Introduction
Biocompatibility is the ability of an external system to perform its function satisfactorily, when introduced into the biological environment of a living organism without producing undesired effects [Z. R. Wang et al 2019]. It is a key concept to understand the response of the host organism towards the implant or any biomaterials introduced within it and is very essential for developing new implants and improving their performance [B. Ratner 2015]. The introduced system should be able to perform its desired function to full potential without developing any adverse response from the recipient biological system [M.S. Zafar et al 2019]. There are many different aspects of biocompatibility tests: cytotoxicity tests, heamo-compatibility tests, sensitization, irritation system wide toxicity, geno-toxicity and implantation etc. Tests are also designed according to the level at which the sample will interact with the host for example: over intact skin, at mucosal membrane, breached or compromised surface, blood path, circulating blood, tissues, bone etc. Tests also depend upon the duration for which the external system will remain in contact with the host, for example less than few minutes as in the case of a syringe to more than 30 days as in the case of permanent implants.
Cyto-toxicity test
A cytotoxicity test is one of the most useful and preliminary testing tools used for screening of external systems before performing more extensive toxicological tests. Cytotoxicity tests measure the number of cells that survive when in contact with the external test system for a specified duration. A cytotoxic test could be performed quantitatively or qualitatively. Qualitative methods include the direct contact cytotoxicity test where cells are adhered to the bottom of a Petri dish up to a confluency of 80%. The test article is placed directly in contact with the cells for 24 hours and then observed afterwards with a phase inverted contrast microscope for any cell reactivity and then marked from 0 to 4 corresponding to no reactivity to severe reactivity. To prevent low density samples from floating away and high density test samples from damaging cell cultures, an agar overlay can be used in between the cell and sample. MEM elution is another qualitative method in which extracts from samples are used instead of samples directly to study the reactivity.
Cytotoxicity of thin films with varying stoichiometric compositions will not be clearly resolved just by 4 grading scales used in qualitative tests, therefore quantitative studies are preferred. In vitro cytotoxicity or cell viability assays have the advantage of being a fast, economic, and efficient method to determine the cell viability and tests can also be done on human cells as well. Biological cells release certain markers when they are viable (living) or when they are non-viable (dead) or when they have completely disintegrated. Appropriate reagents could be added onto the cell medium with test samples to determine these markers which will correlate to the number of viable cells, non-viable cells and/or disintegrated cells. The reaction between the reagent and biological marker results in visual events like colour, fluorescence or luminosity. Cell assays are classified according to these endpoints measured.
Colorimetric based cytotoxic tests are most easy, reliable, and reproducible and are sensitive with cells in high concentration. Most common of these are the Alamar blue, MTT, MTS cytotoxic test assays. The Alamar blues assay depends upon conversion of blue coloured resazurin to purple coloured resorufin in metabolically active cells the resulting change in colour is propotional to the number of viable cells, whereas MTT and MTS assays determine cell viability by determining the mitochondrial function of viable cells, which releases succinate dehydrogenase enzyme. This enzyme reduces the tetrazolium based assays into purple formazan. The colour of solution is directly proportional to the number of viable cells which can be measured by analysing with a spectrophotometer tuned to the correct wavelength.
Alamar Blue assay is the most favoured assay to test the initial effects of β Ti3-Au super hard thin film coatings on biological cell lines because it is efficient, economic, reliable and highly sensitive with cell numbers up to 10,000.
Testing protocols
After establishing the nature (qualitative vs quantitative), endpoint event (colorimetric, Fluorometric, Luminometric) of the cytotoxicity test, it is important to decide the protocols used to perform these tests. Assay plates are set up with cells in a growth conducting medium for a time period to equilibrate the cell line. After this period the test sample is introduced to the cell medium. The exposure time will be determined by the nature of the expected outcome and can extend from 1 hour up to 5 days and in some cases up to 11 days. A short exposure could give information about the immediate effect of sample introduction into the host, whereas prolonged exposure, like up to 5 days, can indicate if the test sample will inhibit the proliferation of the cell line. After exposure time the assay reagent is introduced to the exposed cell culture. Thereafter, depending upon the number of viable cells, the assay would be reduced to its coloured compound. Again, a set amount of time is given so that the assay can react and stabilise to the reducing enzymes from the culture overall.
On the majority of cytotoxicity tests for thin films use a 24 hour equilibration time. For this time period, the selected cell line is maintained in a culture medium at 37°C and 5% Co2 environment and humidity levels varying between 95-100%. Exposure time being the most important parameter is varied between 24 hours up to 11 days depending upon the nature of result expected. 24 hours is mainly used for L929 mouse fibroblast cells and most of the human derived cells like MG63 are exposed for 3-5 days to the test sample. Typically 3 to 5 hours are given for the assay reagent to react with the exposed cell line and develop the endpoint event.
Cell Lines and Medias
According to ISO 10993-5:2009, Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity, cell viability of external material should be proved with L929 mammalian cell (mouse fibroblast). Most of the tests performed on Ti based alloys employ L929 cells as their base cell line for cytotoxicity tests. Another favourite for biocompatibility testes of Ti based alloys is Mg-63 human osteosarcoma cells.
Serum from fetal and calf bovine sources are commonly used to support the growth of cells in culture. Fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells. Although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.
Test method
1. Cell growth: Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate buffer saline (PBS), fetal bovine serum (FBS), trypsin, penicillin/streptomycin and L-glutamine of analytical grade were used to culture stocks of L929 murine fibroblast cells. Cells were cultured for 15-20 passages in a humidified atmosphere at 37oC and 5% CO2, grown as monolayers and sub-cultured at 80-90% confluency.
Preparation of test compounds: The testing samples were disinfected in 70% ethanol solution, air dried and then completely submersed into. They were shaken at low speed using a vortex for 5 seconds and left to leach for 72 hours in humidified conditions. Before removal, each sample was shaken again for 5 seconds and then the test pieces were removed. Cell viability assay L929 cells were seeded into 96-well plates in 2,000 cells/well and left overnight before exposing to the test extract. On the following day, cells were exposed to 20, 50 and 100% (v/v) dilution of test extract and left for 72h. 10% DMSO solution and a Cu substrate of size similar to Ti substrates was used as positive control and untreated DMEM was used as negative control. After 72 hours, fresh medium (containing 0.1 mg/ml resazurin) was added into each well and incubated for 2 hours at 37oC. Centrifuged plates were recorded for absorbance at 570 nm and 600 nm (reference wavelength) in a Spark multimode plate reader (Tecan, Switzerland). The levels of cell viability were estimated and expressed as percentage of control cells.
Stay tuned for the next article on the first batch of thin films deposited in this research….
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