Module 3

MSE 693
Materials Science Technologies for Applications in Life

Sciences
Module 3
Concepts of Biocompatibility
Instructor: Vivek Verma

Topics
• Concept of biocompatibility
– Definition
– Testing (in vitro/ in vivo)
• Host response to implantable device
– Overview of immune system
– Immune response

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

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

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

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
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)

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)

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
Key Terms
• Phagocytosis: specialized cells ingest
macromolecules, particulate matter, cell
debris etc.
http://upload.wikimedia.org/wikipedia/commons/e/e6/Phagocytosis2.png
Key Terms
• Phagocytosis: specialized cells ingest
macromolecules, particulate matter, cell
debris etc.
• Examples
– Blood monocytes
– Neutrophils
– Tissue macrophages

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)
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

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

Adaptive immunity is highly specific
• Recognizes and eliminates specific
microorganisms and molecules

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

Primary/Secondary Response

Overview of the Immune System: Acquired Immunity
• Recognizes, responds and remembers a unique
pathogen
• Primary Vs Secondary response to a new
pathogen
– Graph
• Memory cells catalogue the unique epitope and
produce more rapid response to the pathogen,
should it be encountered again
– Epitope: Part of macromolecule that is recognized by
the immune system, specifically by antibodies
• Estimated 1015 variations of B and T cell receptors
to help specifically identify a broad range of
antigenic material

Overview of the Immune System

• Cell mediated Vs Humoral immunity
– Overlap broadly with cellular component of innate
immunity and acquired immunity in its entirety
– Lymphocytes (type of white blood cells) are the
principle effectors of both cell mediated and humoral
immunity
– Specific receptors on lymphocytes bind to antigens to
initiate a complex imflammatory and immune
response
• Humoral immunity
– Aspect of immunity that is mediated by secreted
antibodies

• Most large lymphocytes are called natural
killer cells
• Small lymphocytes made up T cells and B cells
• T cells
– Have T cell receptors on the surface to recognize
antigen
– Responsible for cell mediated immunity
• B cells are responsible for humoral immunity
– Aspect of immunity that is mediated by secreted
antibodies

• Exogenous (extracellular) antigens are internalized and
degraded by antigen presenting cells (macrophages, B
cells, dendritic cells)
– Resulting antigenic peptides complexed with class II MHC
molecules are then displaces on the cell surface
• Endogenous (intracellular) antigens (viral and tumour
proteins produced in altered self-cells) are degraded in
the cytoplasm and then displaced with class I MHC
molecules on the cell surface
• Humoral system
– Best suited for elimination of exogenous antigens
• Cell mediated response
– Best suited for elimination of endogenous antigens

Host Responses to Implanted Devices
• Implantable devices are engineered to be
biocompatible
– Immune system reacts to some degree with every
surgically implanted device
• Acute Vs Chronic response
• Neutrophils
– Recruited to the site of injury within minutes following
trauma and are the hallmark of acute inflammation
– Most abundant type of white blood cells
– At onset of acute inflammation they are one of first-
responders of inflammatory cells to migrate toward the
site of inflammation
– Predominant cells in pus, accounting for its
whitish/yellowish appearance

Major Group of Cells for Immune Response
Lymphocytes
Antigen presenting cells

T. J. Kindt et al., Kuby Immunology. (WH Freeman

and Company, New York, ed. Sixth, 2007)
Lymphocytes
• Type of white blood cells
– Produced in bone marrow
• Produce and display antigen binding cell
surface receptors
• Two major type of lymphocytes
– B lymphocytes (B Cells)
– T lymphocytes (T Cells)

Lymphocytes
• B lymphocytes (B Cells)
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007) Instructor: Vivek Verma
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
Antibodies

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

Antibodies
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009)
Antibodies
Antibodies
B. Alberts et al., Essential Cell Biology. (Garland Publishing Inc., New York, ed. Third, 2009) Instructor: Vivek Verma
Antibodies
T. J. Kindt et al., Kuby Immunology. (WH Freeman and Company, New York, ed. Sixth, 2007)

Antibodies-To purify molecules
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

Lymphocytes
Lymphocytes
• 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
Lymphocytes
• 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
Antigen presenting cells
• Dendritic cells
• Macrophages
• B cells
http://www.celldextherapeutics.com/img/chart-apc-technology_rev4.jpg Instructor: Vivek Verma

Antigen presenting cells (APC)
• Activation of humoral and cell mediated
immunity require cytokines
• Activation of TH cells highly regulated
– Prevent autoimmune diseases
• APC interact with TH cells
• Antigen receptors of TH cells can recognize
only antigen that is displayed together with
MHC II molecule on the surface of APC

Characteristics of APC
• Express class II MHC molecules on their
membrane
• Can produce cytokines that cause TH cells to
become activated

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

Topics
• Concept of biocompatibility
– Definition
– Testing (in vitro/ in vivo)
• Host response to implantable device
– Overview of immune system
– Immune response

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

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

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
• 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

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

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?
• 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

Biomaterials Testing: In vitro Assays
Direct Contact Test
• Near confluent monolayer of L-929 mouse fibroblast
cells prepared in 35 mm diameter culture plate
• Culture medium replaced by 0.8 ml of fresh culture
medium
• Positive, negative and test specimen is placed in
individual cultures
– Incubated for 24 hours at 37 ± 1 °C in humidified incubator
• Culture medium and specimen is removed
• Cells fixed and stained
– Dead cells can not adhere and are lost in fixation
• Absence of stained cells under and around periphery of
specimen tells about toxicity

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

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
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

• 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 places 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

Advantages and Disadvantages of Cell Culture Methods

• 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

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


Appropriate test selection
• Nature of body contact
• Duration


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

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

Biomaterials Testing: In vivo Assay

Sensitization, Irritation, Intraculatenous 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

• Sensitization: immune system responses to
contact with chemical substances
• Irritation: local tissue inflammation response
to chemical without systemic immunological
component

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

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

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

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

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
Chronic Toxicity:
• Single or multiple exposure
• 10% of lifespan

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

Reproductive and Developmental Toxicity
• Potential effects of medical devices, material,
and/or their extracts
– Reproductive function
– Embryonic development
– Prenatal and postnatal development

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

Immune Response

• First stage of inflammatory response to the
implanted device (in class notes)
• Frustrated phagocytosis
– The device is too big compared to the bacteria to
be engulfed by the cells
• Class notes!

• Total hip arthroplasty
– Arthroplasty: surgical repair of joint
• Designed for long term
• However,
– Device design
– Operative technique
– Mechanical factors
– Cell mediated and humoral immune response
• Nanoscale particles dislodged during wear may lead to
phagocyte activation
• Results in
– Osteolysis
– Loosening of prosthesis

• Organ transplant
– Biological (not mechanical) implant so require
immuno suppression for long term graft function
– Cross matching between donor and patient is
performed to prevent hyperacute rejection of the
graft
– Hyperacute rejection occur when preformed
antibodies exist to the graft prior to implantation
• ABO blood type antibodies

http://en.wikipedia.org/wiki/File:ABO_blood_type.svg

• Organ transplant
– Even with appropriate HLA matching between
donor and recipient, minor antigens present on
the graft may lead to the rejection
• Immunosuppression is required in this case
– HLA: Human Leukocyte Antigen
• Name of MHC in human
• Tissue engineering
– Next generation of replacement
– Cellular constructs are integrated within a biologic
(or inorganic) scaffold to repair the deficient host

• Implanted device must withstand long term exposure
to both the physical and the physiological environment
• Implants must also maintain their function during the
acute and chromic inflammatory response
• Failure to do so is defined as biofouling and results
from
– Chronic oxidation
– Hydrolysis
– Mechanical loading
• As the device becomes smaller and more mechanically
and electronically complex
– The negative impact of the immune response could
increase

• Size threshold may exist for biomaterials that alleviates
frustrated phagocytosis
– Implantable devices would move to the intracellular
environment and thus open new complexities and uncertainities
of the host response to an implantable device
• Nanoscale particles
– Inadvertant product of biomaterial wear
– Product of deliberate and highly technical engineering
• Particle toxicology: studying the impact of fine and ultrafine
materials on the host
• Nanoscale particles may have a toxic profile, even when
larger particles of the same material are nontoxic
– Ultrafine titanium dioxide
• Phagocyte activation after particle ingestion is believed to
contribute to toxicity

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MSE 693

Materials Science Technologies for Applications in Life

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

T. J. Kindt et al., Kuby Immunology. (WH Freeman

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

http://www.celldextherapeutics.com/img/chart-apc-technology_rev4.jpg Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Systemic toxicity (Subchronic toxicity)

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma

Instructor: Vivek Verma