Marina Hasiwa
Detection and removal of surface-bound pyrogenic contaminations
Dissertation
Universität Konstanz July 2006
Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2007/2100/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-21007
Detection and removal of surface- bound pyrogenic contaminations
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
Eingereicht im Fachbereich Biologie an der Universität Konstanz
vorgelegt von
Marina Hasiwa
Tag der mündlichen Prüfung: 26.10.2006 Referent Prof. Dr. Dr. T. Hartung
Referent Prof. Dr. A. Wendel Referent Prof. Dr. M. Leist
FIGURE 0.1.ALVEOLAR MACROPHAGE
(PRINCIPLES OF MICROBIOLOGY,2ND EDITION 1997. PG.504)
Für meine Kinder
Dominic und Corvin
L I S T O F P U B L I C A T I O N S
List of publications:
Major parts of this thesis are published or submitted for publication:
• A new in vitro pyrogen test for surfaces, Marina Hasiwa, Karin Kullmann, Sonja von Aulock, Christoph Klein, Thomas Hartung, in press 2007, Biomaterials
• Plasma-based Depyrogenation, Ondřej Kylián, Marina Hasiwa, Francois Rossi, Plasma Process. Polym. 2006, 3, 272–275
• Effect of Low-Pressure Microwave Discharges on the pyrogen activity, Ondřej Kylián, Marina Hasiwa, Francois Rossi, in press 2006, IEEE
• Plasma-based removal of different immune-stimuli from surfaces, Marina Hasiwa, Ondřej Kylián, Thomas Hartung, Fancois Rossi, in preparation
• Investigation of the influence of Ar:H2 microwave discharge on the Lipid A bioactivity, Ondřej Kylián, Marina Hasiwa, Francois Rossi, in preparation
• Contribution to the Proposal of the ISO 10993, Biological Evaluation of Medical Devices, Principles, and Methods for Pyrogen Testing of Medical Devices. Annex B, Clinical Experience with Pyrogenic Reactions and Medical Devices, Marina Hasiwa
Further publications, not integrated into this thesis:
• Mechanisms of Sterilization and Decontamination of Surfaces by Low Pressure Plasma, Francois Rossi, Ondřej Kylián and Marina
L I S T O F P U B L I C A T I O N S
• Decontamination of surfaces by Low Pressure Plasma discharges, Francois Rossi, Ondřej Kylián and Marina Hasiwa, review, Plasma Process. Polym. 2006, 3, 431-442
• Surfaces Functionalization and Patterning techniques to design Interfaces for Biomedical and Biosensor Applications, Frederic Brétagnol, Andrea Valsesia, Giacomo Ceccone, Pascal Colpo, Douglas Gilliland, Laura Ceriotti, Marina Hasiwa and Francois Rossi, Plasma Process. Polym. 3, 443-455
• Fouling and non-fouling surfaces produced by plasma polymerization of ethylene oxide monomer, Frédéric Brétagnol*, Michaël Lejeune, Andri Papadopoulou-Bouraoui, Marina Hasiwa, Hubert Rauscher, Giacomo Ceccone, Pascal Colpo, François Rossi, Actabiomateriala 2 (2006)165-172
• Micro-patterned surfaces based on plasma modification of PEO-like coating for biological applications, Frédéric Brétagnol*, Ondřej Kylián, Marina Hasiwa, Laura Ceriotti, Hubert Rauscher, Ceccone Giacomo, Douglas Gilliland, Pascal Colpo and Francois Rossi, in press 2006, Sensors and Actuators: Chemistry B
• Functional Micro-patterned Surfaces by Combination of Plasma Polymerization and Lift-Off Processes, Frédéric Brétagnol, Laura Ceriotti, Michaël Lejeune, Andri Papadopoulou-Bouraoui, Marina Hasiwa, Douglas Gilliland, Giacomo Ceccone, Pascal Colpo, François Rossi, Plasma Process. Polym. 2006, 1, 30-38
A C K N O W L E D G E M E N T S
Acknowledgements
This work was carried out between December 2002 and June 2006 as a member of the Chair of Biochemical Pharmacology at the University of Konstanz. Most of the practical part was done in the European Research Centre, Ispra, Italy in cooperation between ECVAM (European Centre for Validation of Alternative Methods) and BMS (Biomaterial Science, Surface Characterization and Processing), both units of the IHCP (Institute for Health and Consumer Protection).
I especially want to thank my supervisor Prof. Dr. Dr. Thomas Hartung. He made this study possible not only by giving me invaluable scientific advice and stimulating ideas, but also by providing excellent working facilities. His continuous support and unbelievable patience gave me the possibility to carry out this interesting work, to improve my scientific knowledge and he prevented me from loosing track of the important things.
Highly appreciated is the support of Prof. Albrecht Wendel, head of the chair of biochemical pharmacology, and Dr. Hermann Stamm, head of unit of BMS, for welcoming me in their groups, following my work and their constructive criticism.
Grateful thanks to Dr. Francois Rossi, my boss and supervisor of the plasma part, for his creative guidance, the motivational support and the critical comments.
Special thanks to Ondrej Kylian, my coworker in most of the experiments. He introduced me to the multi-facetted world of plasma-physics and allowed to plan experiments on the parking place while drinking coffee and smoking cigarettes.
I’m grateful to all my blood donors for giving the most valuable liquid they have for scientific purposes and to the ‘vampires’ from the Medical Service.
A C K N O W L E D G E M E N T S
Thanks a lot to all members of ECVAM, who welcomed me warmly and formed an extraordinary team, which made it possible to enjoy coming everyday to work. In the same way, I want to thank all members of BMS for welcoming me as warmly and being there, whenever I could not solve physics related problems.
Greatest thanks to my all my colleagues, which became friends meanwhile, namely Lars Hareng, Tina Stumann, Helena Hoegberg, Sebastian Hoffmann, Nicola Marquart, Anna Price, Agniezka Kinsner, Siegfried Morath and Erwin van Vliet for sharing good and bad times, for creating an exceptional working atmosphere and for an unforgettable time.
I’m grateful to the ECVAM secretary team for administrative support and organizational work, which made things much easier.
The technical help of Juan Casado, Fernando Fernandez, Nicos Parissis and Matteo Fama is highly appreciated.
Thanks to Patricia Pazos and Antonella Bottini for kissing me whenever we met and making me feel welcome every single day.
I’m grateful to my best friend, Stephanie Traub, for giving me power and courage by having always an open ear. Furthermore, I want to thank my friends Chris, Sebi and Salim for prompt and reliable help and keeping my private things running.
The most important persons in my life are my kids, Dominic und Corvin, and them I thank the most. They were going with me through good times and difficulties but they showed me everyday that it is worth to do all this effort.
Last but not least I want to thank my Mom, who was always there, taking care of my kids, believing in me and encouraging me to go on.
A B B R E V I A T I O N S
Abbreviations
ANOVA analysis of variance Ar Argon
AVS monochromator BET Bacterial endotoxin test
BsLTA Lipoteichoic acid Bacillus subtilis BsPGN Peptidoglycan Bacillus subtilis C Circulator
COX-2 cyclohexagenase-2
CV Coefficient of variation: CV [%]=100.stdev/average D-ala D-alanine
DAP diaminopimelic acid, Dpm D-glu D-glutamic acid DMSO dimethyl sulfoxide Dpm diaminopimelic acid, DAP EcPGN peptidoglycan Escherichia coli
ECVAM European Centre for Validation of Alternative Methods ELISA enzyme-linked immunosorbent assay
EtO ethylene oxide F mass flow controller GlcN D-glucosamine GlcNAc N-acetylglucosamine
H2 hydrogen
Hep L-glycero-D-manno-heptose IFN interferon
IL interleukin
kD kilo Dalton
Kdo 2-keto-3-deoxy-octulosonic acid LAL Limulus amoebocyte lysate
L-ala L-alanine
LBP lipopolysaccharide binding protein
A B B R E V I A T I O N S
LPS lipopolysaccharide LTA lipoteichoic acid
LVAD left ventricular assistant device M N-acetyl-muramic acid;
MDP muramyl dipeptide
MODS multiple organ dysfunction syndrome
MW microwave supply
N2 nitrogen
NK natural killer cell
O2 oxygen
PAMP pathogen-associated molecular pattern PBMC peripheral blood mononuclear cells
PGE2 prostaglandin E2
PGN peptidoglycan
PRR pathogen recognition receptor
pSaLTA lipoteichoic acid ultrapurified Staphylococcus aureus PTFE teflon
R-C rectangular-circular wave-guide transition RPMI cell culture medium
SaLTA lipoteichoic acid Staphylococcus aureus SaPGN peptidoglycan Staphylococcus aureus SEM standard error of the mean
SiW silica window SMOV implant steel TiAl6V4 titanium alloy TH1 T-helper cell 1
TMB 3,3`,5,5`-tetramethylbenzidine TNF-α tumor necrosis factor
WBT human whole blood incubation
3S three-stub impedance matching system
T A B L E O F C O N T E N T S
Table of contents
1. GENERAL INTRODUCTION...1
1.1 The human immune system ... 1
1.2 Pyrogens... 2
1.3 Pyrogen Testing... 6
1.4 Medical Devices ... 10
1.5 Pyrogen testing of medical devices... 11
1.6 Clinical experience with pyrogenic reactions and medical devices ... 12
1.7 Sterilization of Medical Devices ... 15
1.8 Plasma-based Sterilization ... 16
1.9 Aim of the study... 21
2. AN IN VITRO PYROGEN SAFETY TEST FOR IMMUNE-STIMULATING COMPONENTS ON SURFACES...22
2.1 Abstract ... 23
2.2 Introduction ... 24
2.3 Materials and Methods ... 27
2.4 Results ... 31
2.5 Discussion... 39
3. PLASMA-BASED DEPYROGENATION...44
3.1 Abstract ... 45
3.2 Introduction ... 45
3.3 Experimental Part ... 47
3.4 Results and Discussion ... 49
3.5 Conclusion ... 53
4. EFFECT OF THE LOW-PRESSURE MICROWAVE DISCHARGES ON THE PYROGEN BIOACTIVITY...54
4.1 Abstract ... 55
4.2 Introduction ... 55
4.3 Experimental ... 58
4.4 Results ... 60
4.5 Conclusion ... 64
5. REMOVAL OF IMMUNE-STIMULATORY COMPONENTS FROM SURFACES BY PLASMA D ...65
T A B L E O F C O N T E N T S
5.1 Abstract ... 66
5.2 Introduction ... 67
5.3 Materials and Methods ... 71
5.4 Results ... 74
5.5 Discussion... 81
6. INVESTIGATION OF THE INFLUENCE OF AR:H2 MICROWAVE DISCHARGE ON THE LIPID A BIOACTIVITY...85
6.1 Introduction ... 86
6.2 Experimental ... 88
6.3 Results ... 91
6.4 Conclusions ... 95
7. SUMMARIZING DISCUSSION...96
7.1 General... 96
7.2 Demands for a novel pyrogen test... 96
7.3 Test Development... 97
7.4 Plasma-based Depyrogenation ... 100
7.5 Removal of different pyrogens... 103
7.6 Structural changes of Lipid A... 105
8. SUMMARY...107
9. ZUSAMMENFASSUNG...110
10. LITERATURE...113
T A B L E O F F I G U R E S
Table of figures
FIGURE 0.1.ALVEOLAR MACROPHAGE... 1
FIGURE 1.1AGRAM-NEGATIVE BACTERIUM. ... 3
FIGURE 1.2.SCHEMATIC DIAGRAM OF THE PEPTIDOGLYCAN SHEET OF STAPHYLOCOCCUS AUREUS. ... 5
FIGURE 1.3.NUMBERS OF MEDICAL DEVICES PER YEAR WORLDWIDE... 11
TABLE 1.4.STERILIZATION PRINCIPLES FOR MEDICAL DEVICES... 15
FIGURE 1.5.SCHEMATIC PICTURE OF THE PLASMA GENERATOR... 18
FIGURE 2.1.A.CONSTRUCTION SCETCH OF THE INCUBATION CHAMBER... 28
FIGURE 2.1.B.SIDE VIEW... 28
FIGURE 2.2. MATERIALS... 31
FIGURE 2.3.A.LPS RESIDUALS... 32
FIGURE 2.3.B.LPS DRY AND LIQUID... 33
FIGURE 2.4.DIFFERENT MATERIALS... 33
FIGURE 2.5.A.LIPOTEICHOIC ACID... 34
FIGURE 2.5.B.PEPTIDOGLYCAN... 35
FIGURE 2.5.C.ZYMOSAN... 35
FIGURE 2.6. MANUAL CONTAMINATION... 36
FIGURE 2.7.A. CLEANING METHODS A... 37
FIGURE 2.7.B.CLEANIG METHODS B... 37
FIGURE 2.8. CRYO-PRESERVED BLOOD... 38
FIGURE 3.1.DEPYROROGENATION... 49
FIGURE 3.2TIME COURSE... 50
FIGURE 3.3. PRESSURE... 51
FIGURE 3.4. TEMPERATURE... 51
FIGURE 4.1.EXPERIMENTAL SETUP... 58
FIGURE 4.2. HYDROGEN... 60
FIGURE 4.3.UV RADIATION... 61
FIGURE 4.4. ELECTRON DENSITY... 61
FIGURE 4.5. SPECTRAL LINES... 62
FIGURE 4.6. HYDROGEN... 62
FIGURE 5.1.SCHEMATIC PICTURE OF THE PLASMA GENERATOR... 72
FIGURE 5.2. HOMOGENEITY OF THE PLASMA TREATMENT... 74
FIGURE 5.3.REPRODUCIBILITY... 75
FIGURE 5.4. ATOMIC SPECTRAL LINES AND OH BAND INTENSITIES. ... 75
FIGURE 5.5.A.LPS DERIVED FROM E.COLI O111:B4... 76
FIGURE 5.5.B.LPS DERIVED FROM SALMONELLA ABORTUS EQUI... 76
FIGURE 5.5.C.LPS DERIVED FROM E.COLI K-235 ... 76
T A B L E O F F I G U R E S
FIGURE 5.8. ZYMOSAN AND LPS(E.COLI O111:B4) ... 79
FIGURE 5.9.PEPTIDOGLYCAN... 79
FIGURE 5.10.LPS ALONE AND COMBINED WITH MDP... 80
FIGURE 6.1.HYDROGEN... 91
FGURE 6.2. LIPID A LAYER... 92
FIGURE 6.3.AR VERSUS AR:H... 93
FIGURE 6.4.FTIR ... 93
FIGURE 6.5.C-H BANDS... 94
I N T R O D U C T I O N
1. General Introduction
1.1 The human immune system
Microbial organisms, namely bacteria, viruses, molds and protozoa, can overcome the natural barriers of the mammalian body and invade into organs, tissue or the blood stream. Some of them have symbiotic functions like numerous bacteria in our gastro-intestinal tract; others act as pathogens, which can cause local or systemic inflammation and various diseases.
1.1.1 Innate and adaptive immunity
The mammalian immune defense against body-foreign organisms is based on two closely interacting systems, the innate and the adaptive immunity[1]. The front line of the host defense, the innate immune system, is carried by monocytes, macrophages, dendritic cells and neutrophils, which are localized in blood and tissue. They react fast and unspecific by activating their phagocytic capacity and the release of messenger substances, like immune-stimulatory cytokines, chemokines, and acute phase proteins. They effect the body- temperature, the vascular permeability and attract other cells to initiate the second defense line, the adaptive immunity[2]. The recognition of invading pathogens is mediated via specialized receptors, pathogen recognition receptors (PRR)[3] which are able to recognize and bind conserved motifs, pathogen-associated molecular patterns (PAMP)[4] found on microbial pathogens, which do not occur in mammals[5].
Thereby the innate immune system is able to prevent via phagocytosis the spreading of most pathogenic organisms and additionally activate the adaptive immunity to react directly and specific against invading microorganisms. The antigen-specific immune system answers with the production of high-affinity antibodies and the generation of cytotoxic T-cells provides long-lasting protection.
1.1.2 Cytokines
A primary reaction induced by microorganisms is the release of a variety of pro-
I N T R O D U C T I O N
modulate the host’s immune-reaction[6,7]. Cytokines have a molecular weight of 15-30 kD, and represent soluble proteins that are produced in response to stimulation, and function as chemical messengers for regulating the innate and adaptive immune systems. They are produced by all cells involved in immunity and the responses towards cytokines include increasing or decreasing expression of membrane proteins, proliferation, and secretion of effector molecules[8]. It is common for different cell types to secrete the same cytokine or for a single cytokine to act on several different cell types. Cytokines are redundant in their activity, meaning similar functions can be stimulated by different cytokines, and they are often produced in a cascade, as one cytokine stimulates its target cells to make additional cytokines. They can also act synergistically (two or more cytokines acting together) or antagonistically (cytokines causing opposing activities).
A prominent physiological reaction according to pro-inflammatory cytokines, like IL-1β, IL-6 and TNF-α is fever induction. The current understanding of the mechanism of fever in mammals is, that these pro-inflammatory cytokines result in the expression of the enzyme cyclooxygenase-2 (COX-2), which mediates prostaglandin E2 (PGE2) synthesis. PGE2 triggers an intracellular signaling cascade that changes the set point of the body temperature in the hypothalamus and results in fever[9].
1.2 Pyrogens
The so-called pathogen-associated molecular patterns are not only found on living organisms. During the hosts immune-response, antibiotic therapies or spontaneously they are liberated from microbial organisms[10] into the environment. They are then recognized by the human body and the whole cascade of immune defense reactions takes place, like cytokine release, widening of the blood vessels and attraction of other immune cells. Therefore these molecules are also called pyrogens, because the first sign of their presence in the human body is a rise of the body temperature, which can escalate in an overwhelming reaction leading to shock, multiple organ failure and death[6,11,12].
I N T R O D U C T I O N
1.2.1 Lipopolysaccharide (LPS)
Lipopolysaccharide (figure 1.1.C.) is the principal glycolipid component of the outer membrane (figure 1.1.B.) of Gram-negative bacteria (figure 1.1.A.). It can reproduce many of the features of an authentic Gram-negative infection, therefore it is the best studied and the most potent molecule among the already known pyrogens[11]. LPS is released by destroyed bacteria and is captured by the immune system, resulting in immediate host-responses like production of pro-inflammatory cytokines[13] as well as chemokine release and complement activation[14].
LPS is a complex amphiphilic molecule with a molecular weight of about 10 kD.
It consists of a lipid A anchor (figure 1.1.D.), a polysaccharide core and chains of repetitive carbohydrates and with this lipid A anchor LPS is attached to the outer membrane of Gram-negative bacteria. The repeating oligosaccharides are strain-specific for diverse Gram-negative species and appear to be highly variable[15,16].
FIGURE 1.1AGRAM-NEGATIVE BACTERIUM.
ELECTRON MICROGRAPH OF ESCHERICHIA COLI (A), TOGETHER WITH A SCHEMATIC REPRESENTATION OF THE LOCATION OF LIPOPOLYSACCHARIDE (LPS; ENDOTOXIN) IN THE BACTERIAL CELL WALL (B) AND THE ARCHITECTURE OF LPS(C).ALSO SHOWN IS THE PRIMARY STRUCTURE OF THE TOXIC CENTRE OF LPS, THE LIPID A COMPONENT (D).GLCN,D-GLUCOSAMINE;HEP,L- GLYCERO-D-MANNO-HEPTOSE;KDO,2-KETO-3-DEOXY-OCTULOSONIC ACID;P, PHOSPHATE.[14]
Due to its amphiphilic character, LPS is highly attractive to its self and forms huge micelles and clusters[17], which are sticking to different materials
I N T R O D U C T I O N
1.2.2 Lipid A
Lipid A, the membrane anchor of lipopolysaccharides is the biological active principle and free lipid A shows most of the endotoxic activities[19]. It is typically composed of a β-D-GlcN-(1-6)-α-D-GlcN disaccharide carrying two phosphoryl groups. Both phosphates can be substituted with groups such as phosphate, ethanolamine, and others. To this structure up to four acyl chains are attached which can be substituted by fatty acids[20]. Meanwhile it is well-known, that chemical modifications are affecting the bioactivity of Lipid A[21,22].
1.2.3 Lipoteichoic acid (LTA)
The cell wall of Gram-positive bacteria consist about 6 % of LTA, which is embedded in the cytoplasmic membrane via a lipophilic anchor which is linked with a disaccharide to its backbone. The backbone is build of glycerol- phosphate or ribitol-phosphate units, which are repeated up to 50 times. The residues of the backbone are substituted with different groups, e.g. N- acetylglucosamine (GlcNAc), hydroxy-groups or D-alanine, in the case of Staphylococcus aureus (S. aureus)[23]. LTA is also able to induce cytokines and chemokines[24-26]. But most of the commercial LTA preparation are heavily contaminated with endotoxin, nucleic acids and lipoproteins[27].
Additionally, the commonly used hot phenol extraction method leads to loss of bioactivity, especially for LTA of S. aureus[27]. A butanol extraction method made a highly pure (> 99% purity, < 30 pg LPS per mg LTA) and cytokine inducing material available[28]. Additionally, chemical synthesis of LTA derivatives revealed structure-function relationship of cytokine induction[24]. It is still controversially discussed, whether LTA is the main immune stimulatory principle of Gram-positive bacteria or whether peptidoglycan (PGN) alone or in combination with LTA triggers the effects of Gram-positive infections[29].
1.2.4 Peptidoglycan (PGN)
The bacterial cell walls of Gram-negative and Gram-positive bacteria contain PGN. Gram-negative bacteria possess only a small layer while Gram-positive bacteria have a 40 up to 50 layer thick sacculus. PGN is build of sugar chains, which are linked by amino acids. These sugar chains consist of alternating units of N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). The
I N T R O D U C T I O N
cross-linking amino acids are alternating L- and D-isomers, additionally building interpeptide bridges[30]. Peptidoglycan is known to evoke severe immune responses[31,32] which mirrors the properties of a Gram-positive sepsis. It is a well-known target for many clinically useful antibiotics that inhibit cell wall synthesis[30].
FIGURE 1.2.SCHEMATIC DIAGRAM OF THE PEPTIDOGLYCAN SHEET OF STAPHYLOCOCCUS AUREUS.
G=N-ACETYL-GLUCOSAMINE;M=N-ACETYL-MURAMIC ACID;L-ALA =L-ALANINE;D-ALA =D-ALANINE;D-GLU =D-GLUTAMIC ACID;L-LYS =L-LYSINE.
(TODAR’S ONLINE TEXTBOOK OF BACTERIOLOGY)
1.2.5 Muramyl dipeptide (MDP)
The smallest breakdown products of PGN are muropeptides, which are released through lytic enzymes, produced from either the host or the bacteria, during bacterial growth or due to antibiotic administration. The diverse amino acids occurring in the PGN of different species makes a variety of muropeptides possible. The most prominent muropeptide is muramyl dipeptide (MDP), which represents the minimal structure for adjuvant activity[33].
Direct effects of muropeptides on the immune system are rare, most often observed at very high concentrations or in the case of LPS-contaminations. But they seem to be strong immune amplifiers and are therefore responsible for priming and synergistic effects[34]. This illustrates that a combination of immune stimulating components mediates the immune response to bacterial pathogens[35].
1.2.6 Zymosan
Zymosan is a fine, gray powder insoluble in water, which disperses easily to
I N T R O D U C T I O N
(Saccharomyces cerevisiae) and it is composed mainly of carbohydrates as a glucose polymer and is mannan-rich, but contains also small amounts of nitrogen, phosphorus, and magnesium. The removal of most of the nitrogen and magnesium-phosphorus complex is possible without destroying the immunological properties of zymosan[36]. Zymosan activates the alternative complement cascade and is a potent stimulator of alveolar macrophages, which induces the release of cytokines, e.g. IL-8 from human neutrophils and pro- inflammatory cytokines in immune cells. For a lot of physiological and immunological studies, zymosan is used as a fungal model substance[37-39].
1.2.7 Further stimuli
Further possible pyrogenic contaminants can be exotoxins[40], enterotoxins[41], viruses[42] and fungi[43]. Bacterial DNA[44] as well as other components of bacterial origin, like porins and flagellin[45] have been shown to evoke immune- responses. Organic and inorganic dust particles are also able to induce an inflammatory reaction[46], although the nature of their pyrogenic components is not yet known.
1.3 Pyrogen Testing
At the end of the 19th century, fever-inducing contaminations in injectible drugs were found to be heat-stable. The connection was established between pyrogenicity and the endotoxin originating from Gram-negative bacteria. The routine detection became necessary since the development of commercially available drugs in the 1930s and by the introduction of large volume parenterals in World War II. Therefore the rabbit pyrogen test was developed, which was soon incorporated into the United States Pharmacopoeia (USP)[47,48]. The discovery of the coagulation reaction of the hemolymph of the horseshoe crab Limulus polyphemus when in contact with bacterial endotoxin led to the development of the LAL test (Limulus Amoebocyte Lysate)[49], which is used for the detection of LPS contaminations in parenteral drugs. In the 1970s, radiopharmaceutical drugs were introduced into clinical practice, but a pyrogen testing of these short-lived compounds in rabbits is impossible. The solution was the LAL test[50], which has replaced the rabbit pyrogen test for these and numerous other applications[51]. But a full replacement of the rabbit pyrogen
I N T R O D U C T I O N
test by the LAL did not take place because the LAL is not capable of detecting contaminants other than LPS and is disturbed by some protein and lipophilic components.
1.3.1 The Rabbit Pyrogen Test
Even though fever responses to bacterial pyrogens have been observed in a variety of animals, e.g. dogs, cats and horses, rabbits were adopted as the standard animal for the USP test[52]. Briefly, the test consists of the following steps: rabbits are put in restraining boxes and rectal probes to record the body temperature are inserted. Test solutions have to be pre-warmed to 37°C before injected into the ear vein at a maximum volume of 10 ml/kg body weight.
Temperature changes are recorded at least 1, 2 and 3 h after injection. The test is performed on three rabbits per test solution and concentration. It is a “pass- or-fail” test, per definition failed means, that one out of three rabbits shows a temperature rise of 0.6°C or more according to the individual control temperature, or the sum of all three rabbits is higher than 1.4 °C. This leads to a repetition of the test with additional 5 rabbits. If not more than 3 out of the eight show individual rises of 0.6 °C or the sum of all does not exceed 3.7 °C the material meets the requirements for the absence of pyrogens[48].
Drawbacks of the rabbit pyrogen test
The sensitivity of rabbits toward endotoxin preparations depends on the strain used and the experimental conditions, e.g. age, gender and housing conditions[53]. Even if the highest permitted volume (10 ml/kg body weight) is injected, the detection limit is restricted to 50-350 pg (i.e., 0.5-3.5 IU) of LPS/kg, while the human fever threshold is around 30 pg/ml[54].
Also drugs that influence the central or peripheral mechanisms of body temperature regulation cannot be tested by the rabbit pyrogen test, e.g.
antipyretic drugs, steroids, dopamine[55,56]. The same applies to drugs, which can cause immunological reactions (for example, immunoglobulins), oily suspensions, or detergents. Also, the rabbit pyrogen test cannot be used for cellular preparations, such as blood components and stem cells. Drugs that should be injected intravenously, subcutaneous or intramuscularly, e.g.
I N T R O D U C T I O N
quality-control purposes. However, the rabbit pyrogen test is not suitable for the control of such a limit since it is not a quantitative test, i.e. it gives only a pass/fail result[57].
1.3.2 The bacterial endotoxin test (BET)
The "bacterial endotoxin test" (BET) refers to a number of tests, which detect endotoxins from Gram-negative bacteria. The reaction is based on the clotting reaction of the hemolymph of the horseshoe crab, therefore also called “Limulus amoebocyte Lysate (LAL) test”. There are various approaches for measuring the LPS-induced reaction, e.g. by clotting, kinetic turbidimetric measurement, chromogenic endpoint or a kinetic reaction[58].
The main advantage of the LAL test is the availability of an endotoxin standard that permits the semi-quantitative or quantitative measurement of endotoxins[59]. The detection limit is usually about 3 pg/ml, i.e. 0.03 EU/ml (1.5 pg/ml final concentration) for the most sensitive test variants. By quantifying LPS, the LAL detects the most common and most potent pyrogen known with high sensitivity. The LAL test is undoubtedly suitable for the pyrogen testing of a number of products, but not for all products.
Drawbacks of the LAL
The assay can only be performed with liquid samples and only endotoxin originating from Gram-negative bacteria can be detected. Immune-stimulating components from Gram-positive bacteria, such as lipoproteins, peptidoglycan and lipoteichoic acids, or other pyrogens pass the LAL test, without giving a signal. Therefore, the LAL does not reflect the inflammatory potency of a specimen in humans. Drugs which interfere with the clotting system, i.e. through inhibition (binding of divalent cations such as EDTA, citrate, protease inhibitors) or enhancement (high protein content, proteases), cannot be tested with the LAL test[60]. Furthermore, a number of endotoxin-binding components from plasma are known to mask LPS in the LAL test[61]. The not reflected potency of LPS in mammals, and the false positive reactions for glucans, e.g. cellulose filtered materials or herbal products are further limitations. Special problems are evident for solid materials, like medical devices[62].
I N T R O D U C T I O N
1.3.3 Human cell-based assays
The understanding of the human fever reaction led to a development of test systems based on the in vitro activation of human monocytes. Peripheral blood mononuclear cells (PBMC) were used to detect endotoxin by monitoring the release of pyrogenic cytokines[63], like IL-1-β, TNF-α and IL-6. Meanwhile, a number of different test systems, using either whole blood, peripheral blood mononuclear cells (PBMCs) for the monocytoid cell lines MONO MAC 6 (MM6) or THP-1 as a source for human monocytes and various read-outs were established[64-66].
An important feature of the new cell-based test methods is that they are based on the use of monocytoid cells of human origin. The response of humans, horseshoe crabs[67] and rabbits[68] toward Gram-negative endotoxin has been studied. The comparison reveals a good correlation of the whole blood test with the rabbit pyrogen test and the limit of detection is < 50 pg LPS/mI (4.2 pg/ml final concentration). A more closely mirror of the actual response of humans towards pyrogenic contaminations was achieved using cells based assays of human origin. They predict more accurately the pyrogenicity of a substance in humans.
In this study the human whole blood model (In vitro Pyrogen Test, IPT) was used. Fresh human whole blood is diluted (10% v/v in saline) and incubated in the presence of the sample to be tested. The production of IL-1β is subsequently measured in the supernatant by ELISA (enzyme-linked immuno- sorbent assay), which correlates with the content of pyrogens in the sample[65,69-71]. The blood can be used for up to two hours after withdrawal, and the donor-dependent variation in the threshold of IL-1β release is low. The use of cryo-preserved blood is also possible[72] and the test system is not inhibited by up to 10% dimethyl sulphoxide (DMSO); however, cryo-preserved blood gives a much stronger IL-1β response to endotoxin. The human whole blood test for parenterals passed already the validation study[73] and proved to have a lower detection limit than the rabbit test, it is more accurate, cost and time efficient. It is able to detect Gram-positive pyrogens and therefore meets the quality criteria for pyrogen detection[70].
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1.4 Medical Devices
According to the International Organization for Standardization (ISO 10993:
Biological evaluation of medical devices) medical devices are defined as (ISO 10993-1):
“Any instrument, apparatus, appliance, material or other article, including software, whether used alone or in combination, intended by the manufacturer to be used for human beings solely or principally for the purpose of
• diagnosis, prevention, monitoring, treatment or alleviation of disease;
• diagnosis, monitoring, treatment, alleviation of, or compensation for an injury or handicap;
• investigation, replacement or modification of the anatomy or of a physiological process;
• control of conception,
• dental devices”
A medical device consists of a variety of materials, which are one of the factors that play an important role in the question whether a medical device meets the requirements for human use, i.e. whether it is “biocompatible”. To consider the biocompatibility, one must take into account the applied materials and especially the complete medical device as a whole and its intended use in the human body. In general, three categories of contact with a human being are distinguished:
• Surface devices, where contact is made with the skin, intact mucosal membranes and breached, or compromised surfaces, for example ECG electrodes.
• External communicating devices, where indirect contact is made with blood, tissue, or bone, for example dental filling materials.
• Implant devices, where direct contact is made with blood, tissue, or bone, for example breast implants.
The use of medical devices is increasing every year by 10% worldwide. To underline the importance of medical devices numbers of implanted or used devices are given in (table 1.3.). For example 100 000 left ventricular assistant devices (LVAD) are implanted annually worldwide and increase the survival
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possibility of suffering patients by 52% for the first year, compared to the typical medication which gives a 25% chance of surviving[74].
FIGURE 1.3.NUMBERS OF MEDICAL DEVICES PER YEAR WORLDWIDE
BUDDY D.RATNER, HTTP://WWW.UWEB.ENGR.WASHINGTON.EDU/RESEARCH/TUTORIALS/INTROBIOMAT.HT ML UWEB,UNIVERSITY OF WASHINGTON ENGINEERED BIOMATERIALS
Nevertheless, around 10% of all patients have to be reoperated due to implant failure[75].
The ISO 10993 standard plays an important role in the assessment of biocompatibility of a medical device. In principle, a great number of tests, e.g.
cytotoxicity, sensitization, irritation and implantation assessments have to be undertaken depending on the intended use of the medical device. The registration process of medical devices contains basic requirements, including pyrogen-free quality. Pyrogen tests currently used for medical devices have various drawbacks.
1.5 Pyrogen testing of medical devices
The rabbit pyrogen test requires that sample test materials are implanted into rabbits and a change in body temperature indicates a pyrogenic reaction. One problem is that the rabbit may respond differently to some pyrogenic contaminations than humans. Additionally this method can only be performed with small implants. The destruction of tissue and stress during implantation into the animal may itself cause an inflammatory reaction that does not necessarily reflect the pyrogenic contamination of the material.
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The rabbit pyrogen test can be also performed by injecting a solution gained by rinsing the implant. Generally, this procedure is performed using purified water or saline solution. The extraction efficacy probably depends on temperature, shaking intensity and storage conditions. It was shown recently, that pyrogenic contaminations cannot be extracted to its full extent[76-78]. It is also unclear whether adherent pyrogens and pyrogens in solution have different inflammatory properties. There are strong indications for this regarding the inflammatory capacity of the Gram-positive pyrogen lipoteichoic acid (LTA).
Adherence of LTA to a polystyrene surface amplified its capacity to induce pro- inflammatory cytokines like TNF-α by about 3 log orders[79]. Thus, determination of the activity of LTA in a rinsing solution would grossly underestimate its biological potency when adherent to the respective medical device.
A partial alternative method to the rabbit pyrogen test is the Limulus Amoebocyte Lysate (LAL) assay. The assay can only be performed with liquid samples, thus again only a rinsing solution of the biomaterial can be assessed.
The rinsing solution is prepared with purified water, with or without a detergent (Tween 20); therefore, the same difficulties as mentioned before have to be taken into consideration.
1.6 Clinical experience with pyrogenic reactions and medical devices
Pyrogenic effects caused by surface contaminations of medical devices in human beings are not yet investigated to its full extent. Summarizing the facts known already, it becomes clear that there is evidence that pyrogens, medical devices and severe clinical problems are closely related[80]. A pyrogenic surface contamination would affect the body mainly depending on the way of contact between the device and the immune system. Monocytes and macrophages, the first immune-defense of the mammalian body to bacterial, fungal, protozoal and viral components are present in the blood stream and in certain parts of the tissue[14]. Their main contribution to the human body protection against pathogens is the recognition of body-foreign molecules via specialized receptors[11] and the following release of messenger substances,
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called cytokines[6], like IL-1β, IL-6 and TNF-α[81]. They are able to induce a variety of physiological changes like fever, widening of the blood vessels, attraction of other immune-cells, which sometimes can lead to severe consequences like tissue destruction, shock, multiple organ failure and death[14]. Therefore, cytokines are good indicators of a starting inflammation in the body. Several studies were undertaken to show that lipopolysaccharide (LPS), the most potent pyrogen originating from Gram-negative bacteria, on medical devices can induce cytokine release and lead to severe problems, like tissue and device degradation[82], chronic complaints, bone pain, up to the loosening of the device[83-85]. The main problem related to orthopedic joint prosthesis is the aseptic loosening of the implant and this is shown by several authors to be mediated by macrophages[86,87]. Once activated they lead via cytokine release to an acceleration of osteoclast differentiation and maturation which causes osteolysis at the bone-implant interface[88] and bone- resorption[89]. In these studies, the cytokine release was triggered by metal ions or polyethylene particles, which seems to have immune-stimulating competence. Even without any added stimulus a certain probability was found that a cytokine-release occurs e.g. in relation to mesh biomaterials[90,91], which have a clinical importance of estimated 1 million implanted meshes per year worldwide. It is not clear whether the immune reaction is due to the material or possible contaminations. In other studies a LPS-mediated osteoclast differentiation via cytokine production was demonstrated[92,93]. Some authors even conclude that the main reason for implant failure is due to endotoxin contaminations originating from dead bacteria on the device surface[80].
Pyrogens are not easy to detect and once there, hard to remove. Up to now it was not possible to measure properly the contamination grade on the surface.
Tests were done by employing extracts (washing solutions) of the device with or without Tween (detergent) in the LAL (Limulus Amoebocyte Lysate assay) or the Rabbit test. Meanwhile it was shown that the amount washed off and tested is not at all reflecting the amount, which is sticking on the surface[77,78]. It was possible to show via radioactive-labeled LPS that it is highly adherent to various surfaces and different sterilization methods were not capable to remove the LPS from the device, depending also on the surface properties[94]. Therefore,
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freedom of a device and the LAL is clearly underestimating the pyrogenic capacity. But even via LAL testing, which is not reflecting the complete pyrogenic amount present on the surface, it was possible to show LPS contaminations in wound dressings made of natural biomaterials[95] and natural rubber latex products[96].
Gram-positive bacteria are gaining over the last decade more clinical importance. From approximately 500 000 cases of sepsis annually in the US with multiple organ dysfunction syndrome (MODS) and a mortality of 35%, 50%
were caused by Gram-positive bacteria[32]. The Gram-positive bacterium Staphylococcus aureus is the one most common isolated of sepsis patients and also highly involved in biofilm formation[97]. The main immune-stimulating components originating from Gram-positive bacteria are peptidoglycan (PGN)[31] and lipoteichoic acid (LTA)[23], which are sharing many of the pathophysiological properties with LPS, e.g. the pro-inflammatory cytokine- release[98]. For LTA it was shown, that the biological activity is strongly enhanced, when it is coated on the surface[79]. Other immune-stimulating components enhance their biological activity in a synergistic manner[34], which simulates much more the physiological situation than investigating the action of highly purified bacterial compounds alone. There is a variety of immune- stimulating components, which are able to evoke cytokine-release[99], which are not investigated yet. The possibility that these compounds adhere to surfaces and induce severe clinical problems cannot be excluded.
The in vitro pyrogen test (IPT), which is based on the human whole blood reaction towards any pyrogenic substance, is not distinguishing from where the residuals do origin. Every compound, which is able to induce an inflammatory response, activates the monocytes, which are reacting with a release of IL-1β, which is than measured by ELISA and the immune stimulatory capacity of the surface is clearly reflected. Metal ions, particles, surface properties, bacterial, fungal, protozoal or viral residuals can be detected, which is shown for some of these already[78,100]. The fact that the test can be done directly on the surface is taking into account the problem of the molecules, adhering to the surfaces and also leachables, which are released over time. Distinctions between different stimuli cannot be made but to guarantee consumer safety and to
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increase implant success the IPT opens new dimensions of pyrogen and biocompatibility testing.
1.7 Sterilization of Medical Devices
For medical devices, recent statistics show that nosocomial infections are responsible for several thousands of death each year worldwide[101] and are every day responsible for a huge number of post surgical complications.
Moreover, the risk linked to the transmission of the Creutzfeld Jacob Disease (CJD) by contaminated surgical instruments has also been reported recently[102]. Instruments entering the human body have to be therefore sterilized and decontaminated by removal or inactivation of any harmful biological residuals, especially in invasive surgery and dental practice. The contaminations can origin from microorganisms, e.g. bacteria, viruses, molds, or their residues, which are often not sufficiently destroyed and removed by routine sterilization procedures. For medical devices, the principal methods of sterilization and their relative mechanisms are presented in Table 1.4.[103].
Sterilization methods Modes of action Ethylene oxide
Alkylating agent
Affects essential components of the cell Denaturation of proteins and DNA
Heat sterilization (Moist heat)
Denaturation of proteins Inactivation of enzymes Oxidizing agent, alterations of lipids
Spores: denaturation of DNA Inhibition of the germinal system Irradiation by
gamma rays and e-beam radiation
Interferences with DNA, RNA (strand breakage, base damage)
Inhibition of DNA replication Inhibition of the protein synthesis repair
TABLE 1.4.STERILIZATION PRINCIPLES FOR MEDICAL DEVICES
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All above mentioned sterilization methods have their drawbacks, which are not easy to overcome. Ethylene oxide (EtO) gas, for example is used in combination with Freon-12 (CCl2F2) at pressures up to 3 bar in a special sterilization chamber for one to three hours followed by a twelve hours aeration period. Therefore, large-scale industries consider that process as inefficient and costly. Even worse is the fact, that ethylene oxide is declared as mutagenic and carcinogenic and therefore highly toxic for the working personnel and the environment. The gamma sterilization approach is difficult, expensive and interacts with the properties of polymers by breaking bonds and cross-linking chains within the material. Radioactive sources require expensive waste disposal procedures as well as radiation safety precautions. Every sterilization method, which involves high temperatures like dry heat or high-pressure autoclaving, is not suitable for any kind of heat-sensitive material, like corrodible metals, plastic-made devices, or polymers. For example glass vials used by the pharmaceutical industry are sterilized by washing and a heat tunnel (260- 350oC)[104]. The main disadvantage is the very high-energy consumption and the floor space occupied by the installation, which is important for the large heating and cooling zones necessary to avoid thermal shock.
Low-temperature decontamination and sterilization of surfaces gained a major attention in recent years, especially in the medical device and pharmaceutical industry. Therefore, much effort has been devoted in the last years to develop and validate techniques of sterilization and decontamination of wide range of materials. The selected technique should fulfill the following requirements:
• Efficiency of treatment (low treatment duration at low costs)
• Low temperature to treat thermally sensitive materials
• Applicability to a wide range of materials and shapes
• Avoidance of toxic reagents or by-products 1.8 Plasma-based Sterilization
Compared to the conventional sterilization and decontamination methods non- equilibrium plasma discharges offer fundamental advantages - they can be operated at low temperatures suitable for the treatment of heat-sensitive materials and since they are usually sustained in non-toxic gases like hydrogen
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(H2), oxygen (O2), nitrogen (N2), argon (Ar), or air, their operation is harmless both for the operator and the environment. According to these reasons plasma- based sterilization and decontamination attracts increased attention in the last years, which can be seen by the number of publications devoted to this topic[105-111].
1.8.1 Definition of gas plasmas
Following the order of increasing energy, solid, liquid, and gaseous, gas plasmas can be described as the fourth state of matter, like found in stars. They are defined as gas, made of ions and free electrons only. The plasmas employed for sterilization appear to be much colder (< 100°C) and consist of ionized gases. Besides ions and electrons, they contain uncharged particles, such as molecules and radicals. Ions and neutral atoms can be in an excited state and therefore also de-excite, which means they loose their internal energy, either spontaneously by emitting a photon or through collision with other particles or a surface[112]. Collision with a surface can lead to the physical sputtering of their elements or to chemical reactions, such as oxidation. Photons emitted by excited species can also induce chemical reactions on a surface: UV photons are particularly efficient in this respect[113].
1.8.2 Plasma generation
Plasmas are usually produced by subjecting a gas or gas mixtures to an electric field, either of constant or alternating amplitude, therefore called electrical discharges. The charged particles, especially light electrons, are accelerated in the applied electric field and couple their energy via inelastic collisions with other particles[114-116]. These collisions lead to the production of ions, chemically active particles (e.g. O, H, OH) or excited particles, having high internal energy. Such created particles can subsequently interact with the treated substrate, which can lead to physical cleaning of the surface or to introduction of chemical modifications. Moreover, radiative de-excitation of excited particles can result in generation of intense UV radiation that may also effectively alter chemical composition of the treated substances.
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1.8.3 Plasma generator
The plasma reactor in our studies[117-121] is schematically depicted in (figure 1.5.). It consists of a stainless steel cylindrical vacuum chamber (200 mm in diameter and 380 mm in length) equipped with several diagnostics windows and one port for the sample introduction. The processing chamber is connected to the gas inlet system, which is composed of MKS mass flow controllers attached to the gas lines (H2 and O2) and it is evacuated by a primary pump and a roots blower allowing a base vacuum of 0.3 Pa. The plasma is sustained by microwaves with excitation frequency 2.45 GHz. The microwave circuit includes the microwave supply, a circulator protecting the power supply from the reflected power, a three-stub impedance matching system, and a rectangular- circular wave-guide transition. Microwaves are introduced in to the plasma chamber through a silica window placed at the extremity of a circular 100 mm wave-guide.
FIGURE 1.5.SCHEMATIC PICTURE OF THE PLASMA GENERATOR
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1.8.4 Plasma characterizing methods
The physical properties of the generated plasma can be characterized by the following devices:
• Langmuir probe
One or more electrodes are inserted into the plasma chamber and the constant or time-varying electric potential between the electrodes or the electrodes and the surrounding vessel is determined. This enables the detection of the electron temperature, the electron density, and the plasma potential.
• Optical emission spectroscopy
The range of electromagnetic spectra in which a substance radiates is determined. The wavelength defines the identity of the atom while the intensity is directly proportional to the concentration of the atom.
• Infrared pyrometry
The temperature is measured via the object’s capability to emit electromagnetic radiation.
1.8.5 Possible mechanisms, responsible for the sterilization effect
Low pressure plasma sterilization has been the object of many general studies and reviews which discuss different potential mechanisms of its action[105- 108,112,122-124]. The approach is based on non-toxic gas mixtures, to produce reactive species (such as O, OH) and UV light at different wavelengths.
By adjusting the process condition (reactor geometry, pressure, power, frequency, flow and gas composition), several mechanisms can be activated and operate alone or simultaneously, which lead to sterilization with different kinetics and different effects. Typically, these effects relate to the production of UV of different wavelength which interacts with the DNA of spores, of active radicals which react with the outer layer of spores and cause denaturation of protein and lipid components, and of ion and/or electron bombardment which sputters the contaminant material. All these mechanisms can be operative during the surface treatment and lead to different sterilization kinetics[121,124].
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1.8.6 Plasma-interaction with biological molecules
In the case of bio-functional molecules strand scissions in plasmid DNA exposed to low-power plasma were observed, combined with the finding of nicked, linear, cross-linked and multi-fragmented plasmid DNA[107]. Employing bovine serum albumin (BSA), as a model protein to plasma exposure a rapid destruction of the protein structure was detected. Investigations under the same conditions with the enzyme soybean lipoxygenase (SLO) showed an abatement of the enzymatic activity[108].
Compared to the plasma-based sterilization of spores, depyrogenation by means of plasma is a relatively new topic. The possibility of employing non- equilibrium plasmas for depyrogenation had been proposed[124-126], but until recently very little work was published on the subject.
A I M O F T H E S T U D Y
1.9 Aim of the study
• The first part of the thesis is dedicated to the development of a pyrogen test system for solid biomaterials, which represents a suitable model for the human body reaction, by the mean of mimicking the human fever reaction in vitro. The test principle of the in vitro pyrogen test (IPT) should be transferred to this new application, which should allow direct contact of human whole blood with the solid test material. This approach should both address whether the material itself is immunological inert and/or whether it carries inflammatory contaminations.
• In the second part of the study a system employing low-temperature plasmas should be developed showing a significant decrease of the biological activity of LPS or Lipid A. The effect of microwave discharge on the pyrogen bioactivity should be studied in different oxygen and hydrogen containing mixtures with the aim of identifying the crucial depyrogenation agent and to identify possible side effects.
• The third part of the study should employ immune stimulating agents from different origins, to take into account the increasing attributed importance of especially Gram-positive bacteria. It should be verified if firstly it is possible to detect these stimuli on surfaces and secondly if there is a possibility to remove them by using the low-pressure microwave plasma discharge.
• Finally, the mechanisms of action by low-pressure microwave discharges should be investigated. It should be clarified if the removal of the bioactivity is due to pure etching of the material, or if other mechanisms, for example chemical modifications play a major role.
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2. An in vitro pyrogen safety test for immune- stimulating components on surfaces
Abbreviated running title:
In vitro pyrogen test for surfaces
Marina Hasiwaxy, Karin Kullmann, Sonja von Aulocky, Christoph Kleinx and Thomas Hartung*xy
xEU Joint Research Centre, ECVAM, Ispra, Italy
yBiochemical Pharmacology, University of Konstanz, Germany
in press Biomaterials
Keywords
In vitro test, Human Whole Blood, Lipopolysaccharide, Lipoteichoic Acid, Peptidoglycan, Zymosan
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2.1 Abstract
New materials and surgical techniques are constantly increasing the use of biomaterials and medical devices. Any kind of contact with tissue or blood of the human body commonly leads to inflammation of varying extent, sometimes resulting in severe health problems. This could be caused by limited biocompatibility or by pyrogenic contamination of the material.
We adapted the recently validated In vitro Pyrogen Test (IPT) based on human whole blood cytokine release to the safety testing of biomaterials. Human whole blood is brought into direct contact with the surface of the test material and the release of the pro-inflammatory cytokine IL-1β is measured. This procedure represents a human-relevant assay allowing the detection of pyrogens of different origins, e.g. Gram-negative (lipopolysaccharide, LPS) or Gram–positive (lipoteichoic acid, LTA), peptidoglycan (cell-wall components of most bacteria) and fungal zymosan by direct material contact. The sensitivity of the test system allows a starting concentration of 10 pg/ml for LPS, 10 ng/ml for zymosan and 1 µg/ml for LTA and peptidoglycan from different strains.
Furthermore it is shown, that the test for solid materials can be carried out with cryo-preserved blood, which results in an even lower detection limit.
