Download biomaterials ent 311/4

BIOMATERIALS
ENT 311/4
Lecture 8
BIOLOGICAL TESTING OF BIOMATERIALS
Prepared by: Nur Farahiyah Binti Mohammad
Date: 25th August 2008
Email : [email protected]
Teaching Plan
METALLIC
BIOMATERIALS
DELIVERY
MODE
LEVEL OF
COMPLEXITY
COURSE OUTCOME
COVERED
Describe
Lecture
Knowledge
Ability
and
assess in vitro
and in vivo tissue
compatibility of
biomaterials in
biological
environment.
Repetition
Application
Analysis
Evaluation
to explain and
evaluate the
biocompatibility of
biomaterials utilized as
implants or contact
devices with human
tissue.
2
1.0 Introduction
Biomaterials must be evaluated to
determine if they are biocompatible
and will function in a biologically
appropriate manner in the in vivo
environment.
 Biomaterial will be evaluated under in
vitro and in vivo conditions.

3
1.0 Introduction
Evaluation under in vitro conditions
can provide rapid and inexpensive data
on biological reaction.
 However, the question must always be
raised – will the in vitro test measure
parameters relevant to what will occur
in the much complex in vivo
environment? → In vivo evaluation

4
1.0 Introduction

Protocol for biomaterial test provide by:
American Society for Testing Material
(ASTM)
 International Standards Organization (ISO)
 Government agencies, e.g., the FDA

5
2.0 In vitro Assessment of
Tissue Compatibility
The evaluation of biomaterials by
method that use isolated, adherent
cells in culture to measure cytotoxicity
and biological compatibility.
 Cytotoxicity means to cause toxic
effect.

6
2.0 In vitro Assessment of
Tissue Compatibility
2.1 Background Concepts
(Things that observe)
2.1.1 Toxicity
 Toxic
material is defined as a material that
release a chemical in sufficient quantities to
kill cell either directly or indirectly through
inhibition of key metabolic pathway.
 Number of cells that are effected is an
indication of dose and potency of the chemical.
7
2.0 In vitro Assessment of
Tissue Compatibility
2.1.2 Delivered and Exposure Doses
 Delivered
dose refers to the dose of the agent
that is actually absorbed by the cell.
 Exposure dose is the amount of cytotoxic
agent delivered to the test system.
 Example: if an animal is exposed to an
atmosphere containing a noxious substance
(exposure dose), only a small portion of the
inhaled substance will be absorbed and
delivered to the internal organ organs and cell.
8
2.0 In vitro Assessment of
Tissue Compatibility
2.1.3 Solubility Characteristic
 Test
of dissolution of materials
 Investigate either it stimulate the intended
clinical application or may create desirable or
undesirable degradation products.
9
2.0 In vitro Assessment of
Tissue Compatibility
2.2 ASSAY METHODS

Three primary in vitro cell culture
cytotoxicity assay are:
2.2.1 Direct Contact Test
2.2.2 Agar Diffusion
2.2.3 Elution Test
10
2.0 In vitro Assessment of
Tissue Compatibility
2.2.1 Direct Contact
 A near-confluent monolayer of L-929
mammalian fibroblast cells is prepared in a
35mm diameter cell culture plate.
 The culture medium is removed and
replaced with 0.8 ml of fresh culture
medium.
 Specimen of negative or positive controls
and the test article are carefully placed in
prepare culture and incubated for 24 hour.
11
2.0 In vitro Assessment of
Tissue Compatibility
Live cells adhere to the culture plate
and are stained by the cytochemical
stain.
 Toxicity is evaluated by the absence of
stained cells under and around the
periphery of the specimen.

12
2.0 In vitro Assessment of
Tissue Compatibility
2.2.2 Agar Diffusion Test
 A near-confluent monolayer of L-929 is
prepared in a 60mm diameter plate.
 The culture medium is removed and
replaced with a culture medium containing
2% agar.
 After the agar has solidified, specimen of
negative and positive controls and the test
article are placed on the surface of the same
prepared plate and the culture incubated for
at least 24 hours.
13
2.0 In vitro Assessment of
Tissue Compatibility
This assay also contain red stain in the
agar mixture, which allows ready
visualisation of live cells.
 Healthy cells retain red stain.
 Dead or injured cells do not retain
neutral red and remain colourless.
 Toxicity is evaluated by the loss of the
stain under and around the periphery
of the specimens.

14
2.0 In vitro Assessment of
Tissue Compatibility
2.2.3 Elution Test

An extract of the material is prepared by
using 0.9% sodium chloride or serum-free
culture medium.

The extract is placed on prepared nearconfluent monolayer of L-929 mammalian
fibroblast cells.

Toxicity is evaluated after 48 hours.

Live or dead cells may be distinguished by
the use of histochemical or vital stain as
agar diffusion test method.
15
Advantages and Disadvantages
of Cell Culture Methods
Advantages
Disadvantages
Direct
contact
Eliminate extraction preparation
Zone of diffusion
Target cell contact with material
Mimic physiological conditions
Standardize amount of test
material or test indeterminate
shapes
Can extend exposure time by
adding fresh media
Cellular trauma if
material moves
Cellular trauma with
high density materials
Decreased cell
population with highly
soluble toxicants
Agar
Diffusion
Eliminate extraction preparation
Zone of diffusion
Better concentration gradient of
toxicant
Can test one side of a material
Independent of material density
Requires flat surface
Solubility of toxicant
in agar
Limited exposure time
Risk of absorbing
water form agar
16
Advantages and Disadvantages
of Cell Culture Methods
Elution
Advantages
Disadvantages
Separate extraction from testing
Dose response effect
Extend exposure time
Additional time and
step
17
3.0 In Vivo Assessment of
Tissue Compatibility

The goal of in vivo assessment of a
biomaterial, prosthesis or medical
devices is:


to determine that the device performs as
intended and presents no significant harm
to the patient or user.
In vivo test for assessment of tissue
biocompatibility are chosen to
stimulate end-use applications.
18
3.0 In Vivo Assessment of
Tissue Compatibility

To facilitate the selection of
appropriate tests, medical devices with
their components of biomaterial can be
categorized by:
The nature of body contact of the medical
device
 Duration of contact of the medical device

19
3.0 In Vivo Assessment of
Tissue Compatibility
Medical device categorization by tissue contact and
contact duration
Tissue Contact
Surface devices
External communicating
devices
Implant devices
Contact duration
Skin
Mucosal membrane
Breached or compromised surface
Blood path
Tissues/Bone/dentin communicating
Circulating blood
Tissue/bone
Blood
Limited, ≤ 24 hours
Prolonged, ≥ 24 hours and < 30 days
20
Permanent, >30 days
3.0 In Vivo Assessment of
Tissue Compatibility

In vivo test for tissue compatibility
1.
2.
3.
4.
5.
6.
7.
8.
Sensitization
Irritation
Intracutaneous reactivity
Systemic toxicity (acute toxicity)
Subcronic toxicity (subacute toxicity)
Genotoxicity
Implantation
Hemocompatibility
21
3.0 In Vivo Assessment of
Tissue Compatibility
Chronic toxicity
10. Carcinogenicity
11. Reproductive and developmental toxicity
12. Biodegradation
13. Immune responses
9.
22
3.0 In Vivo Assessment of
Tissue Compatibility
1. Sensitization



Sensitization test estimate the potential for
contact sensitization to medical devices or
materials.
Symptom of sensitization are often seen in
skin.
Sensitization is a immune system response
to chemicals
23
3.0 In Vivo Assessment of
Tissue Compatibility
2.


Irritation
Irritant test emphasize utilization of extracts of
biomaterials to determine the irritant effects of
potential leachables
Irritation is a local tissue inflammation response to
chemical.
3. Intracutaneous (intradermal) reactivity
 Determine the localized reaction of tissue to
intracutaneous injection of extracts of medical
devices, biomaterials, or prosthesis in the final
product form.
24
3.0 In Vivo Assessment of
Tissue Compatibility
4.
Systemic toxicity (acute toxicity)


Estimate the potential harmful effects in
vivo on target tissues and organs away
from the point of contact with either
single or multiple exposure to medical
devices or biomaterials.
Acute toxicity is considered to be the
adverse effects occurring after
administration test sample within 24
hours.
25
3.0 In Vivo Assessment of
Tissue Compatibility
5.
Subacute toxicity

6.
Focuses on adverse effect occuring after
administration of a single dose or
multiple doses of a test sample per day
during a period of from 14 to 28 days.
Subcronic toxicity

adverse effect occuring after
administration of a single dose or
multiple doses of a test sample per day
given during a part of the life span,
usually 90 days but not exceeding 10%
of the life span of the animal.
26
3.0 In Vivo Assessment of
Tissue Compatibility
7.


Genotocity
Genocity tests are carried out if in
vitro test results indicate potential
genotoxicity.
The in vitro assay should cover three
levels of genotoxicity effects:



DNA destruction
Gene mutation
Chromosomal aberrations (abnormality)
27
3.0 In Vivo Assessment of
Tissue Compatibility
8.

Implantation
Implantation test assess the local
pathological effects on the structure
and function of living tissue induced
by a sample of a material or final
product at site where it is surgically
implanted.
28
3.0 In Vivo Assessment of
Tissue Compatibility
9.
Hemocompatibility


This test evaluate effect on blood and/or blood
component by blood contacting medical devices
or materials.
From the ISO standard prospective, five test
categories for hemocompatibility evaluation:





Thrombosis (blood coagulation)
Coagulation
Platelets
Haematology
Immunology
29
Alternative scenario that can be applied for interpreting
results of blood-material interaction assay
Alternate
interpretation
Result implying
poor blood
compatibility
Evaluation
method
Result implying Alternate
good blood
interpretation
compatibility
Many platelet
adhere, but the
platelets are not
activated and
form passivating
natural biological
layer on the
surface
Many adherent
platelets
Measure
platelet
adhesion
No adherent
platelets
Platelets
aggregate and
embolize
downstream
The thrombus
layer forms a nonreactive natural
biological film on
the surface
Surface coated
with adherent
thrombus
Measure the
mass of
adherent
thrombus
No adherent
thrombus
Thromus detaches
and embolizes
downstream.
Therefore it not
seen on the
surface
Released factors
stimulated
desirable
endothelial cell
growth
Extensive platelet
granule release
Measure the
platelet
granule
release
No release
Release actually
occurs but its
diluted by the
flowing blood
30
3.0 In Vivo Assessment of
Tissue Compatibility
10.
Carcinogenity

11.
This test determine the tumorigenic
potential of medical devices and
biomaterial.
Reproductive and Developmental
Toxicity

These test evaluate the potential effects
of medical devices and biomaterials on
reproductive function, embryonic
development and prenatal and postnatal
development.
31
3.0 In Vivo Assessment of
Tissue Compatibility
12.


Biodegradation
This test determine the effects of
biodegradation materials and its
biodegradation products on the tissue
response.
This test focus on:




Amount of degradation during a period of time
The nature of the degradation products
The origin of the degradation product
Leachable in adjacent tissue and in distant
organ.
32
3.0 In Vivo Assessment of
Tissue Compatibility
13.
Immune response




Immune response evaluation is not a
component of the standards currently in vivo
tissue compatibility assessment.
However, ASTM, ISO and FDA currently have
working groups developing guidance documents
for immune response evaluation.
Synthetic material are not generally
immunogenic
However, immune response evaluation is
necessary with modified natural tissue implant
such as collagen.
33
Advantages and Limitation of
Biocompatibility Test
Test/Assay
Advantages
Limitations
In vitro tests
Quick turnover (days), high Relevance to in vivo
throughput screening,
standardized with
appropriate protocols
In vivo test
Provide multi-system
interactions, more
comprehensive than
ioutcome inconsistent
In vitro test
Relevance to clinical
use questionable, low
turnover (week to
months), high cost
and low throughput,
animal use concerns,
outcome can be
difficult to interpret
34