Material Matters

Problems with the use of conventional cytotoxicity tests to evaluate biological risk with biomaterials are leading to the development of alternative procedures based on cell function. These tests, recently discussed at a conference in China,¹ provide an attractive option for the future.

 

By: David Williams

China revisited

I am writing this article in Beijing Airport, trying to leave the country before it closes down for a week or so, because the 60th anniversary of the founding of the People’s Republic of China coincides with the mid-autumn moon festival. My two day visit was occasioned by an invitation to give the guest lecture at the first International Conference on the Quality Control of Biomaterials and Tissue Engineering Products sponsored by China’s State Food and Drug Administration (sFDA).1 It may be recalled that I wrote about the emerging position of China in medical device technology in 2005.2 The impression gained from two or three visits a year since then that this position is strengthening all the time was confirmed by this Conference. I do not wish to report here on the Conference itself, but rather to comment on two important, related issues that emerged.

The first of these is a timely reminder that all is not well with the development of tissue engineering scaffold materials. In a previous column on this subject in 20043 I said that the existence of previous regulatory approval for a material to be used in a medical device or drug delivery system was not a good specification for a tissue engineering scaffold material. My long standing concern has been that synthetic biodegradable polymers are usually proinflammatory in the late stages of their degradation. Thus, any specialised tissue generated by a tissue engineering process is likely to be disrupted by the behaviour of inflammatory cells such as neutrophils and macrophages, which probably negates all of the good work already done. In one way it was reassuring, although of course disappointing for the position of tissue engineering in general, to hear findings from Professor Xi Tingfei, who represented the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP) in China. He stated that some early clinical experiments and trials in China on tissue engineering products were revealing responses that were not predicted by the in vitro and other preclinical tests, largely as a result of the effect of degrading polymer systems. This is clearly of major significance for the whole field of biomaterials.

Live cell sensor chips

I recently wrote about some problems with the use of existing standard tests for the evaluation of biomaterials for medical devices and tissue engineering products.4 These are manifested in the above reported findings. It is interesting to note that the sFDA and NICPBP are working hard to incorporate international standards into their procedures for testing and regulatory approval, but are aware of some deficiencies, and the Chinese Government, through various programmes is funding work on the scientific basis for new testing procedures. However, it was one project funded by the Japanese Government that caught my attention with respect to the future. The background to this work may be seen in a paper published in 2008 by Wada et al.,5 which was discussed at the Conference by Dr Taniguchi of the National Institute for Materials Science in Japan.

Let us first consider the basis of traditional tests for cytotoxicity. In reality the process is simple. We take a colony of cells in an appropriate culture medium. These cells will stay alive and healthy, providing they are given the right nutrients and environmental conditions. If a chemical is added to the culture medium, the cells will respond according to the nature of the chemical agent; the extent of any change in cell behaviour equates to the extent of the toxic character of that agent. In the simplest situation, we may use death of the cell as the ultimate marker of toxicity, and measure some parameter of the relationship between death (such as percentage of cells to die) and the dose or concentration of the agent. In biomaterials testing, the agent is usually an extract obtained by storing the test material in a standard solution (such as saline or cottonseed oil); during this process it is assumed that any toxic component will be leached out into that solution. This, of course, is crude, but it is still the basis of many standard cytotoxicity tests. Somewhat better than waiting for a cell to die before recording a cytotoxic event is the type of test that records the state of health of the cell. For example, the widely used MTT test measures the metabolic activity or cell respiration rate of the cells.

Alternative methods mostly try to use live cells whose behaviour can be monitored by far more sophisticated methods. These types of test have been in existence for a few years, especially those that involve measuring electrochemical characteristics of cells in contact with a suitable sensing electrode. The Japanese approach utilises a combination of reporter gene analysis with microfluidic technology. This technology allows microscale channels to be fabricated on silicone substrates and the well defined flow of cell suspensions is generated in these channels. A fibroblast cell line has been prepared in which the cells have been transfected with specific genes that allow extremely minor perturbations to cell function to be readily detected. The principal approach here has been to transfect the cells with green fluorescence plasmid derived from heat shock protein, which is extremely sensitive to minor changes in their environment. Analysis of the fluorescence changes in the microfluidic channels as these cells are exposed to test media provides a precise and reproducible measure of the effect of different chemical agents on the cells.

This technology still has to be validated with respect to biomaterials testing, however, it is viewed as having significant potential for this application as well as for environmental monitoring and drug evaluation. This is especially important because the microfluidic approach is amenable to so-called high throughput screening. To have the potential for reproducible, quantitative analytical methods for the better determination of the potential for biomaterials to do harm is encouraging. It is of no real surprise to see this type of technology emerging from Asia.

David Williams, Morgan & Masterson, Avenue de la Forêt 103, Brussels 1000, Belgium, tel. +32 4 7597 0556, e-mail: peggy@morgan-masterson.com, www.morgan-masterson.com