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Methods of Biomaterials Testing
Special Biology: Blood & Vessel
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Blood Compatibility
Mainly: Inhibition of blood clot formation
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Materials in Bloodflow
Venous catheter
Heart Valves
Vascular StentsCardiac Assist Devices
Artificial Hearts
Dialysis filter
Dialysis shunt
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Blood CompatibilityCells and proteins come in contact with foreign materials
SubendothelialSubendothelial matrixmatrix
PlateletsPlatelets
Blood clotBlood clot Endothelial cellEndothelial cell
RBCRBCWBCWBC FibrinFibrin
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The Triade of Virchow
Clottability
(Endothelium-)Surface Properties
Flow Conditions
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Red Blood Cells
Hemolysis• Reasons
– Important for polymers (plastiziser)– Result of corrosion of metals– Mechanically at heart valves
• Measurement– Absorption of Hemoglobin– Reaction with cyanide and measurement of the
absorption of cyan-methemiglobin λ = 546nm• Consequences
– Activation of blood platelets and clotting cascade
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White Blood CellsActivation of Phagocytes• Release of reactive oxygen species
– 2 O2 + NADPH → 2 O2- + NADP+ + H+
– O2.- + 2H+ → H2O2 + O2
– H2O2 + Cl- → HOCl + H2O• Release of inflammatory cytokines
– Interleukin 1– Interleukin 6– TNF-α
Consequences– Activation of endothelium cells– Proliferation of vascular smooth muscle cells– Recrutation and activation of other cells
Activation of Lymphocytes• So far not described for implants in the blood
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Reactive Oxygen SpeciesBecause of short lifetime (<< 1 second) direct measurement is
often difficult• Hydrogen Peroxide
– Direct measurement
• Other free radicals– Detection with luminescent probes (luminol or lucigenin) in real-time– Chromogenic or fluorogenic substrates
• Detection of free radicals effects– Lipide peroxidation: Malondialdehyde (MDA) formation
• Direct detection with HPLC• Detection with Tiobarbituric acid (pink product)
– Consumption of scavengers (radical antagonists)• Ascorbic acid• SH groups (glutathione)
H2O2 + TMB Light blue product
H2SO4
HRPDeep yellow
productmeasure at 450nm
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Thrombocyte ActivationReasons
– Activated endothelium cells (expression of vWF)– Defect of the endothelium cell layer
• Uncovered collagen, fibronectin und laminin• Negatively charged surfaces
– Fibrin and activated clotting factors– Denatured proteins– Mediators
• Noradrenalin• ADP
– Decreased blood flow
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Platelet Activation Pathways
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Platelet Activation Pathways
Platelet
AdrenalineCollagen
GPIb
vWF
GPIIb/III
a (CD41
)ADP
Endothelium
GpIIb/III
a (CD41
)
P-selectinCD62P
Fibrinogen
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Thrombocyte ActivationConsequences
– Aggregation– Surface adhesion
• Change in morphology/ spreading– Release of Serotonin
→ Vasodilatation– Release of Platelet factor 3
→ Activation of the clotting cascade– Release of Platelet factor 4
→ Heparin-Inactivation– Release of growth factors (PDGF, FGF)
→ Proliferation of vascular smooth muscle cells– Release of RANTES
→ Monocyte recruitment to endothelial cells
Detection
Morphology
ELISA
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Protein Adsorption• Albumin and fibrinogen show competitive behaviour
– In vitro dependent from the salt concentration and buffer system– Albumin binds more to hydrophobic surface– Fibrinogen binds more to hydrophilic surfaces
• Different behaviour of free and adsorbed fibrinogen– Adsorbed fibrinogen activates blood platelets– Increased effect by parallel adsorption of a phospholipide (lecithin)
• Threshold Surface Fibrinogen Concentration for Platelet Activation– 30 ng/cm2
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The Blood Clotting Cascade
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The Clotting Cascade
Thrombin
Fibrinmonomers
FibrinogenFibrinpolymers
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Factor F X
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
Intrinsic Pathway
Ca2+
Ca2+
Ca2+
The Clotting Cascade
Factor F X
F VIIF VIIa
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Extrinsic Pathway
Ca2+
Ca2+
FibrinogenFibrinmonomers
Fibrinpolymers
ThrombinProthrombin I
Factor F Xa
Ca2+
Platelet Factor 3
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
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The Fibrinolytic System
Clotting Cascade
Fibrin degradation productsFibrinogen Fibrin-clot
Plasminogen Plasmin
T-PAF XIIaHMWK
KallikreinUrokinase
Streptokinase
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Effect of Antithrombin III/Heparin
Prothrombin I
FibrinogenFibrinmonomers
Thrombin
Factor F XaFactor F X Factor F X
F VIIF VIIa
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Intrinsic Pathway Extrinsic Pathway
Platelet Factor 3
Ca2+
Ca2+
Ca2+
Ca2+ Ca2+
Fibrinpolymers
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
Ca2+
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Surface Sensitive Steps
FibrinogenFibrinmonomers
Prothrombin I Thrombin
Factor F XaFactor F X Factor F X
F VIIF VIIa
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Intrinsic Pathway Extrinsic Pathway
Platelet Factor 3
Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Fibrinpolymers
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
Ca2+
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Physical Model of Fibrinogen Activation
Source: Baurschmidt & Schaldach, Med Biol Eng Comput 18: 496-502 (1980)
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Ser:O
H
RN
CO
HN
RC
Serine Proteases
H2O
O
RN
CO-
HN
RC
Ser
H Tetraedric Transition State
RN
CO OH :O
HSer
Release of the N-terminal peptide
HN H
RC
O
RN
CO Ser
Break of the Peptide Bonding
Enzyme-acetyl-transition-state
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Testing of the Clotting Cascade
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Tests of the Clotting Cascade• Global Test: Clotting Time
– Start the clotting cascade by addition of CaCl2– Stir the sample and measure the time until a fibrin clot is formed
(visually, change of viscosity, turbidimetry (change of transparence)⇒Can be performed with the plasma on the test material or after some
incubation of the plasma with the sample• Modified Global Tests
– F1+2 fragment: Fragment of prothrombin, which is cleaved off during activation
– TAT (Thrombin-antithrombin) complex: Further downstream indicator of activated thrombin
• Amount/ activity of individual (pro-)factors in the plasma– Mix plasma with factor deficient plasma and determine clotting time
• Activity of some activated factors– Chromogenic substrates are available for some factors,
e.g. FXIIa: Z-Lys-Phe-Arg-pNA; Thrombin: S-2238• Clinical clotting tests (TT, PT, aPTT)
– Mainly test some systems
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Thrombin Time (TT)
FibrinogenFibrinmonomers
Prothrombin I Thrombin
Factor F XaFactor F X Factor F X
F VIIF VIIa
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Intrinsic Pathway Extrinsic Pathway
Platelet Factor 3
Ca2+
Ca2+
Ca2+
Ca2+ Ca2+
Fibrinpolymers
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
Ca2+
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The Quick (PT)-Test
FibrinogenFibrinmonomers
Prothrombin I Thrombin
Factor F XaFactor F X Factor F X
F VIIF VIIa
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Intrinsic Pathway Extrinsic Pathway
Platelet Factor 3
Ca2+
Ca2+
Ca2+
Ca2+ Ca2+
Fibrinpolymers
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
Ca2+
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Partial Thromboplastin Time [(a)PTT]
FibrinogenFibrinmonomers
Prothrombin I Thrombin
Factor F XaFactor F X Factor F X
F VIIF VIIa
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Intrinsic Pathway Extrinsic Pathway
Platelet Factor 3
Ca2+
Ca2+
Ca2+
Ca2+ Ca2+
Fibrinpolymers
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
Ca2+
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Surface Sensitive Steps
FibrinogenFibrinmonomers
Prothrombin I Thrombin
Factor F XaFactor F X Factor F X
F VIIF VIIa
F IXaF IX
F XIaF XI
Surface ContactCollagen
FXII activator
F XIIaF XII
F III (Tissue Thromboplastin)
Tissue/Cell Defect
Intrinsic Pathway Extrinsic Pathway
Platelet Factor 3
Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Fibrinpolymers
CrosslinkedFibrin Meshwork
F XIIIa F XIII
F VF Va
F VIIIaF VIII
Ca2+
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Reaction of the Vessel Wall
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Blood Vessel Cells
Neo-Intima
Smooth Muscle Cell-Proliferation
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Blood Vessel Anatomy
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(Re-)Stenosis Evaluation• Angiography: X-Ray with
contrast media– Length and extent of the
stenosis• Intravascular ultrasound• Histology
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(Re-)Stenosis Evaluation• Angiography: X-Ray with
contrast media• Intravascular ultrasound
– Wall thickness– (Length of stenosis)
• Histology
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(Re-)Stenosis Evaluation• Angiography: X-Ray with
contrast media• Intravascular ultrasound• Histology
– Wall thickness– Measurement of intima
and media area– Scores for injury or
stenosis– Inflammatory cells– Fibrin deposition– Immunochemistry
• Cell types, proliferation markers, various cytokines
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Endothelial CellsResting State• Phospholipids on the surface → optimum blood compatible surfaceStimuli for Activation• Inflammatory cytokines (IL-1, IL-6, TNF etc)• Thrombin (Proteinase Activated Receptor PAR-1)• Low Density Lipoprotein (LDL-Cholesterol)• Mechanically (?)
Consequences of Activation• Expression of E-selectins
– Leukocyte rolling and adhesion and penetration• Expression of von Willebrand Factor (vWF)
– Platelet adhesion and activation• Production of NO
– Relaxation of vascular smooth muscle cells
Antibody techniques
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Nitric Oxide• NO is a free radical with extremely short lifetime• NO has very many signaling functions• NO is produced by Nitric Oxide Synthase (NOS) in an
oxidation of L-arginine to L-citrulline– Neuronal NOS: nNOS– Inducible NOS: iNOS– Endothelial NOS: eNOS
• Detection– NO sensitive electrodes– Measurement of nitrate as stable end product of NO
• Griess Reaction -> photometrical detection• 4,5-diaminofluorescein -> fluorescent detection
– Molecular biology, RNA-methods for iNOS
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Smooth Muscle Cells• Usual cells in the Tunica Media• At inflammatory processes (arteriosclerosis,
mechanical irritation) they proliferate forming a neo-intima
• Restenosis tissue is 30% smooth muscle cells, the rest are inflammatory cells
• Analysis– Characteristic protein: smooth muscle cell actin– Proliferation markers– Inflammatory cytokines– Growth factors
• Transforming growth factor beta (TGF-β) provokes SMC growth• Vascular endothelial growth factor (VEGF) induces endothelialization
and inhibits SMC growth