The interactions between the host and a biomaterial, following the introduction of a prosthesis or medical device, can dictate whether the implant is a success or failure. It is, therefore, vital to have a good understanding of a material’s potential to affect the tissues that it will come into contact with. These studies will lead to enhanced biocompatibility that, in turn, will result in more successful implants and better treatment strategies.
When assessing the biocompatibility of materials, there are several factors that need to be considered. Each potential medical device or implant will vary not just in terms of the material it is made of, but also in terms of its intended purpose. Studies need to consider factors such as where in the body a material is likely to be used. For example, a suitable material for an artificial heart valve will not necessarily make a good intraocular lens.
 There may also be different forms in which the material can be presented, with small variations in material properties, such as surface morphology, net charge, porosity and degradation rate, capable of significantly changing the host’s immune response. Factors such as these will influence, for example, complement activation, cell recruitment and cell attachment, which in turn lead to a wider overall response such as inflammation. The material may then in turn be affected by the host’s response, which can cause accelerated damage and degradation leading to the failure of the medical device.
It should also be taken into account whether the material needs to be passive or interactive. A passive material will not stimulate a specific cellular response in the host, whereas an interactive material is intended to guide or stimulate specific host responses.
Techniques used
As even subtle changes to biomaterials can cause large changes in the host’s response, it is essential to determine the differences between one material and another both accurately and objectively.
 The University of Liverpool’s Division of Clinical Engineering has been working at the forefront of medical device biocompatibility for many years and uses a variety of techniques to characterise the biological response in vivo, ex vivo and in vitro. Microscopically, a combination of the following is used to investigate material/body-tissue interactions:
Immunohistochemistry is used to characterise the response non-subjectively, using a panel of monoclonal antibodies to stain for specific cell types that are potentially involved. The technique may include enzymatic detection, using enzymes such as chloroacetate esterase, which is found almost exclusively in neutrophils and mast cells. Antibody labelling can also be employed, using antibodies directed against T-cell receptors, with a FITC-conjugated secondary antibody to allow the labelling to be examined using fluorescence microscopy. Monoclonal antibodies can additionally distinguish between activated and non-activated cells, for example with activated T-lymphocytes labelled with anti-IL-2 receptor antibody.
Laser scanning confocal microscopy is used to visualise cytoskeletal elements in cells adhered to the surface of biomaterials and the presence of specific cell surface receptors. The division currently uses a Zeiss LSM 510 four channel UV system on an Axioplan 2 Imaging MOT upright imaging microscope with DIC (differential interference contrast) and fluorescence. This employs four photomultipliers that supply images for interpretation.
Subsequently, computer-based image analysis can give quantitative data on the immune response and highlight trends that may have been overlooked using standard qualitative methods. The University of Liverpool uses LSM 510 Imaging Software from Zeiss to assess not only the number of cells present, but also factors such as the spatial distribution of cells in relation to the implant and the ratio of activated to non-activated cells. As this approach requires minimal user input, it greatly reduces the subjectiveness of the assessment. However, computer-based image analysis is by no means foolproof– the user still needs to supply good quality images in order to avoid the “rubbish in, rubbish out” maxim. This means it is essential that good quality images are produced by the microscopy.
When further details are required, flow cytometry can be employed to look in more depth at receptor expression on inflammatory cells and to determine the real size and granularity of the cells.
Lastly, In situ hybridisation (ISH) defines and characterises molecules such as cytokines that mediate immune and inflammatory processes. ISH can be used to study in vivo cytokine expression, yielding information on, for example, the relationship between TNF - α and biomaterial surface charge.
Evaluating the response at a cellular level
 The response to biomaterials may not necessarily be a non-specific or innate immune response, but rather one that involves discrete subpopulations of cells whose phenotype depends on the nature of the material surface. There are, therefore, a number of different cell types that need to be taken into account when evaluating the inflammatory response, namely neutrophils, monocytes, macrophages, T- and B-lymphocytes, mast cells and fibroblasts.
The performance of different materials can then be further compared and categorised according to the spatial distribution of these cells in relation to the implant. The ratio of activated to non-activated cells is also important, as a factor such as the number of activated macrophages is indicative of the magnitude of the inflammatory response. This is one area where the quality of images produced by the microscopy is paramount, as activated cells can be identified morphologically by features such as membrane ruffles and cytoplasmic projections.
Lastly, the source of the sample being tested is important as animal models and human samples may differ greatly. Even when the immune response in an animal model is close to its human equivalent, human samples are only studied when an implant has failed. The immune response cannot be monitored in the same depth for a successful implant.
Conclusions
It is essential to have a good understanding of the relationship between an implanted material and the host tissue in order to maximise the chances of success for an implant or medical device. When used in conjunction with high-quality microscopy, image analysis helps to standardise analysis methods, giving reproducible and objective results. This is invaluable in the evaluation of the performance of biomaterials with a view to improving their clinical performance.
Equipment used
- Zeiss LSM 510 Laser Scanning Microscope, Axioplan 2 Imaging Research microscope, 3-off Axioskop 2 with discussion tubes and 2-0ff Axiovert 25 inverted microscopes all from Carl Zeiss.
- Objectives used: x2.5 to x100 (Carl Zeiss), with almost all techniques in transmitted and reflected light
- Images acquired using an AxioCam from Carl Zeiss, with subsequent image analysis carried out using AxioVision software and LSM 510 Imaging Software from Carl Zeiss.
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