BioSciences, Fresenius Medical Care, Bad Homburg, Germany
Know the pitfalls
“Methodology is everything and the devil is in the details,” remarked Paul Simmons, the current president of the International Society for Stemcell Research, in an article in Nature.1 The article refers to current problems related to the reproducibility of data in stem cell research. Reproducibility in in vitro testing is also mandatory when selecting polymers for medical device applications. Many mechanical and physical engineers are surprised when they realise the enormous standard deviations (sometimes between 50 and 100%) of data found in biological or physiological investigations of biomaterials. The reasons for this are the complexity of physiological parameters such as the nature of blood originating from a variety of donors and hour-to-hour and day-to-day physiological differences. As a consequence, standardisation is a “conditio sine qua non” in biomaterial testing and knowledge of possible pitfalls is absolutely necessary.
Therefore, ISO 10993-4, Biological Evaluation of Medical Devices, Selection of Tests for Interaction With Blood,2 provides a practical tool, including a decision tree for use in the selection of appropriate polymers for biomaterial applications. The interested reader finds in Section 3.1 of ISO 10993-4 the definition of blood-device interaction: “Any interaction between blood or any component of blood and a device, resulting in effects on blood, or on any organ or tissue, or on the device. A note added to this definition further clarifies: “Such effects may or may not have clinically significant or undesirable consequences.” This prompts one to ask if effects leading to undesirable consequences that are not clinically significant would be helpful to the polymer chemist.
This article provides some observations and examples of the misconceptions and pitfalls that exist in testing biomaterials for biocompatibility.
Importance of close simulation
Figure 1: Literature on biocompatibility parameters and their assessment would fill a huge room. However, a clear-cut opinion in favour of one or the other parameter for a concise biomaterial characterisation is still lacking. A solution to this dilemma could be the application of a score model that incorporates a combination of several representative parameters.
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The scientific literature offers a quantity of data on biocompatibility parameters. Polymers have been tested in relation to their capacity to cause coagulation, cell activation, inflammation and hypersensitivity (allergy) as well as effects on the blood vasculature (Figure 1). It is still not yet clear which parameter best describes the final clinical situation. Conditions in testing should always simulate as closely as possible the clinical situation. Conditions such as the polymer surface area in contact with blood, blood flow in hollow fibre devices, and device sterilisation should be matched between the testing and the clinical situation. For example, blood trauma caused by repeated exposure to shear stress in a capillary tube of 1-mm inner diameter led to an irreversible stiffening of red cells, yet this effect was less pronounced in continuous shear conditions.3 These observations may apply to blood recirculation experiments using a peristaltic pump and a low-volume blood reservoir. In addition, miniaturised devices should always be assessed with a blood flow that is proportionally reduced to the actual surface area in contact with blood. By this means, mechanically induced shear forces remain in the same order of magnitude as those found in the original device.
Testing with healthy donors
Biocompatibility testing of polymers for application in medical devices is routinely performed with blood from healthy donors. However, medical devices are normally employed in patients suffering from a variety of diseases or during the administration of medicinal drugs that can interfere with polymer properties. It is questionable whether polymer testing with healthy blood represents the correct final clinical situation. Some examples originating from the application of biomaterials in haemodialysis are provided here to better explain this notion. This is a useful application area to examine because, to date, the majority of publications on the biocompatibility of biomaterials in clinical application deal with haemodialysis. This is no surprise. Chronic exposure of blood to foreign polymer surfaces takes place during haemo-dialysis treatment, because patients are treated thrice weekly for the rest of their lives. Furthermore, polymers for haemodialysis are used for a variety of applications such as capillary membranes, dialyser housings, tubing systems and bags for infusion solutions. Thus, they are the most widely used biomaterials in the world. Their increasing application in single-use devices means that even more biomaterials will be used in haemodialysis in the near future.
Patients with uremia or kidney disease experience disorders of blood physiology, that is, their blood cells and blood composition are different from normal blood. White blood cells from uremic patients show an increased oxidative metabolism,4 a decreased antioxidant capacity depending on the progress of uremia,5 a reduced capacity for phagocytosis,6 and an increased number of reticulated platelets,7 which may lead to disturbances in coagulation behaviour of uremic blood. In addition, lipid composition of uremic platelets differs from normal thrombocytes,8 which may lead to a change in platelet aggregation. The behaviour of platelets in biocompatibility testing is extremely important because these cells determine the initial events of the blood-clotting cascade. For example, metabolically inhibited platelets do not adhere to glass test tubes, whereas their passive adhesion to polypropylene tubes remains unchanged.9 How could this conclusion be made if biocompatibility testing is only performed using blood from healthy donors?
Figure 3: Thromboxane formation by means of blood–material interaction is affected by aspirin, whereby the level of reduction of thromboxane formation depends on the polymer type.11
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Figure 2: Aspirin blocks the action of cyclooxygenase I on arachidonic acid from activated cell membranes. As a result, thromboxane and prostacyclin are not formed. Aspirin administration to blood donors, therefore, affects biocompatibility assessment of biomaterials.
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Experiments on extracorporeal cardiopulmonary bypass are normally assessed with blood from the calf or the pig. Analyses of blood count in the animal model after a prolonged perfusion of up to 7 days proved that species type, whether bovine or porcine, had an impact on the haematology profile.10 Absolute values of red cell count were found to be higher in the calf, and normalised values were higher in the pig. Leukocyte counts did not behave similarly.
Testing and medication
A neglected area in biomaterials testing is the previous or simultaneous administration of medicinal drugs. Drugs such as aspirin or angiotensin-converting-enzyme (ACE) inhibitor, may interfere with results on biocompatibility parameters to the extent that a clear-cut conclusion on whether or not a material is biocompatible cannot be drawn. Two examples illustrate this notion. After the administration of aspirin, the enzyme cyclooxygenase I is blocked. As a consequence, the formation of prostaglandins from arachidonic acid derived from activated cell membranes is also blocked and the thromboxanes A2 or B2 are not formed (Figure 2). The analysis of aspirin effects on the formation of thromboxane by different biomaterials used in haemodialysis such as polymethylmethacrylate (PMMA) and cellulose showed that the formation of thromboxane was halved with PMMA and completely blocked with cellulose when 1000 mg of aspirin had been administered to the blood donor (Figure 3).11
A second example relates to the behaviour of biomaterials bearing a defined negative charge density, for example, the blend between polyacrylonitrile and methallysulphonate employed as a polymer for capillary membranes for haemodialysis. Negative surface charges are able to stimulate the contact phase of coagulation, depending on charge density. As a consequence, a cascade is initiated. This includes the formation of bradykinin, which is a nonapeptide that is able to down regulate blood pressure through prostaglandin formation (Figure 4). It is fortunate that in the healthy human, the half-life of bradykinin is extremely short because of the activity of ACE. However, when ACE inhibitors are administered for medical reasons, the degradation process of ACE is inhibited and blood pressure down regulation is observed. This effect is dose dependent as shown in a sheep model (Figure 5).12
Figure 4: Negatively charged biomaterials activate the contact phase of coagulation. The degree of activation depends on charge density. As a result, bradykinin is formed and blood pressure is down regulated. In the presence of ACE inhibitors, bradykinin degradation is inhibited and blood pressure down regulation occurs.
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This indicates that biocompatibility testing of biomaterials in the future should involve investigations on simultaneously administered medicinal drugs. Old and new medicinal drugs may express synergistic effects between polymer composition and physiological cascades of the body, which can ultimately lead to adverse clinical events.
Anticoagulants may also affect biocompatibility testing. Several physiological cascades such as complement and coagulation, as well as cell activation pathways, depend on the presence of calcium ions or magnesium ions. The use of sodium citrate as an anticoagulant affects the release of the enzyme elastase from white blood cells, reduces complement activation and blocks the coagulation cascade, depending on the polymer under investigation.13
Liquids for testing for extractables
Figure 5 : Down regulation of systolic blood pressure with negatively charged biomaterials depends on the presence of ACE inhibitors in a dose dependent manner as shown in a sheep model. Blood pressure drop is partially counterbalanced here by an increased heart rate.
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“Blood is a very peculiar juice,” stated Mephisto to Faust in the novel “Faust” by the German writer Johann Wolfgang von Goethe. Indeed, blood, which contains electrolytes, enzymes, lipids and proteins apart from water, is capable of extracting leachables from polymers or medical devices in a highly efficient manner. Consequently, biomaterial testing should always examine extraction capacity with the help of appropriate extraction media. One reason for the occurrence of extractables is a shift to broader molecular weight distribution during polymer synthesis (Figure 6). Polymer ageing adds to the source of extractables as well as the degradation of some polymers in a wet atmosphere or after some sterilisation procedures.
ISO 10993-12 prescribes those extraction media that should be used for the isolation of leachables.14 Solvents selected as extractants, “shall simulate the extraction which occurs during clinical use of the device and/or maximise the amount of extract.” For a reliable extraction of all leachables three types of extractants should be used, and using all three allows the blood properties for extraction to be simulated effectively:
- polar media such as water, saline (0.9% sodium chloride solution) or culture media without serum derived from cell culture technologies
- nonpolar media such as vegetable oil (cotton seed or sesame oil)
- mixtures of ethanol–water, polyethyleneglycol 4000, dimethyl-sulphoxide or culture media with serum.
Figure 6 : Gel permeation chromatography (GPC) analysis of polymer formation shows a broadening of the polymer´s molecular weight distribution at the end of reaction. This effect may give rise to extractable material that may leach out in contact with blood.
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Recently, severe adverse clinical events15,16 and even fatal incidences17 originating from extractable material have been reported in the scientific literature. These data show that aged polymers,15 degraded polymers16 or liquids used for device integrity tests during the manufacturing process17 may give rise to extractable material. This can be later extracted from medical devices and accumulated in patients associated with adverse clinical events. In many of these cases analyses are extremely time-consuming because the correct extractant has not been applied at first instance.
It has been shown that a series of factors may interfere with biocompatibility performance of polymers. Among these are blood donor specificities, anticoagulants, physical conditions of blood flow and drugs administered to the blood donor. Consequently, a simple and clear-cut analysis of testing results is difficult and sometimes even unpredictable. Testing polymers and other biomaterials for their application in medical devices should always be performed as a systems approach taking into account a series of side effects. The development of a score model, which summarises several impact factors at a time, may be a solution.
1. E. Check,“The Hard Copy,” Nature, 446,485–486 (2007).
2. Biological Evaluation of Medical Devices, Part 4: Selection of Tests for Interactions With Blood, ISO 10993-4, 2nd Edition.
3. S.S. Lee et al., “Strain Hardening of Red Blood Cells by Accumulated Cyclic Supraphysiological Stress,” Artif. Organs, 31, 80–86 (2007).
4. J. Paul et al., “Influence of Uremia on Polymorphonuclear Leukocytes Oxidative Metabolism in Endstage Renal Disease and Dialysed Patients,” Nephron, 57, 428–432 (1991).
5. J. Mimic-Oka et al., “Alteration in Plasma Antioxidant Capacity in Various Degrees of Chronic Renal Failure,” Clin. Nephrol., 51, 233–241 (1999).
6. J. Alexiewicz et al., “Impaired Phagocytosis in Dialysis Patients: Studies on Mechanisms,” Am. J. Nephrol., 11, 102–111 (1991).
7. J. Himmelfarb et al., “Increased Reticulated Platelets in Dialysis Patients,” Kidney Int., 51, 834–839 (1997)
8. A. Vecino et al., “Lipid Composition of Platelets in Patients with Uremia,” Nephron., 78, 271–273 (1998).
9. N. LaFayette et al., “An In Vitro Method for Assessing Biomaterial-Asociated Platelet Activation,” ASAIO J., 53,159–162 (2007).
10. X. Mueller et al., “Hemolysis and Hematology Profile During Perfusion: Inter-Species Comparison,” Int. J. Artif. Organs, 24, 89–94 (2001).
11. J. Vienken and R. Schäfer, “Acetyl-Salicylsäure (Aspirin) Beeinflusst die Generation von Thromboxan durch Dialysemembranen,” Nieren & Hochdruckkrankheiten, 24, 480 (1995).
12. D. Krieter et al., “Anaphylactoid Reactions During Hemodialysis in Sheep are ACE Inhibitor Dose-Dependent and Mediated by Bradykinin,” Kidney Int., 53, 1026–35 (1998).
13. J. Böhler et al., “Mediators of Complement-Independent Granulocyte Activation During Haemodialysis: Role of Calcium, Prostaglandins and Leukotrienes,” Nephrol. Dial. Transplant., 8, 1359–1365 (1993).
14. Biological Evaluation of Medical Devices, Part 12: Sample Preparation and Reference Materials, ISO 10993-12, 2nd Edition. Corrected version 2003-06-01.
15. J. Hutter et al., “Acute Onset of Decreased Vision and Hearing Loss Traced to Hemodialysis Treatment With Aged Dialysers,” J. Am. Med. Assoc., 26, 2128–2134 (2000).
16. Z. Averbukh et al., “Red Eye Syndrome: Clinical and Experimental Experience in a New Aspect of Diffuse Eosinophilic Infiltration?” Artif. Organs, 25, 437–440 (2001).
17. B. Canaud et al., “Pathochemical Toxicity of Perfluorocarbon, A Liquid Test Performance Fluid Previously Used in Dialyser Manufacturing, Confirmed in an Animal Experiment,” J. Am. Soc. Nephrol., 16,1819–1823 (2005).
Dr. Jörg Vienken is Vice President BioSciences, Department International Marketing and Medicine, Fresenius Medical Care, Else Kroenerstrasse 1, D- 61342 Bad Homburg, Germany tel. +49 6172 609 2463, e-mail: email@example.com, www.fmc-ag.com