotoxicity tests. 

The ocular irritation caused by cosmetic ingredients has been evaluated by the determination of the amount of histamine contained in tears. Contact of surfactants and the eye tissue cause an immediate dose-dependent release of histamine through direct cytotoxic damage of cell membranes. This method has been tested with sodium lauryl sulfate with volunteers [187]. 

Primary irritant-contact dermatitis results from direct cytotoxicity produced on first contact. The cellular injury is characterised by two macro-scopically visible events a reddening of the skin (erythema) and accumulation of fluid (oedema). By observing or measuring these changes, one can estimate the extent of skin damage that has occurred. The most widely used single-exposure irritancy test is based on the Draize rabbit test. ... 

The cytotoxicity, acute hemolysis, skin sensitization results suggest that AI2O3 porous ceramics prepared via freeze casting in vitro biological reactions is biosafe and the porous AI2O3 ceramics have a potential application for implants. Further experiments, such as, direct cells contact in vitro, long-term implantation test in vivo will be conducted to meet the practical application requirements. 

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