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Module 04 Concept of Biocompatibility
Module 04 Concept of Biocompatibility
204
Introduction to Biomaterials
Module 4
Concepts of Biocompatibility
Instructor: Vivek Verma
Topics
• Concept of biocompatibility
– Definition
– Immune response
– Testing (in vitro/ in vivo)
• Biological phenomenon on materials surfaces
– Protein adsorption isotherms
– Kinetics of cell‐material interaction
– Bacterial adhesion and kinetics of biofilm formation
Instructor: Vivek Verma
Biocompatibility
• Biocompatibility is the ability of a material to
perform with an appropriate host response in a
specific application: Memorize above definition
verbatim
– While effectiveness is important, safety is
mandatory
Instructor: Vivek Verma
Biocompatibility
• Links material properties/ characteristics with
performance (biological requirement) with
specific application (medical device or
material used in medical device)
• Appropriate host response: identification and
characterization of tissue reactions and
responses that could prove harmful to the
host and/ or lead to ultimate failure of the
biomaterial, medical device or prosthesis
through biological mechanisms
Instructor: Vivek Verma
Biocompatibility
• Biocompatibility is the attribute of a non‐
autogenous substance that offers a synergetic
environment for a stable and sustained
functionality of both the material and its host
when scientifically employed for its intended
function
• Non‐autogenous:
– Earlier nonviable materials
– Now: living cells also used
Instructor: Vivek Verma
Biocompatibility
• Biocompatibility is the attribute of a non‐
autogenous substance that offers a synergetic
environment for a stable and sustained
functionality of both the material and its host
when scientifically employed for its intended
function
• Synergetic environment:
– While performing effectively, the biomaterial should
not adversely effect the functionality of host tissue and
the host
• Stable and sustained functionality:
– Integrity of biomaterial and host tissue throughout
implant lifetime
Instructor: Vivek Verma
Biocompatibility
• Biocompatibility assessment is considered to
be a measure of magnitude and duration of
the adverse alterations in homeostatic
mechanisms that determine the host response
• Homeostatis: is the property of a system,
either open or closed, that regulates its
internal environment and tends to maintain a
stable, constant condition (Wiki)
Instructor: Vivek Verma
Key Terms
• Implant: material or graft used inside the body
– For: prosthetic, therapeutic, diagnostic or cosmetic
purpose
– Material: synthetic, natural, composite
• Inflammation: response of a tissue to stimuli such
as physical/ chemical irritants, foreign bodies
(infectious microbes), trauma, immune reaction
– Acute or chronic
– Signs:
• Calor (heat)
• Rubor (redness)
• Dolor (pain)
• Tumor (swelling)
• Functio laesa (loss of function)
Instructor: Vivek Verma
Key Terms: Inflammation Signs
• Signs:
– Calor (heat)
– Rubor (redness)
– Dolor (pain)
– Tumor (swelling)
– Functio laesa (loss of function)
http://3.bp.blogspot.com/_Jx78YcF‐F8U/TR5‐_YPKAcI/AAAAAAAAD5w/f9eq2HMyUoM/s1600/inflammation_cartoon.jpg
Instructor: Vivek Verma
Key Terms
• Phagocytosis: specialized cells ingest
macromolecules, particulate matter, cell
debris etc.
http://upload.wikimedia.org/wikipedia/commons/e/e6/Phagocytosis2.png
Instructor: Vivek Verma
Key Terms
• Phagocytosis: specialized cells ingest
macromolecules, particulate matter, cell
debris etc.
• Examples
– Blood monocytes
– Neutrophils
– Tissue macrophages
Instructor: Vivek Verma
Overview of Immune System
• Immune system evolved to protect multicellular
organisms from pathogens
– Highly adaptable
Protection by immune system
• Recognition
– Capacity to distinguish foreign invader (non‐self) from
self component (self‐nonself discrimination)
• Response
– Effector response: eliminate and neutralize invader
– Memory response: more rapid and heightened
immune reaction upon later attack
• Vaccines*
*Constant Gardener (Movie)
Instructor: Vivek Verma
Overview of Immune System
• Infection and Immunity
• Organisms causing disease are termed as
pathogens and their means of attacking the
host is called pathogenesis
• Innate and adaptive immunity
Instructor: Vivek Verma
Innate and Adaptive Immunity
Innate immunity
• Less specific
• Provide first line of defense against infection
• Present before the infection (innate)
• Not specific to a particular pathogen
• Example: lysozyme
• Phagocytic cells are barrier to infection
Instructor: Vivek Verma
Innate and Adaptive Immunity
Adaptive immunity is highly specific
• Recognizes and eliminates specific
microorganisms and molecules
Instructor: Vivek Verma
Innate and Adaptive Immunity
Adaptive immunity is highly specific
Attributes of adaptive immunity
• Antigenic specificity
– Distinguish subtle difference among antigens
• Can distinguish two protein molecules that differ only by
single amino acid
• Diversity
– Recognize billions of unique structures
• Immunologic memory
– Second encounter with same antigen will result in
heightened state of immune reactivity
• Self‐nonself recognition
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes
Antigen presenting cells
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes
• B lymphocytes (B Cells)
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes
• B lymphocytes (B Cells)
• Express unique antigen binding receptor (B cell
receptor) on its membrane
– Antibody
• Antibodies
– Two identical polypeptide (heavy chains)
– Two identical shorter polypeptide (light chain)
– Heavy chains joined to light chain by disulfide bond
– Heavy/light chain pairs are linked together by
additional disulfide bonds
Instructor: Vivek Verma
Antibodies
Instructor: Vivek Verma
Antibodies
• Proteins produced by the immune system in
response to foreign molecules
• Y shaped molecules with two identical binding
sites
– Billions of different antibodies
– Diversity generated by changing only the length
and amino acid sequence of the loops
• Antigens
– Target of antibodies
Instructor: Vivek Verma
Antibodies
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009)
Instructor: Vivek Verma
Antibodies
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009)
Instructor: Vivek Verma
Antibodies
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009) Instructor: Vivek Verma
Antibodies
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009)
Instructor: Vivek Verma
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007)
Instructor: Vivek Verma
Antibodies‐To purify molecules
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009)
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes (B and T)
• T Lymphocytes (T cells)
• Express unique antigen binding molecule
– T cell receptor (TCR)
• Two type of T cells
– T helper (TH) cells
• Have CD4 glycoprotein on their surface
– T cytotoxic (TC) cells
• Have CD8 glycoprotein on their surface
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes
• T Lymphocytes (T cells)
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes
• T Lymphocytes (T cells)
• T‐cell receptors can recognize antigen only when
it is bound to cell membrane protein called major
histocompatibility complex (MHC)
Two types of MHC
• Class I MHC
– Expressed in almost all nucleated cells of vertebrates
• Class II MHC
– Expressed by antigen presenting cells (APCs)
• Upon antigen encounter T cell differentiates into
– Effector T cell
– Memory T cell
Instructor: Vivek Verma
Major Group of Cells for Immune Response
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
Major Group of Cells for Immune Response
Lymphocytes
• T Lymphocytes (T cells)
• TH cell recognizes and interacts with antigen‐
MHC class II molecule complex
– Secretes cytokines
• Activate several cells of immune system
• TH cell can trigger Tc cell to form cytotoxic T
lymphocytes (CTLs)
– Eliminate any cell that display antigen such as
• Virus infected cells
• Tumor cells
• Cells of a foreign tissue graft
Instructor: Vivek Verma
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
Major Group of Cells for Immune Response
Antigen presenting cells
• Dendritic cells
• Macrophages
• B cells
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Antigen presenting cells (APC)
Characteristics of APC
• Express class II MHC molecules on their
membrane
• Can produce cytokines that cause TH cells to
become activated
Instructor: Vivek Verma
Major Group of Cells for Immune Response
Antigen presenting cells (APC)
How they work
• Internalize antigen by phagocytosis
• Display part of antigen on their membrane
bound to class II MHC
• TH cell interact with antigen‐class II MHC on
membrane of APC
• APC produces additional signal leading to
activation of TH cells
Instructor: Vivek Verma
Primary/Secondary Response
Instructor: Vivek Verma
Topics
• Concept of biocompatibility
– Definition
– Immune response
– Testing (in vitro/ in vivo)
• Biological phenomenon on materials surfaces
– Protein adsorption isotherms
– Kinetics of cell‐material interaction
– Bacterial adhesion and kinetics of biofilm formation
Instructor: Vivek Verma
Biomaterials Testing
Test to check:
• Biocompatible?
• Function in biological appropriate manner in
the in vivo environment
In vivo Vs in vitro
• Inside organism Vs in glass
Instructor: Vivek Verma
Biomaterials Testing: In Vitro
• Inexpensive and rapid
• Minimize use of animals in research
• Required by regulatory agencies in device
approval for clinical application
• Gives insight is the material/ device need to
be further tested in in vivo test
• Measure parameters relevant to what will
occur in vivo?
• Does not provide information relevant to
implant situation
Instructor: Vivek Verma
Biomaterials Testing: In Vivo
• Animals are used for testing to model the
environment that might be encountered in
humans
• Animal anatomy, physiology, biochemistry?
• Choice of animal
– Anatomically and physiologically
• Animal model should provide useful data for use
in humans
– Further clinical trials are necessary to test this
• Experiment design
– Minimize number of animals
– Treated humanely
– Maximize relevant information generated by testing
procedure
Instructor: Vivek Verma
Biomaterials Testing
• Biomaterial role (time wise)
– Few seconds to life time
• Test device by implanting six months
– Actual use few seconds to minutes
– Actual use for life time
Instructor: Vivek Verma
Biomaterials Testing
Statistics
• Large variability
• Defensible conclusions from (expensive) tests
– Statistics is used to ensure results are meaningful
withing probability range
• Before experiment: reducing the number of
experiments
• After experiment: extracting maximum
information
Instructor: Vivek Verma
Biomaterials Testing
Toxicity
• Toxic material definition: Material that
releases a chemical in sufficient quantities to
kill cells either directly or indirectly through
inhibition of key metabolic pathways
• Dose!
Delivered and exposure doses
• Actually absorbed Vs amount applied
• Cells most sensitive are called target cells
• Local?
Instructor: Vivek Verma
Biomaterials Testing
• Controls: A scientific control is an experiment or
observation designed to minimize the effects of
variables other than the single independent
variable (Wiki)
• Negative controls are groups where no
phenomenon is expected
– They ensure that there is no effect when there should
be no effect
• Positive controls are groups where a
phenomenon is expected
– they ensure that there is an effect when there should
be an effect
Instructor: Vivek Verma
Biomaterials Testing
Instructor: Vivek Verma
Biomaterials Testing: In vitro Assays
Direct Contact Test
• Interface of living and dead cells show
intermediate zone of damaged cells
– Abnormal morphology
– Degree: varies with toxicant
• Signs
– Increased vacuolization
– Rounding
– Crenation (scalloped shape)
– Swelling
Instructor: Vivek Verma
Biomaterials Testing: In vitro Assays
Direct Contact Test
– Increased vacuolization: occurs with basic
substances
• Due to lysosomal uptake of the toxicant
– Rounding: before disintegration
– Crenation (scalloped shape)
– Swelling
http://www.chem.ufl.edu/~itl/4411/matter/FG13_022.GIF
Instructor: Vivek Verma
Biomaterials Testing: In vitro Assays
Agar Diffusion Test
• Near confluent monolayer of L‐929 mouse fibroblast
cells prepared in 60 mm diameter culture plate
• Culture medium replaced by fresh culture medium
containing 2% agar
– Agar allowed to solidify
• Positive, negative and test specimen is placed in
individual cultures
– Incubated for 24 hours at 37 ± 1 °C in humidified incubator
• Assay include ‘neutral red’ vital stain
– Healthy (viable) cells show
– Dead/ injured cells do not show (colourless)
• Absence of stained cells under and around periphery of
specimen tells about toxicity
Instructor: Vivek Verma
Biomaterials Testing: In vitro Assays
• Elution (Extract Dilution Test)
• Solvent extraction of biomaterial under
consideration and testing of this extract, most
commonly at various dilutions, for evidence of
cytotoxicity and cellular interaction
• Extract is placed on near confluent monolayer
of L‐929 mouse fibroblast cells
– Toxicity evaluates after 48 hours of incubation at
37 ± 1 °C in humidified incubator
• Live or dead cells may be distinguished by
histochemical or vital stains
Instructor: Vivek Verma
Advantages and Disadvantages of Cell Culture Methods
Instructor: Vivek Verma
Biomaterials Testing: In vitro Assays
• In vitro cytotoxicity assays are initial screening
tests for a wide variety of biomaterials
• More application specific tests are performed
to asses the biocompatibility of biomaterial
under end‐use condition
• Biomaterials identified as non‐toxic in in vitro
assays are usually non‐toxic in in vivo assays
• Biomaterials identified as toxic in in vitro
assays must be further investigated for clinical
acceptability
Instructor: Vivek Verma
Biomaterials Testing: In vivo
Why in vivo tests:
• Assess biocompatibility of biomaterial/
prosthesis/ medical devices in biological
environment
– Device performs as intended and posses no harm to
the patient or user
Consideration while testing
• Properties of biomaterials
– Chemical, toxicological, physical, electrical,
morphological, mechanical
• Condition, nature, degree, frequency, duration of
tissue exposure
Instructor: Vivek Verma
Biomaterials Testing: In vivo
Instructor: Vivek Verma
Biomaterials Testing: In vivo
Appropriate test selection
• Nature of body contact
• Duration
Instructor: Vivek Verma
Biomaterials Testing: In vivo
Instructor: Vivek Verma
Biomaterials Testing: In vivo
In vivo test to test general biocompatibility of newly
developed biomaterials for which some knowledge of
the tissue compatibility is necessary for further
research and development
• General tissue response and information regarding
design criteria in production of medical device
• Identification and quantification of extractable
chemical entities precede biological examination
• Preliminary in vivo assessment is carried out to
determine if any chemical entities produce adverse
biological reactions
• Adverse reactions observed at this state would require
further improvement in biocompatibility
Instructor: Vivek Verma
Biomaterials Testing: In vivo
Biocompatibility of the final product
• Determine general biocompatibility of final
product
• Tested in animal models
• All tests need not be necessary
• Manufacturing/ sterilization steps should be
scrutinized
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
• Sensitization: immune system responses to
contact with chemical substances
• Irritation: local tissue inflammation response
to chemical without systemic immunological
component
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Sensitization, Irritation, Intracuteneous reactivity
• Estimates the potential for contact sensitization of
medical devices, materials and/ or their extracts
• Potential leachables in medical devices or biomaterials
can result in allergic or sensitization reactions
• Carried out in guinea pigs
• Reflect intended routes, nature, degree, frequency,
duration and conditions of exposure of biomaterial in
its intended clinical use
• Emphasis on effect of potential leachables
• Intracutaneous reactivity tests determine the localized
reaction of tissue to extracts of medical devices,
biomaterials, or prosthesis in the final product form
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Systemic toxicity: Acute, Subacute, Subchronic
Systemic toxicity (Acute toxicity)
• Estimate the potential harmful effects of either single
or multiple exposures, during a period of 24 hours, to
medical devices, biomaterials, and/or their extracts
• Evaluate systemic toxicity potential of medical devices
which release constituents into the body
• Form, area of material, thickness, surface area to
extraction vehicle volume are critical considerations in
the testing protocol
• Mice, rats and rabbits are usual animals for testing
• Acute toxicity is considered to be the adverse effects
occurring within 24 hours
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Systemic toxicity: Acute, Subacute, Subchronic
Systemic toxicity (Subacute toxicity)
• Focus on adverse effects occurring after
administration of a single dose or multiple doses
of a test sample per day given during a period of
from 14 to 28 days
Systemic toxicity (Subchronic toxicity)
• Estimates adverse effects occurring after
administratoin of a single dose or multiple doses
of a test sample during part of the lifespan,
usually 90 days but less than 10% of total lifespan
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Genotoxicity
• Carried out if indicated by chemistry and/or
composition of the biomaterial or in vitro test
results indicate potential genotoxicity
• Initially, at least three in vitro assays should be
used and two of these assays should utilize
mammalian cells
• Three levels of genotoxic effects should be tested
– DNA effects
– Gene mutations
– Chromosomal aberrations
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Implantation
• Implantation tests assess the local
pathological effects on living tissue of a
sample of a material or final product that is
surgically implanted or places into an implant
site or tissue appropriate to the intended
application of the biomaterial or medical
device
• Evaluation
• Choice of animals
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Hemocompatibility
• Evaluate effect on blood and/or blood
components by blood contacting devices/
materials
• Platelets
– Platelets play a fundamental role in hemostasis and
are a natural source of growth factors
– They circulate in the blood of mammals and are
involved in hemostasis, leading to the formation of
blood clots
• Hematology
– Study concerned with the study of blood, the blood
forming organs, and blood diseases
• Immunology
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Chronic Toxicity:
• Single or multiple exposure
• 10% of lifespan
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Carcinogenicity:
• Cancer causing potential of medical device or
extract
• Single or multiple exposure or contact over a
period of the major portion of the lifespan of
the test animal
• Rare
– Few tests
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Reproductive and Developmental Toxicity
• Potential effects of medical devices, material,
and/or their extracts
– Reproductive function
– Embryonic development
– Prenatal and postnatal development
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Biodegradation
• Effect of biodegradable material and
biodegradation products
– Kinetics of degradation
– Nature of degradation product
– Origin of degradation product
• Impurities, additives, corrosion products, bulk polymer
Instructor: Vivek Verma
Biomaterials Testing: In vivo Assay
Immune Response
Instructor: Vivek Verma