BIOMATERIALS
ENT 311/4
Lecture 8
BIOLOGICAL TESTING OF BIOMATERIALS
Prepared by: Nur Farahiyah Binti Mohammad
Date: 25 th August 2008
Email : farahiyah@unimap.edu.my

Teaching Plan
METALLIC
BIOMATERIALS
DELIVERY
MODE
LEVEL OF
COMPLEXITY
COURSE OUTCOME
COVERED

Describe and assess in vitro and in vivo tissue compatibility of biomaterials in biological environment.
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Lecture 
Knowledge
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Repetition

Application
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Analysis

Evaluation
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Ability to explain and evaluate the biocompatibility of biomaterials utilized as implants or contact devices with human tissue.
2

1.0 Introduction
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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
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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:
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American Society for Testing Material
(ASTM)
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International Standards Organization (ISO)
Government agencies, e.g., the FDA
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2.0 In vitro Assessment of
Tissue Compatibility
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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.
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2.0 In vitro Assessment of
Tissue Compatibility
2.1.2 Delivered and Exposure Doses
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Delivered dose refers to the dose of the agent that is actually absorbed by the cell.
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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.
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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.
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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.
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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.
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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.
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2.0 In vitro Assessment of
Tissue Compatibility
2.2.2 Agar Diffusion Test
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A near-confluent monolayer of L-929 is prepared in a 60mm diameter plate.
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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.
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2.0 In vitro Assessment of
Tissue Compatibility
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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.
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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.
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The extract is placed on prepared nearconfluent monolayer of L-929 mammalian fibroblast cells.
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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.
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Direct contact
Agar
Diffusion
Advantages and Disadvantages of Cell Culture Methods
Advantages
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
Eliminate extraction preparation
Zone of diffusion
Better concentration gradient of toxicant
Can test one side of a material
Independent of material density
Disadvantages
Cellular trauma if material moves
Cellular trauma with high density materials
Decreased cell population with highly soluble toxicants
Requires flat surface
Solubility of toxicant in agar
Limited exposure time
Risk of absorbing water form agar
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Elution
Advantages and Disadvantages of Cell Culture Methods
Advantages
Separate extraction from testing
Dose response effect
Extend exposure time
Disadvantages
Additional time and step
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3.0 In Vivo Assessment of
Tissue Compatibility
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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.
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3.0 In Vivo Assessment of
Tissue Compatibility
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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
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Duration of contact of the medical device
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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
Permanent, >30 days 20

3.0 In Vivo Assessment of
Tissue Compatibility
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1.
2.
3.
4.
5.
6.
7.
8.
In vivo test for tissue compatibility
Sensitization
Irritation
Intracutaneous reactivity
Systemic toxicity (acute toxicity)
Subcronic toxicity (subacute toxicity)
Genotoxicity
Implantation
Hemocompatibility
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3.0 In Vivo Assessment of
Tissue Compatibility
9.
10.
11.
12.
13.
Chronic toxicity
Carcinogenicity
Reproductive and developmental toxicity
Biodegradation
Immune responses
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3.0 In Vivo Assessment of
Tissue Compatibility
1. Sensitization
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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
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3.0 In Vivo Assessment of
Tissue Compatibility
2.
Irritation
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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.
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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.
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3.0 In Vivo Assessment of
Tissue Compatibility
4.
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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.
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3.0 In Vivo Assessment of
Tissue Compatibility
5.
6.
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Subacute toxicity
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.
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3.0 In Vivo Assessment of
Tissue Compatibility
7.
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Genotocity
Genocity tests are carried out if in vitro test results indicate potential genotoxicity.
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The in vitro assay should cover three levels of genotoxicity effects:
DNA destruction
Gene mutation
Chromosomal aberrations (abnormality)
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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.
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3.0 In Vivo Assessment of
Tissue Compatibility
9.
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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)
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Coagulation
Platelets
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Haematology
Immunology
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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 good blood compatibility
Alternate interpretation
Many platelet adhere, but the platelets are not activated and form passivating natural biological layer on the surface
The thrombus layer forms a nonreactive natural biological film on the surface
Many adherent platelets
Surface coated with adherent thrombus
Measure platelet adhesion
Measure the mass of adherent thrombus
Released factors stimulated desirable endothelial cell growth
Extensive platelet granule release
Measure the platelet granule release
No adherent platelets
No adherent thrombus
No release
Platelets aggregate and embolize downstream
Thromus detaches and embolizes downstream.
Therefore it not seen on the surface
Release actually occurs but its diluted by the flowing blood
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3.0 In Vivo Assessment of
Tissue Compatibility
10.

Carcinogenity
This test determine the tumorigenic potential of medical devices and biomaterial.
11.

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.
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3.0 In Vivo Assessment of
Tissue Compatibility
12.
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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.
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3.0 In Vivo Assessment of
Tissue Compatibility
13.
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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.
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Advantages and Limitation of
Biocompatibility Test
Test/Assay Advantages Limitations
In vitro tests Quick turnover (days), high throughput screening, standardized with appropriate protocols
Relevance to in vivo
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
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