BG4231&BG6001-Lect 3- Characterization of biomaterials.pdf

Characterization of Biomaterials Lecture 3 Characterization of biomaterials Asst. Prof. Dang Thuy Tram [email protected] Office N1.3-B3-09 BG 4231/BG6001 – Advanced Biomaterials 1 Characterization of Biomaterials Lecture Outline 1. Physical and chemical characterization of bulk and surface pr...

Characterization of Biomaterials Lecture 3 Characterization of biomaterials Asst. Prof. Dang Thuy Tram [email protected] Office N1.3-B3-09 BG 4231/BG6001 – Advanced Biomaterials 1 Characterization of Biomaterials Lecture Outline 1. Physical and chemical characterization of bulk and surface properties 2. Biological characterization of blood-material interaction 3. Biological characterization of tissue-material interaction - Host response to material implant - In vitro assessment of cell/tissue-material compatibility - In vivo assessment of tissue-material compatibility 2 Characterization of Biomaterials Hipjoint implant Physical and chemical characterization Bulk properties • Mechanical properties for optimal structural compatibility : optimal matching properties of the implant to host tissues  Stiffness, strength to high mechanical loading, fatigue strength, wear resistance  Mismatch can cause poor functionality and pathological complication • Thermal properties : heat conduction • Optical properties : refractive index Contact lenses Dental fillings Vascutek® grafts fabricated from polyesters or ePTFE 3 Characterization of Biomaterials Physical and chemical characterization Surface properties • Important notes about surface characteristics of materials - Surface if different from bulk - Surface has unique reactivity - Mass of material that makes up the surface zone is very small - Surface contaminates easily - Surface molecules can exhibit considerably motility 4 Characterization of Biomaterials Physical and chemical characterization Contact angle measurement of liquid droplets Surface properties • • • - Properties of material surface Roughness Patterns Charge Wettability Surface chemical composition Surface analysis pitfalls Characterization techniques might alter surface properties Potential artifacts, more than 1 characterization techniques Polymeric biomaterials are more easily damaged and exhibit more surface mobility Surface face characterization techniques Contact angle measurement (hydrophobicity/hydrophilicity) Secondary Ion Mass Spect (SIMS) for atomic and molecular surface composition SIMS Pattern of N-hydroxysuccinimide groups (yellow) 5 Characterization of Biomaterials Biological characterization What happens when a device/material is placed in the body? Blood-material interaction Modification of healing (host/foreign-body response) https://www.sganalytics.com/ Surface cracking of implanted material 6 Characterization of Biomaterials Characterization of blood-material interaction Blood coagulation mechanism Platelet structure https://hubpages.com/health/ImmuneThrombocytopenia-Purpura-or-ITP Hemostatic mechanism involves interdependent reactions between (i) Material surface (ii) Platelets (iii) Coagulation proteins  Formation or removal of blood clots (thrombus) 7 Characterization of Biomaterials Characterization of blood-material interaction Blood coagulation mechanism 8 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) Concept of blood-compatibility • “Blood-compatibility” is the property of a material or device that permits it to function in contact with blood without inducing adverse reactions. • “Blood-compatibility” is determined by - Biochemistry of coagulation - Design and function of the device in the blood stream • A “thrombogenic” or “not blood-compatible” induces - Clot formation upon contact with blood - Shedding or nucleation of detached thrombus or “emboli” - Destruction of circulating blood components - Activation of the complement systems and other immunologic pathways • “Thrombogenicity” is defined as the extent to which a device, when employed in its intended use configuration, induces adverse response. • There is no consensus on which materials are blood-compatible. 9 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) BMI measurement • Animal/human sources • Handling protocols • Storage time Initial platelet attachment to material surface increases with increasing blood flow or increasing wall shear stress 10 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) BMI measurement Methods for contacting material and blood Methods for assessment of blood change In vitro - Clotting time - Observe platelet morphology In vivo - Quantify number of platelet adhering to material surface - Quantify platelet consumption rate 11 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) BMI measurement - Example Tetraethylene glycol dimethyl ether (Tetraglyme) Catheter surface modified unmodified Cao et al, J. Biomed. Mat. Res A 2007 12 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) Commonly applied in vitro tests for blood-material interaction in regulatory process (ISO 10993-4) 13 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) Challenges in assessment of blood-compatibility 14 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) Challenges in assessment of blood-compatibility 15 Characterization of Biomaterials Characterization of blood-material interaction Evaluation of Blood-Material Interaction (BMI) Challenges in assessment of blood-compatibility • Numerous designs of blood-contacting devices, each impacting thrombogenicity differently • Manufacturers are unwilling to disclose specific chemical compositions or technical details of materials • Blood response to materials are not well-understood • Difficulty and high cost required for systematic measurement of thrombogenicity of a material in a device, both in humans and animals. • Existing test measures the outcome of blood-material interaction (BMI) but interpreting these results to determine “blood-compatibility” is not straightforward 16 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues Sequence of host response or modified wound healing in the presence of a material Dang TT, Nikkhah M et al, Natural and Synthetic Biomedical Polymers 2014 • Molecular and cellular sequence of host response to implanted biomaterials/devices https://www.youtube.com/watch?v=6qU-0ETo5_s Time 0:47-4:51 17 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues Protein adsorption CH3-terminated SAM COOH-terminated SAM • Computationally simulated interaction of a gamma fragment of fibrinogen on CH3 or COOH-terminated self-assembled monolayer (SAM) • CH3-terminated SAM, hydrophobic groups (side chain of Ala A, Leu L and CH2 of Lys K) lying close to the hydrophobic methyl surface • COOH-terminated SAM, hydrophilic groups of Lys, Glu and Arg are close to COOH 18 surface Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues Protein adsorption • Monolayer model of protein adsorption on solid surface • Langmuir adsorption isotherm 19 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues cell binding to surface Bilayer membrane Integrin receptor Protein in extracellular liquid Protein adsorbed on solid surface • Molecular spreading events : adsorbed proteins change their conformational and biological characteristics • Cell interaction with foreign surface is mediated by integrin receptors with adsorbed adhesion protein layer 20 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues Timeline of cellular recruitment in host response Stages of host response Injury Blood-Material interaction Acute inflammation Chronic inflammation Granulation tissue Foreign-body reaction Fibrosis capsule development • Temporal variation in host response to implanted biomaterials • Intensity and time depend on the extent of injury and characteristics of biomaterials (shape, size, topography, chemical and physical properties) 21 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vitro assessment of cell and tissue compatibility • Xenobiotics are foreign materials or chemicals found in the body e.g those leaching or degrading from a medical device or implanted material. • Assessing cell and tissue compatibility of a device/material oftens involving examination of potential xenobiotics that it might release. • “Cell and tissue compatibility” is NOT “biocompatibility”. • “Cell and tissue compatibility” gives some insight to the safety/toxicology aspect of the material tested but does not give any information on functional performance of a material in a specific context of application 22 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues 23 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vitro assessment of cell and tissue compatibility 24 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vitro assessment of cell and tissue compatibility 25 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vitro assessment of cell and tissue compatibility 26 Characterization of Biomaterials Characterization of tissue-material interaction Effects of implanted biomaterials on host biological systems In vitro assessment of cell and tissue compatibility 27 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vitro assessment of cell and tissue compatibility • In vitro study : Scanning Electron Microscopy image of MC3T3-E1 cells at various stages of adherent to polished titanium surface • Limitation of biomaterial evaluation in vitro :  Reduced complexity : no immune/inflammatory response  No cascade of events resulting from implantation  No recruitment of a variety of cell types • In vitro assessment is useful as a screening step to reduce toxicology risk 28 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vivo assessment of tissue-material compatibility Example : PLGA microparticles subcutaneously implanted in mice Histology sample Mouse sacrifice Histology processing 50µm Day 3 Day 9 Day 15 Day 21 Day 28 50µm Dang TT et al, Biomaterials, 2013 29 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues In vivo assessment of tissue-material compatibility Neutrophils – multinucleated cells Macrophages – foam cells Monocyte (oval, bean-shaped nucleus) 30 Characterization of Biomaterials Characterization of tissue-material interaction Foreign body reaction to polyethylene Effects of material on host tissues In vivo assessment of tissue-material compatibility wear particulate from a total knee prothesis Acute inflammation of an ePTFE vascular graft Fibrin cap Neutrophils Multinucleated foreign body giant cells Acellular fibrous capsule consiting of dense, compacted collagen at the surface of a silicon breast implant 31 Characterization of Biomaterials Characterization of tissue-material interaction Effects of implanted biomaterials on host biological systems In vivo assessment of tissue-material compatibility Non-invasive imaging to evaluate host response to biomaterials Immunocompetent SKH-1E mice IVIS Spectrum imaging • Cathepsins - Inflammatory proteases - Produced by various immune cells such as neutrophils and macrophages • Key advantages - Information on dynamics of cellular enzymatic activity - Parallel analysis of multiple materials Dang TT, Bratlie KM et al Biomaterials 2011 - Easy quantification from images acquired Bratlie KM, Dang TT et al PlosOne 2010 32 Weissleder R et al Nat. Biotech 1999 - Non-invasive Characterization of Biomaterials Characterization of tissue-material interaction Effects of implanted biomaterials on host biological systems In vivo assessment of tissue-material compatibility Non-invasive imaging to evaluate host response to biomaterials • Reactive oxygen species (ROS) - Secreted by neutrophils and macrophages - Important regulator in cellular processes - Contributed to oxidative degradation of biomaterials Liu WF, Ma M, Bratlie KM, Dang TT et al, Biomaterials 2011 Dang TT, Anh VT, Cohen J et al, Biomaterials 2013 33 Characterization of Biomaterials Characterization of tissue-material interaction Effects of implanted biomaterials on host biological systems In vivo assessment of tissue-material compatibility Non-invasive imaging to evaluate host response to biomaterials i.V injection of imaging probe s.q injection of particles Imaging with IVIS system Prosense fluorescent efficiency (x 10e-4) (days) PLGA particles without drug PLGA particles with dexamethasone 1.40 1.20 1.00 0.80 0.60 ** 0.40 P<0.05 0.20 0.00 0 ** 10 20 Time period post-injection (days) 30 • Quantification of activity of inflammatory enzymes in host response to implanted PLGA microparticles Dang TT, Bratlie KM, Bogatyrev SR et al Biomaterials 2011 34 Characterization of Biomaterials Characterization of tissue-material interaction Effects of implanted biomaterials on host biological systems In vivo assessment of tissue-material compatibility What factors influence host response to implanted biomaterials? • Bio-physical characteristics - Size - Shape - Surface roughness - Stiffness • Bio-chemical/molecular characteristics - Surface chemistry - Hydrophobicity/Hydrophilicity - Interaction/binding with cell surface receptor 35 Characterization of Biomaterials Characterization of tissue-material interaction Effects of implanted biomaterials on host biological systems Animal models, surgery and ethics • Animal model selection • Animal model is used to predict clinical efficacy and safety of medical devices/materials in humans • Choice of animal model must consider the advantages and disadvantages of the selected model in this prediction • Ethics approval (guided by 3R principles) • Replacement : nonanimal models should be used to the maximum extent possible • Reduction : using minimum number of animals to achieve statistically meaningful conclusion • Refinement : least invasive and most modern technique to minimize animal pain and distress • Regulatory requirement • Government regulatory agency (FDA/HSA) • Institutional responsibilities (University) 36 Characterization of Biomaterials Characterization of tissue-material interaction Effects of material on host tissues Choice of tests and strategy for biomaterial assessment • Each test can only evaluate one or a few aspects of material-cell/tissue interaction • These tests involve significant cost and time. • Choice of in vitro test and animal models depends significantly on intended applications. • Complexity increased with combination devices such as devices which include drug or cellular therapeutics • Current practice involves in vitro testing first, followed by in vivo testing only when in vitro tests are deemed successful. Must this be strictly adhered to? Eg. Processing of porcine heart valves involves the use glutaraldehyde which is deemed cytotoxic, but these heart valves were successfully used in clinics. 37 Characterization of Biomaterials Characterization of tissue-material interaction Effects of host environment on material • Host environment surrounding an implanted biomaterials contains biochemical molecules such as enzymes, free radicals, peroxides, hydrogen ions secreted by inflammatory cells and bacteria. • Secreted species cause intended/unintended breakdown of implanted materials over prolonged period of time • Degradation process further release biomaterial-associated components which invoke further response by the host system. • For polymeric biomaterial, main in vivo biodegration pathways are hydrolysis and oxidation. 38 Characterization of Biomaterials Characterization of tissue-material interaction Effects of host environment on material Hydrolytic degradation of polymeric implants 2 3 1 Hydrolyzable groups in polymeric biomaterials 39 Characterization of Biomaterials Characterization of tissue-material interaction Effects of host environment on material Hydrolytic degradation of polymeric implants Groups stable to hydrolysis in polymeric biomaterials 40 Characterization of Biomaterials Characterization of tissue-material interaction Effects of host environment on material Oxidative degradation of polymeric implants Sites favored for initial oxidative attack  Allow abstraction of an atom or ion  Provide resonance stablization of resultant radicals or ions Sources of oxidants : neutrophils, macrophages Readily oxidizable functional groups (* site favoring initial attack) 41 Characterization of Biomaterials Characterization of tissue-material interaction Effects of host environment on material Oxidative degradation of polymeric implants Example : Oxidation-induced stress cracking of poly(ester) urethane - Resistant to hydrolysis - Characterized by surface attack - Extensive loss of ether functionality - Slow ductile fracture (not rapid brittle fracture) Cardiac pacemaker 42 4 Introduction to polymeric biomaterials Summary 1. Physical and chemical characterization of bulk and surface properties 2. Biological characterization of blood-material interaction 3. Biological characterization of tissue-material interaction - Host response to material implant - In vitro assessment of cell/tissue-material compatibility - In vivo assessment of tissue-material compatibility 43