Biomaterials and Artificial Organs

Dr. I. Manjubala

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Early Definition (Historical):

“Lack of interaction between material and tissue”

Definition of Biomaterials“A nonviable material used in a medical device, intended to interact with biological systems.”

Contemporary Definition:

“Ability of a material to perform with an appropriate host response, in a specific application”

interdependent mechanisms of interaction between material and tissue“Ability of material to perform” and not just reside in the body“Appropriate host response” must be acceptable given the desired function“Specific application” must be defined

Implies inert, non-toxic, non-carcinogenic, non-allergenic, non-inflammatory, non-degradableThus, material has zero influence…

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Material used to construct artificial organs, rehabilitation devices, or prostheses and replace natural body tissues(The American Heritage® Medical Dictionary, 2007)

Definitions of Biomaterials (modern)

A synthetic material used to replace part of a living system or to function in intimate contact with living tissue (Park, 1995)

A systemically and pharmacologically inert substance designed for implantation within or incorporation with living substances

(The Clemson University Advisory Board for Biomaterials)

A nonviable material used in a medical device, intended to interact with biological systems (Williams, 1987)

A biomaterial is any material, natural or man-made, that comprises whole or part of a living structure or biomedical device which performs, augments, or replaces a natural function (Wikipedia)

bi·o·ma·te·ri·al (n)

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• Historically, biomaterials consisted of materials common in the laboratories of physicians, with little consideration of material properties.

• Early biomaterials :- Gold: Malleable, inert metal (does not oxidize); used in dentistry

by Chinese and Romans--dates 2000 years- Iron, brass: High strength metals; rejoin fractured femur (1775)

BACKGROUND

- Glass: Hard ceramic; used to replace eye (purely cosmetic)- Wood: Natural composite; high strength to weight; used for

limb prostheses and artificial teeth- Bone: Natural composite; uses: needles, decorative piercing-

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• Important dates- 600 BC: Sushruta Samhita, Nose reconstruction- 1860's: Lister develops aseptic surgical technique, wires and nails

made of iron, gold, silver and platinum- early 1900's: W.A. Lane, Bone plates used to fix fracture

- 1930's: Introduction of stainless steel, cobalt chromium alloys- 1938 : P. Wiles, first total hip prosthesis- 1940's: Polymers (Plastics) in medicine: PMMA bone repair; cellulose

for dialysis; nylon sutures- 1944 : W. J. Kolff, Hemodialyser - 1946: J.Judet & R.Judet, Biomechanically designed hip prostheses,

from plastic. - 1952: A.B. Voorhees, First blood vessel, made of cloth

HISTORY

Vinyon N Copolymer, (polyvinyl chloride and polyacrylonitrile)Nylon, Orlon®, Dacron®, Teflon®, Ivalon®

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- 1953: Dacron (polymer fiber) vascular grafts- 1958: J. Charnley, Cemented (PMMA) joint, total hip replacement

- 1958: S.Furman & G.Robinson, First direct stimulation of heart - 1960: A. Starr, M.I. Edwards, first commercial heart valves- 1976: W.J. Kolff et al., Artificial heart

HISTORY (Contd)

- 1976: FDA amendments governing testing & production of biomaterials /devices

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An (ideal) biomaterial must be:

• inert or specifically interactive • biocompatible • mechanically and chemically stable or • biodegradable • process able (for manufacturability) • Non-thrombogenic (if blood-contacting) • sterilizable

Requirements of Biomaterials

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General properties and Criteria

1. Mechanical properties (strength)

2. Toxicity and Biocompatibility

3. Tissue response

4. Interfacial response

5. Performance

6. Regulation and ethics

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1. Mechanical properties of Biological materials and biomaterials

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2. Toxicity and Biocompatibility

A biomaterial should not be toxicIt deals with the substances that migrate out of biomaterials.

• There is no general set of criteria, for a material to qualify as being biocompatible– The time scale over which the host is exposed to the material or device

must be considered

material contact time

syringe needle 1-2 s

tongue depressor 10 s

contact lens 12 hr - 30 days

bone screw / plate 3-12 months

total hip replacement 10-15 yrs

intraocular lens 30 + yrs

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3. Tissue Response to Implants

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3. Tissue Response to Implants - Contd

1. Minimal Response: Thin layer of fibrous tissueSilicon rubber, PTFE, PMMA, Ceramics, alloys

2. Chemically Induced response: Acute, mild inflammationAbsorbable sutures, resins

4. Physically Induced response: Inflammation to particulatesPTFE, PMMA, Nylon, Metals

3. Chemical (2) : Chronic and Severe InflammationDegradable material, Metallic corrosion

5. Physical (2) : Tissue growth into porous material Polymer, ceramic, metal, Composites

6. Necrotic response: Layer of necrotic Debris Bone cement, surgical adhesives

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4. Interfacial Response

Based on Interfacial response, there are four types of biomaterials:

1. Nearly inert, smooth surface 2. Nearly inert, microporous surface3. Controlled reactive surface4. Resorbable

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5. Performance of Implants

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5. Performance of Implants (contd)

Schematic illustration showing probability of failure vs implant period

The performance of an implant after insertion can be considered in terms of reliability.

there are four major factors contributing to the failure of hip joint replacementsfracture, wear, infection, and loosening of implants

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6. Regulation

Different standards:

ISO – International Standards Organisation

ASTM – American Soceity for Testing Materials

BSI – British Standards Institute

AISI – American Institute of Steel and Iron

BIS – The Bureau of Indian Standards

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6. Regulation (contd.)

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Inert, Interactive, Living and replacing materials

Inert biomaterials: Implantable materials with little or no counter reaction from the body.

Interactive biomaterials: Implantable materials designed to elicit a specific benign tissue reaction, such as integration, adhesion, etc.

Living biomaterials: Implantable materials that possibly contain living cells at time of implantation, regarded by the host tissue as tolerable tissue, and are actively resorbed and/or remodeled.

Replacement biomaterial: Implantable materials made of living tissue that has been cultivated from the patient own cells outside the body.

Classification of Biomaterials

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Material Advantages Disadvantages

POLYMERS:Nylon, silicones, PTFE, UHMWPE

Resilient, easy to fabricate

Not strong, deform with time, may degrade

METALS:Titanium, stainless steels, CoCr alloys, gold

Strong, tough, ductile May corrode, high density

CERAMICS:Aluminum oxide, carbon, hydroxyapatite

Highly biocompatible, inert, high modulus and compressive strength, good esthetic properties

Brittle, difficult to make, poor fatigue resistance

COMPOSITES:Various combinations

Strong, tailor-made Difficult to make

Classes of Biomaterials

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Important Definitions

• Biological response: host response towards a material.

• Biodegradation: breakdown of a material in a biological system.

• Bioactive material: A biomaterial intended to cause or modulate a biological activity.

• Bioactivity: Degree of wanted (positive) reaction from tissues.

• Inherent thrombogenicity: establishment of a thrombus that is controlled by material surface properties (thrombus=blood plug, genicity=”cause”).

• Osseointegration: is a description of the clinical performance of bone that interact with a biomaterial (ossus=bone).

• Bone bindning: the establishment of a continuity between implants and living bone through physical/biochemical processes.

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Material properties

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• To introduce the fundamental mechanical and surface chemistry properties of biomaterials

• OUTLINE– Mechanical Properties (bulk)

• elasticity, viscoelasticity, brittle fracture, fatigue– Surface Properties– List of Characterization techniques

Material properties

The bulk of a biomaterial presents physical and chemical properties of the material that remain during the lifetime of the implant.The surface properties is mainly defined by chemical, microstructure and it interacts with the host tissue directly.

Biomaterial Properties• Surface properties• Bulk properties• Biological properties

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Surface Properties

The surface region of a material is known to be uniquely reactive

The surface of a material is inevitably different from the bulk

Surfaces readily contaminate

The surface structure of a material is often mobile.

•Biomaterial surface is extremely important in determining the biological response.•Some of the biomedical devices and materials do not leach undesirable substances in sufficient quantities to influence cells and tissue.•Therefore, the surface structure play an important role in attract the cells and tissue to respond with the materials.

Surface properties

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Parameters: roughness, wettability, surface mobility,chemical composition, electrical charge, crystallinity, and heterogeneity to biological reaction

Surface is a material, near interface, having different properties from bulk. (few nm layers)

Surface Sensitivity

This is usually relative to the penetration depth of the probe.

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Physical Description of Biomaterial SurfacesBiomaterial surfaces exhibit remarkable heterogeneity in physical structure:

Material dependant: Metals vs. Polymers vs. Ceramics vs. Gels

Chemistry: Polar vs. Apolar, Charge, Reactivity, Patterned

Morphology: Smooth, Rough, Stepped, Patterned, Diffuse

Order: Crystalline, Amorphous, Semi-Crystalline, Phases

Environment: Hydration, Solvent Quality

Bumpy with Phases

Hydration

Glassy

• Some Possibilities for Surface Structure

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• Parameters to be measured:

a) Roughness, smoothness b) Chemical composition

(atomic, supramolecular, macromolecular)

Surface properties

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c) Surfaces may be structurally or compositionally inhomogeneous in the plane of the surface such as phase-separated domains or microcontact printed lanes.

d) Surfaces may be inhomogeneous with depth into the specimen or simply over layered with a thin film.

Surface properties

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e) Crystallinity f) Unreconstructed surface or reconstructed surface

Surface properties

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Common Methods to Characterize Biomaterial SurfacesSurface Characterization

SURFACE PROPERTIES

The surface properties for the biomaterials which are being considered for discussion are

Surface Energy

Contact Angle

Critical Surface Tension

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• Interface– boundary between 2 layers

• significance– protein adsorption to materials– blood coagulation/thrombosis due to material contact– cellular response to materials

Surface Energy

SURFACE ENERGY

Surface energy quantifies the disruption of intermolecular bonds that occurs when a surface is created.

In other words surface energy is a measure of the extent to which bonds are unsatisfied at the surface of material. At the surface, there is an asymmetric force field, which results in an attraction of atoms which are there on the surface in to the bulk.

This tends to deplete the surface of atoms putting the surface in tension.

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SURFACE ENERGY

Metals and ceramics have surfaces with high surface energies ranging from 102 to 104

ergs/cm2.

In contrast, most polymers and plastics have much smaller surface energies, usually <100 ergs/cm2.

The surface energy values are subject to much experimental variation due to adsorption of gases or organic species.

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CONTACT ANGLE

The contact angle is the angle at which a liquid/vapor interface meets the solid surface.

The contact angle is specific for any given system and is determined by the interactions across the three interfaces.

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CONTACT ANGLEWhen a liquid drop is placed on to the surface of a solid or the surface of the liquid, the processes which occur are:

1.The liquid may sit on the surface in the form of a droplet or 2. It may spread out over the entire surface depending on the

interfacial free energies of the two substances.At equilibrium contact angle or Young Dupree equation

is given bys / g = s / l + l / g cos

Where s / g, s / l and l / g are the interfacial free energy between the solid and gas; solid and liquid, liquid and gas respectively and the contact angle. 37

CONTACT ANGLE

The wetting characteristic can be generalized as

= 0, complete wetting ; 0 900, partial wetting ; > 900 , no wetting.

The contact angle can be affected greatly by the surface roughness and adsorption of polar gases or organic species or contamination by dirt.

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Superhydrophobicity

CRITICAL SURFACE TENSION

The critical surface tension is defined as that value of surface tension of a liquid below which the liquid will

spread on a solid and is expressed in dynes/cm.

The critical surface tension of a material is determined by measuring the different values of contact angle formed by liquids with different values of l / g.

A plot of cos versus l / g is usually a straight line

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The critical surface tension is the surface tension of a liquid that would completely wet the solid of interest.

Material c (dyne/cm)

Co-Cr-Mo 22.3

Pyrex glass 170

Gold 57.4

poly(ethylene) 31-33

poly(methylmethacrylate)

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Teflon 18

Critical Surface Tension

The l / g at which cos =1 is defined as the critical surface-tension (c).

CRITICAL SURFACE TENSION

Blood compatibility of material surfaces has been shown to vary in the same order as the critical surface tension.

It is found that the amount of thrombus formation increases and blood clotting time decreases as cincreases.

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At the surface (interface) there are intermolecular forces and intramolecular forces of attraction and repulsion.

• van der Waals forces : Hydrogen Bonds : Coulombic :

Surface Chemistry

How surfaces interact with molecules? Nonspecific interaction Specific binding Surface topology

Nonspecific Interaction• attractive van der Waals force that arises from dipole-dipole type interactions;• Electrostatic forces resulting from charged molecules• Hydration or solvation force that results from expulsion of water between the two surfaces• Hydrophobic effects that non‐polar molecules tend to form intermolecular aggregates in an aqueous medium• Repulsive steric forces that arise due to proteins on both surfaces forming spikes of up to 10 nm.

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• surface may become charged by– adsorption of ionic species present in sol’n or preferential

adsorption of OH-

– ionization of -COOH or -NH2 group

------

solid

++++++ hydroxyl ion

Surface Electrical properties

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------

++++++

+-+

+-

+ - -++

-+

-- ++ - +

+

gegenion

Nernst potential

zeta potential

tightlybound

diffuse

electroneutralbulk

Electric Double Layer

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Surface Analysis Techniques for Biomaterials• Contact Angle Measurements

• Electron Spectroscopy for Chemical Analysis (ESCA / XPS)

• Auger Electron Spectroscopy (Auger)

• Near Edge X-ray Absorption Fine Structure (NEXAFS)

• Secondary Ion Mass Spectroscopy (SIMS)

• Scanning Probe Microscopy (AFM)

• Sum Frequency Generation (SFG)

• Surface Plasmon Resonance (SPR)

• Optical Imaging and Spectroscopy (microscopy, TIRF)

• Ellipsometry

• Scanning Electron Microscopy (SEM)

• Infrared Spectroscopy (FTIR)

• Many more...

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Biomaterials Surface Analysis

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Property Technique(s)

Composition ESCA, Auger, SIMS, NEXAFS

Structure SIMS, ESCA, NEXAFS, FTIR, SFG

Orientation NEXAFS, FTIR, SFG

Spatial Distribution Imaging SIMS, AFM, microscopy

Topography AFM

Thickness ESCA, AFM, ellipsometry, SPR

Energetics Contact angle

Surface Information

Biocompatibility and Sterilization

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• Protocol for biomaterial test provide by:– American Society for Testing Material (ASTM)– International Standards Organization (ISO)– Government agencies, e.g., the FDA

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STANDARDS• Recently, extensive efforts have been made by government

agencies, i. e., FDA, and regulatory bodies, i.e., ASTM, IS0, and USP, to provide procedures, protocols, guidelines, and standardsthat may be used in the in vivo assessment of the tissue compatibility of medical devices.

• This chapter draws heavily on the IS0 10993 standard, Biological Evaluation of Medical Devices, in presenting a systematic approach to the in vivo assessment of tissue compatibility of medical devices.

• 20 parts of the ISO 10993 standard• are either accepted or under preparation. Tests that may be used in

the assessment of medical device biocompatibility includeprocedures for cytotoxicity, sensitization, irritation, acute systemictoxicity, subchronic toxicity, mutagenicity, geno toxicity,hemocompatibility etc. The testing procedures can be performed invitro as well as in vivo

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List of the standards in the 10993 series

• ISO 10993-1:2003 Biological evaluation of medical devices Part 1: Evaluation and testing

• ISO 10993-2:2006 Biological evaluation of medical devices Part 2: Animal welfare requirements

• ISO 10993-3:2003 Biological evaluation of medical devices Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity

• ISO 10993-4:2002/Amd 1:2006 Biological evaluation of medical devices Part 4: Selection of tests for interactions with blood

• ISO 10993-5:1999 Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity

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• ISO 10993-6:1994 Biological evaluation of medical devices Part 6: Tests for local effects after implantation

• ISO 10993-7:1995 Biological evaluation of medical devices Part 7: Ethylene oxide sterilization residuals

• ISO 10993-8:2001 Biological evaluation of medical devices. Part 8: Selection and qualification of reference materials for biological tests

• ISO 10993-9:1999 Biological evaluation of medical devices Part 9: Framework for identification and quantification of potential degradation products

• ISO 10993-10:2002/Amd 1:2006 Biological evaluation of medical devices Part 10: Tests for irritation and delayed-type hypersensitivity

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• ISO 10993-11:2006 Biological evaluation of medical devices Part 11: Tests for systemic toxicity

• ISO 10993-12:2002 Biological evaluation of medical devices Part 12: Sample preparation and reference materials (available in English only)

• ISO 10993-13:1998 Biological evaluation of medical devices Part 13: Identification and quantification of degradation products from polymeric medical devices

• ISO 10993-14:2001 Biological evaluation of medical devices Part 14: Identification and quantification of degradation products from ceramics

• ISO 10993-15:2000 Biological evaluation of medical devices Part 15: Identification and quantification of degradation products from metals and alloys

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• ISO 10993-16:1997 Biological evaluation of medical devices Part 16: Toxicokinetic study design for degradation products and leachables

• ISO 10993-17:2002 Biological evaluation of medical devices Part 17: Establishment of allowable limits for leachable substances

• ISO 10993-18:2005 Biological evaluation of medical devices Part 18: Chemical characterization of materials

• ISO/TS 10993-19:2006 Biological evaluation of medical devices Part 19: Physico-chemical, morphological and topographical characterization of materials

• ISO/TS 10993-20:2006 Biological evaluation of medical devices Part 20: Principles and methods for immunotoxicology testing of medical device

Biocompatibility• Defined as the compatibility between a material and the biological

system.

• “the ability of a material to perform with an appropriate host responsein a specific application”

• Two principal elements.

• First there is the absence of a cytotoxic effect and second, there is theaspect of functionality.

• Cytotoxicity - survival of cells and the maintenance of specific cellularfunctions under the influence of a material and/or its degradationproducts.

• Functionality - integration of the biomaterial into a biological system.assumes the absence of impairment of cellular function requires thatthe mechanical, chemical and physical features of the material aresufficient for the performance of cell-specific functions.

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TESTING OF BIOMATERIALS

• How can biomaterials be evaluated to determine if they are biocompatible and will function in a biologically appropriate manner in the in vivo environment?

• in vitro (literally "in glass") conditions: rapid and inexpensivedata on biological interaction – Cells attach and grow on MODIFIED Tissue culture

polystyrene, a surface modified polymer, not on untreated polystyrene.

• in vivo, both materials heal almost indistinguishably with a thin foreign body capsule.

• Thus, the results of the in vitro test do not provide information relevant to the in vivo implant situation.

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Advantages of in vitro:

• In vitro tests minimize the use of animals in research, a desirable goal.

• Also, in vitro testing is required by most regulatory agencies in the device approval process for clinical application.

• When appropriately used, in vitro testing provides useful insights that can dictate whether a device need be further evaluated in expensive in vivo experimental models.

In vivo Experiments:

• Will the animal model provide data useful for predicting how a device performs in humans?

• Without validation to human clinical studies, it is often difficult to draw strong conclusions from performance in animals.

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IN VITRO ASSESSMENT• “Cytotoxicity”: to cause toxic effects (death, alterations in

cellular membrane permeability, enzymatic inhibition, etc.) at the cellular level.

• It is distinctly different from physical factors that affect cellular adhesion (surface charge of a material, hydrophobicity, hydrophilicity, etc.).

• Evaluation of biomaterials by methods that use isolated, adherent cells in culture to measure cytotoxicity and biological compatibility.

• Cells from established cell lines purchased from biological suppliers or cell banks.

• Primary cells are seldom used – less assay repeatability, reproducibility, efficiency, and, in

some cases, availability.

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Toxicity

• A toxic material is defined as a material that releases a chemical in sufficient quantities to kill cells either directly or indirectly through inhibition of key metabolic pathways.

• The number of cells that are affected is an indication of the dose and potency of the chemical.

• Although a variety of factors affect the toxicity of a chemical (e.g., compound, temperature, test system), the most important is the dose or amount of chemical delivered to the individual cell.

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Delivered and Exposure Doses

• Delivered dose: the dose that is actually absorbed by the cell. • Exposure dose: the amount applied to a test system. • The cells that are most sensitive are referred to as the target

cells. • Cell culture methods: evaluate target cell toxicity by using

delivered doses of the test substance. • This distinguishes cell culture methods from whole- animal

studies, which evaluate the exposure dose and do not determine the target cell dose of the test substance.

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ASSAY METHODS• Three primary cell culture assays are used for

evaluating biocompatibility: – direct contact, – agar diffusion, – elution (also known as extract dilution).

• These are morphological assays, meaning that the outcome is measured by observations of changes in the morphology of the cells.

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Direct contact method1. A near confluent layer of fibroblasts are prepared in a culture

plate 2. Fresh media is added 3. Material being tested is placed onto the cultures, which are

incubated for 24 hours at 37 degrees Celsius 4. The material is removed 5. The culture media is removed 6. The remaining cells are fixed and stained, dead cells are lost

during fixation and only the live cells are stained7. The toxicity of the material is indicated by the absence of

stained cells around the material

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Agar diffusion method 1. A near confluent layer of fibroblasts are prepared in a culture plate 2. Old cell culture media is removed 3. The cells are covered with a solution of 2% agar, which often

contains red vital stain 4. When the agar solidifies the cells will have dispersed throughout

its volume 5. The material is then placed on the surface of the agar and

incubated for 24 hours at 37 degrees Celsius 6. Live cells take up the vital stain and retain it, dead cells do not 7. The toxicity of the material is evaluated by the loss of vital stain

under and around the material 8. Surface microscopy is also needed to evaluate the material-cell

interface

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Elution method 1. A near confluent layer of fibroblasts are prepared in a

culture plate 2. An extract of the material which is being tested is

prepared using physiological saline or serum free media 3. Extraction conditions are used which are appropriate for

the type of exposure which the cells would receive in the in vivo environment if the material were to be implanted

4. The extract is placed on the cells and incubated for 48 hours at 37 degrees Celsius

5. After 48 hours the toxicity is evaluated using either a histochemical or vital stain

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ASSAY METHODS• To standardize the methods and compare the results of

these assays, the variables – number of cells, – growth phase of the cells (period of frequent cell

replication), – cell type, – duration of exposure, – test sample size (e.g., geometry, density, shape,

thickness), and – surface area of test sample

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IN VIVO ASSESSMENT BIO-COMPATIBILITY

• The goal of in vivo assessment of tissue compatibility of a biomaterial, prosthesis, or medical device is to determine the biocompatibility or safety of the biomaterial, prosthesis, or medical device in a biological environment.

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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.

• 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

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Medical device categorization by tissue contact and contact duration

Tissue ContactSurface devices

External communicating devices

Implant devices

Contact duration

SkinMucosal membraneBreached or compromised surfaceBlood pathTissues/Bone/dentin communicatingCirculating bloodTissue/boneBloodLimited, ≤ 24 hoursProlonged, ≥ 24 hours and < 30 daysPermanent, >30 days

In Vivo Assessment of Tissue Compatibility

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In vivo test for tissue compatibility1. Sensitization2. Irritation3. Intracutaneous reactivity4. Systemic toxicity (acute toxicity)5. Subcronic toxicity (subacute toxicity)6. Genotoxicity7. Implantation8. Hemocompatibility9. Chronic toxicity10. Carcinogenicity11. Reproductive and developmental toxicity12. Biodegradation13. Immune responses

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In Vivo Assessment of Tissue Compatibility1. 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 chemicals2. 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.

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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.

5. 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.

6. 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.

In Vivo Assessment of Tissue Compatibility

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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)

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.

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

In Vivo Assessment of Tissue Compatibility

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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

Many adherent platelets

Measure platelet adhesion

No adherent platelets

Platelets aggregate and embolize downstream

The thrombus layer forms a non-reactive 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

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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.

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.

In Vivo Assessment of Tissue Compatibility

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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.

In Vivo Assessment of Tissue Compatibility

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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 inconsistentIn 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

STERILIZATION

• One of the greatest challenges for devices is ensuring sterility

• Many in-vivo degradation schemes have been linked to loss of mechanical properties due to post-sterilization aging

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SterilizationSterility Definition: Sterility can be defined as the absence of all living organisms, particularly microorganisms. “the state in which the probability of any one bacterial endosporesurviving is 10-6 or lower”

The sterility assurance level (SAL) minimum, that the probability of a given implant will remain non-sterile after exposure to a given sterilization process, is one in a million implants.

Example: In operating theatre area: Under aseptic conditions,5,000–50,000 skin particles are delivered daily from each physician’s flora in intensive care units and pathogenic bacteria such as Staphylococcus aureus can be recovered from about 90% of “clean” wounds at the time of closure 81

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STERILIZATION SCHEMES

• Steam • Autoclaving• Eto Gas• E-beam radiation• Gamma Radiation

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Steam Autoclaves

• Steam sterilizers (autoclaves) are instruments that producesuperheated steam under high pressure, and are used for bothdecontamination and sterilization

• Material to be sterilized must come in contact with steam andheat

• Tight-fitting containers do not permit steam penetration, and thusare not acceptable for use in autoclaves.

• The use of autoclave tape alone however is not an adequatemonitor of efficiency.

• Indicator such as Bacillus stearothermophilus spore strips. Thespores, which can survive 121 ◦C for 5min, but are killed at 121◦C in 13min,

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Dry Heat• Dry heat is less efficient than wet heat and requires longer times

and/or higher temperatures to achieve sterilization.• Sterilization of glassware by dry heat can usually be accomplished

at 160–170 ◦C for 2–4 h.

Radiation• Gamma and electron-beam irradiation are among the most popular

and well established

• Gamma radiation kills microorganisms by attacking their DNA.

• Processes for sterilizing polymer-based medical devices.• These techniques can lead to significant alterations in the materials

being treated

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Ethylene Oxide• 100% ethylene oxide (EtO) gas remains one of the most popular.• kills microorganisms by destruction of proteins and nucleic acids

• EtO is suitable for heat-labile instruments such as endoscopes with sensitive optics, medical utensils or implants.

• Disadvantage: Ethylene oxide is an explosive and toxic gas, and a long degassing period

New Technologies• gaseous chlorine dioxide, low-temperature gas plasma, gaseous

ozone and vapor-phase hydrogen peroxide• machine-generated X-rays are being investigated as a substitute to

gamma radiation

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Infections of Biomaterials• First contact of biomaterial in the body- fluids• Bacterial organisms have the ability to adhere to all types of materials

that are made of polymers, glasses, ceramics and metals.• Biofilms account accounts or over 65% of human bacterial infections• Biofilms: Three major problems.

– First, a reservoir of bacteria that can be shed into the body, - a chronicinfection.

– Second, biofilm bacteria are highly resistant to treatment withantibiotics; extremely difficult to eliminate with conventionalantimicrobial therapies.

– Finally, unable to eliminate bacteria growing in a biofilm, a chronicinflammatory response at the site of the biofilm may be produced.-

– may account for tissue damage due to host response– A non-shedding surface on a medical device encourages the

establishment of biofilms.

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Biofilm-Classes of Bacteria• Staphylococci• streptococci• Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and• Pseudomonas aeruginosa

These organisms may originate from the skin of patients or health-care workers, tap water to which entry points are exposed, or other sources in the environment.

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