biomedical engineering biomaterials

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BIOMEDICAL ENGINEERING BIOMATERIALS

Biological Analyses & Sterilization of Biomaterials

SECTION 3

Biological analyses For Bachelor Science(B.Sc.) Students

Esmaeil BiazarPh.D in

Biomedical Engineering

Biomaterials Eng.

Biomaterials Eng.

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Material attributes for biomedical applications

Property Desirables

Biocompatibility Noncarcinogenic, nonpyrogenic,

nontoxic, nonallergenic, blood

compatible, non-inflammatory

Sterilizability Not destroyed by typical sterilizing

techniques such as autoclaving, dry heat,

radiation, ethylene oxide

Physical characteristics Strength, elasticity, durability

Manufacturability Machinable, moldable, extrudable

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Adverse Effects Of Biomaterial/Bio-device

• B = f (X1,X2......Xn)• Where X: Material, Design, Application etc.

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r = 1-frt = r1 × r2 …… rn

r = (1-0.05).(1-0.03).(1-0.02).(1-0.04) = 0.89

Example

r ? For Knee replacement after 1 yearsFi = 5% , Fw = 3% , Fl= 2% , Fsur= 1% and Ffr=4%

Adverse Effects Of Biomaterial/Bio-device

Reliability

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Human Biomedical Devices

Class I: crutches, bedpans, tongue depressors,

adhesive bandages etc. minimal invasiveness, does not contact the user internally.

Class II: hearing aids, blood pumps, catheters, contact lens, electrodes etc. higher degree of invasiveness and risk, but relatively short duration.

Class III: cardiac pacemakers, intrauterine devices, intraocular lenses, heart valves, orthopedic implants, etc. considerably more invasive and can pose immense risk to the user-implantables.

Based on the duration of the device use, invasiveness and risk to the user.

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FIRST GENERATION IMPLANTS • “ad hoc” implants.• Specified by physicians using common and borrowed

materials.• Most successes were accidental rather than by design.

Examples

• Gold fillings, wooden teeth, dental prosthesis.• Steel, gold, ivory, etc., bone plates.• Glass eyes and other body parts.• Dacron and parachute cloth vascular implants.

Human Biomaterials Devices

Materials

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SECOND GENERATION IMPLANTS

• Engineered implants using common and borrowed materials.• Developed through collaborations of physicians and engineers.• Built on first generation experiences.• Used advances in materials science (from other fields).

• Titanium alloy dental and orthopaedic implants.• Cobalt-chromium-molybdinum orthopaedic implants.• UHMW polyethylene bearing surfaces for total joint replacements.• Heart valves and pacemakers.

Examples

Third generation implants

• Bioengineered implants using bioengineered materials.• Few examples on the market.• Some modified and new polymeric devices.• Many under development.

Example• Tissue engineered implants designed to regrow rather than replace tissues.• Integra LifeSciences artificial skin.• Genzyme cartilage cell procedure.• Some resorbable bone repair cements.• Genetically engineered “biological” components (Genetics Institute and Creative Biomolecules BMPs).

Objective is to minimize inflammatory responses and toxic effects.

• Biocompatibility has been defined as the ability of a medical device to perform with an appropriate host response in a specific application, and biocompatibility assessment is considered to be a measurement of the magnitude and duration of the adverse alterations in homeostatic mechanisms that determine the host response.

• Bioactivity– The characteristic that allows the material to form a bond with living tissue

(Hench, 1971)– The ability of a material to stimulate healing and trick the tissue system into

responding as if it were a natural tissue (Hench 2002).– Advantages: Bone tissue – implant interface, enhanced healing response,

extends implant life• Biodegradability

– Breakdown of implant due to chemical or cellular actions– If timed to rate of tissue healing transforms implant to scaffold for tissue

regeneration– Negates issues of stress shielding, implant loosening, long term stability

Biological Analyses

• Biology is a science of surfaces and interfaces• Understanding and controlling performance

PhysicalChemicalBiological

• Relevant material performance is under biological conditions– 37 ºC, aqueous, saline, Extra Cellular Matrix (ECM)– Material properties as a function of time

• Initial negative biological response - toxicity• Long term biological response – rejection

– Seldom (never) at equilibrium

Biological Analyses

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Physically Abrasive, adhesive, delamination wear Fatigue and Fracture Stress Corrosion cracking General corrosion.

Biologically Absorption of substances from the tissues Enzymatic degradation Calcification

Host can affect the implant

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Host Factors:

Age and health status Immunological/metabolic status Choice of surgeon: minimize tissue damage and contamination,

proper implantation.

Host can affect the implant

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Biological Analyses

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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Biological Analyses

Biocompatibility factors Components and natural properties of materials. Tissue type

Implantation time Limited X ≤ 24 hrs. Prolonged 24 hrs. < X < 30 days. Permanent X > 30 days.

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Biological Analyses Screening tests

Cytotoxicity Sensitization Irritation or intracutaneous reactivity Acute systemic toxicity Subacute toxicity Genotoxicity Implantation effect Hemocompatibility Chronic toxicity Carcinogenocity Reproductive and developmental toxicity Biodegradation

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• Assistance in the design of many biomaterials tests is available through national and international standards-organizations.

• Thus, the American Society for Testing Materials (ASTM) and

the International Standards Organization (IS0) can often provide detailed protocols for widely accepted, carefully thought out testing procedures.

• Other testing protocols are available through government agencies (e. g., the FDA) and through commercial testing laboratories.

Standards

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USP (U.S. Pharmacopeia). ISO (International Standard Organization), ISO 10993. FDA ( Food and Drug Administration), Blue Book. AAMI (The Association for the Advancement of Medical

Instrumentation). ANSI (American National Standards Institute). BSI (British Standard Institute). NHLBI (National Heart , Lung and Blood Institute). UIPAC (Union for Pure and Applied Chemistry). IACUC (Institutional Animal Care and Use Committee).

Standards

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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 Part 2: Animal welfare requirements. • ISO 10993-3:2003Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity.

• ISO 10993-4:2002/Amd 1:2006 Part 4: Selection of tests for interactions with blood.

• ISO 10993-5:1999Part 5: Tests for in vitro cytotoxicity.

Standards

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• ISO 10993-6:1994 Part 6: Tests for local effects after implantation.

• ISO 10993-7:1995Part 7: Ethylene oxide sterilization residuals.

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

• ISO 10993-9:1999 Part 9: Framework for identification and quantification of potential degradation products. • ISO 10993-10:2002/Amd 1:2006 Part 10: Tests for irritation and delayed-type hypersensitivity.

Standards

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• ISO 10993-11:2006

Part 11: Tests for systemic toxicity.

• ISO 10993-12:2002

Part 12: Sample preparation and reference materials.

• ISO 10993-13:1998

Part 13: Identification and quantification of degradation products from polymeric medical devices.

• ISO 10993-14:2001

Part 14: Identification and quantification of degradation products from ceramics.

• ISO 10993-15:2000

Part 15: Identification and quantification of degradation products from metals and alloys.

Standards

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• ISO 10993-16:1997 Part 16: Toxicokinetic study for degradation products and leachable.

• ISO 10993-17:2002 Part 17: Establishment of allowable limits for leachable substances. • ISO 10993-18:2005 Part 18: Chemical characterization of materials. • ISO 10993-19:2006 Part 19: Physico-chemical, morphological and topographical characterization of materials. • ISO 10993-20:2006 Part 20: Principles and methods for immunotoxicology testing of medical devices.

Standards

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Standards

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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.

Biological Analyses

BACKGROUND CONCEPTS

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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.

• For 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 organs and cells

(delivered dose).

Biological Analyses

BACKGROUND CONCEPTS

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DELIVERED AND EXPOSURE DOSES

o The cells that are most sensitive are referred to as the target cells.

o 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.

Biological Analyses

BACKGROUND CONCEPTS

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SAFETY FACTORS

• This practice requires being able to exaggerate the anticipated human clinical dosage

in the nonhuman test system.

• Local toxicity model in animals: there is ample opportunity for reducing the target

cell dose by distribution, diffusion, metabolism, and changes in the number of

exposed cells (because of the inflammatory response).

• Cell culture models: the variables of metabolism, distribution, and absorption are

minimized, the dosage per cell is maximized to produce a highly sensitive test

system.

Biological Analyses

BACKGROUND CONCEPTS

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

This introduction: common to all biomaterials biological testing.

Biological Analyses

BACKGROUND CONCEPTS

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Tests

IN VITRO

EX VIVO

IN VIVO

Biological Analyses

BACKGROUND CONCEPTS

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• Evaluation under in vitro (literally "in glass") conditions: rapid and inexpensive data on biological interaction .

No one material will be appropriate for all medical device application. The material, its composition and degradation products may affect host cells

and tissues. The host environment may also affect material properties and device

performance.

• The question: will the in vitro test measure parameters relevant to what will occur in vivo environment?

IN VITRO

Biological Analyses

BACKGROUND CONCEPTS

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IN VITRO TESTING

Types of in - Vitro tests for estimating biocompatibility :

Cytotoxicity – Elution or extract test (MEM Elution) Direct contact test Agar or agarose overlay test

Hemocompatibility - Hemolysis assay

Clotting and complement activation Mutagenecity – Ames test Hypersensitivity – Lymphocyte transformation test

Leukocyte migration & inhibition test

Biological Analyses

BACKGROUND CONCEPTS

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• Three primary cell culture assays are used for evaluating biocompatibility (Cytotoxicity):

o Direct contact o Agar diffusion o 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.

IN VITROCytotoxicity

Biological Analyses

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In this method, a piece of test material is placed directly onto cells growing on culture medium.

Cell cultures are grown to a standard monolayer. During incubation, leachable chemicals in the test material can diffuse into

the culture medium and contact the cell layer. Subsequently, the monolayers are examined microscopically for the

presence of morphological changes, reduction in cell density or lysis of cells around the test material.

DIRECT CONTACT METHOD

Biological Analyses IN VITRO

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DIRECT CONTACT METHOD• A near-confluent monolayer of L-929 mammalian fibroblast cells is prepared in a

35 mm diameter cell culture plate. • Old cell culture media (agar generally) is removed .• Fresh media is added .• Material being tested is placed onto the cultures, which are incubated for 24 hours

at 37 degrees Celsius (Incubator in 37 degrees Celsius, 5% CO2 , Humidity 90-95%).

(Specimen of negative or positive controls and the test article are carefully placed in prepare culture and incubated for 24 hour).• The material is removed .• The culture media is removed .• The remaining cells are fixed and stained by the cytochemical stain. • Dead cells are lost during fixation and only the live cells are stained .• The toxicity of the material is indicated by the absence of stained cells around the

material .


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In this method, a thin layer of nutrient-supplemented agar is placed over the cultured cells (L-929 mouse fibroblast cells ).The test material (or an extract of the test material dried on filter paper/sample) is placed on top of the agar layer, and the cells are incubated for 24 hours. Cytotoxic leachates diffuse into the cell layer via the agar, and a zone of malformed, degenerative or lysed cells under and around the test material indicates cytotoxicity.

AGAR DIFFUSION METHOD


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AGAR DIFFUSION METHOD • A near confluent layer of L-929 mammalian fibroblast cells are prepared in a culture

plate .• Old cell culture media is removed .• The cells are covered with a solution of 2% agar, which often contains red vital stain. • 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 (Incubator in 37 degrees Celsius, 5% CO2 , Humidity 90-95%).

• This assay also contain red stain in the agar mixture, which allows ready visualization of live cells.

• Healthy cells retain red stain.• Dead or injured cells do not retain neutral red and remain colorless.• Toxicity is evaluated by the loss of the stain under and around the periphery of the

specimens.


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ELUTION METHOD • A near confluent layer of L-929 mammalian fibroblast cells are prepared in a

culture plate .• An extract of the material which is being tested is prepared using

physiological saline (0.9% sodium chloride) or serum-free culture medium (the latter is generally preferred) .

• The extract is placed on the cells and incubated for 48 hours at 37 degrees Celsius (Incubator in 37 degrees Celsius, 5% CO2 , Humidity 90-95%).

• 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.– Cell type. – Duration of exposure. – Test sample size (e.g., geometry, density, shape, thickness). – Surface area of test sample.

– Cell morphologyTrypan blue (enters dead cells), neutral red (actively taken up by living cells)


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ASSAY METHODS

• In general, cell lines that have been developed for growth in vitro are preferred to primary cells that are freshly harvested from live organisms because the cell lines improve the reproducibility of the assays and reduce the variability among laboratories.

• That is, a cell line is the in vitro counterpart of inbred animal strains used for in vivo studies.

• Cell lines maintain their genetic and morphological characteristics throughout a long (sometimes called infinite) life span.


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ASSAY METHODS

• Cell lines from other tissues or species may also be used.

• Selection of a cell line is based upon the type of assay, the investigator’s experience, measurement endpoints (viability, enzymatic activity, species specific receptors, etc.), and various other factors.

• It is not necessary to use human cell lines for this testing because, by definition, these cells have undergone some dedifferentiation and lost receptors and metabolic pathways in the process of becoming cell lines.


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Organ of origin Primary cultures or isolated cells Cell lines

Nervous system Chick embryo ganglia; chick embryo brain cells;

mouse and rat cerebellum cells

C 1300 (mouse);C 6 (rat)

Lung Human, rabbit and rat alveolar macrophages

P 388Dl (mouse);A 549 (human)

Reticuloendothelialsystem

Human, mouse lymphocytes and erythrocytes;

rat and mouse peritoneal macrophages

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Liver Rat and chick embryo hepatocytes Chang (human);CC1144 (rat);

ARL (rat);RLC-GA (rat)


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SPECIAL CELLS

Chondrocyte (Cartilage) Epithelial (Skin) Hepatocyte (Liver) Endothelial (Vascular) Osteoblast (Bone) Fibroblast (Connective tissue) Schwann cell (Nerve)


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CELL TOXICITY

MTT Neutral red assay Calcein assay Alamar Blue Radioisotope 3-H-Leucine assessment Bromodeoxyuridine (BrdU) assessment Lactate dehydrogenase (LDH) assessment

MTT (Quantitative cytotoxic assays)Dehydrogenase enzyme: conversion of the water-soluble yellow dye MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble purple formazan by the action of mitochondrial reductase (ELISA).Dead cells do not have active mitochondrial reductases (as the cellular reduction is only catalyzed by living cells), MTT is not reduced and the purple formazan is not formed.


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Neutral Red (NR) assay

Lysosomal activity Membrane permeability

The neutral red (NR (3-amino-7-dimethyl-2-methylphenazine

hydrochloride)) assay is a cell survival chemo sensitivity assay.

This assay is based on the incorporation of NR into the lysosomes of

viable cells after being incubation with test agents.

Therefore, it is possible to distinguish between viable, damaged or dead

cells as viable cells take up the NR dye, damaged or dead cells do not.


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• Positive and negative controls are often included in the assays to ensure the operation and suitability of the test system.

• The negative control of choice: high-density polyethylene material or TCPS (Tissue Culture Poly Styrene).

• The positive controls: low-molecular-weight organo tin-stabilized poly (vinyl chloride), gum rubber, and dilute solutions of toxic chemicals, such as phenol and benzalkonium chloride.


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The in vitro cytotoxicity assays are the primary biocompatibility screening tests for a wide variety of elastomeric, polymeric, and other materials used in medical devices.

After the cytotoxicity profile of a material has been determined, then more application specific tests are performed to assess the biocompatibility of the material.


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HEMOCOMPATIBILITY TESTS

Are used to evaluate the effect of a material on blood coagulation processes, thrombus formation and hemolysis (destruction of red blood cells).

Materials or theirs extracts are incubated with red blood cells, isolated from rabbits, mice, or rats for three hours with intermittent shaking to keep samples mixed and in contact with blood.

The amount of hemoglobin released into the supernatant from the cells is determined spectrophotometrically and reported as percent hemolysis with respect to negative controls.

To evaluate the effect of materials and their surfaces on blood clotting, materials are exposed to whole blood serum.

Turbulent flow of blood may increase hemolysis and or clotting – the number of adherent platelets may be determined per unit area after exposure to whole blood.


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MUTAGENECITY AND GENOTOXICITY

Mutagenes – modify the genome of a host, so materials may be classified as genotoxic. It is widely accepted that carcinogenic behavior proceeds via a mutation in the genome.

Ames test – uses a mutant bacterial cell line (Salmonella typhimurium or Escherichia coli) that must be supplied with histidine to growth. The cells are cultured in histidine-free environment and only those material that mutate the cells back to state of histidine indipendence will allow the cells back to grow.Styles test (Fibroblast cell deformation , Agar gel)

Hypersensitivity Tests – the leukocyte migration inhibition and lyphocyte transformation tests have been used as in-vitro models to estimate delayed hypersensitivity reaction to implant materials and their released components.


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CONSIDERATIONS

Choice of cells Conformity of Biomaterials to tissue Repetition (Number of replicates) Statically evaluations (P-value)CAS (Cell Analysis System) or Image Pro Plus


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• 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.


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• 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.

• Current experience indicates that a material that is judged to be nontoxic in vitro will be nontoxic in in vivo assays.

• This does not necessarily mean that materials that are toxic in vitro could not be used in a given clinical application.

• The clinical acceptability of a material depends on many different factors, of which target cell toxicity is but one.

For example, glutaraldehyde-fixed porcine valves produce adverse effects in vitro owing to low residues of glutaraldehyde; however, this material has the greatest clinical efficacy for its unique application.


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Low expense Low work High speed High reproducibility Cell and tissue (biopsy) access High Performability

Advantages


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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.• From a practical perspective, the in vivo assessment of tissue

compatibility of medical devices is carried out to determine that the device performs as intended and presents no significant harm to the patient or user.

• Thus, the goal of the in vivo assessment of tissue compatibility is to predict whether a medical device presents potential harm to the patient or user by evaluations under conditions simulating clinical use.

Biological Analyses IN VIVO

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SENSITIZATION, IRRITATION, AND INTRACUTANEOUS (INTRADERMAL) REACTIVITY

• Exposure to or contact with even minute amounts of potential leachables from medical devices or biomaterials can result in allergic or sensitization reactions.

• Sensitization tests estimate the potential for contact sensitization to medical devices, materials, and/or their extracts.

• Symptoms of sensitization are often seen in skin and tests are

often carried out topically in guinea pigs.


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SENSITIZATION, IRRITATION, AND INTRACUTANEOUS (INTRADERMAL) REACTIVITY

• The most severely irritating chemical leachable may be discovered prior to in vivo studies by careful material characterization and in vitro cytotoxicity tests.

• Irritant tests emphasize utilization of extracts of the biomaterials to determine the irritant effects of potential leachable.

• Intracutaneous (intradermal) reactivity tests determine the localized reaction of tissue to intracutaneous injection of extracts of medical devices, biomaterials, or prostheses in the final product form.

• Intracutaneous tests may be applicable where determination of irritation by dermal or mucosal tests are not appropriate.

• Albino rabbits are most commonly used.


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Systemic Toxicity Acute, Subacute, and Subchronic Toxicity

Mice, rats, or rabbits are the usual animals of choice for the conduct of these tests and oral, dermal, inhalation, intravenous, intraperitoneal, or subcutaneous application of the test substance may be used, depending on the intended application of the biomaterial.


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SYSTEMIC TOXICITY ACUTE, SUBACUTE, AND SUBCHRONIC TOXICITY

• Acute toxicity is considered to be the adverse effects that occur after administration of a single dose or multiple doses of a test sample given within 24 hours.

• Subacute toxicity (repeat-dose toxicity) focuses on adverse effects occurring after administration of a single dose or multiple doses of a test sample per day during a period of from 14 to 28 days.


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SYSTEMIC TOXICITY ACUTE, SUBACUTE, AND SUBCHRONIC TOXICITY

• Pyrogenicity tests are also included in the systemic toxicity category to detect material-mediated fever-causing reactions to extracts of medical devices or materials.

• It is noteworthy that no single test can differentiate pyrogenic

reactions that are material-mediated from those due to endotoxin contamination.


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GENOTOXICITY• In vivo genotoxicity tests are carried out if indicated by the

chemistry and/or composition of the biomaterial or if in vitro test results indicate potential genotoxicity [changes in deoxyribonucleic acid (DNA)].

• Initially, at least three in vitro assays should be used and two of these assays should utilize mammalian cells.

• The initial in vitro assays should cover the three levels of genotoxic effects:

– DNA destruction. – Gene mutations.– Chromosomal aberrations.


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IMPLANTATION


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IMPLANTATION

• For short-term implantation evaluation out to 12 weeks, mice, rats, guinea pigs, or rabbits are the usual animals utilized in these studies.

• For longer-term testing in subcutaneous tissue, muscle, or bone, animals such as rats, guinea pigs, rabbits, dogs, sheep, goats, pigs, and other animals with relatively long life expectancy are suitable.


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IMPLANTATION

• If a complete medical device is to be evaluated, larger species may be utilized so that human-sized devices may be used in the site of intended application.

For example– substitute heart valves are usually tested as heart valve

replacements in sheep, – whereas calves are usually the animal of choice for ventricular

assist devices and total artificial hearts.


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Haematoxylin and eosin (H&E) staining

Haematoxylin and eosin (H&E) stainingZiehl-Neelsen stainPapanicolaou stainingMasson's trichrome….

IMPLANTATION


http://en.wikipedia.org/wiki/File:Emphysema_H_and_E.jpg

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HEMOCOMPATIBILITY OR BLOOD COMPATIBILITY

• Hemocompatibility tests evaluate effects on blood and/or blood components by blood-contacting medical devices or materials.

• In vivo hemocompatibility tests are usually designed to simulate the geometry, contact conditions, and flow dynamics of the device or material in its clinical application.

• From the IS0 standards perspective, five test categories are indicated

for hemocompatibility evaluation : Thrombosis, coagulation, platelets, hematology, and immunology

(complement and leukocytes).


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HEMOCOMPATIBILITY

In vivo testing in animals may be convenient, but species' differences in blood reactivity must be considered and these may limit the predictability of any given test in the human clinical situation.


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CHRONIC TOXICITY

Chronic toxicity tests determine the effects of either single or multiple exposures to medical devices, materials, and/or their extracts during a period of at least 10 % of the lifespan of the test animal, e.g. over 90 days in rats.

Chronic toxicity tests may be considered an extension of subchronic (subacute) toxicity testing and both may be evaluated in an appropriate experimental protocol or study.


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CARCINOGENICITY

• Carcinogenicity tests determine the tumorigenic potential of medical devices, materials, and/or their extracts from either single or multiple exposures or contacts over a period of the major portion of the lifespan of the test animal.

• Since tumors associated with medical devices have been rare carcinogenicity tests should be conducted only if data from other sources suggest a tendency for tumor induction.

• In addition, both carcinogenicity (tumorigenicity) and chronic toxicity may be studied in a single experimental study.


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REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

• These tests evaluate the potential effects of medical devices, materials, and/or their extracts on reproductive function, embryonic development (teratogenicity), and prenatal and early postnatal development.

• The application site of the device must be considered and tests and/or bioassays should only be conducted when the device has a potential impact on the reproductive potential of the subject.


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BIODEGRADATION

Biodegradation tests determine the effects of a biodegradable material and its biodegradation products on the tissue response.

• They focus on: – The amount of degradation during a given period of time (the

kinetics of biodegradation). – The nature of the degradation products. – The origin of the degradation products (e. g., impurities,

additives, corrosion products, bulk polymer).– The qualitative and quantitative assessment of degradation

products and leachable in adjacent tissues and in distant organs.


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IMMUNE RESPONSE

• Immune response evaluation is not a component of the standards currently available for in vivo tissue compatibility assessment.

• However, ASTM, IS0, and the FDA currently have working groups developing guidance documents for immune response evaluation where pertinent.

• Synthetic materials are not generally immunotoxic. • However, immune response evaluation is necessary with modified

natural tissue implants such as collagen, which has been utilized in a number of different types of implants and may elicit immunological responses.


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SELECTION OF ANIMAL MODELS FOR IN VIVO TESTS

• Animal models are used to predict the clinical behavior, safety, and biocompatibility of medical devices in humans.

• The selection of animal models for the in vivo assessment of

tissue compatibility must consider the advantages and disadvantages of the animal model for human clinical application.

• Several examples follow, which exemplify the advantages and

disadvantages of animal models in predicting clinical behavior in humans.


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SELECTION OF ANIMAL MODELS FOR IN VIVO TESTS

Thus, the choice of this animal model for bio prosthetic heart valve evaluation is made on the basis of accelerated calcification in rapidly growing animals, which has its clinical correlation in young and adolescent humans.

Nevertheless, normal sheep may not provide a sensitive assessment of the propensity of a valve to thrombosis.


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