1 BME Experiment 2010 Summer Ming-Long Yeh 8/25/2010 Biological Testing of Biomaterials From:...

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BME ExperimentBME Experiment2010 Summer2010 Summer

Ming-Long YehMing-Long Yeh8/25/20108/25/2010

Biological Testing of BiomaterialsBiological Testing of Biomaterials

From: Biomaterials Science by RatnerFrom: Biomaterials Science by Ratner

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5. Biological Testing of Biomaterials5. Biological Testing of Biomaterials

5.1 INTRODUCTION TO TESTING BIOMATERIALS5.1 INTRODUCTION TO TESTING BIOMATERIALS5.2 IN VITRO ASSESSMENT OF TISSUE 5.2 IN VITRO ASSESSMENT OF TISSUE

COMPATIBILITYCOMPATIBILITY5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITY5.4 EVALUATION OF BLOOD-MATERIALS 5.4 EVALUATION OF BLOOD-MATERIALS

INTERACTIONSINTERACTIONS5.5 LARGE ANIMAL MODELS IN CARDIAC AND 5.5 LARGE ANIMAL MODELS IN CARDIAC AND

VASCULAR BIOMATERIALS RESEARCH AND VASCULAR BIOMATERIALS RESEARCH AND TESTINGTESTING

5.6 MICROSCOPY FOR BIOMATERIALS SCIENCE5.6 MICROSCOPY FOR BIOMATERIALS SCIENCE

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5.1 INTRODUCTION TO 5.1 INTRODUCTION TO TESTING BIOMATERIALSTESTING BIOMATERIALS

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5.1 INTRODUCTION TO TESTING 5.1 INTRODUCTION TO TESTING BIOMATERIALSBIOMATERIALS

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

This introduction: common to all This introduction: common to all biomaterials biological testing.biomaterials biological testing.

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Evaluation under Evaluation under in vitroin vitro (literally "in (literally "in glass") conditions: glass") conditions: rapid and inexpensiverapid and inexpensive data on biological interaction (Chapter 5. data on biological interaction (Chapter 5. 2). 2).

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

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For example, tissue culture For example, tissue culture polystyrenepolystyrene, a , a surface modified polymer, will surface modified polymer, will readily attach and readily attach and grow most cells in culturegrow most cells in culture. .

Untreated Untreated polystyrene will polystyrene will neither attach nor neither attach nor growgrow mammalian cells. mammalian cells.

Yet when implanted Yet when implanted in vivoin vivo,, both both materials heal materials heal almost almost indistinguishablyindistinguishably with a with a thin foreign body thin foreign body capsulecapsule. .

Thus, the results of the Thus, the results of the in vitroin vitro test test do do notnot provide provide information relevant to the implant information relevant to the implant situationsituation. .

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

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

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

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AnimalsAnimals are used for testing biomaterials to are used for testing biomaterials to model the environment that might be model the environment that might be encountered in humans (Chapter 5. 3). encountered in humans (Chapter 5. 3).

However, there is However, there is great rangegreat range in animal in animal anatomy, physiology, and biochemistry. anatomy, physiology, and biochemistry.

Will the Will the animal modelanimal model provide data useful for provide data useful for predictingpredicting how a device performs in humans? how a device performs in humans?

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

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The The first step in designing animal testingfirst step in designing animal testing procedures is to choose procedures is to choose an animal modelan animal model that that offers a reasonable parallel anatomically or offers a reasonable parallel anatomically or biochemically to the situation in humans. biochemically to the situation in humans.

Experiments designedExperiments designed to to minimize the number of animals neededminimize the number of animals needed, , ensure that the ensure that the animals are treated humanelyanimals are treated humanely (e. g., (e. g.,

NIH guidelines for the use of laboratory animals), and NIH guidelines for the use of laboratory animals), and maximize the relevant information generatedmaximize the relevant information generated by the by the

testing procedure are essential.testing procedure are essential.

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Some Some biomaterials biomaterials fulfill their intended function in fulfill their intended function in seconds.seconds.

Others are Others are implanted for a lifetimeimplanted for a lifetime (10 years ? 70 (10 years ? 70 years?). years?).

Are Are 6-month implantation6-month implantation times times useful to learnuseful to learn about a about a device intended for device intended for 3-minute insertion3-minute insertion? ?

Will six months implantation in a test model provide Will six months implantation in a test model provide adequate informationadequate information to draw conclusions about the to draw conclusions about the performance of a device intended for performance of a device intended for lifetime lifetime implantation? implantation?

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

Thus, the Thus, the American Society for Testing Materials American Society for Testing Materials (ASTM)(ASTM) and the and the International Standards International Standards Organization (IS0)Organization (IS0) can often provide detailed can often provide detailed protocols for widely accepted, carefully thought protocols for widely accepted, carefully thought out testing procedures.out testing procedures.

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

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5.2 5.2 IN VITRO ASSESSMENTIN VITRO ASSESSMENT OF TISSUE COMPATIBILITYOF TISSUE COMPATIBILITY

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5.2 IN VITRO ASSESSMENT OF 5.2 IN VITRO ASSESSMENT OF TISSUE COMPATIBILITYTISSUE COMPATIBILITY

““CytotoxicityCytotoxicity”: to cause toxic effects (death, alter”: to cause toxic effects (death, alterations in cellular membrane permeability, enzymations in cellular membrane permeability, enzymatic inhibition, etc.) at the cellular level. atic inhibition, etc.) at the cellular level.

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

Evaluation of biomaterials by methods that use Evaluation of biomaterials by methods that use iisolated, adherent cells in culture to measure solated, adherent cells in culture to measure cytcytotoxicity and biological compatibility.otoxicity and biological compatibility.

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HISTORICAL REVIEWHISTORICAL REVIEW

Cell culture methods: more than Cell culture methods: more than two two decadesdecades (Northup, 1986). (Northup, 1986).

Most often today the cells used for culture Most often today the cells used for culture are from are from established cell lines established cell lines purchased purchased from biological suppliers or cell banks.from biological suppliers or cell banks.

Primary cellsPrimary cells are are seldom seldom used used less assay repeatability, reproducibility, less assay repeatability, reproducibility,

efficiency, and, in some cases, availability.efficiency, and, in some cases, availability.

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BACKGROUND CONCEPTBACKGROUND CONCEPT

ToxicityToxicity A toxic material is defined as a material that A toxic material is defined as a material that releases a creleases a c

hemical in sufficient quantities to kill cellshemical in sufficient quantities to kill cells either directly o either directly or indirectly through r indirectly through inhibition of key metabolic pathwainhibition of key metabolic pathways.ys.

The The number of cells that are affectednumber of cells that are affected is an indication of t is an indication of the he dose and potencydose and potency 效力效力 of the chemical. of the chemical.

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

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BACKGROUND CONCEPTBACKGROUND CONCEPT

Delivered and Exposure DosesDelivered and Exposure Doses Delivered doseDelivered dose: the dose that is actually absorbed by the : the dose that is actually absorbed by the

cellcell. . Exposure dose:Exposure dose: the the amount applied to a test systemamount applied to a test system. .

For example, if an animal is exposed to an atmosphere cFor example, if an animal is exposed to an atmosphere containing a ontaining a noxiousnoxious 有毒的有毒的 substancesubstance (exposure dose(exposure dose), o), only nly a small portion of the inhaled substancea small portion of the inhaled substance will be absor will be absorbed and delivered to the bed and delivered to the internal organsinternal organs and and cells cells (delive(delivered dose). red dose).

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Delivered and Exposure DosesDelivered and Exposure Doses

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

Cell culture methods:Cell culture methods: evaluate evaluate target cell toxicitytarget cell toxicity by using by using delivered dosesdelivered doses of the test substance. of the test substance.

This distinguishes This distinguishes cell culturecell culture methods from methods from whole- whole- animal studiesanimal studies, which evaluate the , which evaluate the exposure doseexposure dose and do not determine the target and do not determine the target cell dosecell dose of the test substance. of the test substance.

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Safety FactorsSafety Factors

A A highly sensitive test systemhighly sensitive test system is desirable for is desirable for evaluating the potential hazards of biomaterials evaluating the potential hazards of biomaterials because the because the inherent characteristics of the inherent characteristics of the materialsmaterials often often do not allow the dosedo not allow the dose to be to be exaggeratedexaggerated. .

There is a There is a great deal of uncertaintygreat deal of uncertainty in in extrapolating from one extrapolating from one test system to anothertest system to another, , such as from such as from animals to humansanimals to humans. .

To allow for this, toxicologists have used the To allow for this, toxicologists have used the concept of concept of safety factorssafety factors to take into account to take into account intra- and interspecies variation. intra- and interspecies variation.

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Safety FactorsSafety Factors This practice requires being able to This practice requires being able to exaggerate texaggerate t

he anticipatedhe anticipated 預期預期 human clinical dosagehuman clinical dosage in the in the nonhuman test system. nonhuman test system.

Local toxicity model in animals:Local toxicity model in animals: there is ample o there is ample opportunity for pportunity for reducing the target cell dosereducing the target cell dose by by disdistribution, diffusion, metabolism, and changestribution, diffusion, metabolism, and changes in t in the number of exposed cells (because of the inflahe number of exposed cells (because of the inflammatory response). mmatory response).

Cell culture models:Cell culture models: the the variables of metabolism, variables of metabolism, distribution, and absorption are minimizeddistribution, and absorption are minimized, the , the ddosage per cellosage per cell is maximized to produce a highly is maximized to produce a highly sensitive test system.sensitive test system.

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ASSAY METHODSASSAY METHODS

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Three primary Three primary cell culturecell culture assays assays are are used for evaluating biocompatibility: used for evaluating biocompatibility: direct contact, direct contact, agar diffusion, agar diffusion, elutionelution (also known as extract dilution). (also known as extract dilution).

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

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cell culturecell culture assays assays

http://en.wikipedia.org/wiki/Cell_Culture_Assayshttp://en.wikipedia.org/wiki/Cell_Culture_Assays

direct contact, direct contact, agar diffusion, agar diffusion, elutionelution

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Direct contact methodDirect contact method

1.1. A near confluent layer of A near confluent layer of fibroblastsfibroblasts are prepared in a are prepared in a culture plate culture plate

2.2. Old cell culture media (Old cell culture media (agaragar generally) is removed generally) is removed 3.3. Fresh media is added Fresh media is added 4.4. Material being tested is placed onto the cultures,Material being tested is placed onto the cultures, which which

are incubated for 24 hours at 37 degrees Celsius are incubated for 24 hours at 37 degrees Celsius 5.5. The material is removed The material is removed 6.6. The culture media is removed The culture media is removed 7.7. The remaining cells are fixed and stained, The remaining cells are fixed and stained, dead cells dead cells

are lost during fixation and only the live cells are stainedare lost during fixation and only the live cells are stained 8.8. The toxicity of the material is indicated by the absence The toxicity of the material is indicated by the absence

of stained cells around the material of stained cells around the material

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Agar diffusion method Agar diffusion method 1.1. A near confluent layer of A near confluent layer of fibroblastsfibroblasts are prepared in a cu are prepared in a cu

lture plate lture plate 2.2. Old cell culture media is removed Old cell culture media is removed 3.3. The cells are covered with a The cells are covered with a solution of 2% agarsolution of 2% agar, which , which

often contains red often contains red vital stainvital stain 4.4. When the agar solidifies the cells will have dispersed thrWhen the agar solidifies the cells will have dispersed thr

oughout its volume oughout its volume 5.5. The The material is then placed on the surface of the agarmaterial is then placed on the surface of the agar a a

nd incubated for 24 hours at 37 degrees Celsius nd incubated for 24 hours at 37 degrees Celsius 6.6. Live cells take up the Live cells take up the vital stainvital stain and retain it, dead cells and retain it, dead cells

do not do not 7.7. The toxicity of the material is evaluated by the loss of vitThe toxicity of the material is evaluated by the loss of vit

al stain under and around the material al stain under and around the material 8.8. Surface Surface microscopymicroscopy is also needed to evaluate the mate is also needed to evaluate the mate

rial-cell interfacae rial-cell interfacae

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Elution method Elution method

1.1. A near confluent layer of A near confluent layer of fibroblastsfibroblasts are prepared in a cu are prepared in a culture plate lture plate

2.2. An extract of the material which is being tested is preparAn extract of the material which is being tested is prepared using ed using physiological salinephysiological saline or serum free media (the l or serum free media (the latter is generally preferred) atter is generally preferred)

3.3. Extraction conditions are used which are appropriate for Extraction conditions are used which are appropriate for the type of exposure which the the type of exposure which the cells would receive in thcells would receive in the e in vivoin vivo environment if the material were to be implante environment if the material were to be implanted d

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

5.5. After 48 hours the toxicity is evaluated using either a hisAfter 48 hours the toxicity is evaluated using either a histochemical or tochemical or vital stainvital stain

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To To standardize the methodsstandardize the methods and and compare the compare the results of these assaysresults of these assays, , the variables the variables number of cells, number of cells, growth phase of the cellsgrowth phase of the cells (period of frequent (period of frequent

cell replication), cell replication), cell typecell type, , duration of exposureduration of exposure, , test sample sizetest sample size (e.g., geometry, density, (e.g., geometry, density,

shape, thickness), and shape, thickness), and surface area of test samplesurface area of test sample

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ASSAY METHODSASSAY METHODS In general, In general, cell linescell lines that have been developed f that have been developed f

or growth or growth in vitroin vitro are are preferred preferred to primary cellsto primary cells th that are at are freshly harvestedfreshly harvested from from live organismslive organisms bec because the ause the cell lines improve the reproducibilitycell lines improve the reproducibility of t of the he assays and reduce the variability amongassays and reduce the variability among labo laboratories. ratories.

That is, That is, a cell linea cell line is the is the in vitroin vitro counterpart of counterpart of ininbredbred 近親交配的近親交配的 animal strainsanimal strains used for used for in vivoin vivo stud studies. ies.

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

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ASSAY METHODSASSAY METHODS Cell lines from Cell lines from other tissues or speciesother tissues or species may also may also

be used. be used. Selection of a cell line is based upon the type of Selection of a cell line is based upon the type of

assayassay, the , the investigator’s experienceinvestigator’s experience, , measurement endpointsmeasurement endpoints (viability, enzymatic (viability, enzymatic activity, species specific receptors, etc.), and activity, species specific receptors, etc.), and various other factors. various other factors.

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

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

The The negative control of choicenegative control of choice: high-density : high-density polypolyethylene materialethylene material. .

The positive controls:The positive controls: low-molecular-weight orgalow-molecular-weight organotin-stabilized poly (vinyl chloride),notin-stabilized poly (vinyl chloride), gum rubber, gum rubber, and dilute solutions of toxic chemicals, such as and dilute solutions of toxic chemicals, such as pphenol and benzalkonium chloridehenol and benzalkonium chloride. .

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CLINICAL USECLINICAL USE

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

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

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CLINICAL USECLINICAL USE Current experience indicates that a material that is Current experience indicates that a material that is

judged to be judged to be nontoxic nontoxic in vitroin vitro will be nontoxic in will be nontoxic in in vivoin vivo assays. assays.

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

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

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

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5.3 5.3 IN VIVOIN VIVO ASSESSMENT ASSESSMENT OF TISSUE OF TISSUE

COMPATIBILITYCOMPATIBILITY

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5.3 IN VIVO ASSESSMENT OF 5.3 IN VIVO ASSESSMENT OF TISSUE COMPATIBILITYTISSUE COMPATIBILITY

INTRODUCTIONINTRODUCTIONThe goal of The goal of in vivoin vivo assessment of tissue assessment of tissue

compatibility of a biomaterial, prosthesis, compatibility of a biomaterial, prosthesis, or medical device is to determine the or medical device is to determine the biocompatibility or safetybiocompatibility or safety of the of the biomaterial, prosthesis, or medical device biomaterial, prosthesis, or medical device in a biological environment.in a biological environment.

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INTRODUCTIONINTRODUCTION

Biocompatibility has been defined as the Biocompatibility has been defined as the ability of a medical device to perform with ability of a medical device to perform with an appropriate host response in a specific an appropriate host response in a specific applicationapplication, and , and

biocompatibility assessmentbiocompatibility assessment is considered is considered to be a to be a measurement of the magnitude measurement of the magnitude and durationand duration of the adverse alterations in of the adverse alterations in homeostatic mechanisms that determine homeostatic mechanisms that determine the host response. the host response.

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From a practical perspective, the From a practical perspective, the in vivoin vivo assessment of tissue compatibilityassessment of tissue compatibility of medical of medical devices is carried out to determine that the devices is carried out to determine that the device performs as intended and presents no device performs as intended and presents no significant harm to the patient or usersignificant harm to the patient or user. .

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

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Recently, extensive efforts have been made by Recently, extensive efforts have been made by government agencies, i. e., FDA, and regulatory government agencies, i. e., FDA, and regulatory bodies, i.e., ASTM, IS0, and USP, to provide bodies, i.e., ASTM, IS0, and USP, to provide procedures, protocols, guidelines, and standardsprocedures, protocols, guidelines, and standards that may be used in the that may be used in the in vivoin vivo assessment of assessment of the tissue compatibility of medical devices. the tissue compatibility of medical devices.

This chapter draws heavily on the This chapter draws heavily on the IS0 10993 standardIS0 10993 standard, , Biological Evaluation of Biological Evaluation of Medical DevicesMedical Devices, in presenting a systematic , in presenting a systematic approach to the approach to the in vivoin vivo assessment of tissue compatibility of medical de assessment of tissue compatibility of medical devices.vices.

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List of the standards in the 10993 seriesList of the standards in the 10993 series ISO 10993-1:2003 Biological evaluation of ISO 10993-1:2003 Biological evaluation of

medical devices Part 1: medical devices Part 1: Evaluation and testingEvaluation and testing ISO 10993-2:2006 Biological evaluation of ISO 10993-2:2006 Biological evaluation of

medical devices Part 2: medical devices Part 2: Animal welfare Animal welfare requirements requirements

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

ISO 10993-18:2005 Biological evaluation of medical ISO 10993-18:2005 Biological evaluation of medical devices Part 18: devices Part 18: Chemical characterization of materChemical characterization of materialsials

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

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

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BIOMATERIALS AND DEVICE BIOMATERIALS AND DEVICE PERSPECTIVES IN PERSPECTIVES IN IN VIVOIN VIVO TESTINGS TESTINGS

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BIOMATERIALS AND DEVICE BIOMATERIALS AND DEVICE PERSPECTIVES IN IN VIVO TESTINGSPERSPECTIVES IN IN VIVO TESTINGS

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Sensitization, irritationSensitization, irritation 刺激刺激 , and intracutane, and intracutaneous (intradermal) Reactivityous (intradermal) Reactivity

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

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

Symptoms of sensitization are often seen in Symptoms of sensitization are often seen in skin skin and tests are often carried out topically in and tests are often carried out topically in guinea guinea pigs. pigs.

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Sensitization, irritation, and intracutaneous (iSensitization, irritation, and intracutaneous (intradermal) Reactivityntradermal) Reactivity

The most severely irritating chemical leachables may be The most severely irritating chemical leachables may be discovered discovered prior to prior to in vivoin vivo studies studies by careful by careful material chamaterial characterization and racterization and in vitroin vitro cytotoxicity tests. cytotoxicity tests.

Irritant testsIrritant tests emphasize utilization of emphasize utilization of extracts of the biomextracts of the biomaterialsaterials to determine the to determine the irritant effects of potentialirritant effects of potential leach leachables. ables.

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

Intracutaneous testsIntracutaneous tests may be applicable where determina may be applicable where determination of irritation by dermal or mucosal tests are not approtion of irritation by dermal or mucosal tests are not appropriate. priate.

Albino rabbitsAlbino rabbits are most commonly used. are most commonly used.

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Systemic Toxicity: Systemic Toxicity: Acute, Subacute, and Subchronic ToxicityAcute, Subacute, and Subchronic Toxicity

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

Peritoneal Peritoneal 腹膜的

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

Subacute toxicitySubacute toxicity (repeat-dose toxicity) focuses on adver (repeat-dose toxicity) focuses on adverse effects occurring after administration of a single dose se effects occurring after administration of a single dose or multiple doses of a test sample per day during a perioor multiple doses of a test sample per day during a period of from d of from 14 to 28 days14 to 28 days. .

Sub-chronic toxicitySub-chronic toxicity is considered to be the adverse effe is considered to be the adverse effects occurring after administration of a single dose or multcts occurring after administration of a single dose or multiple doses of a test sample per day given iple doses of a test sample per day given during a part of during a part of the life spanthe life span, usually 90 days , usually 90 days but not exceeding 10 % of tbut not exceeding 10 % of the life span of the animal.he life span of the animal.

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

It is noteworthy that It is noteworthy that no single testno single test can diffe can differentiate rentiate pyrogenic reactionspyrogenic reactions that are that are matermaterial-mediatedial-mediated from those due to endotoxin c from those due to endotoxin contaminationontamination

Pyrogenicity:Pyrogenicity: producing or produced by heat or fever

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GenotoxicityGenotoxicity In vivoIn vivo genotoxicity tests are carried out if indicated by th genotoxicity tests are carried out if indicated by th

e chemistry and/or composition of the biomaterial (see Te chemistry and/or composition of the biomaterial (see Table 1) or if able 1) or if in vitroin vitro test results indicate potential genotoxi test results indicate potential genotoxicity [changes in deoxyribonucleic acid (DNA)]. city [changes in deoxyribonucleic acid (DNA)].

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

The initial The initial in vitroin vitro assays should cover the three levels of assays should cover the three levels of genotoxic effectsgenotoxic effects: : DNA destruction, DNA destruction, gene mutations, and gene mutations, and chromosomal aberrations .chromosomal aberrations .

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ImplantationImplantation

Dystrophic: 營養不良的

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

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

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

For example, For example, substitute substitute heart valvesheart valves are usually tested as are usually tested as

heart valve replacements in heart valve replacements in sheepsheep, , whereaswhereas calves calves are usually the animal of are usually the animal of

choice for choice for ventricular assist devices and total ventricular assist devices and total artificial hearts.artificial hearts.

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HemocompatibilityHemocompatibility Hemocompatibility tests evaluate effects on Hemocompatibility tests evaluate effects on bloobloo

d and/or blood componentsd and/or blood components by by blood-contacting blood-contacting medical devices or materialsmedical devices or materials. .

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

From the IS0 standards perspective, five test catFrom the IS0 standards perspective, five test categories are indicated for hemocompatibility evalegories are indicated for hemocompatibility evaluation : uation : thrombosisthrombosis 血栓血栓 , coagulation, coagulation 凝結凝結 , platel, platelets, hematologyets, hematology 血液學血液學 , and immunology, and immunology (compl (complement and leukocytes). ement and leukocytes).

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HemocompatibilityHemocompatibility

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

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Chronic ToxicityChronic Toxicity

Chronic toxicity tests determine the effects of eitChronic toxicity tests determine the effects of either her single or multiple exposures to medical devicsingle or multiple exposures to medical devices, materials, and/or their extractses, materials, and/or their extracts during a perio during a period of d of at leastat least 10 % of the lifespan10 % of the lifespan of the test anim of the test animal, e.g. over 90 days in rats. al, e.g. over 90 days in rats.

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

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CarcinogenicityCarcinogenicity Carcinogenicity tests determine the Carcinogenicity tests determine the tumorigenic potential tumorigenic potential

of medical devices, materials, and/or their extracts from of medical devices, materials, and/or their extracts from either single or multiple exposures or contacts over a pereither single or multiple exposures or contacts over a period of theiod of the major portion of the lifespan of the test animal. major portion of the lifespan of the test animal.

Since tumors associated with medical devices have been Since tumors associated with medical devices have been rare rare (see Chapter 4. 7) carcinogenicity tests should be c(see Chapter 4. 7) carcinogenicity tests should be conducted onducted only if data from other sourcesonly if data from other sources suggest a tende suggest a tendency for tumor induction. ncy for tumor induction.

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

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Reproductive and Developmental ToxicityReproductive and Developmental Toxicity

These tests evaluate the potential effects of medThese tests evaluate the potential effects of medical devices, materials, and/or their extracts on ical devices, materials, and/or their extracts on rreproductive function, embryonic development (teeproductive function, embryonic development (teratogenicity), and prenatalratogenicity), and prenatal 產前的產前的 and early postand early postnatal developmentnatal development. .

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

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BiodegradationBiodegradation Biodegradation tests determine the effects of a Biodegradation tests determine the effects of a

biodegradable material and its biodegradation biodegradable material and its biodegradation productsproducts on the tissue response. on the tissue response.

They focus on They focus on the the amount of degradation during a given period of amount of degradation during a given period of

timetime (the kinetics of biodegradation), (the kinetics of biodegradation), the nature of the degradation productsthe nature of the degradation products, , the the origin origin of the degradation products (e. g., of the degradation products (e. g.,

impurities, additives, corrosion products, bulk impurities, additives, corrosion products, bulk polymer), and polymer), and

the the qualitative and quantitative assessment of qualitative and quantitative assessment of degradation products and leachablesdegradation products and leachables in adjacent in adjacent tissues and in distant organs. tissues and in distant organs.

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Immune ResponseImmune Response Immune response evaluation Immune response evaluation is not a componentis not a component of the of the

standards currently available for in vivo tissuestandards currently available for in vivo tissue compatibility assessment. compatibility assessment.

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

SyntheticSynthetic materials are materials are not generally immunotoxicnot generally immunotoxic. . However, immune response evaluation is necessary with However, immune response evaluation is necessary with

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

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過敏的

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SELECTION OF ANIMAL MODELS FOR IN SELECTION OF ANIMAL MODELS FOR IN VIVO TESTSVIVO TESTS

蕁麻疹

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Immune ResponseImmune Response

Direct measures of immune system activityDirect measures of immune system activity by by fufunctional assaysnctional assays are the most important types of t are the most important types of tests for immunotoxicity. ests for immunotoxicity.

Functional assaysFunctional assays are generally more are generally more important important tthan tests for han tests for soluble mediatorssoluble mediators, which are more i, which are more important than mportant than phenotypingphenotyping. .

Signs of illnessSigns of illness may be important in may be important in in vivoin vivo exper experiments but symptoms may also have a significaniments but symptoms may also have a significant role in studies of t role in studies of immune functionimmune function in clinical tria in clinical trials and ls and postmarket studiespostmarket studies..

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SELECTION OF ANIMAL MODELS SELECTION OF ANIMAL MODELS FOR IN VIVO TESTSFOR IN VIVO TESTS

Animal models are used to predict the clinical Animal models are used to predict the clinical behavior, safety, and biocompatibility of medical behavior, safety, and biocompatibility of medical devices in humans devices in humans (Table7(Table7). ).

The selection of animal models for the in vivo The selection of animal models for the in vivo assessment of tissue compatibility must consider assessment of tissue compatibility must consider the advantages and disadvantages of the animal the advantages and disadvantages of the animal model for human clinical application. model for human clinical application.

Several examples follow, which exemplify the Several examples follow, which exemplify the advantages and disadvantages of animal advantages and disadvantages of animal models in predicting clinicalmodels in predicting clinical behavior in humans behavior in humans (also see Chapter 5. 5).(also see Chapter 5. 5).

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

Nevertheless, normal sheep may Nevertheless, normal sheep may notnot provide a provide a sensitive assessment of the sensitive assessment of the propensity of a valve to propensity of a valve to thrombosisthrombosis, ,

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The The in vivoin vivo assessment assessment of tissue of tissue responses to responses to vascular graft materialsvascular graft materials is is an example in which an example in which animal models animal models presents particularly presents particularly misleading misleading picturepicture of what generally occurs in of what generally occurs in humans. humans.

Virtually all animal models, including Virtually all animal models, including nonhuman primates, heal rapidly and nonhuman primates, heal rapidly and completely with an endothelial blood-completely with an endothelial blood-contacting surface.contacting surface.

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Humans, on the other hand, Humans, on the other hand, do not show extensivdo not show extensive endothelializatione endothelialization of vascular graft materials and of vascular graft materials and the the resultant pseudointima from the healing responsresultant pseudointima from the healing response in humans has potential thrombogenicity. e in humans has potential thrombogenicity.

Consequently, despite Consequently, despite favorable results in animalsfavorable results in animals, , small-diameter vascular grafts (less than small-diameter vascular grafts (less than 4mm4mm in int in internal diameter) yield early thrombosis in humansernal diameter) yield early thrombosis in humans, th, the mayor mechanism of failure, which is secondary to e mayor mechanism of failure, which is secondary to the the lack of endothelialization in the luminal surface hlack of endothelialization in the luminal surface healing response.ealing response.

intima 內膜

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STANDARDSSTANDARDS

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Preoperative CarePreoperative Care

AnesthesiaAnesthesia Dogs should be Dogs should be fasted for 12fasted for 12 hour hours prior to their operation to s prior to their operation to reduce the risk of aspreduce the risk of aspirationiration 肺內異物的吸入肺內異物的吸入 during anesthetic induction anduring anesthetic induction and endotracheal intubationd endotracheal intubation 氣管內插管氣管內插管 . .

Preparation of the animal involves administration Preparation of the animal involves administration of a mild of a mild intramuscular (IMintramuscular (IM) sedative such as ) sedative such as acacepromazine,epromazine, a a phenothiazine tranquillizerphenothiazine tranquillizer. .

Peripheral Peripheral intravenous (IVintravenous (IV) accesses can then b) accesses can then be established. e established.

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Preoperative Care Preoperative Care AnesthesiaAnesthesia

Prior to intubation, Prior to intubation, 0.05 mg/kg of atropine0.05 mg/kg of atropine 阿托品阿托品 ,, an anticholinergican anticholinergic 抗副交感神經藥抗副交感神經藥 .,., may be administer may be administered to reduce the amount of oral secretions and eed to reduce the amount of oral secretions and ease endotracheal (ET) intubation. ase endotracheal (ET) intubation.

Once a secure airway has been obtained, Once a secure airway has been obtained, anestanesthesiahesia can be fully induced with a barbiturate suc can be fully induced with a barbiturate such as thiopental sodium (12.5 mg / kg IV). h as thiopental sodium (12.5 mg / kg IV).

Table 3 lists several of the recommended Table 3 lists several of the recommended drugs drugs and dosesand doses for sedation and anesthesia in dogs. for sedation and anesthesia in dogs.

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Preoperative CarePreoperative Care

AnesthesiaAnesthesia Dogs should be Dogs should be fasted for 12fasted for 12 hour hours prior to their operation to s prior to their operation to reduce the risk of aspreduce the risk of aspirationiration 肺內異物的吸入肺內異物的吸入 during anesthetic induction anduring anesthetic induction and endotracheal intubationd endotracheal intubation 氣管內插管氣管內插管 . .

Preparation of the animal involves administration Preparation of the animal involves administration of a mild of a mild intramuscular (IMintramuscular (IM) sedative such as ) sedative such as acacepromazine,epromazine, a a phenothiazine tranquillizerphenothiazine tranquillizer. .

Peripheral Peripheral intravenous (IVintravenous (IV) accesses can then b) accesses can then be established. e established.

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Preoperative Care Preoperative Care AnesthesiaAnesthesia

Prior to intubation, Prior to intubation, 0.05 mg/kg of atropine0.05 mg/kg of atropine 阿托品阿托品 ,, an anticholinergican anticholinergic 抗副交感神經藥抗副交感神經藥 .,., may be administer may be administered to reduce the amount of oral secretions and eed to reduce the amount of oral secretions and ease endotracheal (ET) intubation. ase endotracheal (ET) intubation.

Once a secure airway has been obtained, Once a secure airway has been obtained, anestanesthesiahesia can be fully induced with a barbiturate suc can be fully induced with a barbiturate such as thiopental sodium (12.5 mg / kg IV). h as thiopental sodium (12.5 mg / kg IV).

Table 3 lists several of the recommended Table 3 lists several of the recommended drugs drugs and dosesand doses for sedation and anesthesia in dogs. for sedation and anesthesia in dogs.

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Swine vascular graftSwine vascular graft

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1 BME Experiment 2010 Summer Ming-Long Yeh 8/25/2010 Biological Testing of Biomaterials From:...

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